Re:미토콘드리아 질환: from molecular mechanisms to therapeutic 2025

[문형철]

1. nature  
2. signal transduction and targeted therapy  
3. review articles  
4. article

• Review Article
• Open access
• Published: 10 January 2025

Mitochondrial diseases: from molecular mechanisms to therapeutic advances

• Haipeng Wen, 
• Hui Deng, 
• Bingyan Li, 
• Junyu Chen, 
• Junye Zhu, 
• Xian Zhang, 
• Shigeo Yoshida & 
• Yedi Zhou 

Signal Transduction and Targeted Therapy volume 10, Article number: 9 (2025) Cite this article

•  
•  
•  

Abstract

Mitochondria are essential for cellular function and viability, serving as central hubs of metabolism and signaling. They possess various metabolic and quality control mechanisms crucial for maintaining normal cellular activities. Mitochondrial genetic disorders can arise from a wide range of mutations in either mitochondrial or nuclear DNA, which encode mitochondrial proteins or other contents. These genetic defects can lead to a breakdown of mitochondrial function and metabolism, such as the collapse of oxidative phosphorylation, one of the mitochondria’s most critical functions. Mitochondrial diseases, a common group of genetic disorders, are characterized by significant phenotypic and genetic heterogeneity. Clinical symptoms can manifest in various systems and organs throughout the body, with differing degrees and forms of severity. The complexity of the relationship between mitochondria and mitochondrial diseases results in an inadequate understanding of the genotype-phenotype correlation of these diseases, historically making diagnosis and treatment challenging and often leading to unsatisfactory clinical outcomes. However, recent advancements in research and technology have significantly improved our understanding and management of these conditions. Clinical translations of mitochondria-related therapies are actively progressing. This review focuses on the physiological mechanisms of mitochondria, the pathogenesis of mitochondrial diseases, and potential diagnostic and therapeutic applications. Additionally, this review discusses future perspectives on mitochondrial genetic diseases.

초록

미토콘드리아는 

세포 기능과 생존에 필수적이며, 대사 및 신호전달의 중심 역할을 합니다. 

이들은 

정상적인 세포 활동을 유지하는 데 중요한 

다양한 대사 및 품질 관리 메커니즘을 갖추고 있습니다. 

미토콘드리아 유전 질환은 

미토콘드리아 단백질이나 기타 내용을 암호화하는 미토콘드리아 또는 

핵 DNA의 다양한 돌연변이로 인해 발생할 수 있습니다. 

이러한 유전적 결함은 

미토콘드리아의 가장 중요한 기능 중 하나인 산화적 인산화 반응의 붕괴와 같은 

미토콘드리아 기능 및 대사 장애를 초래할 수 있습니다. 

미토콘드리아 질환은 

흔한 유전 질환으로, 표현형 및 유전적 이질성이 두드러지는 것이 특징입니다. 

임상 증상은 

신체 전반의 다양한 시스템과 장기에서 나타날 수 있으며, 

심각도의 정도와 형태가 다양합니다. 

미토콘드리아와 미토콘드리아 질환 간의 복잡한 관계는 

이러한 질환의 유전자형-표현형 상관관계에 대한 이해가 부족하게 만들었으며, 

역사적으로 진단과 치료가 어려웠고 

종종 불만족스러운 임상 결과를 초래했습니다. 

그러나 

최근 연구와 기술의 발전은 

이러한 질환에 대한 이해와 관리 능력을 크게 향상시켰습니다. 

미토콘드리아 관련 치료법의 임상적 적용이 

활발히 진행 중입니다. 

미토콘드리아 유전 질환은 세포의 에너지 생산 공장인 미토콘드리아의 기능 이상으로 발생하는 광범위한 질병 그룹입니다. 이러한 질환은 미토콘드리아 자체의 유전체인 **미토콘드리아 DNA(mtDNA)**의 돌연변이 또는 미토콘드리아 기능에 필요한 단백질을 암호화하는 **핵 DNA(nDNA)**의 돌연변이에 의해 발생할 수 있습니다.

이 질환들은 매우 다양한 증상을 보이며, 거의 모든 기관에 영향을 미칠 수 있지만, 특히 에너지 요구량이 높은 기관(뇌, 근육, 심장, 간, 신장 등)에 더 심각한 영향을 미치는 경향이 있습니다.

다음은 주요 미토콘드리아 유전 질환의 종류입니다:

1. 미토콘드리아 DNA (mtDNA) 돌연변이 관련 질환 (모계 유전)

• 멜라스 증후군 (MELAS Syndrome):
  • 정식 명칭: Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes
  • 특징: 뇌병증(간질, 치매), 젖산 산증(lactate acidosis), 뇌졸중 유사 에피소드 등이 나타납니다. 가장 흔한 mtDNA 질환 중 하나입니다.
• 머프 증후군 (MERRF Syndrome):
  • 정식 명칭: Myoclonic Epilepsy with Ragged Red Fibers
  • 특징: 근간대성 간질(myoclonic epilepsy), 근병증, 운동실조, 얼룩덜룩한 붉은 섬유(근육 생검에서 관찰되는 비정상 미토콘드리아 축적) 등이 나타납니다.
• 레버 시신경 위축증 (LHON):
  • 정식 명칭: Leber's Hereditary Optic Neuropathy
  • 특징: 주로 젊은 성인 남성에게서 급성 또는 아급성으로 발생하는 양측성 시력 상실이 특징입니다. 시신경의 손상으로 인해 발생합니다.
• 나르프 증후군 (NARP Syndrome):
  • 정식 명칭: Neuropathy, Ataxia, Retinitis Pigmentosa
  • 특징: 신경병증, 운동실조, 망막색소변성증(retinitis pigmentosa), 인지 장애 등이 나타날 수 있습니다. 같은 mtDNA 돌연변이라도 더 심한 경우 Leigh 증후군으로 나타나기도 합니다.
• 컨스-세이어 증후군 (Kearns-Sayre Syndrome, KSS) / 만성 진행성 외안근 마비 (Chronic Progressive External Ophthalmoplegia, CPEO):
  • 특징: KSS는 눈꺼풀 처짐(ptosis), 외안근 마비, 망막색소변성증의 세 가지 주요 증상을 포함하며, 심장 전도 장애 및 소뇌 실조증 등 다양한 증상을 동반할 수 있습니다. CPEO는 KSS의 경증 형태로 눈꺼풀 처짐과 안구 운동 제한이 주로 나타납니다. mtDNA의 대규모 결실(large deletion)이 원인인 경우가 많습니다.

2. 핵 DNA (nDNA) 돌연변이 관련 질환 (상염색체 우성/열성, X-연관 유전)

• 리 증후군 (Leigh Syndrome):
  • 특징: 영아 또는 유아기에 발병하는 심각한 신경 퇴행성 질환입니다. 뇌간 및 기저핵 병변이 특징이며, 경련, 발달 지연, 운동 능력 상실, 호흡 부전 등을 보입니다. mtDNA 또는 nDNA 돌연변이 모두에 의해 발생할 수 있는 이질적인 질환입니다.
• 미토콘드리아 신경위장 뇌병증 (Mitochondrial Neurogastrointestinal Encephalomyopathy, MNGIE):
  • 특징: 위장 운동 이상(설사, 구토, 복통), 신경병증, 안검하수, 안근 마비, 백색질 뇌병증(leukoencephalopathy) 등이 나타납니다. mtDNA 유지에 관여하는 핵 유전자(TYMP)의 돌연변이로 인해 발생합니다.
• 피루브산 탈수소효소 복합체 (PDH) 결핍증:
  • 특징: 피루브산 탈수소효소 복합체는 탄수화물 대사 산물인 피루브산이 미토콘드리아로 들어가 에너지 생산에 사용되도록 아세틸-CoA로 전환하는 데 필수적인 효소입니다. 이 효소의 결핍은 젖산 산증, 신경학적 이상(발달 지연, 운동실조, 경련)을 초래합니다.
• 호흡 사슬 복합체 결핍증:
  • 미토콘드리아의 전자전달계(electron transport chain)를 구성하는 복합체 I, II, III, IV, V 중 하나 이상의 기능 이상으로 발생합니다. 이들은 핵 DNA 또는 mtDNA에 의해 암호화될 수 있으며, 결핍되는 복합체와 손상 정도에 따라 다양한 증상(근력 약화, 신경학적 문제, 심장 질환 등)을 유발합니다.
  • 예: 복합체 I 결핍, 복합체 IV 결핍 등.
• 코엔자임 Q10 (CoQ10) 결핍증:
  • CoQ10은 전자전달계의 중요한 구성 요소입니다. CoQ10의 생합성 경로에 관여하는 핵 유전자의 돌연변이로 인해 CoQ10이 부족해지면, 근병증, 뇌병증, 신장 질환 등 다양한 증상이 나타날 수 있습니다.

이 리뷰는 

미토콘드리아의 생리적 메커니즘, 

미토콘드리아 질환의 병리 메커니즘, 

잠재적인 진단 및 치료 응용에 초점을 맞춥니다. 

또한 이 리뷰는 

미토콘드리아 유전성 질환의 미래 전망에 대해 논의합니다.

Similar content being viewed by others

Mitochondrial heterogeneity in diseases

Article Open access23 August 2023

Mitochondrial proteome research: the road ahead

Article 29 September 2023

Mitochondrial dysfunction: mechanisms and advances in therapy

Article Open access15 May 2024

Introduction

Mitochondria, often referred to as the powerhouses of cells, perform their essential function through oxidative phosphorylation (OXPHOS), which generates ATP as a vital energy source.1 Mitochondrial diseases are genetic disorders resulting from abnormalities of mitochondrial function.2 These disorders arise from mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA), both of which encode subunits of OXPHOS as well as structural or functional mitochondrial proteins.3 These proteins are not only integral to classical mitochondrial metabolism—such as OXPHOS, the Krebs cycle, lipid metabolism, and nucleotide metabolism—but also play key roles in mitochondrial quality control, calcium homeostasis, cell death, and inflammation. Deficiencies in these proteins can lead to mitochondrial dysfunction and subsequent energy failure.4 Given mitochondria’s ubiquitous presence and critical role in cellular metabolism, any tissue in the body can be affected.5 However, organs and tissues with high energy demands, such as the brain, nerve, eye, cardiac, and skeletal muscles, are particularly susceptible to energy failure due to OXPHOS defects, with phenotypes often manifesting in neurological, ophthalmological, and cardiological systems.6 The symptoms of mitochondrial diseases are diverse, with developmental delay, seizure (encephalopathy), hypotonia (myopathy), and visual impairment (retinopathy) being prominent indicators.6,7 Despite recent advances, the molecular mechanisms underlying these diseases remain incompletely understood. The extreme phenotypic and genetic heterogeneity of mitochondrial diseases further complicates diagnosis, making misdiagnosis a common issue.8

Mitochondrial diseases have been recognized as pathway-based diseases rather than merely energy-deficit diseases.7 The variable clinical presentations and tissue specificity suggest that there are contributing factors beyond energy deficit during disease development.9 The reduction of ATP produced from OXPHOS can be compensated by enhanced anaerobic glycolysis, and thus mitochondrial genetic defects may not reduce ATP production.10,11 Furthermore, genetic defects are not always sufficient to cause cellular dysfunction as mitochondria can buffer against mitochondrial lesions, making environmental insults sometimes important to trigger these genetic disorders.12 Recently, the mitochondrial stress responses have gained close attention.9 Mitochondria have a comprehensive quality control system to maintain homeostasis, preventing dysfunction when facing stress. At the molecular level, mitochondria possess the quality control mechanisms of the proteome, such as mitochondrial integrated stress response (mt-ISR).13 At the organelle level, mitochondria can alter their morphology or sub-location through fusion, fission, and transport to adapt to stress or damage. At the cellular level, mitophagy coordinates with mitochondrial biogenesis, controlling the health of the mitochondrial population.14,15 Intercellular mitochondria transfer also plays a role in maintaining mitochondrial homeostasis.16 However, excessive stress can trigger mitochondria-related inflammation or apoptosis as well.17 In the context of mitochondrial diseases, genetic defects can lead to mitochondrial dysfunction. The subsequent responses to the stress induced by mitochondrial dysfunction may aid in understanding these genetic diseases.9,18 Hence, this review concludes the physiological processes of mitochondria and the potential pathogenesis of mitochondrial diseases. Significant progress in diagnosis and treatment is also summarized in this review.

소개

미토콘드리아는

세포의 에너지 공장이라고도 불리는 기관으로,

산화적 인산화(OXPHOS)를 통해 ATP를 생성하여 생명 활동에 필수적인 에너지를 공급합니다.1

미토콘드리아 질환은

미토콘드리아 기능의 이상으로 인해 발생하는 유전적 질환입니다.2

이러한 질환은

미토콘드리아 DNA(mtDNA) 또는 핵 DNA(nDNA)의 돌연변이로 인해 발생하며,

두 DNA는 OXPHOS의 서브 유닛과 미토콘드리아의 구조적 또는 기능적 단백질을 암호화합니다.3

이러한 단백질은

OXPHOS, 크렙스 사이클, 지질 대사, 뉴클레오티드 대사 등

고전적인 미토콘드리아 대사 과정에 필수적인 요소일 뿐만 아니라

미토콘드리아의 품질 관리, 칼슘 항상성, 세포 사멸 및 염증에도 중요한 역할을 합니다.

이 단백질의 결핍은

미토콘드리아 기능 장애와 후속적인 에너지 결핍을 초래할 수 있습니다.4

미토콘드리아의 보편적인 존재와 세포 대사에서의 핵심 역할로 인해

신체 내 모든 조직이 영향을 받을 수 있습니다.5

그러나

뇌, 신경, 눈, 심장, 골격근과 같은 에너지 요구량이 높은 장기 및 조직은

OXPHOS 결함으로 인한 에너지 결핍에 특히 취약하며,

증상은 신경학적, 안과적, 심장학적 시스템에서 자주 나타납니다.6

미토콘드리아 질환의 증상은 다양하며,

발달 지연, 발작(뇌병증), 근육 이완(근육병), 시력 장애(망막병증)가 주요 증상입니다.6,7

최근 진전에도 불구하고,

이러한 질환의 분자적 메커니즘은 여전히 완전히 이해되지 않았습니다.

미토콘드리아 질환의 극심한 표현형 및 유전적 이질성은

진단을 복잡하게 만들며, 오진 문제가 흔히 발생합니다.8

미토콘드리아 질환은

단순히 에너지 결핍 질환이 아닌 경로 기반 질환으로 인식되고 있습니다.7

다양한 임상적 증상과 조직 특이성은

질환 발달 과정에서 에너지 결핍을 넘어서는 기여 요인이 존재함을 시사합니다.9

OXPHOS에서 생성되는 ATP의 감소는

무산소 당분해의 증가로 보상될 수 있으며,

따라서 미토콘드리아 유전적 결함이 ATP 생산을 감소시키지 않을 수 있습니다.10,11

또한 유전적 결함만으로는

세포 기능 장애를 유발하지 않을 수 있습니다.

미토콘드리아는

미토콘드리아 손상에 대한 완충 역할을 할 수 있으며,

환경적 스트레스가 이러한 유전적 질환을 유발하는 데 중요한 역할을 할 수 있습니다.12

최근 미토콘드리아 스트레스 반응이 주목받고 있습니다.9

미토콘드리아는

항상성을 유지하기 위한 포괄적인 품질 관리 시스템을 갖추고 있어

스트레스에 직면했을 때 기능 장애를 방지합니다.

분자 수준에서 미토콘드리아는

프로테옴의 품질 관리 메커니즘인 미토콘드리아 통합 스트레스 반응(mt-ISR)을 갖추고 있습니다.13

기관체 수준에서는

미토콘드리아가 융합, 분열, 운반을 통해 형태나 하위 위치를 변경하여

스트레스나 손상에 적응할 수 있습니다.

세포 수준에서 미토파기는

미토콘드리아 생성과 협응력을 발휘하여 미토콘드리아 개체군의 건강을 제어합니다.14,15

세포 간 미토콘드리아 전달도

미토콘드리아의 항상성 유지에 중요한 역할을 합니다.16

그러나

과도한 스트레스는

미토콘드리아 관련 염증이나 세포 사멸을 유발할 수도 있습니다.17

미토콘드리아 질환의 경우,

유전적 결함이 미토콘드리아 기능 장애를 유발할 수 있습니다.

미토콘드리아 기능 장애로 인한 스트레스에 대한 후속 반응은

이러한 유전적 질환을 이해하는 데 도움을 줄 수 있습니다.9,18

따라서 이 리뷰는

미토콘드리아의 생리적 과정과 미토콘드리아 질환의 잠재적 병리 메커니즘을 결론지으며,

진단 및 치료 분야의 중요한 진전도 요약합니다.

Historical review and Epidemiology of mitochondrial diseases

The history of mitochondrial diseases dates back to 1871 when Theodor Leber documented hereditary and congenital optic nerve diseases, marking the first known description of a genetic mitochondrial disorder, now recognized as Leber hereditary optic neuropathy (LHON).19 The concept of mitochondrial diseases was later introduced in 1962 by Luft et al.20, who identified a young woman with severe hypermetabolism caused by mitochondrial dysfunction due to defective OXPHOS coupling in skeletal muscle mitochondria.20,21 This pivotal discovery brought mitochondrial diseases into the scientific spotlight.

During the 1960s, research primarily focused on mitochondrial myopathies. Milton Shy and Nicholas Gonatas described megaconial and pleoconial congenital myopathies,22,23 hypothesizing that these conditions were linked to mtDNA defects.24 In 1963, Engel et al. introduced an improved Gomori trichrome staining method for muscle histopathology, which enabled the detection of abnormal mitochondrial proliferation as ragged-red fibers, thus advancing histochemical studies of mitochondrial diseases.25 The 1970s saw significant progress in identifying mitochondrial metabolism defects through histochemical assays, including deficiencies in pyruvate dehydrogenase, carnitine, cytochrome c oxidase, and carnitine palmitoyltransferase.26,27,28,29 In 1977, Shapira et al. coined the term “mitochondrial encephalomyopathies” to describe a group of neuromuscular disorders characterized by defects in oxidative metabolism.30 A major breakthrough came in 1981 when Anderson et al. successfully mapped the entire mitochondrial genome, establishing a foundation for subsequent mitochondrial research.31

In 1988, the discovery of single large-scale deletions of up to 7 kilobases in patients with mitochondrial myopathies32 and a point mutation in the NADH dehydrogenase subunit 4 gene in families with LHON33 underscored the importance of mtDNA mutations, heralding the beginning of the molecular era in mitochondrial research.34 By 1989, multiple mtDNA deletions had been identified in the muscle tissues of members from a family with autosomal dominant mitochondrial myopathy.35 Further advancements were made in 1991 when Moraes et al. confirmed mtDNA depletion in the affected muscle or liver tissues of infants with autosomal recessive disorders.36 This period also saw increased attention to the role of nDNA in mitochondrial diseases, particularly with the identification of Mendelian mitochondrial disorders. A landmark discovery in 1995 revealed the first nuclear gene mutation causing mitochondrial respiratory chain deficiency in humans: a mutation in the nuclear-encoded flavoprotein subunit gene of succinate dehydrogenase led to complex II deficiency in two sisters with Leigh syndrome.37 The creation of the first comprehensive mtDNA database, MITOMAP, in 1996 further facilitated the study of mitochondrial diseases.38 Soon after, Nishino et al. attributed mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) to a defect in communication between nuclear and mitochondrial genomes.39 The 2000s saw the introduction of next-generation sequencing (NGS) technology in the diagnosis of mitochondrial diseases.40 By the 2010s, transcriptomics and other omics analyses had gained increasing attention, leading to the emergence of multi-omics approaches in the diagnosis of mitochondrial disorders.41

Scientists are actively pursuing potential treatments to address mitochondrial diseases. In 1997, Taylor et al. pioneered the use of peptide nucleic acid (PNA) in gene therapy to selectively inhibit the replication of mutated human mtDNA, thereby increasing the proportion of wild-type mtDNA and correcting defective phenotypes through heteroplasmy alteration.42 In 2006, Spees et al. discovered that intercellular mitochondrial transfer could restore aerobic respiration in mammalian cells.43 By 2009, Tachibana et al. had successfully separated the spindle-chromosome complex from mature metaphase II (MII) oocytes and transferred it into enucleated oocytes, resulting in the birth of healthy primate offspring with nDNA from the spindle donor and mtDNA from the cytoplasmic donor.44 In 2015, idebenone received approval from the European Medicine Agency (EMA) for treating LHON under specific conditions.45 In 2017, Zhang et al. reported the application of the spindle-chromosome complex transfer (ST) method in a woman carrying the m.8993 T > G mutation associated with Leigh syndrome, leading to the birth of a healthy child.46 In 2018, a gene therapy employing an allogeneic expression‎ strategy was tested in a clinical trial for patients with LHON, demonstrating both safety and good tolerability.47 More recently, in 2023, Omaveloxolone became the first drug approved by the Food and Drug Administration (FDA) for treating Friedreich’s ataxia.48

As our understanding of mitochondria deepens, so does our knowledge of the mutant genes and pathogenesis underlying mitochondrial genetic disorders. Figure 1 presents a timeline summarizing key milestones in mitochondrial disease research. Beyond the focus on primary or secondary OXPHOS, significant attention is being directed toward gene mutations that impair mitochondrial structure and function.2,3,41

미토콘드리아 질환의 역사적 검토 및 역학

미토콘드리아 질환의 역사는 1871년 Theodor Leber가 유전성 및 선천성 시신경 질환을 기록한 것으로 거슬러 올라갑니다. 이는 현재 Leber 유전성 시신경 신경병증(LHON)으로 알려진 유전성 미토콘드리아 장애의 첫 번째 알려진 설명입니다.19 미토콘드리아 질환의 개념은 1962년 Luft 등20에 의해 처음 소개되었습니다. 이들은 골격근 미토콘드리아의 OXPHOS 결합 결함으로 인한 심각한 과대대사증을 보이는 젊은 여성 환자를 식별했습니다.20,21 이 획기적인 발견은 미토콘드리아 질환을 과학계의 주목을 받게 했습니다.

1960년대에는 주로 미토콘드리아 근육 질환에 대한 연구가 진행되었습니다. Milton Shy와 Nicholas Gonatas는 메가코니얼 및 플레오코니얼 선천성 근육병을 기술했으며,22,23 이 질환이 mtDNA 결함과 연관되어 있을 가능성을 제기했습니다.24 1963년, Engel 등 연구진은 근육 조직 병리학에 적용 가능한 개선된 Gomori 삼색 염색법을 소개했습니다. 이 방법은 미토콘드리아 과다 증식을 낡은 빨간 섬유로 관찰할 수 있게 하여 미토콘드리아 질환의 조직화학적 연구를 진전시켰습니다.25 1970년대에는 피루vate dehydrogenase, 카르니틴, 사이토크롬 c 산화효소, 카르니틴 팔미토일트랜스퍼레이즈 결핍 등을 포함한 미토콘드리아 대사 결함을 식별하는 조직화학적 검사에 있어 중요한 진전이 이루어졌습니다.26,27,28,29 1977년, Shapira 등 연구진은 산화 대사 결함을 특징으로 하는 신경근육 장애 그룹을 설명하기 위해 “미토콘드리아 뇌근육병증”이라는 용어를 창안했습니다.30 1981년, Anderson 등 연구진은 미토콘드리아 유전체 전체를 성공적으로 지도화하여 후속 미토콘드리아 연구의 기반을 마련했습니다.31

1988년, 미토콘드리아 근육병 환자에게서 최대 7킬로베이스 규모의 단일 대규모 삭제가 발견되었으며32, LHON을 가진 가족에서 NADH 탈수소효소 서브유닛 4 유전자의 점 돌연변이가 확인되었습니다. 이는 mtDNA 돌연변이의 중요성을 강조하며 미토콘드리아 연구의 분자 시대를 알리는 계기가 되었습니다.34 1989년까지, 상염색체 우성 미토콘드리아 근육병을 가진 가족의 근육 조직에서 다중 mtDNA 삭제가 확인되었습니다.35 1991년에는 Moraes 등 연구진이 상염색체 열성 장애를 가진 영아의 영향을 받은 근육 또는 간 조직에서 mtDNA 고갈을 확인했습니다.36 이 기간에는 미토콘드리아 질환에서 nDNA의 역할에 대한 관심도 높아졌으며, 특히 멘델의 미토콘드리아 질환이 확인되었습니다. 1995년에 인간에서 미토콘드리아 호흡 사슬 결핍을 유발하는 최초의 핵 유전자 돌연변이가 발견된 것은 획기적인 발견이었습니다. 리 증후군을 가진 두 자매에서, 핵에서 암호화된 플라보프로테인 서브유닛 유전자의 돌연변이가 복합체 II 결핍을 유발한 것이 밝혀졌습니다.37 1996년 첫 번째 종합 mtDNA 데이터베이스인 MITOMAP의 구축은 미토콘드리아 질환 연구를 더욱 촉진했습니다.38 이후 Nishino 등 연구진은 미토콘드리아 신경위장관 뇌근육병증(MNGIE)을 핵과 미토콘드리아 유전체 간의 통신 결함에 기인시켰습니다.39 2000년대에는 미토콘드리아 질환 진단에 차세대 시퀀싱(NGS) 기술이 도입되었습니다.40 2010년대에는 트랜스크립토믹스와 다른 오믹스 분석이 주목받기 시작하며, 미토콘드리아 장애 진단에 다중 오믹스 접근법이 등장했습니다.41

과학자들은 미토콘드리아 질환을 치료하기 위한 잠재적 치료법을 적극적으로 연구하고 있습니다. 1997년, Taylor 등 연구진은 유전자 치료에 펩타이드 핵산(PNA)을 최초로 적용해 변이된 인간 미토콘드리아 DNA(mtDNA)의 복제를 선택적으로 억제함으로써 야생형 mtDNA의 비율을 증가시키고 이형성 변이 조절을 통해 결함 있는 표현형을 교정했습니다.42 2006년, Spees 등 연구진은 세포 간 미토콘드리아 전달이 포유류 세포의 산소 호흡을 회복시킬 수 있음을 발견했습니다.43 2009년까지 Tachibana 등 연구진은 성숙한 메타상 II기(MII) 난자에서 spindle-chromosome 복합체를 분리해 핵을 제거한 난자에 이식하는 데 성공했으며, 이로써 spindle 기증자의 nDNA와 세포질 기증자의 mtDNA를 가진 건강한 영장류 후손이 태어났습니다.44 2015년, 이데베논은 유럽 의약품청(EMA)으로부터 특정 조건 하에서 LHON 치료에 대한 승인을 받았습니다.45 2017년, Zhang 등 연구진은 Leigh 증후군과 관련된 m.8993 T > G 돌연변이를 가진 여성에게 spindle-chromosome complex transfer (ST) 방법을 적용해 건강한 아이의 출생을 보고했습니다.46 2018년, LHON 환자를 대상으로 한 임상 시험에서 이종 표현 전략을 활용한 유전자 치료법이 안전성과 우수한 내약성을 보여주었습니다.47 최근 2023년에는 오마벨록솔론이 Friedreich의 운동 실조증 치료를 위해 식품의약국(FDA)으로부터 승인받은 첫 번째 약물이 되었습니다.48

미토콘드리아에 대한 이해가 깊어짐에 따라, 미토콘드리아 유전 질환의 원인인 돌연변이 유전자와 병리 메커니즘에 대한 지식도 함께 발전하고 있습니다. 그림 1은 미토콘드리아 질환 연구의 주요 이정표를 요약한 타임라인을 제시합니다. OXPHOS의 일차적 또는 이차적 장애에 대한 초점을 넘어, 미토콘드리아 구조와 기능을 손상시키는 유전자 돌연변이에 대한 관심이 크게 증가하고 있습니다.2,3,41

Fig. 1

Timeline of Major Historical Events in the Study of Mitochondrial Diseases. From the initial discovery to current advancements, our understanding of the mechanisms underlying mitochondrial diseases has continually deepened. Over time, research explorations and progress have contributed to the development of diagnostic and treatment methods, ultimately providing insights into more efficient and accurate diagnostic and therapeutic strategies

미토콘드리아 질환 연구의 주요 역사적 사건 연표. 초기 발견부터 현재의 진전까지, 미토콘드리아 질환의 기전을 이해하는 우리의 지식은 지속적으로 심화되어 왔습니다. 시간이 지나며 연구 탐구와 진전은 진단 및 치료 방법의 개발에 기여해 왔으며, 결국 더 효율적이고 정확한 진단 및 치료 전략에 대한 통찰을 제공했습니다.

Previous studies estimate the global preval‎ence of mitochondrial diseases at approximately 1 in 5,000 births,49 with pathogenic mtDNA mutations affecting at least 12.48 per 100,000 individuals.50 Table 1 lists the regional and global incidences of specific mitochondrial diseases.

이전 연구들은

미토콘드리아 질환의 전 세계 유병률을 약 5,000명 중 1명으로 추정하며,49 

병인성 미토콘드리아 DNA(mtDNA) 변이가

100,000명당 최소 12.48명에게 영향을 미친다고 보고했습니다.50 

표 1은 특정 미토콘드리아 질환의 지역별 및 전 세계 발생률을 정리했습니다.

Table 1 The preval‎ence of overall or individual mitochondrial diseases

CategoryPreval‎ence (95% CI)PopulationRegionReference

Mitochondrial diseases (caused by mtDNA mutation)	12.48(10.75–14.23)/100,000	Female (age<60) Male (age<65)	Northeast England	50
	9.2 (6.5–12.7)/100,000	Age≥18	Southwest Finland	881
Mitochondrial diseases	12.5 (11.1–14.1)/100,000	Female (16<age<60) Male (16<age<65)	Northeast England	882
	4.7 (4.1–5.4)/100,000	Total	New Zealand	883
	1.02 (0.81–1.28)/100,000	Total	Hong Kong, China	884
	2.9 (2.8–3.0)/100,000	Total	Japan	885
	2.3 (2.14–2.47)/1,000,000	Total	Spain	886
	7.5 (5.0–10.0)/100,000	Age≤18	Northwest Spain	887
LHON	2491 (1996–2986)/126,167,000	Total	Japan	888
	3.22 (2.47–3.97)/100,000	Age<65	Northeast England	889
	2.06 (1.8–2.4)/100,000	Age≥5	Finland	890
	1/54,000	Total	Denmark	891
	1/68,403	Age<85	Australia	892
	1/39,000	Total	Netherlands	893
	1.9/1,000,000	Total	Serbia	894
MELAS	0.18 (0.02–0.34)/100,000	Total	Japan	895
	4.7 (2.8–7.6)/100,000	Age<16	Western Sweden	896
Mitochondrial myopathy	0.58 (0.54–0.62)/100,000	Total	Japan	895
Leigh syndrome	2.05 (0.72–3.40)/100,000	Age≤18	Northwest Spain	887
Friedreich’s ataxia	1/176,000	Total	Norway	897
	1.2 (0.9–1.6)/100,000	Total	Italy	898
ADOA	2.87 (2.54–3.20)/100,000	Total	North England	899
	1/12,301	Total	Denmark	900
MNGIE	1–9/1,000,000	Total	World	488
MIDD	0.5–2.8/100	Diabetic patients	World	901
Barth syndrome	1/1,000,000	Male	World	902

Determining the exact global incidence of mitochondrial diseases is challenging due to their rarity, high mortality, and clinical and genetic heterogeneity.51 Additionally, symptoms typically manifest only when a certain mutation threshold is reached—usually 80–90%—though this threshold can vary between different cells and patients.52,53 As a result, the clinical phenotypes of mitochondrial diseases caused by mtDNA mutations can differ significantly among individuals and are influenced by the level of heteroplasmy, making these diseases difficult to diagnose.54 Notably, mtDNA mutations are not exclusive to those with mitochondrial diseases; they are present in the general population as well. At least 1 in 200 healthy individuals carries a pathogenic mtDNA mutation, often with no or only mild symptoms.55 These mutations can be maternally inherited, and it is estimated that nearly 2473 women in the UK and 12,423 women in the US, aged 15 to 44, carry pathogenic mtDNA mutations.56 Interestingly, approximately 80% of mitochondrial diseases in adults are linked to mtDNA mutations, while most mitochondrial diseases in children are associated with nDNA mutations, with only 20–25% stemming from mtDNA mutations.2 These factors underscore the complexity and preval‎ence of mitochondrial diseases, which are more common and intricate than previously understood. Consequently, further epidemiological studies are essential to improve our understanding and prediction of mitochondrial disease preval‎ence.

미토콘드리아 질환의 정확한 전 세계 발생률을 결정하는 것은

그 희귀성, 높은 사망률, 임상적 및 유전적 이질성 때문에 어려움이 있습니다.51

또한 증상은 일반적으로 특정 돌연변이 임계값에 도달했을 때만 나타납니다—보통 80–90%—하지만 이 임계값은 세포와 환자之间에 다를 수 있습니다.52,53 결과적으로, mtDNA 변이로 인한 미토콘드리아 질환의 임상적 표현형은 개인 간에 크게 다를 수 있으며, 이질성 수준에 영향을 받아 진단이 어려울 수 있습니다.54

특히, mtDNA 변이는 미토콘드리아 질환 환자에게만 국한되지 않으며 일반 인구에서도 존재합니다.

건강한 개인 중 200명 중 1명 이상이 병리적 mtDNA 변이를 보유하고 있으며, 대부분 증상이 없거나 경미합니다.55

이러한 변이는

모계로 유전될 수 있으며,

영국과 미국에서 15세에서 44세 사이의 여성 중 각각 약 2,473명과 12,423명이

병리적 mtDNA 변이를 보유하고 있는 것으로 추정됩니다.56 

흥미롭게도

성인에서 발생하는 미토콘드리아 질환의 약 80%는

mtDNA 변이와 관련되어 있지만,

어린이에서 발생하는 미토콘드리아 질환의 대부분은

nDNA 변이와 연관되어 있으며,

mtDNA 변이로 인한 경우는 20–25%에 불과합니다.2 

--> 어린이의 경우 핵 DNA변이가 흔한 이유는 '세포분열'이 활발하기 때문'

이러한 요인들은 미토콘드리아 질환의 복잡성과 유병률을 강조하며, 이는 이전에 이해되었던 것보다 더 흔하고 복잡하다는 점을 보여줍니다. 따라서 미토콘드리아 질환의 유병률을 이해하고 예측하기 위해 추가적인 역학 연구가 필수적입니다.

Molecular basis of mitochondria

General characteristics of mitochondria

The mitochondrion is a double-membrane organelle present in nearly all eukaryotic organisms.57 It is widely believed that mitochondria originated from bacteria, specifically α-proteobacteria.58 The human mitochondrion contains a genome of 16,569 base pairs, distinct from the nuclear genome.31 Notably, fragments of mtDNA can integrate into the nuclear genome, forming nuclear-mitochondrial segments (NUMTs).59 The mtDNA is a circular, double-stranded molecule with multiple copies and is maternally inherited. It encodes 37 genes, including 2 rRNAs, 22 tRNAs, and 11 mRNAs. Of these, 14 tRNAs, 2 rRNAs, and 10 mRNAs are encoded on the heavy (H) strand, while the remaining 1 mRNA and 8 tRNAs are encoded on the light (L) strand.60,61,62

Mitochondrial non-coding RNAs (ncRNAs), such as microRNAs, long non-coding RNAs, circular RNAs, and piwi-interacting RNAs, have been identified as potential mediators of mitochondrial homeostasis.63 These ncRNAs are key messengers in mito-nuclear communication and have garnered significant attention.64 Most mitochondrial ncRNAs originate from the nuclear genome and are translocated into mitochondria via Ago2, PNPase, or associated mitochondrial proteins. These nuclear-derived ncRNAs can indirectly regulate mitochondrial homeostasis by influencing nDNA-encoded mitochondrial proteins.63 Conversely, mitochondria-derived ncRNAs, which include a limited number of long non-coding RNAs and circular RNAs, can directly regulate mtDNA expression‎ or mitochondrial protein transport.65,66,67 The biogenesis, processing, and functional mechanisms of ncRNAs encoded by mtDNA remain largely unclear.63 Intriguingly, recent studies suggest the epigenetic inheritance-influenced transfer of mitochondrial tRNA (mt-tRNA) from sperm to oocyte at fertilization, highlighting the potential importance of paternal factors in mitochondrial inheritance.68 Additionally, mt-tRNA fragments have been implicated in mitochondrial diseases.69

Mitochondrial proteomes, comprising approximately 1000 to 1500 proteins, are encoded by both nDNA and mtDNA.70,71 Among these, metabolism-related proteins constitute the largest number and abundance.72 While mtDNA encodes only 13 proteins involved in OXPHOS, the vast majority of mitochondrial proteins (>99%) are encoded by the nuclear genome, synthesized on ribosomes, and subsequently imported into mitochondria.73

Mitochondrial-derived peptides (MDPs) are encoded by short open reading frames within mtDNA, including humanin, small humanin-like peptides 1-6 (SHLP1-6), MOTS-c, and CYTB-187AA.74,75,76 These MDPs play a pivotal role in cellular protection by maintaining homeostasis and cellular function.75 Each peptide exhibits distinct biological effects; for instance, humanin, SHLP2, and SHLP3 inhibit apoptosis and promote cell viability, whereas SHLP6 induces apoptosis.75,77 SHLP2 and SHLP4 also enhance cell proliferation.75 Humanin is essential for maintaining mitochondrial homeostasis and function by increasing mtDNA copy number and mitochondrial mass and promoting mitochondrial biogenesis.77 Similarly, SHLP2 and SHLP3 contribute to mitochondrial biogenesis, enhancing mitochondrial metabolism and function.74,75,78 MOTS-c, the first MDP discovered to enter the cell nucleus, plays a role in mito-nuclear communication.74 MOTS-c transcripts originate in mitochondria, are exported to the cytosol for translation into peptides, and then return to mitochondria.74 Under stress, AMPK activation triggers the translocation of MOTS-c to the nucleus, where it binds to nuclear DNA and interacts with transcription factors such as NRF2 and ATF1, modulating nuclear gene expression‎ to restore cellular metabolic homeostasis.74,79 Recently, Hu et al. demonstrated that the cytochrome b transcript, encoded by mtDNA, is translated by cytosolic ribosomes using the standard genetic code to produce a 187-amino acid protein, CYTB-187AA.76 CYTB-187AA localizes to the mitochondrial matrix and interacts with SLC25A3 to regulate ATP production.76 Single nucleotide polymorphisms in the mtDNA coding regions for MDPs may facilitate the discovery of new MDPs, such as SHMOOSE.80

Without the protective presence of histones, mtDNA is more susceptible to external factors, leading to a higher likelihood of mutations. These mutations can result in various diseases, given the critical role of mitochondria in nucleated cells. The severity of such conditions often depends on the ratio of mutant to wild-type mtDNA.81 Because mtDNA exists in multiple copies, two scenarios are possible: homoplasmy, where all mtDNA copies are identical, or heteroplasmy, where the copies differ.82

Mitochondrial membrane potential (Δψm) is a fundamental property generated during OXPHOS by the respiratory chain. The stability of Δψm is essential for cell viability. Under normal conditions, Δψm may fluctuate slightly in the short term; however, prolonged changes in Δψm can lead to pathological outcomes. As a result, cells activate mechanisms to eliminate mitochondria with abnormal Δψm.83,84

미토콘드리아의 분자적 기반

미토콘드리아의 일반적 특성

미토콘드리아는

거의 모든 진핵생물에 존재하는 이중막 소기관입니다.57 

미토콘드리아는

박테리아, 특히 α-프로테오박테리아에서 유래했다고 널리 알려져 있습니다.58 

인간 미토콘드리아는

핵 게놈과 다른 16,569 염기쌍의 게놈을 포함합니다.31 

주목할 점은

mtDNA의 일부가 핵 게놈에 통합되어

핵-미토콘드리아 세그먼트(NUMTs)를 형성할 수 있다는 것입니다.59 

mtDNA는

원형 이중 가닥 분자로 다중 복사본을 가지고 있으며

모계로 유전됩니다.

이 유전자는

2개의 rRNA, 22개의 tRNA 및 11개의 mRNA를 포함한 37개의 유전자를 암호화합니다.

이 중 14개의 tRNA, 2개의 rRNA 및 10개의 mRNA는 중쇄(H)에 암호화되어 있으며,

나머지 1개의 mRNA 및 8개의 tRNA는 경쇄(L)에 암호화되어 있습니다.60,61,62

미토콘드리아 비코딩 RNA(ncRNA)는

마이크로RNA, 장쇄 비코딩 RNA, 원형 RNA, 피위 상호작용 RNA 등이며,

미토콘드리아 항상성 조절의 잠재적 매개체로 식별되었습니다.63 

이러한

ncRNA는

미토-핵 간 통신에서 핵심 메신저 역할을 하며

상당한 관심을 받고 있습니다.64 

대부분의 미토콘드리아 ncRNA는

핵 게놈에서 유래하며

Ago2, PNPase 또는 관련 미토콘드리아 단백질을 통해 미토콘드리아로 전좌됩니다.

이러한

핵 유래 ncRNA는

nDNA에 암호화된 미토콘드리아 단백질에 영향을 미침으로써

미토콘드리아의 항상성을 간접적으로 조절할 수 있습니다.63 

반대로,

제한된 수의 긴 비코딩 RNA 및 원형 RNA를 포함하는 미토콘드리아 유래 ncRNA는

mtDNA 발현 또는 미토콘드리아 단백질 수송을 직접 조절할 수 있습니다.65,66,67 

mtDNA에 의해 암호화된 ncRNA의 생성과 처리, 기능 메커니즘은

아직 대부분 밝혀지지 않았습니다.63 

흥미롭게도,

최근의 연구에서는 수정 시 정자에서 난자로

미토콘드리아 tRNA(mt-tRNA)가 유전적 영향에 의해 전달된다는 것이 밝혀졌으며,

이는 미토콘드리아 유전에 아버지의 요인이 중요한 역할을 할 가능성을 시사합니다.68 

또한, mt-tRNA 단편은

미토콘드리아 질환과 관련이 있는 것으로 밝혀졌습니다.69

약 1,000~1,500개의 단백질로 구성된 미토콘드리아 단백질체(proteome)는

nDNA와 mtDNA에 의해 암호화되어 있습니다.70,71 

이 중 대사 관련 단백질이 가장 많고 풍부합니다.72 

mtDNA는

OXPHOS에 관여하는 13개의 단백질만을 암호화하지만,

미토콘드리아 단백질의 대부분(99% 이상)은 핵 게놈에 의해 암호화되어

리보솜에서 합성된 후 미토콘드리아로 수입됩니다.73

미토콘드리아 유래 펩티드(MDP)는 인간인, 소형 인간인 유사 펩티드 1-6(SHLP1-6), MOTS-c 및 CYTB-187AA를 포함하여 mtDNA 내의 짧은 오픈 리딩 프레임에 의해 암호화됩니다.74,75,76 이러한 MDP는 항상성 및 세포 기능을 유지하여 세포 보호에 중요한 역할을 합니다.75 각 펩타이드은 독특한 생물학적 효과를 나타냅니다. 예를 들어, 휴머니닌, SHLP2, 및 SHLP3은 세포 사멸을 억제하고 세포 생존력을 증진시키지만, SHLP6은 세포 사멸을 유도합니다.75,77 SHLP2 및 SHLP4는 또한 세포 증식을 촉진합니다.75 휴머니인은 mtDNA 복제 수와 미토콘드리아 질량을 증가시키고 미토콘드리아 생성을 촉진함으로써 미토콘드리아 균형과 기능을 유지하는 데 필수적입니다.77 마찬가지로 SHLP2와 SHLP3는 미토콘드리아 생성에 기여하며 미토콘드리아 대사 및 기능을 향상시킵니다.74,75,78 MOTS-c는 세포 핵으로 들어가는 첫 번째 MDP로, 미토-핵 통신에 역할을 합니다.74 MOTS-c 전사체는 미토콘드리아에서 기원하여 세포질로 수출되어 펩타이드로 번역된 후 다시 미토콘드리아로 돌아갑니다.74 스트레스 조건에서 AMPK 활성화는 MOTS-c의 핵 내 이동을 유발하며, 이는 핵 DNA에 결합하여 NRF2 및 ATF1과 같은 전사 인자와 상호작용하여 핵 내 유전자 발현을 조절하여 세포 대사 균형을 회복합니다.74,79 최근 Hu 등은 mtDNA에 의해 암호화된 시토크롬 b 전사물이 표준 유전 코드를 사용하여 세포질 리보솜에 의해 번역되어 187-아미노산 단백질인 CYTB-187AA를 생성한다는 것을 증명했습니다.76 CYTB-187AA는 미토콘드리아 매트릭스에 국한되어 SLC25A3과 상호 작용하여 ATP 생성을 조절합니다.76 mtDNA의 MDP 코딩 영역에 존재하는 단일 뉴클레오티드 다형성(SNP)은 SHMOOSE와 같은 새로운 MDP의 발견을 촉진할 수 있습니다.80

히스톤의 보호 작용이 결여된 mtDNA는 외부 요인에 더 취약해지며, 이는 돌연변이의 발생 가능성을 높입니다. 이러한 돌연변이는 미토콘드리아가 핵세포에서 수행하는 중요한 역할 때문에 다양한 질환을 유발할 수 있습니다. 이러한 상태의 심각성은 변이형과 야생형 mtDNA의 비율에 따라 달라집니다.81 mtDNA는 다중 사본으로 존재하기 때문에 두 가지 시나리오가 가능합니다: 모든 mtDNA 사본이 동일한 동형접합(homoplasmy) 또는 사본이 서로 다른 이형접합(heteroplasmy).82

미토콘드리아 막 전위(Δψm)는 호흡 사슬에 의해 OXPHOS 과정에서 생성되는 기본적 특성입니다. Δψm의 안정성은 세포 생존에 필수적입니다. 정상 조건에서 Δψm은 단기적으로 약간 변동될 수 있지만, Δψm의 장기적인 변화는 병리적 결과를 초래할 수 있습니다. 이에 따라 세포는 이상적인 Δψm을 가진 미토콘드리아를 제거하기 위한 메커니즘을 활성화합니다.83,84

Mitochondrial metabolism

Mitochondria play a central role in substance metabolism, overseeing a vast array of metabolic processes as depicted in Fig. 2.

미토콘드리아 대사

미토콘드리아는 물질 대사에서 중심적인 역할을 하며, 그림 2에 표시된 것처럼 다양한 대사 과정을 조절합니다.

Fig. 2

Overview of Mitochondrial Metabolism.

As the central hub of bioenergetics, the mitochondrion utilizes NADH and FADH2 produced by the TCA cycle to generate ATP through electron transfer and the H+ gradient across the respiratory chain complexes. Complexes I and III are the primary sources of mtROS, which cause oxidative damage or signaling transduction. The mtROS also can induce the opening of mPTP. Glucose and lipids (via β-oxidation) both contribute to the TCA cycle. Citrate can cross the mitochondrial membrane, allowing acetyl-CoA to be transported into the cytoplasm for various functions. TCA cycle tricarboxylic acid cycle; CACT carnitine-acylcarnitine translocase; CPT I/II carnitine palmitoyltransferase I/II; mtROS mitochondrial reactive oxygen species; ANT adenine nucleotide translocator; VDAC voltage-dependent anion channel; mPTP mitochondrial permeability transition pore

미토콘드리아 대사 개요.

생체 에너지 대사의 중심 허브인 미토콘드리아는

TCA 회로에서 생성된 NADH와 FADH2를

전자 전달과 호흡 사슬 복합체 간의 H+ 농도 차이를 통해 ATP를 생성합니다.

복합체 I과 III는

미토콘드리아 활성 산소 종(mtROS)의 주요 원천으로,

산화 손상이나 신호 전달을 유발합니다.

mtROS는

또한 mPTP의 개방을 유발할 수 있습니다.

포도당과 지질(β-산화 경로를 통해)은

모두 TCA 회로에 기여합니다.

시트르산은

미토콘드리아 막을 통과하여

아세틸-CoA가 세포질로 운반되어

다양한 기능에 사용됩니다.

TCA 회로 트리카르복실산 회로; CACT 카르니틴-아실카르니틴 트랜스포터; CPT I/II 카르니틴 팔미토일트랜스퍼레이스 I/II; mtROS 미토콘드리아 활성산소종; ANT 아데닌 핵산 트랜스포터; VDAC 전압 의존성 아ニオン 채널; mPTP 미토콘드리아 투과성 전환 구멍

Full size image

Oxidative phosphorylation

The primary function of mitochondria is energy production, with the majority of ATP being generated through OXPHOS.85 The OXPHOS system, essential for mitochondrial respiration, consists of five multimeric protein complexes located in the cristae of the inner mitochondrial membrane (IMM).86 The respiratory chain complexes (Complexes I–IV), collectively known as the electron transport chain (ETC), facilitate the transfer of electrons from nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) to oxygen through a series of redox reactions. This process contributes to the formation of an electrochemical (proton) gradient across the IMM, ultimately reducing oxygen to H2O.87 To enhance stability and efficiency, these respiratory chain complexes often assemble into supramolecular structures.88 The proton gradient drives the translocation of protons from the intermembrane space (IMS) to the matrix via ATP synthase (Complex V), which catalyzes the conversion of ADP to ATP.85,89 Of the polypeptides involved in OXPHOS, 13 are encoded by mtDNA, while the remainder are encoded by the nuclear genome.53,89

산화적 인산화

미토콘드리아의 주요 기능은

에너지 생산이며,

ATP의 대부분은 OXPHOS를 통해 생성됩니다.85

미토콘드리아 호흡에 필수적인 OXPHOS 시스템은

내막 미토콘드리아 막(IMM)의 크리스타에 위치한 다중 단백질 복합체 5개로 구성됩니다.86

호흡 사슬 복합체(복합체 I–IV)는

전자 전달 사슬(ETC)로 알려져 있으며,

니코틴아미드 아데닌 디뉴클레오티드(NADH)와 플라빈 아데닌 디뉴클레오티드(FADH2)에서

산소로 전자를 전달하는 일련의 환원-산화 반응을 촉진합니다.

이 과정은

IMM을 가로지르는 전기화학적(프로톤) 그라디언트의 형성에 기여하며,

최종적으로 산소를 H₂O로 환원시킵니다.87

안정성과 효율성을 높이기 위해 이러한 호흡 사슬 복합체는

종종 초분자 구조로 조립됩니다.88 

양성자 기울기 proton gradient는

ADP를 ATP로 전환하는 ATP 합성 효소(복합체 V)를 통해 

양성자를 막간 공간(IMS)에서 매트릭스로 이동시킵니다.85,89 

OXPHOS에 관여하는 폴리펩티드 중 13개는

mtDNA에 의해 암호화되고,

나머지는 핵 게놈에 의해 암호화됩니다.53,89

Glucose metabolism

In glucose metabolism, glucose is initially converted to pyruvate through glycolysis in the cytoplasm. Pyruvate is then either transported into the mitochondria and converted to acetyl-CoA by pyruvate dehydrogenase or converted to lactate by lactate dehydrogenase in the cytoplasm.90 Acetyl-CoA, the principal substrate, enters the tricarboxylic acid (TCA) cycle.91 Within mitochondria, citrate synthase catalyzes the condensation of acetyl-CoA and oxaloacetate to form citrate. Citrate can either proceed through the TCA cycle, generating NADH, FADH2, and guanosine triphosphate (GTP), or be transported to the cytoplasm where it regenerates acetyl-CoA and oxaloacetate.92 

When carbohydrate supply is excessive, acetyl-CoA is converted to citrate, which can then exit the mitochondria to participate in lipid synthesis or histone acetylation in the cytoplasm or nucleus.92,93,94 Additionally, certain gluconeogenesis processes occur in mitochondria, such as the conversion of pyruvate to oxaloacetate, followed by its conversion to malic acid and aspartic acid to facilitate gluconeogenesis.95

포도당 대사

포도당 대사에서 포도당은 세포질에서 글리코lysis를 통해 피루vate로 전환됩니다. 피루vate는 미토콘드리아로 운반되어 피루vate dehydrogenase에 의해 아세틸-CoA로 전환되거나, 세포질에서 젖산 dehydrogenase에 의해 젖산으로 전환됩니다.90 

아세틸-CoA는

주요 기질로 트리카르복실산(TCA) 순환에 진입합니다.91 

미토콘드리아 내에서는

시트르산 합성효소가 아세틸-CoA와 옥살아세트산을 결합시켜

시트르산을 형성합니다.

시트르산은

TCA 회로를 통해 NADH, FADH2, 및 구아노신 삼인산(GTP)을 생성하거나,

세포질로 운반되어 아세틸-CoA와 옥살아세트산을 재생성합니다.92

탄수화물 공급이 과다할 경우

아세틸-CoA는 시트르산으로 전환되어

미토콘드리아를 벗어나

세포질이나 핵에서 지질 합성 또는 히스톤 아세틸화에 참여할 수 있습니다.92,93,94

When carbohydrate supply is excessive,

acetyl-CoA is converted to citrate,

which can then exit the mitochondria to participate in lipid synthesis or histone acetylation in the cytoplasm or nucleus.

과도한 탄수화물 공급이 지질 합성 및 히스톤 아세틸화로 이어지는 과정

우리 몸이 탄수화물(주로 포도당)을 과도하게 섭취하면, 이 포도당은 에너지 생산을 위해 여러 대사 경로를 거치게 됩니다. 하지만 에너지가 이미 충분히 공급되거나 넘치는 상황에서는, 남은 포도당은 단순한 에너지원 이상의 역할(주로 저장)을 하게 됩니다.

과정은 다음과 같습니다:

1. • 섭취된 탄수화물(포도당)은 세포질에서 해당과정(glycolysis)을 통해 **피루브산(pyruvate)**으로 분해됩니다.
   • 이 피루브산은 미토콘드리아 안으로 들어가기 전에 피루브산 탈수소효소 복합체(pyruvate dehydrogenase complex)에 의해 산화되어 **아세틸-CoA(acetyl-CoA)**로 전환됩니다. 아세틸-CoA는 지방산, 콜레스테롤, 케톤체 등 다양한 분자의 핵심 전구체(precursor)이자, TCA 회로의 주된 연료입니다.
2. • 생성된 아세틸-CoA는 미토콘드리아 기질(matrix)로 들어갑니다. 미토콘드리아는 세포의 에너지 생산 공장이며, 아세틸-CoA는 여기서 **TCA 회로(구연산 회로, Citric Acid Cycle)**로 진입하여 ATP(에너지)를 생산하는 데 사용됩니다.
   • TCA 회로의 첫 번째 단계에서 아세틸-CoA는 옥살로아세트산(oxaloacetate)과 결합하여 **구연산(citrate, 시트르산)**을 형성합니다. 이 반응은 구연산 합성효소(citrate synthase)에 의해 촉매됩니다.
   • 핵심: 우리 몸에 에너지가 이미 풍부한 (탄수화물 공급이 과도한) 상황에서는, TCA 회로가 더 이상 많은 에너지를 생산할 필요가 없습니다. 따라서 TCA 회로의 중간 대사체들이 축적되게 되는데, 특히 구연산이 미토콘드리아 내부에 많이 쌓이게 됩니다.
3. • 아세틸-CoA는 미토콘드리아 내막을 직접 통과할 수 없습니다. 하지만 구연산은 미토콘드리아 내막에 있는 **구연산 운반체(citrate transporter)**를 통해 미토콘드리아 외부인 세포질(cytosol)로 쉽게 운반될 수 있습니다.
   • 미토콘드리아 내 구연산의 농도가 높아지면, 이 농도 기울기에 따라 구연산은 세포질로 활발하게 유출됩니다. 이는 과잉의 아세틸-CoA가 미토콘드리아 밖으로 나갈 수 있는 "우회로" 역할을 합니다.
4. • 세포질로 나온 구연산은 **ATP-구연산 분해효소(ATP-citrate lyase)**라는 효소에 의해 다시 아세틸-CoA와 옥살로아세트산으로 분해됩니다.
   • 이렇게 세포질에서 다시 생성된 아세틸-CoA는 미토콘드리아에서 에너지를 생산하는 데 사용되는 아세틸-CoA와는 다른 용도로 사용됩니다. 즉, 이 아세틸-CoA는 이제 주로 "합성" 과정에 참여하게 됩니다.
5. • 세포질 내 아세틸-CoA는 **지방산 합성(fatty acid synthesis)**의 핵심 전구체입니다. 아세틸-CoA 카르복실화효소(acetyl-CoA carboxylase)와 지방산 합성효소(fatty acid synthase)와 같은 효소들을 통해 아세틸-CoA 분자들이 연결되어 긴 사슬의 지방산이 만들어집니다.
   • 이렇게 합성된 지방산은 글리세롤과 결합하여 **중성지방(triglycerides)**으로 저장됩니다. 이것이 바로 우리가 탄수화물을 과도하게 섭취할 때 지방으로 축적되는 주된 메커니즘입니다.
6. • 세포질 내 아세틸-CoA는 단순히 지질 합성뿐만 아니라, 세포핵으로 이동하여 히스톤 아세틸화(histone acetylation) 과정에도 참여합니다.
   • 히스톤은 DNA가 감겨 있는 단백질로, 염색질(chromatin) 구조를 형성합니다. 히스톤에 아세틸기(acetyl group)가 붙는 것을 히스톤 아세틸화라고 합니다. 이 반응은 **히스톤 아세틸전달효소(HATs, Histone Acetyltransferases)**에 의해 촉매되며, 아세틸-CoA가 아세틸기의 공급원입니다.
   • 중요성: 히스톤 아세틸화는 염색질의 구조를 느슨하게 만듭니다(즉, DNA를 더 쉽게 접근 가능하게 만듭니다). 이는 RNA 중합효소(RNA polymerase)와 같은 전사 인자들이 DNA에 더 쉽게 결합할 수 있게 하여 유전자 발현(gene expression‎)을 촉진합니다.
   • 의미: 따라서 과도한 탄수화물 공급으로 인해 세포질 내 아세틸-CoA가 증가하면, 이는 특정 유전자들의 발현을 변화시킬 수 있는 후성유전학적(epigenetic) 조절 신호로 작용하게 됩니다. 예를 들어, 지질 합성 관련 효소 유전자들의 발현을 증가시켜 지방 축적을 더욱 촉진할 수 있습니다.

---

요약하자면, 과도한 탄수화물 섭취 → 포도당 과잉 → 아세틸-CoA 과잉 (미토콘드리아 내) → 구연산 축적 (미토콘드리아 내) → 구연산의 세포질 유출 → 세포질 내 아세틸-CoA 재 생성 → 지질 합성 (에너지 저장) 및 히스톤 아세틸화 (유전자 발현 조절)로 이어지는 일련의 대사 경로입니다. 이 과정은 세포가 에너지 과잉 상태에 적응하고, 필요한 경우 지방으로 에너지를 저장하며, 이 대사 신호에 반응하여 유전자 발현 패턴까지 조절하는 복잡하고 정교한 메커니즘을 보여줍니다.

또한

미토콘드리아에서 글루코네오게네시스 과정이 발생하며,

이는 피루브산이 옥살아세테이트로 전환된 후

말산과 아스파르트산으로 전환되어 글루코네오게네시스를 촉진합니다.95

Lipid metabolism

The β-oxidation of fatty acids is another major energy source.96 Initially, free fatty acids (FFAs) are activated to fatty acyl-CoA in the cytosol by fatty acyl-CoA synthetase. Fatty acyl-CoA then combines with carnitine to form acylcarnitine before crossing the outer mitochondrial membrane (OMM) and IMM to enter the mitochondrial matrix.97,98 In the mitochondrial matrix, acylcarnitine regenerates into fatty acyl-CoA, which then undergoes β-oxidation to produce NADH/FADH2 and acetyl-CoA.97 The transport of fatty acyl-CoA is facilitated by carnitine palmitoyltransferase I (CPTI) on the OMM, carnitine palmitoyltransferase II (CPTII), and carnitine-acylcarnitine translocase (CACT) on the IMM.99 Some acetyl-CoA generated through β-oxidation is converted into ketone bodies in the liver, which serve as a key energy source.98

지질 대사

지방산의 β-산화는

또 다른 주요 에너지 원천입니다.96 

초기 단계에서 자유 지방산(FFAs)은

세포질에서 지방산-CoA 합성효소에 의해 지방산-CoA로 활성화됩니다.

지방산-CoA는 카르니틴과 결합하여

아실카르니틴을 형성한 후 미토콘드리아 외막(OMM)과 내막(IMM)을 통과하여

미토콘드리아 매트릭스로 들어갑니다.97,98

미토콘드리아 매트릭스에서 아실카르니틴은

지방산-CoA로 재생성되며,

이는 β-산화 과정을 거쳐 NADH/FADH2와 아세틸-CoA를 생성합니다.97

지방산 아실-CoA의 운반은

OMM에 위치한 카르니틴 팔미토일트랜스퍼레이스 I (CPTI),

IMM에 위치한 카르니틴 팔미토일트랜스퍼레이스 II (CPTII), 및

카르니틴-아실카르니틴 트랜스록레이스 (CACT)에 의해 촉진됩니다.99

β-산화를 통해 생성된 일부 아세틸-CoA는

간에서 케톤체로 전환되어 주요 에너지 원으로 작용합니다.98

베타-산화(β-oxidation)를 통해 생성된 아세틸-CoA가 간에서 케톤체로 전환되어 주요 에너지원으로 사용되는 과정

우리 몸이 에너지원, 특히 탄수화물이 부족할 때 지방을 분해하여 에너지를 얻는 중요한 대사 경로를 설명

1. 배경: 지방산의 분해 (베타-산화)와 아세틸-CoA 생성

• 언제 발생하는가? 우리 몸은 주로 포도당(탄수화물)을 에너지원으로 사용합니다. 하지만 공복 상태가 길어지거나(예: 단식), 저탄수화물 식단을 유지하거나, 장시간 운동을 할 때처럼 포도당 공급이 부족해지면, 몸은 비축해둔 지방을 에너지원으로 사용하기 시작합니다.
• 어떻게 발생하는가? 저장된 중성지방(triglycerides)은 지방산과 글리세롤로 분해됩니다. 이 지방산은 세포의 미토콘드리아 안으로 들어가 **베타-산화(β-oxidation)**라는 과정을 거칩니다.
• 베타-산화의 결과: 베타-산화는 지방산 사슬을 두 탄소 단위인 아세틸-CoA(acetyl-CoA) 분자들로 반복적으로 잘라내는 과정입니다. 이렇게 생성된 아세틸-CoA는 원래 TCA 회로(구연산 회로)에 들어가 에너지(ATP)를 생성하는 데 사용되어야 합니다.

2. 간에서의 특별한 아세틸-CoA 처리 (케톤체 형성)

• 왜 간인가? 대부분의 조직(근육, 뇌 등)에서는 베타-산화를 통해 생성된 아세틸-CoA가 해당 조직의 미토콘드리아에서 직접 TCA 회로로 들어가 에너지로 사용됩니다.
  • 하지만 **간(liver)**은 포도당 공급이 부족할 때 케톤체를 생산하는 유일한 기관입니다. 간은 지방산을 매우 활발하게 베타-산화하여 많은 양의 아세틸-CoA를 생성합니다.
  • 이때, 간은 동시에 **포도당 신생합성(gluconeogenesis)**이라는 과정을 통해 비탄수화물(예: 아미노산, 글리세롤)로부터 새로운 포도당을 만들려고 합니다. 포도당 신생합성은 TCA 회로의 중간체인 옥살로아세트산(oxaloacetate)을 사용하는데, 옥살로아세트산은 아세틸-CoA가 TCA 회로로 진입하는 데 필수적인 파트너입니다.
  • 따라서 포도당이 부족한 상황에서는 간에서 옥살로아세트산이 포도당 신생합성으로 전환되면서 그 양이 부족해집니다. 결과적으로 간의 미토콘드리아 내부에 아세틸-CoA는 넘쳐나지만, 이를 TCA 회로로 돌릴 옥살로아세트산이 부족해지는 상황이 발생합니다.
• 케톤체 형성(Ketogenesis) 과정: 아세틸-CoA가 TCA 회로로 들어가지 못하고 과도하게 축적되면, 간 미토콘드리아는 이 아세틸-CoA를 이용하여 **케톤체(ketone bodies)**라는 대사 물질을 합성합니다.
  • 두 분자의 아세틸-CoA가 결합하여 아세토아세틸-CoA를 만들고, 여기에 다시 아세틸-CoA가 추가되어 HMG-CoA가 됩니다.
  • HMG-CoA는 최종적으로 **아세토아세트산(acetoacetate)**과 아세틸-CoA로 분해됩니다. 아세토아세트산은 케톤체의 주된 형태입니다.
  • 아세토아세트산은 또한 자발적으로 탈탄산화되어 소량의 **아세톤(acetone)**으로 변하거나, 환원되어 **베타-하이드록시부티르산(β-hydroxybutyrate)**으로 변할 수 있습니다. 이 세 가지(아세토아세트산, 아세톤, 베타-하이드록시부티르산)를 통틀어 케톤체라고 합니다.

3. 케톤체의 에너지원으로서의 역할

• 이동: 간에서 생성된 케톤체(주로 아세토아세트산과 베타-하이드록시부티르산)는 물에 잘 녹는 특성 덕분에 혈액을 통해 간 밖의 다른 조직들로 쉽게 운반됩니다.
• 사용(Ketolysis): 간을 제외한 거의 모든 조직(특히 근육, 신장, 그리고 가장 중요하게 뇌)은 케톤체를 에너지원으로 사용할 수 있습니다.
  • 이들 조직에서 케톤체는 다시 아세틸-CoA로 전환됩니다.
  • 이렇게 재 생성된 아세틸-CoA는 해당 조직의 미토콘드리아에서 TCA 회로로 진입하여 ATP(에너지)를 생산하는 데 사용됩니다.
• 왜 중요한가? 특히 뇌는 지방산(탄수화물)을 직접 연료로 사용할 수 없습니다 (혈액-뇌 장벽 때문에). 정상적인 상황에서는 포도당이 뇌의 주된 연료입니다. 하지만 공복이 길어지거나 탄수화물 섭취가 극도로 제한될 때, 뇌는 포도당 대신 케톤체를 주요 에너지원으로 사용하게 됩니다. 이는 뇌 기능 유지를 위해 포도당이 절대적으로 필요한 상황에서, 단백질(근육 등)이 과도하게 분해되어 포도당 신생합성에 사용되는 것을 막아주는 중요한 적응 기전입니다.

---

결론적으로, 이 과정은 우리 몸이 탄수화물 부족 상황에서 지방을 효과적으로 에너지로 전환하고, 특히 뇌와 같은 중요한 기관에 대체 연료를 공급하여 생존을 가능하게 하는 정교한 대사 적응 메커니즘입니다.

Oxidative stress

Oxidative stress occurs when the formation of reactive oxygen species (ROS) exceeds the capacity of the antioxidant defense system, which includes enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx).100 This imbalance, primarily due to an excess of ROS, leads to cellular damage. ROS includes molecules such as superoxide anion (O2−•), hydrogen peroxide (H2O2), singlet oxygen (1O2), and hydroxyl radicals (OH•),101,102 which can impair mitochondrial function by damaging mtDNA, proteins, membrane lipids, and other cellular components.103

Mitochondria are a major source of ROS, and even under normal conditions, the ETC generates mitochondrial ROS (mtROS) as a byproduct.104,105 However, ROS can also originate from other sources, such as NADPH oxidase (Nox), monoamine oxidase (MAO), p66Shc, α-glycerophosphate dehydrogenase, electron transfer flavoprotein (ETF) and ETF dehydrogenase, and aconitase.102 The contribution of these sources varies depending on the type of ROS, the cell type, and the specific physiological or pathological conditions.106 Despite these other sources, mitochondria remain a significant producer of ROS and therefore merit considerable attention.

ROS plays a complex and essential role in cellular physiology. Although high concentrations of ROS are typically viewed as harmful byproducts of aerobic metabolism, at physiological levels, ROS function as key secondary messengers. They regulate various signaling pathways, including PI3K, MAPK, AMPK, NRF2, NF-κB, and p53, and influence enzyme activity, gene expression‎, cell proliferation, differentiation, immune responses, apoptosis, and mitochondrial quality control, allowing cells to adapt to environmental changes.107,108,109 For example, low ROS levels are critical for maintaining the self-renewal potential of stem cells.110 In addition, moderate ROS concentrations are vital for promoting axonal and dendritic growth, maintaining neuronal function, and supporting the self-renewal of neural stem cells and neurogenesis.107,109

A significant consequence of OXPHOS dysfunction is the increased production of mtROS due to decreased electron transfer efficiency in the ETC, which results in more electrons leaking and interacting with O2.88 Oxidative stress is known to play a role in the pathophysiology of mitochondrial diseases.111 Complexes I and III are the primary sites of mtROS production (Fig. 2). At Complex I, flavin mononucleotide (FMN) and possibly CoQ can transfer an electron to O2, generating O2−•, while at Complex III, ubisemiquinone (CoQH•) in the Qo site also contributes to O2−• production.101 However, the amount of mtROS produced at these sites varies between tissues: the brain primarily generates mtROS at Complex I, while the heart and lungs mainly rely on Complex III.101,104,112 O2−• can be dismutated to H2O2 by SOD or react with NO• to form peroxynitrite. H2O2 can be fully reduced to H2O or partially reduced to OH•.113

ROS disrupts cellular homeostasis by damaging lipids, proteins, and DNA.114 Due to their proximity, mtROS pose a significant threat to mtDNA, which, lacking histone protection, is especially vulnerable. When mtROS accumulate beyond a certain threshold, they can lead to a reduction in Δψm, which in turn may trigger the opening of the mitochondrial permeability transition pore (mPTP).115 The mPTP is a complex structure which spans the IMM and OMM, comprising the voltage-dependent anion channel (VDAC), adenine nucleotide translocator (ANT), ATP synthetase, and cyclophilin D.88,116,117 Additionally, β-tubulin regulates mPTP opening through its interaction with VDAC.118

Transient opening of the mPTP allows mitochondria to release excess ROS and Ca2+, preventing the harmful accumulation of these molecules. However, prolonged mPTP opening can lead to a secondary burst of ROS, a process known as ROS-induced ROS release.102 While RIRR can help eliminate irreversibly damaged mitochondria to maintain cellular homeostasis, it can also result in pathological consequences. The mPTP can also be activated by elevated Ca2+ levels.102,119 Persistent mPTP opening severely disrupts mitochondrial membrane function, becoming an important factor in mitochondrial dysfunction and ultimately leading to the activation of mitochondrial apoptosis.118 Beyond apoptosis, the mPTP plays a role in regulating other forms of cell death.17 The tendency for mPTP opening increases with aging, which further exacerbates the reduction in Δψm.84

산화 스트레스

산화 스트레스는

활성 산소 종(ROS)의 생성량이 항산화 방어 시스템의 용량을 초과할 때 발생합니다.

이 시스템에는

슈퍼옥사이드 디스뮤타제(SOD), 카탈라제(CAT), 글루타티온 환원효소(GR), 글루타티온 과산화효소(GPx)와 같은

효소가 포함됩니다.100 

이 불균형은

주로 ROS의 과다로 인해 세포 손상을 초래합니다.

ROS에는

슈퍼옥사이드 음이온(O2−•), 과산화수소(H2O2), 단일 산소(1O2), 하이드록실 라디칼(OH•) 등이 포함되며,101,102 

이러한 물질은 미토콘드리아 DNA(mtDNA), 단백질, 세포막 지질 및 기타 세포 구성 요소를 손상시켜

미토콘드리아 기능을 저해할 수 있습니다.103

미토콘드리아는

ROS의 주요 발생원으로,

정상 조건에서도 ETC는 미토콘드리아 ROS(mtROS)를 부산물로 생성합니다.104,105 

그러나

ROS는 NADPH 산화효소(Nox), 모노아민 산화효소(MAO), p66Shc,

α-글리세로포스페이트 탈수소효소, 전자 전달 플라보단백질(ETF) 및 ETF 탈수소효소, 아코니타제와 같은

다른 원천에서도 발생할 수 있습니다.102 

이러한 원천의 기여도는

ROS의 유형, 세포 유형, 특정 생리적 또는 병리적 조건에 따라 달라집니다.106 

이러한 다른 원천에도 불구하고

미토콘드리아는 여전히 ROS의 주요 생산원으로 남아 있으며

따라서 상당한 주목을 받을 가치가 있습니다.

ROS는

세포 생리학에서 복잡하고 필수적인 역할을 합니다.

일반적으로 높은 농도의 ROS는

산소 호흡 대사 과정의 유해한 부산물로 간주되지만,

생리적 수준에서는 핵심적인 제2 신호 전달체로 기능합니다.

이들은 PI3K, MAPK, AMPK, NRF2, NF-κB, 및 p53 신호 전달 경로를 조절하며,

효소 활성, 유전자 발현, 세포 증식, 분화, 면역 반응, 세포 사멸, 및 미토콘드리아 품질 관리에 영향을 미쳐

세포가 환경 변화에 적응할 수 있도록 합니다.107,108,109 

예를 들어,

낮은 ROS 수준은

줄기세포의 자기 재생 잠재력을 유지하는 데 필수적입니다.110 

또한 중간 수준의 ROS 농도는

축삭 및 수상돌기 성장 촉진, 신경 기능 유지, 신경 줄기세포의 자기 재생 및

신경 발생 지원에 필수적입니다.107,109

OXPHOS 기능 장애의 주요 결과는

전자 전달 효율 감소로 인해 ETC에서 mtROS 생산이 증가하는 것입니다.

이는 더 많은 전자들이 누출되어

O2와 상호작용하게 됩니다.88 

산화 스트레스는

미토콘드리아 질환의 병리생리학에 역할을 하는 것으로 알려져 있습니다.111 

복합체 I과 III는

mtROS 생산의 주요 부위입니다(그림 2).

복합체 I에서는

플라빈 모노뉴클레오티드(FMN)와 아마도 코엔자임 Q(CoQ)가

O2에 전자를 전달하여 O2−•를 생성하며,

복합체 III에서는 Qo 부위의 우비세미퀴논(CoQH•)도 O2−• 생성에 기여합니다.101 

그러나

이러한 부위에서 생성되는 mtROS의 양은 조직에 따라 다릅니다.

뇌는 주로 복합체 I에서 mtROS를 생성하는 반면,

심장과 폐는 주로 복합체 III에 의존합니다.101,104,112 

O2−•는 SOD에 의해 H2O2로 분해되거나 NO•와 반응하여 퍼옥시니트라이트를 형성할 수 있습니다.

H₂O₂는 완전히 H₂O로 환원되거나 부분적으로 OH•로 환원될 수 있습니다.113

ROS는

지질, 단백질, DNA를 손상시켜

세포 내 항상성을 방해합니다.114 

mtROS는

히스톤 보호가 없는 mtDNA에 특히 취약하기 때문에,

그 근접성으로 인해 심각한 위협을 가합니다.

mtROS가

특정 임계치를 초과해 축적되면 Δψm이 감소할 수 있으며,

이는 다시 미토콘드리아 투과성 전환 구멍(mPTP)의 개방을 유발할 수 있습니다.115 

mPTP는 IMM과 OMM을 가로지르는 복잡한 구조로,

전압 의존성 음이온 채널(VDAC),

아데닌 뉴클레오티드 트랜스포터(ANT),

ATP 합성효소,

사이클로필린 D로 구성됩니다.88,116,117 

또한 β-튜불린은

VDAC와의 상호작용을 통해 mPTP 개방을 조절합니다.118

mPTP의 일시적인 개방은

미토콘드리아가 과도한 ROS와 Ca2+를 방출하여

이러한 분자의 유해한 축적을 방지합니다.

그러나

mPTP의 장기적인 개방은

ROS-유발 ROS 방출(ROS-induced ROS release)로 알려진 과정으로 이어질 수 있습니다.102 

RIRR은

세포 내 항상성을 유지하기 위해 영구적으로 손상된 미토콘드리아를 제거하는 데

도움을 줄 수 있지만,

병리학적 결과를 초래할 수도 있습니다.

mPTP는

또한 증가된 Ca2+ 수준에 의해 활성화될 수 있습니다.102,119 

지속적인 mPTP 개방은 미토콘드리아 막 기능을 심각하게 방해하여

미토콘드리아 기능 장애의 중요한 요인이 되며,

결국 미토콘드리아 아포토시스 활성화로 이어집니다.118 

아포토시스 외에도 mPTP는 다른 형태의 세포 사멸을 조절하는 역할을 합니다.17 

mPTP 개방 경향은 노화와 함께 증가하며,

이는 Δψm의 감소가 더욱 악화됩니다.84

Mitochondrial quality control

Mitochondrial quality control (MQC) is a complex and integrated network that monitors mitochondrial integrity, responds to damage or stress, and maintains mitochondrial homeostasis. This system coordinates various processes, including mt-ISR, biogenesis, dynamics, mitophagy, and intercellular mitochondria transfer (Fig. 3). MQC plays a pivotal role in a wide range of human diseases, including cancer, cardiovascular diseases, and neurodegenerative disorders.120,121,122,123,124

미토콘드리아 품질 관리

미토콘드리아 품질 관리(MQC)는

미토콘드리아의 무결성을 모니터링하고 손상 또는 스트레스에 대응하며

미토콘드리아 항상성을 유지하는 복잡하고 통합된 네트워크입니다.

이 시스템은

mt-ISR, 생합성, 역학, 미토파지, 세포 간 미토콘드리아 전달 등

다양한 과정을 조정합니다(그림 3).

MQC는

암, 심혈관 질환, 신경 퇴행성 질환 등

다양한 인간 질병에서 중요한 역할을 합니다.120,121,122,123,124

Fig. 3

Mitochondrial Quality Control Network.

Mitochondria employ both intercellular and intracellular quality control mechanisms to maintain homeostasis and redox balance. These mechanisms include mitochondrial biogenesis, fusion, fission, axonal transport, docking, mitophagy, the mitochondrial integrated stress response, and intercellular mitochondrial transfer. IMM inner mitochondrial membrane; OMM outer mitochondrial membrane; Δψm mitochondrial membrane potential; Ub ubiquitin; mt-ISR mitochondrial integrated stress response; mt-UPR mitochondrial unfolded protein response; MDV(s) mitochondria-derived vesicle(s)

미토콘드리아 품질 관리 네트워크.

미토콘드리아는

세포 간 및 세포 내 품질 관리 메커니즘을

모두 활용하여 항상성과 산화환원 균형을 유지합니다.

이러한 메커니즘에는

미토콘드리아 생합성, 융합, 분열, 축삭 수송, 도킹, 미토파지, 미토콘드리아 통합 스트레스 반응,

세포 간 미토콘드리아 전달이 포함됩니다.

mitochondrial biogenesis,

fusion, fission,

axonal transport, docking, mitophagy, the mitochondrial integrated stress response, and

intercellular mitochondrial transfer

IMM 내막; OMM 외막; Δψm 미토콘드리아 막 전위; Ub 유비퀴틴; mt-ISR 미토콘드리아 통합 스트레스 반응; mt-UPR 미토콘드리아 미접힘 단백질 반응; MDV(s) 미토콘드리아 유래 소체(들)

Mitochondrial integrated stress response

The mt-ISR in mammals is a multifaceted mechanism that includes a transcriptional response, metabolic remodeling, and the mitochondrial unfolded protein response (mt-UPR).125 This response can be triggered by various mitochondrial stressors such as OXPHOS defects, reduction in Δψm, increased ROS level, amino acid deprivation, or the accumulation of unfolded proteins.126,127 The mt-ISR is essential for maintaining OXPHOS function and mitochondrial homeostasis through mito-nuclear communication. Key to mt-ISR are the eIF2α kinases—HRI, PKR, PERK, and GCN2—which are activated in response to mitochondrial stress.127,128 Activation of these kinases leads to the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α), which reduces global protein synthesis while selectively enhancing the translation of stress-related transcription factors like ATF4, ATF5, and CHOP, thereby activating the mt-UPR.129,130,131 This translational reprogramming helps reduce cellular energy consumption and protects cells from mitochondrial dysfunction. However, mt-ISR can also trigger apoptosis to eliminate severely damaged cells under intense stress.128 In the context of mitochondrial diseases, genetically defective respiratory chain complexes or mitochondrial dysfunction can activate mt-ISR, which aims to enhance ATP production and antioxidant capacity, thereby attempting to restore mitochondrial homeostasis before mitophagy is activated.132 While mt-ISR activation could be a common event in mitochondrial diseases, its overactivation may be detrimental, exacerbating the disease condition.125

미토콘드리아 통합 스트레스 반응

포유류의 mt-ISR은

전사 반응, 대사 재편성,

미토콘드리아 미접힘 단백질 반응(mt-UPR)을 포함하는

다면적인 메커니즘입니다.125 

이 반응은 OXPHOS 결함, Δψm 감소, ROS 수준 증가, 아미노산 결핍, 또는 미접힘 단백질 축적과 같은 다양한 미토콘드리아 스트레스 요인에 의해 유발될 수 있습니다.126,127 mt-ISR은 미토-핵 통신을 통해 OXPHOS 기능과 미토콘드리아 항상성을 유지하는 데 필수적입니다. mt-ISR의 핵심은 미토콘드리아 스트레스에 반응하여 활성화되는 eIF2α 키나제—HRI, PKR, PERK, 및 GCN2—입니다.127,128 이 키나제의 활성화는 진핵생물 번역 시작 인자 2 알파(eIF2α)의 인산화를 유발하여 전체 단백질 합성을 감소시키며, 동시에 ATF4, ATF5, CHOP와 같은 스트레스 관련 전사 인자의 번역을 선택적으로 증강시켜 mt-UPR을 활성화합니다.129,130,131 이 번역 재프로그래밍은 세포 에너지 소비를 감소시키고 미토콘드리아 기능 장애로부터 세포를 보호합니다. 그러나 mt-ISR은 극한 스트레스 하에서 심하게 손상된 세포를 제거하기 위해 아포토시스를 유발할 수도 있습니다.128 미토콘드리아 질환의 맥락에서 유전적 결함으로 인한 호흡 사슬 복합체 또는 미토콘드리아 기능 장애는 mt-ISR을 활성화시켜 ATP 생산과 항산화 능력을 강화함으로써 미토파지가 활성화되기 전에 미토콘드리아 항상성을 회복하려는 시도를 합니다.132 mt-ISR 활성화는 미토콘드리아 질환에서 흔한 현상일 수 있지만, 과도한 활성화는 질환 상태를 악화시킬 수 있습니다.125

Mitochondrial biogenesis

Mitochondrial biogenesis is the process by which mitochondria increase in number and size.133 This process is primarily regulated by PGC-1α (PPAR-γ coactivator-1α), a key regulator that activates nuclear respiratory factors NRF-1 and NRF-2, as well as oestrogen-related receptor-α (ERR-α).134 These factors enhance the expression‎ of mitochondrial transcription factor A (TFAM, also known as mtTFA),135,136 which is critical for the replication and transcription of mtDNA and subsequent mitochondrial function.135 PGC-1β, which shares structural and functional similarities with PGC-1α, also promotes mitochondrial biogenesis.133 Additional regulators of this process include AMPK (AMP-activated protein kinase), nitric oxide (NO), SIRTs (sirtuins), TORCs (transducers of regulated CREB-binding protein), CaMK (calcium/calmodulin-dependent protein kinase), calcineurin, p38 MAPK, RIP140 (receptor-interacting protein 140), and Sin3A, which all influence mitochondrial biogenesis through the activation of PGC-1α.15,133

미토콘드리아 생합성

미토콘드리아 생성은

미토콘드리아의 수와 크기가 증가하는 과정입니다.133 

이 과정은 주로

PGC-1α (PPAR-γ 코액티베이터-1α)에 의해 조절되며,

이는 핵 호흡 인자 NRF-1과 NRF-2를 활성화하는 핵심 조절인자이자

에스트로겐 관련 수용체-α (ERR-α)를 조절합니다.134 

이러한 인자들은

미토콘드리아 전사 인자 A(TFAM, mtTFA로도 알려져 있음)의 발현을 증진시키며,135,136 

이는 mtDNA의 복제와 전사, 그리고 후속 미토콘드리아 기능에 필수적입니다.135 

PGC-1α와 구조적 및 기능적 유사성을 공유하는 PGC-1β도

미토콘드리아 생성을 촉진합니다.133 

이 과정의 추가 조절 인자에는

AMPK(AMP 활성화 단백질 키나제), 질소산화물(NO), SIRTs(sirtuins),

TORCs(규제된 CREB 결합 단백질의 전달체),

CaMK(칼슘/칼모듈린 의존성 단백질 키나제),

칼시뉴린, p38 MAPK, RIP140(수용체 상호작용 단백질 140), 및 Sin3A가 포함되며,

이들은 모두 PGC-1α의 활성화를 통해 미토콘드리아 생성에 영향을 미칩니다.15,133

Mitochondrial dynamics

The structure of mitochondria includes the mitochondrial matrix, OMM, IMM, and IMS. The IMM is further divided into the inner boundary membrane and mitochondrial cristae. The shape and size of mitochondria vary and are closely linked to their function.137 Mitochondrial dynamics involve two primary processes: fusion and fission, both of which are essential for maintaining mitochondrial homeostasis and are associated with mtDNA stability, oxidative stress, apoptosis, mitophagy, and cell division.138,139

Fission is the process by which a single mitochondrion divides into two or more daughter mitochondria. This process allows for the segregation of healthy mitochondria from those that are old or damaged, thereby eliminating mitochondria with irreversible mtDNA damage or low Δψm to maintain cellular homeostasis.140 Fission is closely associated with mitophagy, the selective degradation of damaged mitochondria, which will be discussed further in the next section.141 Additionally, fission helps meet increased energy demands by producing more daughter mitochondria.142 In endothelial cells, fission also aids in the localization of mitochondria near cytoskeletal proteins to support metabolic needs.142 Functional fission can mitigate mitochondrial damage in mitochondrial diseases.143 However, excessive fission is linked to pathological outcomes, including impaired mitochondrial bioenergetics as well as induction of oxidative stress and cell death.144 The GTPase dynamin-related protein 1 (Drp1) is a key regulator of fission, and the endoplasmic reticulum also plays a role in this process.145 Drp1 is recruited to mitochondria from the cytoplasm, where it interacts with mitochondrial fission factor (MFF), mitochondrial dynamics protein of 49 kDa (MID49), MID51, and mitochondrial fission 1 protein (FIS1) on the OMM to drive the fission process.146,147,148 Additionally, FIS1 inhibits mitochondrial fusion by interacting with mitofusins, thereby inhibiting their

미토콘드리아 동역학

미토콘드리아의 구조는

미토콘드리아 매트릭스, OMM, IMM, IMS로 구성됩니다.

IMM은 내막과 미토콘드리아 크리스타로 추가로 구분됩니다.

미토콘드리아의 모양과 크기는 다양하며 기능과 밀접하게 연관되어 있습니다.137

미토콘드리아 동역학은 융합과 분열이라는 두 가지 주요 과정을 포함하며,

이 두 과정은 미토콘드리아 항상성 유지에 필수적이며

mtDNA 안정성, 산화 스트레스, 아포토시스, 미토파지, 세포 분열과 연관되어 있습니다.138,139

분열은

단일 미토콘드리아가 두 개 이상의 딸 미토콘드리아로 분열되는 과정입니다.

이 과정은

건강한 미토콘드리아를 노화되거나 손상된 미토콘드리아로부터 분리하여,

회복 불가능한 mtDNA 손상이나 낮은 Δψm을 가진 미토콘드리아를 제거함으로써

세포 내 항상성을 유지합니다.140

분열은

손상된 미토콘드리아의 선택적 분해인 미토파지와 밀접하게 연관되어 있으며,

이는 다음 섹션에서 자세히 논의될 것입니다.141

또한 분열은 더 많은 딸 미토콘드리아를 생성함으로써 증가된 에너지 수요를 충족시키는 데 도움을 줍니다.142 내피 세포에서 분열은 세포 골격 단백질 근처에 미토콘드리아를 국소화하여 대사 요구를 지원합니다.142 기능적인 분열은 미토콘드리아 질환에서 미토콘드리아 손상을 완화할 수 있습니다.143 그러나 과도한 분열은 미토콘드리아 생체 에너지 대사 장애, 산화 스트레스 유도, 세포 사멸 등 병리학적 결과와 연관되어 있습니다.144 GTPase 동역학 단백질 1(Drp1)은 분열의 주요 조절인자이며, 내소기관도 이 과정에 역할을 합니다.145 Drp1은 세포질에서 미토콘드리아로 이동하여 OMM에 위치한 미토콘드리아 분열 인자(MFF), 미토콘드리아 동역학 단백질 49kDa(MID49), MID51, 미토콘드리아 분열 1 단백질(FIS1)과 상호작용하여 분열 과정을 촉진합니다.146,147,148 또한 FIS1은 미토퓨신과 상호 작용하여 미토퓨신의

GTPase activity.149

Mitochondrial fusion is the process where two or more mitochondria come into close contact and merge their IMM and OMM. This process relies on two key GTPases: optic atrophy protein 1 (OPA1) and mitofusins 1 and 2 (Mfn1 and Mfn2).150,151 Fusion begins with the merging of the OMM, driven by Mfn1 and Mfn2, which are localized on the OMM. After the OMM fusion, long OPA1 (L-OPA1) interacts with cardiolipin to facilitate the fusion of the IMM.152,153 There is also a short form of OPA1 (S-OPA1) produced by the proteolytic cleavage of L-OPA.153 The interaction between S-OPA1 and L-OPA1 promotes the fusion of the IMM.154,155 Fusion plays a pivotal role in mitochondrial heteroplasmy, which refers to the co-existence of mutant and wild-type (healthy) mitochondria.156 Defective mitochondria with mutant mtDNA can fuse with healthy mitochondria, compensating for defects by sharing components such as transcripts, thus mitigating the effects of mutations (heteroplasmy). Similarly, two defective mitochondria can fuse to cross-complement each other. Therefore, mitochondrial fusion can rescue certain dysfunctions if the mutation remains within a critical threshold.157,158 Fusion is often viewed as a defensive response, enabling mitochondria to adapt to cellular stress by reducing mtDNA heteroplasmy, bridging Δψm, and exchanging various metabolic intermediates.142

Mitochondrial transport is an ATP-dependent process that is especially critical in neurons. This transport occurs in both anterograde and retrograde directions along microtubules.159 Anterograde transport provides healthy and robust mitochondria from soma for distal axon, while damaged mitochondria in these distal regions are retrogradely transported to the soma for repair and degradation.160 Long-distance mitochondrial transport along microtubules is facilitated by two types of motor proteins: kinesin and dynein. In neurons, axonal microtubules are oriented with their minus ends toward the soma and their plus ends toward the distal axon. The minus-end-directed dynein drives retrograde transport, while the plus-end-directed kinesin (mainly KIF5) controls anterograde transport.161 The kinesin and dynein should interact with their motor adaptors before transport. For anterograde transport, the motor adaptor complex consisting of Miro (an atypical Rho GTPase) and Milton (TRAK in mammals) connects kinesin to mitochondria. Retrograde transport is primarily mediated by dynein and its motor adaptor, dynactin. The initiation of retrograde transport is believed to involve cooperation between the dynein-dynactin complex, VDAC on the OMM, and the Milton-Miro complex.162 Additionally, Drp1, Mfn1, and Mfn2 are thought to play roles in mitochondrial transport.160 There is also short-distance mitochondrial movement along actin filaments within dendritic spines, which is mediated by myosins.162

In contrast to transport, mitochondrial docking ensures that mitochondria remain in place to maintain stable mitochondrial numbers, adequate ATP production, and meet metabolic demands, particularly in regions with high energy requirements and metabolic stress.161 Syntaphilin, an anchoring protein, binds to the OMM and attaches axonal mitochondria to microtubules, resulting in mitochondrial docking.160

GTPase 활성을 억제하여 미토콘드리아 융합을 억제합니다.149

미토콘드리아 융합은

두 개 이상의 미토콘드리아가 밀접하게 접촉하여 IMM과 OMM을 융합하는 과정입니다.

이 과정은 두 가지 주요 GTPase인 광신경 위축 단백질 1(OPA1)과 미토퓨신 1 및 2(Mfn1 및 Mfn2)에 의존합니다.150,151 융합은 OMM에 국소화된 Mfn1 및 Mfn2에 의해 OMM의 융합으로 시작됩니다. OMM 융합 후, 장쇄 OPA1(L-OPA1)은 카르디올리핀과 상호작용하여 IMM의 융합을 촉진합니다.152,153 L-OPA의 프로테아제적 분해로 생성되는 단쇄 OPA1(S-OPA1)도 존재합니다.153 S-OPA1과 L-OPA1의 상호작용은 IMM의 융합을 촉진합니다.154,155 융합은 돌연변이형과 야생형(건강한) 미토콘드리아의 공존을 의미하는 미토콘드리아 이형성에서 핵심적인 역할을 합니다.156 돌연변이 mtDNA를 가진 결함 미토콘드리아는 건강한 미토콘드리아와 융합하여 전사체 등 구성 요소를 공유함으로써 결함을 보상하고 돌연변이의 영향을 완화합니다(이형성). 마찬가지로 두 개의 결함 미토콘드리아는 서로를 보완하기 위해 융합할 수 있습니다. 따라서, 미토콘드리아 융합은 돌연변이가 중요한 임계값 이내에 남아 있는 경우 특정 기능 장애를 회복시킬 수 있습니다.157,158 융합은 종종 방어적 반응으로 간주되며, 미토콘드리아가 세포 스트레스에 적응하기 위해 mtDNA 이형성을 감소시키고 Δψm을 연결하며 다양한 대사 중간체를 교환함으로써 이를 가능하게 합니다.142

미토콘드리아 운반은 ATP에 의존하는 과정으로, 특히 신경세포에서 매우 중요합니다. 이 수송은 미세관沿으로 전방 및 후방 방향으로 발생합니다.159 전방 수송은 소마에서 건강한 미토콘드리아를 원위 축삭으로 공급하며, 원위 부위의 손상된 미토콘드리아는 수리 및 분해를 위해 소마로 후방 수송됩니다.160 미세관沿의 장거리 미토콘드리아 수송은 두 종류의 모터 단백질인 키네신과 다이닌에 의해 촉진됩니다. 신경세포에서 축삭 미세관은 마이너스 끝이 세포체 방향으로, 플러스 끝이 원위 축삭 방향으로 정렬되어 있습니다. 마이너스 끝을 향한 다이닌은 역방향 수송을 주도하며, 플러스 끝을 향한 키네신(주로 KIF5)은 전방 수송을 조절합니다.161 키네신과 다이닌은 수송 전에 모터 적응체와 상호작용해야 합니다. 전방 수송을 위해 모터 적응 단백질 복합체(미로(비전형적 Rho GTPase)와 밀턴(포유류의 TRAK))가 키네신을 미토콘드리아에 연결합니다. 역방향 수송은 주로 다이닌과 그 모터 적응 단백질인 다이나틴에 의해 매개됩니다. 역방향 수송의 시작은 다이닌-다이나틴 복합체, OMM상의 VDAC, 밀턴-미로 복합체 간의 협력을 통해 이루어지는 것으로 추정됩니다.162 또한 Drp1, Mfn1, Mfn2가 미토콘드리아 수송에 역할을 하는 것으로 알려져 있습니다.160 ден드리틱 스파인 내 액틴 필라멘트沿의 단거리 미토콘드리아 이동은 마이오신에 의해 매개됩니다.162

운송과 달리 미토콘드리아 도킹은 미토콘드리아가 안정적인 수를 유지하고 적절한 ATP 생산을 보장하며, 특히 에너지 요구량이 높고 대사 스트레스가 큰 지역에서 대사 요구를 충족시키기 위해 미토콘드리아가 제자리에 머무르도록 합니다.161 앵커링 단백질인 시냅필린은 OMM에 결합하여 축삭 미토콘드리아를 미세관(microtubules)에 고정시켜 미토콘드리아 도킹을 유발합니다.160

Mitophagy

Mitophagy is the process by which damaged mitochondria are delivered to lysosomes for degradation, a concept first introduced by Lemasters as a specific form of organelle autophagy.163 Unlike mitochondrial biogenesis, which generates new mitochondria, mitophagy removes damaged or unnecessary mitochondria to maintain cellular homeostasis, often through a selection process mediated by mitochondrial fission.164 However, excessive mitophagy can lead to a significant loss of mitochondrial content, potentially triggering cell death.165,166 The interaction between Drp1 and Zip1 (a mitochondrial zinc transporter) at the fission site facilitates Zn2+ entry into the mitochondrial matrix, resulting in a localized reduction of Δψm, which subsequently initiates mitophagy in the affected mitochondria.167 Interestingly, mtDNA mutations alone may not be sufficient to trigger mitophagy.168 ROS plays a critical role in activating mitophagy, and in turn, mitophagy helps regulate ROS levels.169 Excessive ROS can induce non-selective autophagy in response to oxidative stress, while mild oxidative stress typically triggers selective mitophagy that is dependent on mitochondrial fission.170 The mitophagy process involves several key steps: the reduction of Δψm, formation of the mitophagosome, delivery of the mitophagosome to the lysosome, and finally, the degradation and recycling of mitochondrial components.171,172

Mitophagy mechanisms are generally categorized into ubiquitin (Ub)-dependent and Ub-independent pathways.

Among the Ub-dependent pathways, the phosphatase and tensin homolog-induced putative kinase 1 (PINK1)-Parkin pathway is the most extensively studied. In mitochondria with normal Δψm, PINK1 is transported to the IMM, where it is cleaved and degraded.173 However, when Δψm is reduced, PINK1 cannot reach the IMM and instead accumulates on the OMM. There, PINK1 phosphorylates ubiquitin and recruits and phosphorylates the E3 ubiquitin ligase Parkin.174,175 Once phosphorylated, Parkin binds to Ser65-phosphorylated ubiquitin on the OMM, fully activating its E3 ubiquitin ligase activity. This activation amplifies the pathway, promoting mitophagy.176 Mitochondria tagged with phosphorylated poly-Ub chains by Parkin are recognized by ubiquitin-binding receptor proteins such as OPTN, NDP52, SQSTM1/p62, TAX1BP1, and NBR1,177,178,179 which then bind with LC3 to initiate mitophagy. Additionally, TBK1 and Mfn2 have been shown to participate in this pathway.180,181 Beyond the PINK1-Parkin pathway, there are Parkin-independent, ubiquitin-dependent pathways where PINK1 directly recruits NDP52 and OPTN.177

Mitophagy receptors such as Fun14 domain containing 1 (FUNDC1), BCL2 interacting protein 3 (BNIP3), BCL2 interacting protein 3 like (BNIP3L/NIX), FKBP prolyl isomerase 8 (FKBP8), and ATAD3B can directly interact with LC3 through their LIR (LC3-interacting region) motifs, thereby initiating ubiquitin-independent mitophagy. Additionally, PHB2 and cardiolipin also participate in the ubiquitin-independent pathway due to their translocation to the OMM.171,182,183

미토파지

미토파기는 손상된 미토콘드리아를 리소좀으로 전달하여 분해하는 과정으로, Lemasters가 세포 기관의 자가포식의 특정 형태로 처음 소개한 개념입니다.163 새로운 미토콘드리아를 생성하는 미토콘드리아 생성과는 달리, 미토파기는 손상되거나 불필요한 미토콘드리아를 제거하여 세포의 항상성을 유지하며, 이는 종종 미토콘드리아 분열에 의해 매개되는 선택 과정을 통해 이루어집니다.164 그러나 과도한 미토파지는 미토콘드리아 내용물의 심각한 손실을 초래할 수 있으며, 이는 세포 사멸을 유발할 수 있습니다.165,166 Drp1과 미토콘드리아 아연 운반체 Zip1이 분열 부위에서 상호작용하여 Zn2+가 미토콘드리아 매트릭스로 유입되도록 촉진하며, 이는 Δψm의 국소적 감소로 이어져 영향을 받은 미토콘드리아에서 미토파지를 유발합니다.167 흥미롭게도, mtDNA 돌연변이만으로는 미토파지을 유발하기에 충분하지 않을 수도 있습니다.168 ROS는 미토파지 활성화에 중요한 역할을 하며, 미토파지는 ROS 수치를 조절하는 데 도움을 줍니다.169 과도한 ROS는 산화 스트레스에 반응하여 비선택적 자가포식을 유도할 수 있으며, 경미한 산화 스트레스는 일반적으로 미토콘드리아 분열에 의존하는 선택적 미토파지를 유발합니다.170 미토파지 과정에는 몇 가지 주요 단계가 있습니다. Δψm의 감소, 미토파고소체의 형성, 미토파고소체의 리소좀으로의 전달, 그리고 최종적으로 미토콘드리아 구성 요소의 분해 및 재활용입니다.171,172

미토파지 메커니즘은 일반적으로 유비퀴틴(Ub) 의존적 경로와 Ub 독립적 경로로 분류됩니다.

Ub 의존적 경로 중에서는 인산화효소 및 텐신 동형체 유도 잠재적 키나아제 1(PINK1)-파크린 경로가 가장 광범위하게 연구되었습니다. 정상적인 Δψm을 가진 미토콘드리아에서 PINK1은 IMM으로 운반되어 절단 및 분해됩니다.173 그러나 Δψm이 감소하면 PINK1은 IMM에 도달하지 못하고 대신 OMM에 축적됩니다. 여기서 PINK1은 유비퀴틴을 인산화하고 E3 유비퀴틴 리가제 파킨을 모집 및 인산화합니다.174,175 인산화된 파킨은 OMM에 결합된 세린 65 인산화 유비퀴틴과 결합하여 E3 유비퀴틴 리가제 활성을 완전히 활성화합니다. 이 활성화는 경로를 증폭시켜 미토파지를 촉진합니다.176 파킨에 의해 인산화된 폴리-Ub 사슬로 표지된 미토콘드리아는 OPTN, NDP52, SQSTM1/p62, TAX1BP1, NBR1과 같은 유비퀴틴 결합 수용체 단백질에 의해 인식되며,177,178,179 이 단백질들은 LC3와 결합하여 미토파지를 시작합니다. 또한 TBK1과 Mfn2가 이 경로에 참여한다는 것이 밝혀졌습니다.180,181 PINK1-Parkin 경로 외에도 Parkin에 독립적인 유비퀴틴 의존적 경로가 존재하며, 이 경로에서 PINK1은 직접 NDP52와 OPTN을 모집합니다.177

미토파지 수용체인 Fun14 도메인 함유 1(FUNDC1), BCL2 상호작용 단백질 3(BNIP3), BCL2 상호작용 단백질 3 유사체(BNIP3L/NIX), FKBP 프로릴 이소머라제 8(FKBP8), 및 ATAD3B는 LIR(LC3 상호작용 영역) 모티프를 통해 LC3와 직접 상호작용합니다. 이로써 유비퀴틴 독립적 미토파지를 유발합니다. 또한 PHB2와 카르디올리핀은 OMM으로의 이동으로 인해 유비퀴틴 독립적 경로에 참여합니다.171,182,183

Intercellular mitochondria transfer

Intercellular mitochondria transfer, where mitochondria are exchanged between donor and recipient cells, is another key component of MQC. The mitochondria transfer between cells with normal mitochondria and cells with dysfunctional mitochondria can rescue mitochondrial respiration defects.43 Mitochondria transfer is believed to aid in coping with cellular stress by facilitating intercellular communication under both physiological and pathological conditions.16 In this process, stressed donor cells transfer damaged mitochondria to healthy recipient cells. The recipient cells, upon receiving the damaged mitochondria, trigger mitochondrial biogenesis and fission to regenerate healthy mitochondria, which can then be re-transferred to the stressed donor cells. Additionally, stressed donor cells can transfer damaged mitochondria to other cells to initiate transcellular mitophagy (autophagy), especially when the stress or damage exceeds their metabolic capacity.16,184,185 There are three major routes of mitochondria transfer: tunneling nanotubes (TNT), mitochondrial extracellular vesicles (mitoEVs), and free mitochondria release.

Mitochondria can be shuttled across TNTs along either microtubules or actin filaments. Kinesin and its motor adaptor, the Miro-Milton complex, facilitate movement along microtubules, while myosin mediates transfer along actin filaments by interacting with Miro and anchoring mitochondria to the actin filaments.184,185

In addition to conventional extracellular vesicles (EVs), mitochondria produce specialized vesicles known as mitochondria-derived vesicles (MDVs), which encapsulate mtDNA and other mitochondrial components.186 MDVs primarily fuse with multivesicular bodies, such as late endosomes and lysosomes, although a select few are secreted into the extracellular space via a process driven by OPA1 and Snx9.187

Mitolysosome exocytosis, a mitoEVs-related MQC mechanism first observed in flunarizine-induced Parkinsonism-like symptoms, eliminates mitochondria through a mitophagy-independent pathway.188 During this process, mitochondria are directly engulfed by lysosomes and extruded from the cell without the formation of autophagosomes.189 Flunarizine-induced impairment of OXPHOS and the collapse of Δψm are believed to trigger this process. Proteins such as BAX, a mediator of mitochondrial outer membrane permeabilization (MOMP), and NDUFS4, a complex I subunit, may facilitate mitochondrial entry into lysosomes.188 Once inside lysosomes, the extracellular secretion of mitochondria is mediated by a VAMP2 (vesicle-associated membrane protein 2)-STX4 (syntaxin-4)-dependent mechanism.188

Migrasome-mediated exocytosis is also an emerging MQC mechanism involving mitoEVs.190 Migrasomes, defined as vesicles containing cytosolic contents, form on retraction fibers during cell migration.191,192 This process enables cells to clear damaged mitochondria, which may harbor detrimental mutant mtDNA, reduced Δψm, or elevated ROS levels, ensuring mitochondrial quality. Key factors in this process include Myosin19 (Myo19), KIF5B, and Drp1. Damaged mitochondria are transported to the plasma membrane by KIF5B, where Myo19 anchors them to cortical actin before Drp1-driven fission occurs. The reduced recruitment of dynein, the inward motor on microtubules, prevents damaged mitochondria from retracting back, ultimately leading to their incorporation into migrasomes.190 Migrasome formation relies on a reconstituted membrane system rich in tetraspanins and cholesterol.193 Migrasome-mediated exocytosis plays a role in maintaining homeostasis, particularly when the damage is insufficient to trigger mitophagy, and is especially important in migrating cells.

Mitopherogenesis is a specialized form of mitochondria-specific ectocytosis identified in sperm, which functions to control mitochondrial quantity. Unlike other exocytosis processes, mitopherogenesis involves the secretion of healthy mitochondria through EVs, with each vesicle carrying a single healthy mitochondrion. Proper actin-filament dynamics, extracellular protease activity, and the tyrosine kinase SPE-8 significantly influence this process,194 highlighting its potential impact on MQC.

Finally, free mitochondria released without membrane encapsulation occur in cases of mitophagy dysfunction.195 Damaged mitochondria within mitophagosomes lacking mammalian ATG8 conjugation cannot be degraded by lysosomes and instead undergo autophagic secretion, transferring the damaged mitochondria to healthy cells for transcellular degradation.195

세포 간 미토콘드리아 전달

세포 간 미토콘드리아 전달은 기증 세포와 수신 세포 사이에서 미토콘드리아가 교환되는 과정으로, MQC의 또 다른 핵심 구성 요소입니다. 정상적인 미토콘드리아를 가진 세포와 기능 장애를 가진 미토콘드리아를 가진 세포 사이의 미토콘드리아 전달은 미토콘드리아 호흡 결함을 회복시킬 수 있습니다.43 미토콘드리아 전달은 생리적 및 병리적 조건 하에서 세포 간 통신을 촉진함으로써 세포 스트레스에 대응하는 데 도움을 주는 것으로 믿어집니다.16 이 과정에서 스트레스를 받은 기증 세포는 손상된 미토콘드리아를 건강한 수신 세포로 전달합니다. 수용 세포는 손상된 미토콘드리아를 받으면 미토콘드리아 생성과 분열을 촉진하여 건강한 미토콘드리아를 재생하며, 이는 다시 스트레스 받은 기증 세포로 재전달될 수 있습니다. 또한, 스트레스를 받은 기증 세포는 손상된 미토콘드리아를 다른 세포로 전달하여 세포 간 미토파지 (자가포식)를 시작할 수 있으며, 특히 스트레스나 손상이 대사 능력을 초과하는 경우에 그러합니다.16,184,185 미토콘드리아 전달에는 세 가지 주요 경로가 있습니다: 터널링 나노튜브 (TNT), 미토콘드리아 세포외 소포 (mitoEVs), 그리고 자유 미토콘드리아 방출.

미토콘드리아는 미세관이나 액틴 필라멘트를 따라 TNT를 통해 이동할 수 있습니다. 키네신과 그 모터 적응체인 미로-밀턴 복합체는 미세관沿으로의 이동을 촉진하며, 마이오신은 미로와 상호작용하고 미토콘드리아를 액틴 필라멘트에 고정시켜 액틴 필라멘트沿으로의 전달을 매개합니다.184,185

전통적인 세포외 소포(EVs) 외에도 미토콘드리아는 mtDNA와 다른 미토콘드리아 구성 요소를 포함하는 특수 소포인 미토콘드리아 유래 소포(MDVs)를 생성합니다.186 MDVs는 주로 후기 엔도좀과 리소좀과 같은 다중 소포체와 융합되지만, 일부는 OPA1과 Snx9에 의해 조절되는 과정으로 세포외 공간으로 분비됩니다.187

미토리소좀 분비(mitolysosome exocytosis)는 플루나리진 유발 파킨슨증 유사 증상에서 처음 관찰된 mitoEVs 관련 MQC 메커니즘으로, 미토파지 독립적 경로를 통해 미토콘드리아를 제거합니다.188 이 과정에서 미토콘드리아는 오토파고소체 형성 없이 리소좀에 직접 포식되어 세포 밖으로 배출됩니다.189 플루나리진에 의한 OXPHOS 장애와 Δψm의 붕괴가 이 과정을 유발하는 것으로 추정됩니다. 미토콘드리아 외막 투과성(MOMP)의 매개체인 BAX와 복합체 I 서브유닛인 NDUFS4와 같은 단백질은 미토콘드리아가 리소좀으로 들어가는 것을 촉진할 수 있습니다.188 리소좀 내부에 들어간 후, 미토콘드리아의 세포외 분비는 VAMP2(vesicle-associated membrane protein 2)-STX4(syntaxin-4) 의존적 메커니즘을 통해 매개됩니다.188

미그라소ーム 매개 분비(migrasome-mediated exocytosis)는 mitoEVs와 관련된 새로운 MQC 메커니즘으로 부상하고 있습니다.190 미그라소ーム은 세포 이동 중 수축 섬유에서 형성되는 세포질 내용물을 포함한 소포로 정의됩니다.191,192 이 과정은 손상된 미토콘드리아를 제거하여 유해한 돌연변이 mtDNA, 감소된 Δψm, 또는 증가된 ROS 수준을 포함할 수 있는 미토콘드리아를 제거함으로써 미토콘드리아 품질을 유지합니다. 이 과정의 주요 요인에는 마이오신19(Myo19), KIF5B, 및 Drp1이 포함됩니다. 손상된 미토콘드리아는 KIF5B에 의해 세포막으로 운반되며, 여기서 Myo19가 코르티컬 액틴에 고정시킨 후 Drp1에 의해 분열이 발생합니다. 미세관 내부의 내향성 모터인 다이닌의 모집 감소는 손상된 미토콘드리아가 다시 수축되는 것을 방지하여 결국 미그라소메에 통합되도록 합니다.190 미그라소ーム 형성은 테트라스판인과 콜레스테롤이 풍부한 재구성된 막 시스템에 의존합니다.193 미그라소ーム 매개 분비(exocytosis)는 특히 미토파지가 유발되지 않을 정도로 손상이 충분하지 않을 때 항상성 유지에 역할을 하며, 특히 이동 중인 세포에서 중요합니다.

미토포게네시스(mitopherogenesis)는 정자에서 발견된 미토콘드리아 특이적 엑소시토시의 특수 형태로, 미토콘드리아 수를 조절하는 기능을 합니다. 다른 엑소시토시 과정과 달리 미토포게네시스는 건강한 미토콘드리아를 엑소좀(EVs)을 통해 분비하며, 각 소포는 단일 건강한 미토콘드리아를 운반합니다. 액틴 필라멘트 동역학, 세포외 프로테아제 활성, 티로신 키나제 SPE-8은 이 과정에 크게 영향을 미치며,194 이는 MQC에 미치는 잠재적 영향을 강조합니다.

마지막으로, 막으로 둘러싸이지 않은 자유 미토콘드리아는 미토파지 기능 장애 시 발생합니다.195 포유류 ATG8 결합이 결여된 미토파고소체 내 손상된 미토콘드리아는 리소좀에 의해 분해되지 않고 대신 자식작용 분비를 통해 손상된 미토콘드리아를 건강한 세포로 전달하여 세포 간 분해됩니다.195

미토콘드리아 아포토시스

스트레스 상황에서 세포는 이러한 악조건을 관리하기 위한 메커니즘으로 아포토시스를 유발할 수 있습니다.196 아포토시스는 미토콘드리아 경로를 통해 발생할 수 있으며(그림 4), 미토콘드리아 질환의 병리학적 기전에 역할을 합니다.111,197,198

Mitochondrial apoptosis

Under stress, cells may initiate apoptosis as a mechanism to manage these adverse situations.196 Apoptosis can occur through mitochondrial pathway (Fig. 4), thereby playing a role in the pathogenesis of mitochondrial diseases.111,197,198

Fig. 4

Mitochondrial Apoptosis and Inflammation. Components within the IMS or matrix can trigger apoptosis or inflammation if leaked into the cytosol, primarily due to the mPTP and MOMP. Upon MOMP formation and mPTP opening, cytochrome C, SMAC, and mtDNA are released into the cytosol. Cytochrome C interacts with APAF1, activating caspase 9 to initiate the caspase cascade leading to apoptosis. SMAC accelerates this process by inhibiting XIAP. After binding with mtDNA, the cGAS enzyme produces cGAMP from ATP and GTP, activating the cGAS-STING signaling pathway and inducing type I interferon expression‎ and NF-κB activation. The NLRP3 inflammasome can also bind with (oxidized) mtDNA to promote IL-1β and IL-18 cleavage. However, caspase 3 cleaves cGAS and IRF3 during apoptosis, inhibiting inflammation. MOMP mitochondrial outer membrane permeabilization; mPTP mitochondrial permeability transition pore; SMAC second mitochondrial-derived activator of caspases; APAF1 apoptosis protease activating factor 1; XIAP X-linked inhibitor of apoptosis protein; cGAS cyclic GMP-AMP synthase; STING stimulator of interferon genes; cGAMP cyclic guanosine monophosphate–adenosine monophosphate; TBK1 TANK binding kinase 1; IKK IκB kinase; IRF3 interferon regulatory factor 3

미토콘드리아 아포토시스 및 염증.

IMS 또는 매트릭스 내의 구성 요소가 세포질로 유출될 경우

아포토시스 또는 염증을 유발할 수 있으며,

이는 주로 mPTP 및 MOMP에 의해 발생합니다.

MOMP 형성 및 mPTP 개방 시

사이토크롬 C, SMAC, 및 mtDNA가 세포질로 방출됩니다.

사이토크롬 C는

APAF1과 상호작용하여 카스파제 9를 활성화시켜

아포토시스 유발을 위한 카스파제 캐스케이드를 시작합니다.

SMAC은 XIAP를 억제함으로써 이 과정을 가속화합니다. mtDNA와 결합한 후, cGAS 효소는 ATP와 GTP로부터 cGAMP를 생성하여 cGAS-STING 신호전달 경로를 활성화시키고 유형 I 인터페론 발현 및 NF-κB 활성화를 유도합니다. NLRP3 염증체는 산화된 mtDNA와 결합하여 IL-1β 및 IL-18의 분해를 촉진합니다. 그러나 아포토시스 과정에서 카스파제 3은 cGAS와 IRF3를 분해하여 염증을 억제합니다.

MOMP 미토콘드리아 외막 투과성; mPTP 미토콘드리아 투과성 전환 구멍; SMAC 제2 미토콘드리아 유래 카스파제 활성화 인자; APAF1 아포토시스 프로테아제 활성화 인자 1; XIAP X-연관 아포토시스 억제 단백질; cGAS 사이클릭 GMP-AMP 합성효소; STING 인터페론 유전자 자극인자; cGAMP 사이클릭 구아노신 모노포스페이트–아데노신 모노포스페이트; TBK1 TANK 결합 키나제 1; IKK IκB 키나제; IRF3 인터페론 조절 인자 3

The mitochondrial apoptosis pathway is intricately linked to MOMP, which is predominantly regulated by BCL-2 family proteins, though it can also be triggered by the mPTP.116,196 The BCL-2 family is divided into pro-apoptotic and anti-apoptotic members. Pro-apoptotic BCL-2 proteins include BH3-only proteins (such as BID, BIK, and BIM) and effectors like BAK, BAX, and BOX, while anti-apoptotic proteins include BCL-2, BCL-X, BCL-W, A1, and MCL1. BH3-only proteins respond to apoptotic stimuli by activating effectors, typically BAX and BAK.199,200 Once activated, BAX and BAK accumulate on the OMM, dimerize, and form higher-order oligomers that create pores in the OMM, leading to MOMP.201 MOMP results in the release of IMS proteins, such as cytochrome c and the second mitochondrial-derived activator of caspases (SMAC), into the cytoplasm. Cytochrome c binds to apoptosis protease activating factor 1 (APAF1) in the cytoplasm, and together, they activate caspase 9. Activated caspase 9 then activates caspases 3 and 7, which ultimately drive the apoptotic process. Additionally, SMAC enhances the caspase cascade by inhibiting the X-linked inhibitor of apoptosis protein (XIAP).199,202,203 Other forms of programmed cell death, such as necroptosis, pyroptosis, and ferroptosis, are also related to mitochondria.204

미토콘드리아 아포토시스 경로는

주로 BCL-2 가족 단백질에 의해 조절되는 MOMP와 복잡하게 연결되어 있으며,

mPTP에 의해 유발될 수도 있습니다.116,196

BCL-2 가족은

프로아포토시스 및 안티아포토시스 구성원으로 나뉩니다.

아포토시스 촉진 BCL-2 단백질에는

BH3-only 단백질(예: BID, BIK, BIM)과 BAK, BAX, BOX와 같은 효능 단백질이 포함되며,

아포토시스 억제 단백질에는

BCL-2, BCL-X, BCL-W, A1, MCL1이 포함됩니다.

BH3-only 단백질은 아포토시스 자극에 반응하여 효과인자(주로 BAX와 BAK)를 활성화합니다.199,200 활성화된 BAX와 BAK는 OMM에 축적되어 이량체화되고 고차 올리고머를 형성하여 OMM에 구멍을 생성해 MOMP를 유발합니다.201 MOMP는 사이토크롬 c와 두 번째 미토콘드리아 유래 카스파제 활성화 인자(SMAC)와 같은 IMS 단백질을 세포질로 방출합니다. 사이토크롬 c는 세포질에서 아포토시스 프로테아제 활성화 인자 1(APAF1)과 결합하여 함께 카스파제 9를 활성화합니다. 활성화된 카스파제 9는 카스파제 3과 7을 활성화시켜 최종적으로 아포토시스 과정을 촉진합니다. 또한 SMAC는 X-연관 아포토시스 억제 단백질(XIAP)을 억제함으로써 카스파제 캐스케이드를 강화합니다.199,202,203

네크로토시스, 피로토시스, 페로토시스 등

다른 형태의 프로그램된 세포 사멸도

미토콘드리아와 관련이 있습니다.204

Mitochondrial inflammation

Mitochondria can initiate inflammation by releasing damage-associated molecular patterns (DAMPs) due to their evolutionary similarities to bacterial pathogen-associated molecular patterns when subjected to stress or damage. These DAMPs include N-formyl peptides, TFAM, cardiolipin, ATP, ROS, and mtDNA.205 In addition to the inflammatory effects of mtDNA, which will be discussed in detail, other DAMPs have also been recognized for their roles in promoting inflammation.206,207

Under conditions of mitochondrial stress or dysfunction, mtDNA can be released into the cytosol or extracellular space.208,209 MtDNA can trigger pro-inflammatory and type I interferon responses, which vary depending on the cell type and context.210 The release of mtDNA into the cytoplasm occurs via MOMP and the opening of the mPTP.210 It is likely that mPTP and MOMP work together to facilitate mtDNA release.208 The inflammation mediated by mtDNA is primarily driven by the cGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) signaling pathway (Fig. 4). When cGAS binds to mtDNA, it catalyzes the production of cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) from ATP and GTP.211 cGAMP then activates STING, which subsequently recruits and activates TANK binding kinase 1 (TBK1). TBK1 phosphorylates STING, leading to the activation of transcription factors interferon regulatory factor 3 (IRF3), leading to type I interferon expression‎.212,213,214 STING also phosphorylates IκB kinase (IKK) to initiate NF-κB pathway, resulting in increased production of pro-inflammatory cytokines.208 In addition to the cGAS-STING pathway, the mtROS and mtDNA can activate NLRP3 inflammasome to promote IL-1β and IL-18 cleavage.17 Notably, low levels of mtDNA-induced inflammation, caused by minor MOMP, can aid in fighting infections,215 while simultaneous activation of caspase 3 following MOMP can inhibit mtDNA-induced inflammation by cleaving cGAS and IRF3.216,217

A connection between inflammation and mitochondrial bioenergetics has been established, indicating that mitochondrial dysfunction can exacerbate inflammation, which in turn impairs OXPHOS and disrupts MQC.218 An inflammatory transcriptomic profile has been observed in peripheral blood mononuclear cells of patients with mitochondrial diseases.219 Although research on the role of inflammation in mitochondrial diseases is still limited, inflammation is a critical factor in many human diseases, such as neurodegenerative disorders.220 Its potential pathological effects on mitochondrial diseases warrant further investigation.

In summary, mitochondria are central to metabolism, stress response, inflammation, and other critical cellular processes. Energy stress, characterized by reduced intracellular ATP levels, is a key feature of many human diseases.221,222 Mitochondrial genetic defects that disrupt energy metabolism lead to protein synthesis defects, ATP insufficiency, and mtROS overproduction.81 Despite the genetic heterogeneity of mitochondrial diseases, the resulting protein, energy, and oxidative stress are consistent.223 Cells initiate various MQC mechanisms to restore normal bioenergetics and redox balance in response to stress.224 However, when damage exceeds the capacity of MQC system, stress-induced mPTP opening and MOMP can release mtDNA or other DAMPs into the cytoplasm, triggering inflammation and cell death.209,225 Understanding how cells respond to stress induced by mitochondrial genetic defects may provide insights into the onset and progression of these diseases.

미토콘드리아 염증

미토콘드리아는

스트레스나 손상을 받을 때

세균성 병원체 관련 분자 패턴(PAMPs)과 진화적 유사성을 가지고 있어

손상 관련 분자 패턴(DAMPs)을 방출함으로써

염증을 유발할 수 있습니다.

이러한 DAMPs에는

N-포르밀 펩타이드, TFAM, 카르디올리핀, ATP, ROS, 및 mtDNA가 포함됩니다.205

mtDNA의 염증 효과는

후술할 것이며,

다른 DAMPs도 염증 촉진 역할이 인정되었습니다.206,207

미토콘드리아 스트레스나 기능 장애 조건에서 mtDNA는

세포질이나 세포외 공간으로 방출될 수 있습니다.208,209

mtDNA는

세포 유형과 맥락에 따라 달라지는 염증 촉진 및 유형 I 인터페론 반응을 유발할 수 있습니다.210

mtDNA의 세포질 방출은

MOMP와 mPTP의 개방을 통해 발생합니다.210

mPTP와 MOMP가

mtDNA 방출을 촉진하기 위해 협력할 가능성이 높습니다.208

mtDNA에 의해 매개되는 염증은

주로 cGAS (사이클릭 GMP-AMP 합성효소)-STING (인터페론 유전자 자극인자) 신호전달 경로를 통해

주로 유발됩니다 (그림 4).

cGAS가 mtDNA에 결합하면 ATP와 GTP로부터 cyclic guanosine monophosphate–adenosine monophosphate (cGAMP)를 생성합니다.211 cGAMP는 STING을 활성화하며, 이는 차례로 TANK 결합 키나제 1 (TBK1)을 모집하고 활성화합니다. TBK1은 STING을 인산화하여 인터페론 조절 인자 3(IRF3) 전사 인자의 활성화를 유도하며, 이는 제1형 인터페론 발현으로 이어집니다.212,213,214 STING은 또한 IκB 키나제(IKK)를 인산화하여 NF-κB 경로를 활성화시켜 염증성 사이토카인의 생산을 증가시킵니다.208 cGAS-STING 경로 외에도 mtROS와 mtDNA는 NLRP3 염증체(inflammasome)를 활성화시켜 IL-1β와 IL-18의 분해를 촉진합니다.17 특히, 미미한 MOMP에 의해 유발된 낮은 수준의 mtDNA-유발 염증은 감염과 싸우는 데 도움을 줄 수 있습니다.215 반면, MOMP에 따른 caspase 3의 동시 활성화는 cGAS와 IRF3를 분해함으로써 mtDNA-유발 염증을 억제할 수 있습니다.216,217

염증과 미토콘드리아 생체 에너지 대사 사이의 연관성이 확립되었으며,

미토콘드리아 기능 장애가 염증을 악화시키고,

이는 다시 OXPHOS를 손상시키고

MQC를 방해합니다.218

미토콘드리아 질환 환자의 말초 혈액 단핵구에서

염증성 전사체 프로파일이 관찰되었습니다.219

미토콘드리아 질환에서 염증의 역할에 대한 연구는 여전히 제한적이지만,

염증은 신경퇴행성 질환 등 많은 인간 질환에서 중요한 요인입니다.220

미토콘드리아 질환에 대한 잠재적 병리학적 영향은

추가 연구가 필요합니다.

요약하면,

미토콘드리아는

대사, 스트레스 반응, 염증 및 기타 중요한 세포 과정의 중심에 있습니다.

세포 내 ATP 수준 감소로 특징지어지는 에너지 스트레스는

많은 인간 질환의 핵심 특징입니다.221,222

미토콘드리아 유전적 결함은

에너지 대사 장애를 초래하여

단백질 합성 장애, ATP 부족, 및 mtROS 과다 생산을 유발합니다.81

미토콘드리아 질환의 유전적 이질성에도 불구하고,

결과적으로 발생하는 단백질, 에너지, 산화 스트레스는 일관됩니다.223

세포는 스트레스에 대응하여 정상적인 생체 에너지 대사와 환원-산화 균형을 회복하기 위해 다양한 MQC 메커니즘을 활성화합니다.224 그러나 손상이 MQC 시스템의 용량을 초과하면 스트레스에 의한 mPTP 개방과 MOMP가 미토콘드리아 DNA(mtDNA)나 다른 손상 관련 분자 신호(DAMPs)를 세포질로 방출해 염증과 세포 사멸을 유발합니다.209,225 미토콘드리아 유전적 결함에 의한 스트레스에 대한 세포의 반응 메커니즘을 이해하는 것은 이러한 질환의 발병과 진행 메커니즘을 규명하는 데 중요한 단서를 제공할 수 있습니다.

Molecular mechanisms of mitochondrial diseases

Mitochondrial diseases are characterized by primary or secondary defects in mitochondrial function or structure, resulting from mutations in either nDNA or mtDNA.226 Specifically, 36 pathogenic genes (11%) are encoded by mtDNA, while 302 (89%) are encoded by nDNA.227 Heteroplasmy is present in most healthy individuals without leading to mitochondrial disease.228 However, symptoms manifest only when the proportion of mutant mtDNA surpasses a certain threshold.229 Due to the considerable genetic and clinical heterogeneity, mitochondrial diseases can affect either single or multiple organ systems, leading to a wide range of clinical manifestations (Fig. 5). Tissues with high energy demands are particularly susceptible to energy deficits and are therefore most commonly affected.230 Additionally, the age of onset and severity of these diseases can vary significantly depending on the degree of heteroplasmy and individual differences.4

미토콘드리아 질환의 분자 메커니즘

미토콘드리아 질환은

nDNA 또는 mtDNA의 돌연변이로 인한

미토콘드리아 기능 또는 구조의 1차적 또는 2차적 결함이 특징입니다.226

구체적으로,

36개의 병원성 유전자(11%)는 mtDNA에 의해 암호화되고,

302개(89%)는 nDNA에 의해 암호화됩니다.227

이종 플라스미는 대부분의 건강한 개인에게 존재하지만 미토콘드리아 질환을 유발하지는 않습니다.228 그러나 증상은 돌연변이 mtDNA의 비율이 특정 임계치를 초과할 때만 나타납니다.229 유전적 및 임상적 이질성으로 인해 미토콘드리아 질환은 단일 또는 다중 장기 시스템에 영향을 미쳐 다양한 임상 증상을 유발할 수 있습니다(그림 5). 에너지 수요가 높은 조직은 에너지 결핍에 특히 취약하며 따라서 가장 흔히 영향을 받습니다.230 또한, 이 질환의 발병 연령과 중증도는 헤테로플라즘의 정도와 개인 차이에 따라 크게 달라질 수 있습니다.4

Fig. 5

Multisystem Clinical Presentation of Mitochondrial Diseases. Due to the ubiquitous presence of mitochondria, mitochondrial diseases can present in any tissue of the body. Tissues and organs with high energy demands, such as skeletal muscle and brain, are particularly susceptible to oxidative phosphorylation defects, leading to common manifestations like myopathy and encephalopathy in mitochondrial diseases. The diverse and variable symptoms associated with these conditions increase the risk of misdiagnosis

Full size image

The primary mitochondrial diseases caused by mtDNA mutations

The hallmark of mitochondrial diseases resulting from mtDNA mutations is the primary disturbance of OXPHOS. This disturbance typically leads to energy deficits, increased oxidative stress, and a collapse of the Δψm.

Leber hereditary optic neuropathy

LHON is the most preval‎ent mitochondrial disease, characterized by maternal inheritance and a pronounced male sex bias.231 It was the first disease definitively linked to mtDNA mutations.33 The primary clinical manifestation of LHON is the bilateral, severe loss of central vision, caused by degeneration of retinal ganglion cells (RGCs).232 The high energy demands of RGCs may explain the cell-specific vulnerability in the eye. Retina is one of the most energy-consuming tissues in the body.233 RGCs are located in the retina, and their axons form the optic nerve. Based on mitochondrial distribution and energy consumption, RGCs can be divided into four subcellular components: dendrites, cell body, unmyelinated axon (including intraocular and optic nerve head segments), and myelinated axon (post-lamina cribrosa).233 RGCs, with their exceptionally long axons and high frequency of action potentials, require substantial energy. Mitochondrial transport along these extensive axons consumes significant amounts of ATP to sustain axonal function.197,233 Additionally, the continuous conduction of action potentials in unmyelinated axons demands more energy than the saltatory conduction in myelinated axons,197,234 making the unmyelinated segments before the lamina cribrosa particularly susceptible to energy deficits.235 Mitochondrial density correlates with energy demand,233 resulting in an uneven distribution where mitochondria are abundant in the cell body and unmyelinated axons but sparse in the myelinated axons.236 Compounding this vulnerability, exposure to light, particularly short-wave or blue light (400–480 nm),237 can negatively impact OXPHOS in RGCs, leading to decreased ATP production and increased ROS levels.237

Over 95% of patients with LHON carry one of three common mtDNA point mutations: m.3460 G > A, m.11778 G > A, or m.14484 T > C.231 These mutations occur in the MT-ND1, MT-ND4, and MT-ND6 genes, respectively, all of which encode subunits of complex I.238 While ATP synthesis deficits are evident, energy failure alone may not be the primary cause of RGC degeneration in LHON.231 Instead, the dysfunctional complex I likely increases mtROS production, which in turn induces cellular apoptosis, playing a critical role in RGC degeneration.235,239 Mitochondrial apoptosis is pivotal in the pathogenesis of LHON.240 For instance, the m.3460 G > A mutation in MT-ND1 is associated with elevated mtROS production and increased levels of pro-apoptotic proteins such as cytochrome c, BAK, BAX, PARP, and cleaved caspases 3, 7, and 9.241 In addition to ROS, energy failure can activate apoptosis-inducing factors and endonuclease G, triggering apoptosis through a caspase-independent pathway.242 Overproduction of mtROS can directly induce cytochrome c release by prolonging mPTP opening or indirectly trigger cytochrome c release through caspase cascade activation and MOMP, leading to apoptosis in RGCs.243 Mito-nuclear communication also plays a role in LHON pathogenesis, as evidenced by the activation of mt-ISR transcripts in LHON, which leads to chronic inhibition of protein synthesis, affecting synaptic and oligodendroglial function, and contributing to disease progression.244,245 Besides, the respiratory chain complex I defect can cause remodeling of one-carbon metabolism through mt-ISR.125,246 This one-carbon metabolic remodeling undermines NADPH production, which sensitizes affected cells to oxidative stress and may facilitate inflammation and cell death.10 However, the anti-apoptotic protein XIAP can prevent RGC apoptosis by inhibiting mitochondrial apoptosis.247

Excessive mitophagy, driven by AMPK activation, has been shown to promote apoptosis and may lead to the widespread and near-synchronous death of RGCs in subacute LHON. In contrast, overexpression‎ of PGC-1α, which facilitates mitochondrial biogenesis, has been found to prevent cell apoptosis.248 Increased mitochondrial biogenesis is thought to contribute to the phenomenon of incomplete penetrance in LHON and serves as a compensatory mechanism to restore mitochondrial turnover in LHON carriers.249 Thus, balancing mitochondrial biogenesis and mitophagy could represent a potential therapeutic target. Additionally, mitochondrial transport, an ATP-dependent process critical for maintaining axonal mitochondrial homeostasis, has been found to be impaired in LHON, potentially contributing to axonal loss.250

Significant progress has been made in developing therapies for LHON. Techniques such as allotopic expression‎ and gene editing hold great promise for future treatments. Currently, Idebenone is the only drug approved by the European Medicines Agency for improving visual impairment in patients with LHON.198,251

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) manifest as lactic acidosis, headaches accompanied by nausea and vomiting, epilepsy, and stroke-like episodes, all inherited maternally.252 Additional symptoms observed in MELAS include deafness, diabetes mellitus, gastrointestinal disturbances, ataxia, growth failure, myopathy, and cardiomyopathy.253,254 The primary genetic cause of MELAS is the m.3243 A > G mutation in the MT-TL1 gene, which encodes mt-tRNALeu(UUR),255 accounting for over 80% of cases.255 Another mutation, m.3271 T > C in MT-TL1, is responsible for 7–15% of MELAS cases,256,257 while other mtDNA mutations associated with MELAS are rare. Mutations in the MT-TL1 gene can lead to defects in the aminoacylation of mt-tRNALeu(UUR), disrupting the interaction between mRNA and ribosomes.258 This abnormal translation impairs the synthesis of respiratory chain complexes, particularly complexes I and IV.259

Oxidative stress is believed to play a significant role in MELAS pathogenesis.111 Increased levels of ROS, apoptosis, and inflammation have been observed in diseased endothelial cells from patients with MELAS carrying the m.3243 A > G mutation.260 Similar endogenous oxidative stress has been found in fibroblasts and induced pluripotent stem cells (iPSCs) from patients with MELAS.183

In neuronal cells, complex I defects predominate, and active degradation of complex I via mitophagy is a protective response to mitochondrial dysfunction during neuronal differentiation.261 This degradation reduces mtROS production, acting as a protective mechanism.261 However, normal lysosomal function and the sequestration of cytosolic components during autophagy depend heavily on adequate ATP levels.262 In MELAS fibroblasts, although mitophagy is activated, autophagic flux is reduced, and autophagosome elimination is defective, likely due to ATP deficiency.263 This reduction in mitophagy can lead to the accumulation of damaged mitochondria with defective complex I. Interestingly, mt-tRNA fragments, a class of ncRNAs, have been implicated in extracellular lactate accumulation in MELAS, possibly through the downregulation of mitochondrial pyruvate carrier 1.69 Furthermore, the oxidative stress induced by primary tRNALeu(UUR) defects can exacerbate impaired tRNA modification.264 Stress-induced microRNA-9/9* has been shown to post-transcriptionally suppress mt-tRNA-modification enzymes, leading to reduced U34 modification of non-mutant tRNAs and promoting mitochondrial dysfunction.264

Current treatments for MELAS are symptomatic and include the supplementation of antioxidants and cofactors, anti-epileptic drugs, and lactate-lowering agents. However, there remains no consensus on the optimal treatment approach for MELAS.265,266

Maternally inherited diabetes and deafness

Maternally inherited diabetes and deafness (MIDD) is another mitochondrial disease frequently associated with the m.3243 A > G mutation in the MT-TL1 gene.267 The most prominent clinical features of MIDD are diabetes and hearing loss,268 although other complications such as myopathy, neuropathy, oculopathy, cardiac disease, and nephropathy are also observed.269 Approximately 85% of MIDD cases are caused by the m.3243 A > G point mutation.269 Notably, this same mutation is also responsible for MELAS. Interestingly, there have been case reports of MIDD evolving into MELAS over time,270 suggesting that these conditions represent different phenotypes within the same disease spectrum influenced by the level of mtDNA heteroplasmy: MELAS typically occurs with higher levels of mtDNA heteroplasmy (typically >85%), while MIDD is associated with lower levels (typically <45%).271

Insulin production demands significant ATP, and the decreased ATP generation coupled with increased ROS production in pancreatic β-cells with dysfunctional mitochondria leads to a gradual decline in insulin secretion, eventually resulting in insulin deficiency.272 Oxidative stress also plays a critical role in hearing loss,273 with ROS-induced activation of the AMPK-E2F1 pathway promoting apoptosis in the stria vascularis and spiral ganglion neurons.274 Additionally, the role of nuclear factors in the phenotypic variability of the m.3243 A > G mutation has been increasingly recognized.275

Managing blood glucose levels is the primary focus of treatment for MIDD. Since insulin sensitivity is generally preserved, oral hypoglycemic drugs, including insulin secretagogues, should be considered. However, metformin should be avoided due to the increased risk of lactic acidosis.272

Myoclonic epilepsy with ragged red fiber

Myoclonic epilepsy with ragged red fibers (MERRF) is a mitochondrial disorder defined by the presence of progressive myoclonus epilepsy and ragged-red fibers (RRF) observed in muscle biopsies.276 This disease also manifests with symptoms like ataxia, cardiomyopathy, lipomatosis, and dementia.277 In 1990, a significant association was identified between MERRF and the m.8344 A > G point mutation in the MT-TK gene, responsible for encoding mt-tRNALys(UUR).278 This mutation, m.8344 A > G, is the most preval‎ent pathogenic variant, accounting for over 80% of cases.279 It hinders the N1-methyladenosine (m1A) modification at position 58 in mt-tRNALys(UUR),280 leading to defects in mt-tRNALys(UUR), which subsequently disrupts the synthesis of respiratory chain complexes, primarily complexes I and IV, resulting in mitochondrial dysfunction.281 Recently, mutations in the MT-RNR1 and MT-RNR2 genes, which encode 12S rRNA and 16S rRNA, respectively, have also been linked to MERRF.282 These rRNAs are integral to mitochondrial ribosomes and the mtDNA-encoded mRNAs.282

The neurological defects observed in MERRF may be related to compromised synaptic plasticity in excitatory neurons, a consequence of mitochondrial dysfunction and synaptic impairment.283 The study utilizing human iPSCs derived from patients harboring the m.8344 A > G mutation reveals mitochondrial fragmentation, reduced oxygen consumption, and elevated ROS production.284 Furthermore, neurons induced from patients with MERRF exhibit smaller, rounded, and fragmented mitochondria with decreased Δψm, increased ROS. The activated mitophagy and impaired autophagy flux are also present in these neurons.285 Despite the activation of autophagy and mitophagy in MERRF, their flux remains impaired, which is associated with ATP deficiency.286 Treatment with coenzyme Q10 (CoQ10) has been shown to enhance mitochondrial function in MERRF fibroblasts and cybrids by promoting autophagy flux,286 indicating that reduced mitophagy and autophagy flux might be critical contributors to MERRF pathogenesis. Notably, no apoptotic changes are observed in the muscles of mitochondrial encephalomyopathies, possibly due to sarcoplasmic expression‎ of XIAP.287,288 However, the exact mechanisms underlying this apoptosis suspension require further investigation. Importantly, defective tRNAs can lead to the accumulation of damaged and unfolded mitochondrial proteins,289,290 suggesting that targeting mt-ISR could be a potential therapeutic strategy for MERRF and other mitochondrial disorders caused by tRNA defects. Continued research into the molecular etiology of these conditions is essential.

Neurogenic muscle weakness, ataxia, and retinitis pigmentosa syndrome

Neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome is a maternally inherited disorder characterized by a range of symptoms including muscle weakness, sensory neuropathy, ataxia, seizures, dementia, retinitis pigmentosa, optic atrophy, and developmental delay.291

Mutations m.8993 T > G or m.8993 T > C in the MT-ATP6 gene are implicated in the pathogenesis of NARP.291,292,293 These mutations are also associated with Leigh syndrome,294,295 with the specific phenotype determined by the level of heteroplasmy. A heteroplasmy level exceeding 85% for the m.8993 T > G mutation is predominantly linked to childhood-onset Leigh syndrome, whereas a level of 60–70% typically results in adult-onset NARP. Both diseases may occur with heteroplasmy levels between 70–85%.234,296 The m.8993 T > G mutation leads to the substitution of a highly conserved leucine with arginine (while m.8993 T > C replaces leucine with proline) in the ATPase6-encoded proton channel, impairing the proton translocation mechanism of ATP synthase (complex V) and subsequently disturbing OXPHOS.234

In addition to role in OXPHOS, the ATP synthase subunits e and g are important to the formation of mitochondrial cristae.297 Dissolution of mitochondrial cristae has been observed in patients with the m.8993 T > G mutation.298 Additionally, ATP synthase is a component of mPTP.88 Aberrant mitochondrial cristae and mPTP opening may lead to the release of mtDNA and IMS proteins, such as cytochrome c, thereby triggering mitochondrial apoptosis and inflammation.299 This hypothesis warrants further investigation. In yeast cells harboring defective MT-ATP6, IMM fusion is inhibited. It is plausible that dysfunctional IMM fusion could be a common feature in all diseases with genetically defective OXPHOS, potentially initiating mitophagy to eliminate defective mtDNA.300 The molecular basis underlying cerebellar atrophy in patients with the m.8993 T > G or m.8993 T > C mutations remains unclear. However, in a mouse model of inherited Purkinje cell degeneration, increased mitophagy and autophagy have been associated with Purkinje cell loss.301

Progressive external ophthalmoplegia

Progressive external ophthalmoplegia (PEO), also known as chronic progressive external ophthalmoplegia (CPEO), is a preval‎ent clinical syndrome within mitochondrial diseases, characterized by progressive bilateral ptosis and diffuse, symmetric ophthalmoparesis.302 PEO can be classified into three phenotypes: pure PEO (isolated occurrence), Kearns-Sayre syndrome, and PEO-plus syndrome, where myopathy or other extraocular symptoms are present.303 The most common cause of PEO is a single large-scale mtDNA deletion,303 placing it within the category of mitochondrial diseases caused by mtDNA mutations.

Typically, single large-scale mtDNA deletions arise sporadically rather than through maternal inheritance, resulting from the amplification of a single mutation associated with spontaneous errors in DNA polymerase γ during early embryonic development.304,305 The age of onset and severity of the disease are correlated with the size of the deletion, the level of heteroplasmy, and the specific region affected within the mtDNA.306 These deletions, whether singular or multiple, can impair the function of one or more mtDNA-encoded proteins, resulting in OXPHOS disturbance.

PEO can also be attributed to multiple mtDNA deletions or depletion, secondary to nDNA mutations, which follow either autosomal recessive or dominant inheritance patterns and involve genes such as POLG, POLG2, SLC25A4, C10orf2, SPG7, DNA2, RNASEH1, TOP3A, TK2, DGUOK, RRM2B, GMPR, LIG3, and RRM1.307 Generally, mtDNA depletion is associated with early-onset disorders that typically have a fatal course, whereas adult-onset disorders are more commonly linked to multiple mtDNA deletions.308

The minimal mitochondrial replisome consists of polymerase γ, Twinkle, the mitochondrial single-stranded DNA-binding protein (mtSSB), and the mitochondrial RNA polymerase.309 DNA polymerase γ, the only polymerase responsible for mtDNA replication,310 comprises three subunits encoded by two nuclear genes: the p140 catalytic subunit encoded by POLG (POLG1) and the p55 accessory subunit encoded by POLG2.311 Mutations in POLG or POLG2 genes can lead to mtDNA defects. Specifically, mutations in POLG compromise mitochondrial genetic integrity, resulting in multiple deletions that contribute to PEO.310 Similarly, POLG2 mutations impair the proper stimulation of p140, disrupting mtDNA replication.312 The deletion pattern associated with POLG mutations may result from faulty strand displacement replication, initiated by replication fork stalling.313

Twinkle, encoded by C10orf2, is essential for nascent H-strand synthesis in the D-loop and is thus indispensable for mtDNA replication, despite the existence of other potential mtDNA helicases.314 A PEO mouse model with a C10orf2 defect has been established,315 demonstrating that type IIB fibers of extraocular muscles are particularly vulnerable to mtDNA deletions, likely due to their relatively low mitochondrial content, which allows even a few mutant mtDNA to surpass the onset threshold.316 The endosomal-mitophagy pathway, involving ATAD3, VPS35, SAMM50, and BAK/BAX, plays a role in mtDNA deletion caused by C10orf2 mutations.317 Furthermore, persistent activation of the mTORC1-mt-ISR pathway due to mtDNA replication defects disrupts cellular metabolic homeostasis, thereby contributing to disease progression.125 Thymidine kinase 2 (TK2) is a mitochondrial enzyme responsible for catalyzing the conversion of deoxycytidine and deoxythymidine nucleosides to their nucleoside monophosphates, which are then converted into deoxynucleoside triphosphates.318 Mutations in the TK2 gene disrupt the maintenance of the mitochondrial deoxyribonucleotide pool, leading to mtDNA depletion or multiple mtDNA deletions, and subsequently resulting in PEO.318,319

For patients with pure PEO, surgery remains the primary treatment option, whereas symptomatic treatment is recommended for extraocular symptoms in PEO-plus syndrome.302

Kearns-Sayre syndrome

Kearns-Sayre syndrome was first identified in 1958 by Kearns and Sayre, characterized by the triad of retinitis pigmentosa, PEO, and complete heart block.320 It is classified as one of the mitochondrial encephalomyopathies and presents with additional common symptoms, including cerebellar ataxia, cerebrospinal fluid protein levels exceeding 100 mg/dL, deafness, dementia, diabetes, delayed puberty, and amenorrhea.321,322 An onset before the age of 20 is a defining feature of Kearns-Sayre syndrome.234

The syndrome is predominantly caused by spontaneous single large-scale deletions of mtDNA,323,324 with the 4977 bp deletion being the most preval‎ent.325 Symptom manifestation requires the accumulation of mtDNA deletions beyond a pathogenic threshold, implying that the defective mtDNA must retain replication capability. This likely explains why most deletions occur within the long arc between heavy and light strands but preserve the replication sites.326 Additionally, during cellular differentiation, mtDNA deletions are preferentially replicated over wild-type mtDNA, potentially due to a kinetic advantage.327 These deletions often result in the loss of genes encoding respiratory chain proteins and tRNAs essential for translation, leading to ATP deficiency and multi-tissue dysfunction.328 For instance, the common 4977 bp deletion typically disrupts complexes I, III, IV, and mt-tRNA.323

Oxidative stress is evident in cells harboring the 4977 bp deletions.329 Respiratory chain complex defects may be independent of the deletion sites, with aberrant translation playing a key role in the pathogenesis of single large-scale mtDNA deletions.289 Increased oxidative damage and misfolded mitochondrial proteins inhibit both the ubiquitin-proteasome system and the OXPHOS system.330 Inhibition of ubiquitin-proteasome leads to decreased amino acid salvage, which triggers eIF2-α phosphorylation and induces mt-ISR. Under conditions of energy deficit and oxidative stress, genes involved in mt-ISR and autophagy are upregulated.330 Prolonged mt-ISR activation propagates and maintains mtDNA deletions, exacerbating the disease condition.331 Moreover, amino acid depletion, combined with ATP insufficiency, collectively inhibits the mTOR pathway, thereby increasing autophagy.330 The protein synthesis inhibition and autophagy increase, reducing mitochondrial contents, could be pathomechanisms of Kearns-Sayre syndrome.330

Notably, alterations in tau protein levels are observed in the cerebrospinal fluid of patients with Kearns-Sayre syndrome.332 Tau protein plays a role in ROS generation, mitochondrial dynamics, and mPTP opening,333 making its impact on Kearns-Sayre syndrome particularly intriguing. Cardiomyocytes in patients with Kearns-Sayre syndrome display increased and enlarged mitochondria.334 Arrhythmias in mitochondrial diseases are closely linked to dysfunctional ion channels, transporters, and membrane excitability caused by ATP deficiency, excessive ROS production, and Δψm collapse.335,336 Future research should focus on ionic dysregulation mediated by the mitochondrial Ca2+ uniporter complex, uncoupling proteins, and mPTP.

Furthermore, nDNA mutations in RRM2B, which encodes the ribonucleotide reductase p53R2 subunit, can lead to multiple mtDNA deletions in Kearns-Sayre syndrome through defective ribonucleotide reductase assembly.337 This defective assembly disrupts deoxynucleotide provision and the maintenance of dNTP pools.338

For patients with heart block, pacemaker implantation or implantable cardioverter defibrillators are recommended.339 In cases where heart failure develops, heart transplantation has been employed.340 The long-term safety and feasibility of human retinal progenitor cell transplantation for retinitis pigmentosa have been demonstrated.341 Gene therapy also holds promise as a potential treatment for retinitis pigmentosa associated with Kearns-Sayre syndrome.342

Pearson syndrome

Pearson syndrome, a fatal multisystem mitochondrial disorder, was first identified in 1979 by Pearson, who described it as a condition primarily affecting the bone marrow and exocrine pancreas.343 This disease is linked to defects in OXPHOS caused by sporadic single large-scale deletions (or rearrangements) of mtDNA.344,345 These deletions vary in size and location, ranging from 1.3 to 10 kb, with the size of the deletion potentially serving as a predictor for disease progression.306,346 Approximately 40–50% of patients with Pearson syndrome harbor the “common deletion,” which is 4977 bp in length.345,347 Sideroblastic anemia is typically the first and most prominent symptom of Pearson syndrome.348 Additional symptoms may include intracerebral bleeding, pancreatic exocrine insufficiency, lactic acidosis, and congenital malformations.348,349 The prognosis for Pearson syndrome is poor, with an average age of death being 5.44 years in individuals aged 0–15 and 7.41 years in those aged 0–19.350 Patients with the 4977 bp deletion have a higher mortality risk.349

Single large-scale mtDNA deletions exhibit phenotypic heterogeneity and contribute to a spectrum of diseases, including Pearson syndrome, Kearns-Sayre syndrome, and PEO.351 It is hypothesized that the timing of the mtDNA deletion during fetal development influences the clinical phenotype: late-stage deletions may result in PEO,4 while earlier-stage deletions may manifest as Kearns-Sayre syndrome or Pearson syndrome, affecting multiple systems.4 Interestingly, Pearson syndrome can evolve into Leigh syndrome or Kearns-Sayre syndrome over time,352,353 indicating that the phenotype of an mtDNA deletion disorder may change with age and is influenced by the concentration of mtDNA with deletions.354 Patients with Pearson syndrome typically have a higher proportion of mtDNA deletions compared to those with Kearns-Sayre syndrome or PEO.347,355

Spontaneous hematological recovery is observed in some Pearson syndrome cases, with a decrease in the amount of deleted mtDNA in blood cells corresponding with an improvement in anemia.356 This recovery is attributed to the positive selection of hematopoietic stem cells (HSCs), where HSCs with a higher load of deleted mtDNA are hard to survive, while those with a lower load are selected for survival.347,348,357 This concept aligns with findings from a mouse model study, which showed a decrease in the mtDNA deletion load with age in affected tissues, such as peripheral blood and liver.358 However, the study also indicated that mtDNA deletions may accumulate in muscle and other tissues, potentially leading to the development of Kearns-Sayre syndrome.358 This tissue-specific change in mtDNA deletion load partially explains the progression from Pearson syndrome to Kearns-Sayre syndrome.

In addition to ATP deficiency and oxidative stress caused by mtDNA deletions, iron accumulation may play a significant role in Pearson syndrome. The abnormal iron deposit is a feature in patients with Pearson syndrome, while the molecular mechanisms behind abnormal iron metabolism remain unclear.359 In a mouse model of large-scale mtDNA deletion, Pearson syndrome-like anemia worsened with the knockout of Drp1, and Drp1 knockout alone also caused anemia.359 Drp1 knockout decreases the pathogenic threshold of mtDNA deletion in erythrocytes,359 which drives us to think about the role of MQC in this anemia. Loss of Drp1 leads to HSC quiescence, reducing their regenerative potential.360 Interestingly, mitochondria with impaired fission ability are retained and accumulate with HSC divisions, potentially preventing unlimited self-renewal of HSCs.360 During erythropoiesis, mitochondria exhibit a specific pattern: increased fusion at early stages and heightened fission at later stages, associated with mPTP opening.361 Besides, the quiescence of HSCs is partly mediated by the regulation of mitochondrial content and activity.362 Thus, mitochondrial content and activity, which are influenced by mitophagy, are closely linked to HSC differentiation.363 A low Δψm is a key trigger for mitophagy, and there is a distinct difference in Δψm between quiescent and cycling-primed HSCs, with quiescent HSCs exhibiting low Δψm.362 Moreover, HSC quiescence is supported by an abundance of large lysosomes, and maintaining this quiescence requires restrained lysosomal activity.362 Interestingly, while suppressed mitophagy may specifically impair terminal erythrocyte maturation without affecting erythroid progenitor differentiation, hyperactivated mitophagy can hinder the differentiation of erythroid lineages.364 Thus, the fine regulation of mitophagy appears crucial during HSC differentiation, highlighting the potential importance of MQC in the anemia associated with Pearson syndrome. Further investigation is needed to determine whether and how energy and oxidative stress lead to MQC alterations, impacting anemia in Pearson syndrome.

Despite the possibility of spontaneous recovery from anemia in some patients with Pearson syndrome, others may require transfusions during infancy and early childhood.347 Hematopoietic stem cell transplantation is a potential option for those with persistent transfusion dependency or severe neutropenia.347,365 Additionally, mitochondrial augmentation therapy—where autologous CD34+ hematopoietic cells are augmented with maternally derived healthy mitochondria—has shown promising outcomes for mtDNA deletion syndromes like Pearson syndrome and Kearns-Sayre syndrome. This therapy has been observed to increase mtDNA content and improve aerobic function, suggesting it may be a viable treatment option.366

The primary mitochondrial diseases caused by nDNA mutations

Given that the majority of mitochondrial proteins are encoded by nDNA, mitochondrial diseases resulting from nDNA mutations encompass not only disturbances in OXPHOS but also defects in various structural or functional proteins essential for mtDNA maintenance, mitochondrial function and structure, and mito-nuclear communication. Consequently, the pathogenesis of mitochondrial diseases caused by nDNA mutations is inherently more complex than those arising from mtDNA mutations.

Autosomal dominant optic atrophy

Autosomal dominant optic atrophy (ADOA) is marked by progressive bilateral vision loss and color vision deficits, typically manifesting before the age of 20.231 The primary histopathological features of ADOA include the degeneration and demyelination of RGCs.367 Mutations in the OPA1 gene, which encodes a dynamin-related GTPase involved in IMM fusion, are the most common cause of ADOA.197 In many cases, haploinsufficiency is the primary pathogenic mechanism, as these mutations often result in premature translation termination.368 OPA1 mutations are typically heterozygous, as bi-allelic homozygous mutations, which lead to a complete loss of OPA1 function, are likely embryonically lethal.369,370 Although no cases of bi-allelic homozygous OPA1 mutations have been reported, bi-allelic compound heterozygous mutations have been observed and are associated with ADOA-plus (or Behr syndrome),371,372 which presents with additional multisystemic features beyond the optic neuropathy.372 Other genes involved in mitochondrial dynamics, such as OPA3, Drp1, and Mfn2, can also cause ADOA or ADOA-plus.197

The specific vulnerability of RGCs to OPA1 mutations remains unclear. However, research has shown that OPA1 mutations lead to defective differentiation and impaired mitochondrial function in RGCs, as demonstrated in human retinal organoids.373 The distinct mitochondrial morphology observed in RGCs and optic nerves in mouse models may provide some insights. In the unmyelinated segments of RGC axons, mitochondria are typically round before the lamina cribrosa, whereas they become elongated after crossing this structure in the myelinated segments, suggesting dynamic changes during mitochondrial transport.197 Elongated mitochondria are generally associated with enhanced ATP production, decreased fission, or increased fusion.374 It is hypothesized that OPA1 mutations may impair mitochondrial fusion after crossing the lamina cribrosa, leading to mitochondrial fragmentation. The clustering of fragmented mitochondria could cause traffic jams, obstructing the axonal transport of mitochondria.375 This theory aligns with observations that small RGCs with thin axons, which have limited mitochondrial transport capacity, are the first to be lost in ADOA.197 This suggests that disrupted mitochondrial transport may contribute to the RGC-specific susceptibility to OPA1 mutations. OPA1 also plays a role in intercellular mitochondria transfer.187 A significant proportion of mitochondria in RGC axons are not degraded by lysosomes within the RGC soma but are instead transferred to astrocytes at the optic nerve head for transcellular degradation.376 The potential impairment of mitochondrial transfer and transport due to OPA1 mutations should be considered as a contributing factor to the vulnerability of RGCs. Further research is needed to verify whether these mitochondrial properties specifically facilitate the susceptibility of RGCs to OPA1 mutations.

The OPA1 mutation has been shown to increase Drp1 expression‎, thereby promoting mitochondrial fission. Inhibiting Drp1 can help balance mitophagy and improve mitochondrial abnormalities associated with ADOA.377 Mitophagy, which can be activated by AMPK, is another critical factor; inhibiting both AMPK and mitophagy has been found to preserve mitochondrial content in RGC axons and mitigate visual loss caused by OPA1 mutations.378 Additionally, OPA1 mutations can lead to an increase in PINK1-Parkin-independent mitophagy, which may be directly driven by the excessive presence of fragmented mitochondria.375 This overactivation of mitochondrial fission and mitophagy could be central to the pathogenesis of ADOA caused by OPA1 mutations. Chronic inhibition of mitochondrial fusion due to OPA1 loss results in mtDNA depletion, exacerbating mitochondrial dysfunction,379 and excessive mitophagy further contributes to this depletion.375 The haploinsufficient OPA1 mutations not only impact mtDNA maintenance but also downregulate nuclear genes encoding mitochondrial components, implying the potential role of mito-nuclear communication in ADOA.373 Given OPA1’s role in mediating intercellular mitochondrial transfer via MDVs,187 the ability to secrete damaged mitochondria into the extracellular space might be impaired in ADOA. The dysfunctional MQC resulting from OPA1 loss reduces mitochondrial content and leads to secondary mtDNA depletion, disrupting OXPHOS.

Beyond its influence on mitochondrial fusion, OPA1 also plays a key role in preventing remodeling of cristae structure and mobilization of cytochrome c during apoptosis.380,381 Loss of OPA1 disturbs the structure and integrity of the IMM, leading to the release of cytochrome c in a which is mainly sequestered within the tight cristae junctions.380,381,382 Cytochrome c release is a well-known trigger of apoptosis. Excessively fragmented mitochondria can induce mtROS overproduction,144 making oxidative stress correlated with ADOA.383,384 The secondary mtDNA depletion may exacerbate OXPHOS disturbance, increasing the production of mtROS. Consequently, OPA1 mutations might facilitate stress-induced cytochrome c release by promoting the remodeling of cristae and mobilization of cytochrome c, thereby leading to RGCs degeneration.380,381,383,385

Given the critical role of mitochondrial cristae architecture in preventing mtDNA release and inflammation,299 mitochondrial inflammation in ADOA warrants significant attention. OPA1 loss has been associated with muscle inflammation due to mitochondrial dysfunction,386 even in the absence of mtDNA leakage into the cytosol. In this context, OPA1 deficiency induces NF-κB-mediated inflammation through the TLR9 pathway.386 Additionally, OPA1 defects reduce muscle mass and lead to premature death.379 The stress-induced mt-ISR, triggered by OPA1 loss, also contributes to muscle loss, and the reduction of FGF21 (a downstream hormone of mt-ISR) can mitigate this muscle wasting.379 Interestingly, while OPA1 loss impairs mitophagy in muscle cells,386 it increases mitophagy in RGCs. Understanding these tissue-specific differences in response to OPA1 mutations could help explain the phenotypic heterogeneity and tissue-specificity observed in ADOA.

Alpers-Huttenlocher syndrome

Alpers-Huttenlocher syndrome is the most preval‎ent mitochondrial disease caused by nDNA mutations. It is an autosomal recessive hepatocerebral syndrome with early onset,387 typically presenting with a triad of intractable seizures, developmental regression, and liver dysfunction.388 Notably, hepatic dysfunction can be aggravated by exposure to valproate, sometimes leading to a misdiagnosis of valproate hepatotoxicity.389 The disease is primarily caused by mutations in the POLG gene, such as p.A467T, p.W748S, and p.G848S, which result in mtDNA depletion.387,390 Alpers-Huttenlocher syndrome is likely the most common POLG-related disorder.391 Recessive POLG mutations can be homozygous or compound heterozygous, with compound heterozygous mutations in trans often associated with a more severe and earlier-onset phenotype, whereas homozygous recessive mutations tend to result in a milder and later-onset presentation.392 Additionally, ecogenetic structural nucleotide variants can influence the clinical phenotype.393 It’s worth noting that compound heterozygous mutations in C10orf2, which encodes the mitochondrial replicative helicase Twinkle, can also cause mtDNA depletion and present as Alpers-Huttenlocher syndrome.394 Mutations in C10orf2 disrupt mtDNA maintenance, leading to secondary mtDNA depletion and subsequent mitochondrial dysfunction.309

Another POLG-related disorder, Myocerebrohepatopathy spectrum (MCHS), presents with a triad of hypotonia, developmental delay, and hepatopathy.395 Other clinical manifestations may include renal tubulopathy, choreoathetosis, neuropathy, ataxia, and cataracts.2,396 MCHS is the earliest POLG-related disorder with mtDNA depletion, with a median onset age of 4.7 months.391,396 A case report documented the clinical progression of a child from infantile MCHS to Alpers-Huttenlocher syndrome, suggesting that MCHS and Alpers-Huttenlocher syndrome may represent different stages or severities of the same disorder.395

The histopathological features of mitochondrial encephalopathies include astrocyte activation, cortical degeneration, and neuron loss.397 In iPSC-derived neural stem cells with compound heterozygous POLG mutations, BNIP3-mediated mitophagy is increased, likely due to elevated ROS levels and a low NAD+/NADH ratio. Concurrently, decreased SIRT1 signaling and increased UCP2 signaling contribute to neuronal senescence.398 The combination of mitochondrial dysfunction and senescence leads to neuron loss in POLG-related disorders.398 Astrocytes, which play a pivotal role in supporting neurons,399 also suffer from dysfunction due to mtDNA defects, contributing to the development of mitochondrial encephalopathy. Indeed, astrocyte dysfunction has been demonstrated in patients with Alpers-Huttenlocher syndrome,400 and astrocytic neurotoxicity caused by mitochondrial dysfunction associated with POLG mutations also has been observed.401 The failure of mtDNA maintenance due to POLG mutations results in the loss of complexes I and IV in astrocytes,402 impairing their ability to proliferate and respond effectively to neuronal damage.403 POLG mutations can also disrupt mitochondrial biogenesis, as well as mitophagy, leading to astrocytic neurotoxicity and a loss of supportive functions.402 Similarly, mtDNA depletion caused by C10orf2 mutations leads to chronic astrocyte activation and dysfunction, which may produce neurotoxic factors and promote neuronal morphology changes and progressive spongiotic encephalopathy.404

Valproic acid is contraindicated in Alpers-Huttenlocher syndrome due to its hepatotoxicity, which is linked to increased apoptotic sensitivity through mPTP opening.405 In iPSC-derived hepatocytes from patients with Alpers-Huttenlocher syndrome, mitochondria exhibit reduced mtDNA content, cristae disorganization, and OXPHOS disturbances.405 However, the full extent of functional changes in hepatocytes remains unknown.

Currently, there are no effective treatments for Alpers-Huttenlocher syndrome. Symptomatic treatments, such as anti-epileptic therapy (excluding valproic acid and divalproex) and ventilation support, should be considered.393 Due to the multisystem involvement, liver transplantation alone is contraindicated.393 Supportive therapies, such as a ketogenic diet, may provide some benefit.406

Ataxia neuropathy spectrum

Ataxia Neuropathy Spectrum encompasses a group of mitochondrial disorders, including sensory ataxia, neuropathy, dysarthria, and ophthalmoplegia (SANDO), as well as mitochondrial recessive ataxia syndrome (MIRAS). SANDO was first identified as a syndrome involving multiple mtDNA deletions in muscle and peripheral nerve tissues by Fadic et al. in 1997.407 It is primarily caused by recessive POLG mutations and is considered part of the PEO-plus syndrome spectrum.408,409 While SANDO is most commonly associated with multiple mtDNA deletions due to POLG mutations, it can also be caused by mutations in the C10orf2 and RNASEH1 genes.410,411,412,413 RNase H1, which cleaves the RNA component of RNA: RNA hybrids as an endonuclease,414 plays a critical role not only in nDNA replication but also in mtDNA replication.415 In mitochondria, RNase H1 is essential for directing RNA primer formation for origin-specific initiation of mtDNA replication and for removing primers at the origin of replication to complete mtDNA replication.416,417 The absence of RNase H1 activity leads to defective mtDNA replication, resulting in linear deletions and depletion of mtDNA.418

MIRAS, an adult-onset mitochondrial disease, is characterized by clinical manifestations including ataxia, headache, axonal neuropathy, late-onset ophthalmoplegia, partial epilepsy in the occipital lobe, and a high risk of status epilepticus.410 Mutations in the POLG gene, such as W748S and A467T, have been identified in patients with MIRAS exhibiting multiple mtDNA deletions.410,419,420,421 Recent research has shown that POLG also plays a role in antiviral defense, and mutations in this gene can compromise antiviral tolerance, leading to epilepsy and liver disease in MIRAS and other POLG-related disorders.422

The spectrum of POLG-related disorders includes conditions such as myoclonic epilepsy myopathy sensory ataxia (MEMSA, characterized by epilepsy, myopathy, and ataxia without ophthalmoplegia), MCHS, Alpers-Huttenlocher syndrome, SANDO, MIRAS, and PEO.423 Despite being linked by common POLG mutations, these disorders differ in their onset age and the specific mtDNA defects they involve, leading to distinct clinical presentations.423 For example, MCHS typically manifests earliest, in neonates or infants, Alpers-Huttenlocher syndrome appears in infancy or childhood, and the remaining disorders are more likely to present in adolescence or adulthood.396 Early-onset and juvenile-onset POLG-related disorders generally result from biallelic pathogenic variants with autosomal recessive inheritance, whereas late-onset disorders (mainly PEO) may arise from a heterozygous POLG pathogenic variant with autosomal dominant inheritance.424

Another mitochondrial ataxia, infantile-onset spinocerebellar ataxia (IOSCA), is an autosomal recessive disorder characterized by sensory axonal neuropathy and progressive atrophy of the cerebellum, brain stem, and spinal cord.425,426,427 IOSCA is caused by two point mutations in the C10orf2 gene, which encodes the mitochondrial helicase Twinkle, as well as a rarer splice variant known as Twinky.428 These mutations result in a preponderance of messenger RNAs encoding Y508C polypeptides, leading to a Y508C alteration in the helicase domain of Twinkle or Twinky.428,429 IOSCA is classified as a mtDNA depletion syndrome, as patients’ brains show significant mtDNA depletion without mtDNA deletions or an increased number of mtDNA point mutations.429

The pathogenesis of POLG-related disorders has been discussed previously, but it’s important to note that different mutant genes involved in mtDNA replication may lead to distinct patterns of mtDNA deletions, varying in size and location. These variations can affect mitochondrial function differently depending on the nature of the mutations.313 Mitochondrial dysfunction in cerebellar tissue, mediated by impaired mitophagy, has been confirmed as a key factor in the development of ataxic diseases.430 Further research is needed to elucidate the molecular and functional changes in mitochondria that underlie mitochondrial ataxias.

As with most mitochondrial diseases, there are no specific treatments or cures for MIRAS, SANDO, IOSCA, and MEMSA. Treatment is primarily supportive and symptomatic, with clinical management focused on alleviating symptoms and improving quality of life.

Barth syndrome

Barth syndrome, an X-linked mitochondrial disorder, is characterized by a clinical triad of cardiomyopathy, skeletal myopathy, and neutropenia, along with aberrant cristae morphology and respiratory chain abnormalities.431 The gene responsible for Barth syndrome, TAZ (also known as G4.5), was identified as an X-linked gene encoding an acyl-specific phospholipid transacylase involved in remodeling cardiolipin acyl chains within mitochondrial membranes.432,433 In highly oxidative tissues such as the heart and skeletal muscle, tetralinoleoyl cardiolipin is the predominant form.434,435 Mutations in TAZ lead to a deficiency of tetralinoleoyl cardiolipin and an accumulation of monolysocardiolipins, which lack a linoleoyl acyl group.436,437 The elevated ratio of monolysocardiolipins to tetralinoleoyl-cardiolipin represents not only a biochemical hallmark but also the underlying molecular mechanism of Barth syndrome.438 This cardiolipin imbalance disrupts the phospholipid composition of the IMM, compromising the function of respiratory chain complexes and other mitochondrial proteins.439 Cardiolipin is essential for the stability of respiratory chain supercomplexes, which are crucial for the efficient operation of OXPHOS.88 In Barth syndrome, TAZ mutations disrupt supercomplex formation, resulting in reduced ATP production.440 Moreover, cardiolipin imbalances may interfere with the coupling of respiration to ATP synthesis, further diminishing energy production.439 The loss of respiratory chain supercomplexes also contributes to increased ROS, a phenomenon observed in Barth syndrome.441 Cardiolipin also plays a pivotal role in mitophagy, acting as a mediator in the process.442 Defective cardiolipin remodeling due to TAZ mutations hinders the initiation of mitophagy, leading to dysfunctional OXPHOS and heightened oxidative stress.443 Restoration of mitophagy through mTOR complex 1 inhibition has been shown to alleviate cardiomyopathy in Barth syndrome.444 The failure of dysfunctional mitophagy to remove and recycle mitochondria with defective components may exacerbate mitochondrial stress.

Current treatment strategies for Barth syndrome focus on managing heart failure, cardiac arrhythmias, and neutropenia.445 Physical therapy and nutritional support are also important considerations. Beyond gene or cell therapy, lipid replacement therapy aimed at restoring mitochondrial cardiolipin levels presents a promising therapeutic approach.445

Friedreich’s ataxia

Friedreich’s ataxia is the most common spinocerebellar ataxia with autosomal recessive inheritance.446 The disease is classically characterized by progressive and unremitting ataxia of the limbs and trunk, typically presenting before the age of 25.447 Additional symptoms include dysarthria, absent tendon reflexes in the lower extremities, loss of deep sensation, scoliosis, and cardiomyopathy.447 This condition is most commonly associated with a homozygous unstable guanine-adenine-adenine (GAA) trinucleotide expansion in the first intron of the Frataxin gene (X25) on chromosome.446,448 The age of onset is correlated with the number of GAA repeats.449 Some cases also result from a compound heterozygous expansion combined with a point mutation or deletion.450,451 Atypical phenotypes of Friedreich’s ataxia have also been reported.450 The GAA expansion within the intron silences the Frataxin gene, leading to reduced production of Frataxin protein and the associated disease phenotypes.452 The silencing mechanism may involve the formation of sticky DNA (a novel DNA structure) and epigenetic modifications.452,453

Frataxin is a mitochondrial protein essential for maintaining mitochondrial iron homeostasis.454 Its critical role in the assembly or transport of iron-sulfur (Fe-S) clusters means that Frataxin deficiency leads to aconitase and mitochondrial Fe-S respiratory enzyme deficiencies (respiratory chain complexes I, II, and III), resulting in mitochondrial iron accumulation.455,456,457 The Fe-S clusters mediate electron transfer and ROS production in complexes I, II, and III.458 Specifically, in response to Frataxin deficiency, the activation of iron-responsive element binding protein 1 (Fe-S protein) increases cellular iron uptake, with the iron being translocated into mitochondria via mitochondrial iron transporters in an attempt to compensate for impaired Fe-S cluster biogenesis.459 However, due to the Frataxin defect, mitochondrial iron cannot be effectively utilized, leading to iron accumulation and oxidation, which in turn causes severe oxidative stress and subsequent cell death.459,460

The development of Friedreich’s ataxia within the nervous system is believed to be driven by oxidative stress, iron neurotoxicity, and neuroinflammation.461 In Drosophila models, reduced Frataxin expression‎ impairs mitochondrial transport in neuronal regions, potentially affecting axonal function.462 Other MQC processes appear robust, with increased mitochondrial turnover and dynamics observed in the hearts of Frataxin-knockout mouse models, suggesting an effort to re-establish mitochondrial energetic and redox homeostasis.463 The interplay of oxidative stress and disordered iron metabolism raises questions about the role of ferroptosis, a form of cell death linked to iron-dependent lipid peroxidation, in Friedreich’s ataxia. While ferroptosis is indeed implicated in this disease,464,465 the precise contribution of mitochondrial dysfunction to ferroptosis in Friedreich’s ataxia remains unclear.

Fatty acid oxidation disorder

Fatty acid oxidation disorder represents a spectrum of syndromes primarily caused by defects in β-oxidation, inherited in an autosomal recessive manner.466 Mutations in specific genes result in defective enzymes, such as acylcarnitine translocase, carnitine palmitoyltransferase, and medium-chain acyl-CoA dehydrogenase.467 Among these, medium-chain acyl-CoA dehydrogenase deficiency, due to mutations in the ACADM gene, is the most preval‎ent form of fatty acid oxidation disorder.468 Clinically, the disorder is characterized by metabolic symptoms, including non-ketotic hypoglycemia, vomiting, encephalopathy, and acidosis, as well as muscular symptoms such as rhabdomyolysis and exercise intolerance.466 The critical role of β-oxidation in energy production means that its failure results in an ATP deficit. Beyond energy stress, mitochondrial dysfunction—driven by the lipotoxicity of accumulating fatty acids and carnitine derivatives—plays a central role in disease progression.467,469 Those toxic lipids can lead to respiratory chain complex inhibition, OXPHOS uncoupling, ROS overproduction, and persistent mPTP opening, resulting in ultimate cell death.467,469

Leigh syndrome

Leigh syndrome, also known as subacute necrotizing encephalopathy, is the most common mitochondrial disorder in childhood. It typically manifests before the age of 2 years and presents with a range of symptoms including hypotonia, epilepsy, respiratory distress, neurodevelopmental delay, ataxia, ophthalmological abnormalities, and lactic acidosis.470 Over 75 genes in both nDNA and mtDNA have been identified as causes of this condition,471 most of which are involved in OXPHOS or other energy production processes.470

Complex I deficiency is the leading cause of Leigh syndrome,472 with mutations in the nuclear gene NDUFS4 being the most common cause of complex I-associated Leigh syndrome. These mutations directly impair complex I function.470 Recent research has shown that NDUFS4 mutations also disrupt direct neuronal reprogramming of proliferating astrocytes through mechanisms involving endoplasmic reticulum stress, mt-UPR, and mt-ISR.473 Besides NDUFS4, other nuclear genes contribute to Leigh syndrome by causing deficiencies in assembly factors, cofactors, or biosynthetic pathways essential for energy metabolism.474 For instance, pyruvate dehydrogenase complex deficiency, often caused by mutations in PDHA1 (which encodes the E1 alpha subunit of pyruvate dehydrogenase),475,476 is another significant contributor to the disease.477 This deficiency disrupts the TCA cycle by depleting substrates needed for OXPHOS, leading to a collapse of energy production. Mutations in nDNA that cause mtDNA depletion are also implicated in Leigh syndrome, particularly those affecting the SUCLA2 or SUCLG1 genes, which encode subunits of succinyl-CoA ligase.470 Defective succinyl-CoA ligase not only hinders the TCA cycle by blocking the conversion of succinyl-CoA but also interferes with mitochondrial nucleotide salvage pathways by disrupting interactions with mitochondrial nucleotide diphosphate kinase, resulting in both metabolic dysfunction and mtDNA depletion.478,479 Primary mtDNA mutations can also lead to Leigh syndrome, with mutations in MT-ND5 (encoding a complex I subunit) and MT-ATP6 (encoding a complex V subunit) being common culprits.476,480 These mutations universally cause a breakdown in mitochondrial energy metabolism, leading to energy failure. MQC mechanisms are also implicated in the pathogenesis of Leigh syndrome-related gene mutations.143,473,481,482,483,484,485

Notably, although hypoxia might reduce energy production of OXPHOS, it alleviates Leigh syndrome in disease models by reducing mtROS generation and activating hypoxia-inducible factor pathway,12 indicating the downstream pathways of genetic mitochondrial dysfunction are promising therapeutic targets.

Mitochondrial neurogastrointestinal encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disorder first identified in 1978,486 is characterized by a range of clinical symptoms including cachexia, gastrointestinal dysmotility, peripheral neuropathy, ophthalmoparesis, and leukoencephalopathy.487 Mutations in the TYMP gene, which result in the loss of thymidine phosphorylase (TP) activity, are the primary cause of MNGIE.39 TP is responsible for catalyzing the reversible phosphorylation of deoxythymidine and deoxyuridine into thymine, uracil, and 2-deoxyribose 1-phosphate.39,488 The deficiency in TP activity leads to the accumulation of deoxythymidine and deoxyuridine, which disrupts the balance of mitochondrial nucleotide pools, thereby impairing mtDNA replication.489 This imbalance results in the accumulation of point mutations, multiple deletions, and depletion of mtDNA,490,491 ultimately leading to a failure in OXPHOS. The defective TP caused by TYMP mutations leads to the excessive accumulation of nucleosides in both mitochondria and lysosomes.492 The buildup of nucleosides, which possess weak alkaline properties, can alter the acidic environment of lysosomes, potentially suppressing lysosomal activity.492 Consequently, defective mitochondria are not adequately degraded and recycled, undermining the mitochondria’s ability to cope with stress, which may exacerbate mitochondrial dysfunction.

In addition to TYMP mutations, MNGIE-like phenotypes can also arise from mutations in other genes. For example, mutations in RRM2B can cause MNGIE-like symptoms by disrupting the docking interface of the ribonucleoside reductase small subunit homodimer, thereby impairing ribonucleoside reductase activity and damaging the mitochondrial nucleotide pool, which leads to mtDNA depletion.493 Mutations in LIG3, the only mtDNA ligase essential for mtDNA replication and repair, also affect mtDNA maintenance and have been reported in patients with MNGIE, leading to mtDNA depletion.494

Current treatment options for MNGIE include hemodialysis and peritoneal dialysis, enzyme replacement therapy, orthotopic liver transplantation, hematopoietic stem cell transplantation (HSCT), celiac plexus neurolysis, and splenic nerve blockage.495 However, while HSCT can restore biochemical homeostasis, it often fails to alleviate gastrointestinal symptoms.496 Encouragingly, gene therapy has shown efficacy in MNGIE murine models,497,498 and its potential effectiveness in human patients is highly anticipated.

Myopathy, lactic acidosis and sideroblastic anemia

Myopathy, lactic acidosis, and sideroblastic anemia (MLASA) is an autosomal recessive mitochondrial disorder affecting skeletal muscle and bone marrow, characterized by mitochondrial myopathy, lactic acidosis, and sideroblastic anemia.499,500 The condition is primarily caused by mutations in the PUS1 gene, which encodes pseudouridine synthase 1.500 PUS1 mutations result in a defective PUS1p, the catalytic center of pseudouridine synthase 1, which is responsible for pseudouridylating mt-tRNAs.500 In patients with MLASA, the loss of pseudouridylation at tRNA sites typically modified by PUS1p has been observed.501 Recent research using MLASA patient-specific iPSCs and mouse models has shown that PUS1p defects lead to a reduction in mt-tRNA due to the loss of pseudouridylation, which subsequently causes abnormal mitochondrial translation.502,503 Moreover, the pseudouridylation of mt-tRNA-derived fragments is also affected by PUS1 mutations, further contributing to defective mitochondrial protein synthesis.504 This mitochondrial dysfunction, stemming from abnormal mitochondrial proteins, disrupts erythropoiesis, leading to anemia.502,503 Another mutation associated with MLASA involves the YARS2 gene, which encodes mitochondrial tyrosyl-tRNA synthetase, an enzyme that catalyzes the covalent linkage of tyrosine to its corresponding tRNA.505 Mutations in YARS2 reduce the aminoacylation activity of this enzyme, leading to faulty translation of OXPHOS subunits, particularly complexes I and IV.506 Both PUS1 and YARS2 mutations ultimately result in the collapse of OXPHOS.

Sengers syndrome

Sengers syndrome is another autosomal recessive mitochondrial disease, characterized by hypertrophic cardiomyopathy, myopathy, lactic acidosis, and congenital cataracts.507 Mutations in the AGK gene, located in nDNA, have been identified as the cause of this syndrome.508 The AGK gene encodes mitochondrial acylglycerol kinase (AGK), a multi-substrate lipid kinase that phosphorylates monoacylglycerol and diacylglycerol to produce lysophosphatidic acid and phosphatidic acid, thus playing a role in phospholipid synthesis and various signaling pathways.508 AGK is also involved in the synthesis of IMM-specific cardiolipin.509 Beyond lipid metabolism, AGK acts as a subunit of the TIM22 complex, promoting the import of mitochondrial carrier proteins independently of its kinase activity.510,511 AGK is also thought to interact with complex I of the respiratory chain, a function that appears to be disrupted by AGK deficiency rather than mutation, leading to complex I dysfunction.512 This interaction requires further validation. The pathogenesis of Sengers syndrome likely involves disruptions in mitochondrial membrane phospholipid metabolism and the protein import machinery, with further studies needed to elucidate the detailed molecular mechanisms.

Perrault Syndrome

Perrault syndrome is an autosomal recessive disorder characterized by sensorineural hearing loss and ovarian dysgenesis, though other neurological symptoms may also be present.513 The syndrome is associated with mutations in several genes, including CLPP, ERAL1, HARS2, LARS2, and C10orf2.514 The CLPP protein is an endopeptidase component of a mitochondrial ATP-dependent proteolytic complex involved in the degradation of defective proteins through mt-UPR.515 The functional CLPP tetradecamer interacts with the hexameric caseinolytic peptidase X to proteolyze specific protein substrates.516 Mutations in CLPP that reduce its proteolytic activity or disrupt its interaction with caseinolytic peptidase X lead to a breakdown in mitochondrial protein quality control, resulting in mitochondrial dysfunction.517 CLPP plays a critical role in the turnover of complex I, providing protection against mtROS overproduction and related damage.518 This protective function of CLPP may serve as an important supplementary mechanism to mitophagy in clearing ROS-damaged mitochondria.519 However, the role of CLPP in mt-UPR in mammals is debated.520 Some studies suggest that CLPP loss may even alleviate mitochondrial diseases caused by defective DARS2, the mitochondrial aspartyl tRNA synthase.520,521 CLPP also participates in other aspects of mitochondrial metabolism, though its precise role in Perrault syndrome remains unclear.522

HARS2 and LARS2 encode mitochondrial histidyl tRNA synthetase and leucyl tRNA synthetase, respectively, which are responsible for catalyzing the covalent attachment of histidine and leucine to their corresponding tRNAs. The activity of these aminoacyl tRNA synthetases is essential for mtDNA translation.523,524 Mutations in HARS2 or LARS2 reduce the aminoacylation activity of these tRNA synthetases, resulting in impaired mitochondrial translation and subsequent mitochondrial dysfunction.523,524 ERAL1 binds to mitochondrial 12S rRNA as a chaperone and is essential for the assembly of the small 28S subunit of the mitochondrial ribosome.525 Mutations in ERAL1 impair RNA processing and mitochondrial translation.526 In other words, the collapse of mitochondrial protein homeostasis appears to be the predominant mechanism in Perrault syndrome.514

Symptomatic treatments for Perrault syndrome include cochlear implantation for hearing loss and estrogen replacement therapy. For women desiring pregnancy, options such as in vitro fertilization and oocyte cryopreservation may be considered.514

Age-related diseases

Beyond mitochondrial diseases directly caused by hereditary mtDNA and nDNA mutations, aging significantly contributes to the gradual decline in mitochondrial function and the efficiency of oxidative phosphorylation, resulting in decreased ATP production and elevated ROS generation.527 This mitochondrial dysfunction is intricately linked to the onset and progression of various age-related diseases, particularly neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD).528,529 Pathogenic factors, including mitochondrial genome defects, increased oxidative stress, disrupted MQC, impaired mitochondrial proteostasis, and neuroinflammation, are central to the development and progression of these neurodegenerative disorders.529,530 Targeted mitochondrial therapies show promise as potential treatments for these conditions.529 Furthermore, MDPs and mtDNA single nucleotide polymorphism within coding regions are strongly associated with age-related diseases.78,531 For example, humanin has been found to prevent synaptic loss and reduce inflammation, offering therapeutic potential in AD.78 Similarly, SHLP2 and its variants can mitigate mitochondrial dysfunction and protect dopaminergic neurons, thereby lowering the risk of PD.531 Additionally, dysregulation of mitochondrial microRNAs is implicated in mitochondrial dysfunction and is associated with neurodegenerative diseases.532

Damage to mitochondrial antioxidant enzymes, combined with excessive ROS, triggers oxidative stress that leads to mtDNA point mutations and deletions accumulating over time. These genetic alterations disturb MQC, and ultimately result in energy depletion, oxidative damage, and apoptosis.527,529,532 Moreover, ROS accelerates the accumulation of oxidative byproducts through mitochondrial proteases and the mt-UPR.527 Released mtDNA, fragmented mitochondria, and other substances from dying neurons can initiate inflammatory responses, further driving the progression of neurodegenerative diseases.209 Elevated ROS levels, along with mtDNA polymerase mutations that cause replication errors, contribute to mtDNA mutations, multiple deletions, and reduced copy numbers, all of which accumulate with age, leading to mitochondrial dysfunction and cell death.209,530 Notably, AD is associated with mtDNA mutations and reduced copy numbers, while PD is closely linked to mtDNA deletions.530,533

In AD, neuronal damage and dysfunction are closely associated with increased oxidative stress due to amyloid-β accumulation and tau aggregation, as well as impaired mitochondrial bioenergetics and MQC networks.532,533 Mitochondrial and autophagic dysfunctions also contribute to microglial activation and neuroinflammation, further advancing AD pathogenesis.534 Similarly, PD is characterized by increased oxidative stress, abnormal mitochondrial dynamics, impaired biogenesis, and autophagy defects.535,536 These dysfunctions are primarily associated with mutations in the genes of certain proteins, such as α-synuclein, Parkin, and PINK1.3,529 Accumulation of α-synuclein in mitochondria can lead to the formation of oligomers that interact with mitochondrial membranes, inhibiting complex I activity and causing excessive mtROS production, which induces neuronal apoptosis.3,529 Mutations in PINK1 or Parkin disrupt PINK1-Parkin-dependent mitophagy, leading to the accumulation of defective mitochondria.533,536 Moreover, PD-related neurotoxins and mutations can induce mitochondrial fission, exacerbating neuroinflammation.537 The examples of AD and PD illustrate the multifaceted pathological roles that mitochondria play in age-related diseases.

In addition to genetic factors and aging, several other elements contribute to the progression of neurodegenerative diseases, including lifestyle choices and environmental exposures. Genomic instability, telomere attrition, and epigenetic modifications may all increase disease susceptibility.528 Moreover, factors such as dysregulated nutrient sensing, stem cell exhaustion, altered intercellular communication, and immune dysfunction also play significant roles in the development of these diseases.528 Future research should focus on further exploring these factors and their interactions to enhance understanding of the underlying pathogenic mechanisms.

It is evident that age-related diseases are intricately linked to mitochondrial dysfunction associated with aging. Their pathogenesis typically results from the long-term accumulation of factors such as pathological protein aggregation and disrupted MQC. In contrast, inherited mitochondrial diseases arise from specific mutations in mtDNA or nDNA that directly impair mitochondrial function, often leading to significant clinical symptoms in infancy or adolescence. Although both types of diseases share mitochondrial dysfunction as a common feature, they differ in their pathogenesis, age of onset, and clinical phenotypes.

Overall, mitochondrial diseases are increasingly recognized as pathway-based disorders rather than merely energy-related conditions.7 Numerous biological processes, such as autophagy, are energy-dependent, and severe energy stress can suppress these processes.538 Consequently, a genetic deficit in ATP can induce cellular dysfunction. ATP deficit can also serve as a signal to trigger downstream pathways such as AMPK activation, resulting in alterations of mitochondrial function.330 The pathological consequences of the downstream pathways activated by ATP shortage may be of importance for disease development, despite the currently incomplete understanding. As previously discussed, the overproduction of mtROS plays a critical signaling role in triggering MQC, apoptosis, and inflammation. Moreover, elevated mtROS levels can exacerbate the maintenance of mtDNA.539 Oxidative stress may be a common contributing factor in mitochondrial diseases. Additionally, the maintenance of a normal Δψm is essential for ATP synthesis, mitochondrial protein and ion transport, and mito-nuclear communication signaling.83 For example, reduction in Δψm disturbs mitochondrial calcium homeostasis, which may be a pathomechanism in mitochondrial diseases.11 Certain MQC processes, such as IMM fusion, also rely on a healthy Δψm.300 A reduction in Δψm is a key initiator of mt-ISR and mitophagy; however, chronic activation of mt-ISR or mitophagy may accelerate disease progression.540 Thus, the collapse of Δψm can further deteriorate cellular functions. Changes in MQC are observed in mitochondrial diseases. Although these processes are intended to help affected cells resist stress, pathological and chronic activation of them can aggravate mitochondrial dysfunction and promote disease progression.125,248 Despite the significant attention given to MQC in mitochondrial diseases, the underlying molecular mechanisms behind these changes remain largely unknown. The roles of mitochondrial apoptosis and inflammation in these diseases are also not fully understood now. Oxidative stress has been emphasized in the fields.10 Given their established influence in age-related diseases, the potential contributions of mitochondrial apoptosis and inflammation to primary mitochondrial diseases warrant close investigation.

Under OXPHOS defects or mitochondrial stress, the mito-nuclear communication pathways like mt-ISR will be activated. These activations could be significant in those mitochondrial diseases caused by mutations in mt-tRNAs due to defects of mitochondrial protein synthesis.9 Notably, research by Burr et al. has demonstrated cell lineage-specific mitochondrial resilience to mutations during mammalian organ development.541 The specific modes of mito-nuclear communication in response to genetic mitochondrial dysfunction, which determine tissue-specific vulnerability to mtDNA defects, are independent of mt-UPR and mt-ISR.541 Different triggers and degrees of mt-ISR activation in response to identical mutational loads in various cell types may also contribute to this specificity,540 potentially in the later phases.541 A deeper exploration of mito-nuclear communication is essential for understanding the pathogenesis and heterogeneity underlying mitochondrial diseases. In addition to the roles of mt-UPR and mt-ISR in facilitating mito-nuclear communication to manage cellular stress,542 the role of epigenetics, an integral component of mito-nuclear communication, is gaining increasing attention.543 Future research should investigate the pathological role of epigenetic modifications, such as lactylation, particularly given that elevated lactate levels are a common characteristic of mitochondrial diseases.544

While various phenotypic syndromes and their associated gene mutations have been discussed, the genetic and phenotypic heterogeneity of mitochondrial diseases is notably complex. The relationship between common pathogenic variants and the phenotypic syndromes of mitochondrial diseases is summarized in Table 2.

Table 2 The common mutant genes of mitochondrial phenotypic syndrome

Full size table

Diagnostic methodology of mitochondrial diseases

Given the genetic and phenotypic heterogeneity of mitochondrial genetic disorders, accurately diagnosing these diseases remains a significant challenge.545 Beyond careful and detailed clinical observation, comprehensive testing is essential, including biochemical analyses of body fluids, neuroimaging, DNA and RNA sequencing, as well as biochemical or pathological testing of tissues (Fig. 6). When a mitochondrial disease is suspected, initial biochemical testing should be performed on blood, urine, and cerebrospinal fluid.545 For instance, a blood count can reveal anemia or neutropenia, which are critical indicators in certain mitochondrial genetic disorders, such as Pearson syndrome.343

미토콘드리아 질환의 진단 방법론미토콘드리아 유전 질환의 유전적 및 표현형적 이질성으로 인해 이러한 질환을 정확히 진단하는 것은 여전히 중요한 과제입니다.545 

세심하고 자세한 임상 관찰을 넘어, 

신체 액체의 생화학적 분석, 신경 영상 검사, DNA 및 RNA 시퀀싱, 조직의 생화학적 또는 병리학적 검사 등을 

포함한 포괄적인 검사가 필수적입니다(그림 6). 

미토콘드리아 질환이 의심될 경우, 

혈액, 소변, 뇌척수액에 대한 초기 생화학적 검사가 수행되어야 합니다.545 

예를 들어, 

혈액 검사는 특정 미토콘드리아 유전 질환(예: 피어슨 증후군)에서 중요한 지표인 빈혈이나 중성구 감소증을 확인할 수 있습니다.343

Fig. 6

Diagnostic Methodology for Mitochondrial Diseases. Diagnostic strategies have evolved from a biopsy-first approach to a genetic-first approach. Initial screenings should utilize blood, urine, and cerebrospinal fluid samples. Biomarker testing is essential in this process. For suspected mitochondrial diseases, mtDNA sequencing and analysis are the preferred methods, while nDNA sequencing should be considered in cases of mtDNA multiple deletions, depletion, or early-onset symptoms. RNA sequencing (transcriptomics) and respirometry also contribute to accurate diagnosis. Biopsy specimens, typically obtained from muscle or skin, remain valuable for confirm‎ing tissue-specific mtDNA mutations that may not be detected in blood or urine samples. Histopathological examination and respiratory chain enzyme analysis can be applied to these tissue samples, revealing abnormal mitochondrial structure, morphology, and function. Thus, biopsy retains significant diagnostic value. mtDNA mitochondrial DNA; nDNA nuclear DNA; ccf-mtDNA circulating cell-free mitochondrial DNA; Δψm mitochondrial membrane potential; SDH succinate dehydrogenase; COX cytochrome c oxidase; NADH-TR nicotinamide adenine dinucleotide tetrazolium reductase

Full size image

Biomarkers

Certain biomarkers for mitochondrial diseases are garnering increasing attention due to their potential diagnostic value. The rise in anaerobic glycolysis, a compensatory response to energy shortages caused by OXPHOS dysfunction, leads to lactate accumulation, making lactic acidemia or lactic acidosis a common feature of these diseases.544 Since lactate is produced from pyruvate by lactate dehydrogenase, elevations in lactate are typically paralleled by increases in pyruvate.546 While lactate elevation exhibits high specificity (83–100%) among patients with mitochondrial diseases, its sensitivity is relatively low (34–62%), compared to pyruvate elevation, which shows a sensitivity of 75% and a specificity of 87.2%.547,548 The balance between lactate and pyruvate is regulated by the cytosolic NAD+/NADH ratio.549 Impaired oxidation of NADH, a function of respiratory complex I, increases NADH levels, driving the equilibrium toward lactate accumulation, which differs from the proportional increase in lactate and pyruvate seen in pyruvate dehydrogenase deficiency.549 Therefore, the lactate/pyruvate ratio offers excellent diagnostic accuracy for distinguishing pyruvate dehydrogenase deficiency from other mitochondrial diseases.550 Additionally, transaminase testing is essential as hepatopathy can be an early sign of mitochondrial diseases like Alpers-Huttenlocher syndrome.394

OXPHOS function measured in blood cells is emerging as a potential biomarker, with an estimated sensitivity of 68.3%, directly reflecting OXPHOS defects.546 The dysfunction of OXPHOS also leads to long-term alterations in Δψm as respiratory chain complexes fail to transfer protons.83 Notably, significant changes in Δψm were consistently observed in blood cell tests, suggesting that Δψm quantification could be a superior diagnostic method.546 Although less sensitive, biomarkers like creatine, creatine kinase, free carnitine, and acylcarnitine in the blood can aid in diagnosing mitochondrial diseases caused by fatty acid β-oxidation disorders.546,547 Additionally, amino acid analysis of both plasma and cerebrospinal fluid, along with organic acid analysis of urine, is valuable for diagnosing specific mitochondrial metabolic disorders, although patient selection prior to testing is recommended.551 For instance, an elevated monolysocardiolipins/tetralinoleoyl-cardiolipin ratio in blood and increased 3-methylglutaconic acid in urine (a precursor to cardiolipin) are strong biochemical indicators of Barth syndrome.438 Notably, two cytokines involved in the mt-ISR, FGF-21 and GDF-15, have shown exceptional diagnostic value for mitochondrial myopathy and tRNA gene mutation-related mitochondrial diseases, and they may also serve as biomarkers for monitoring therapeutic efficacy.548,552,553

The significance of extracellular mitochondrial content has been highlighted by Miliotis et al.554. Recent studies suggest that mitoEVs are involved in encephalopathy associated with mitochondrial disorders, potentially serving as indicators of mitochondrial encephalopathy.555 MitoEVs containing mitochondria or mtDNA are emerging as promising biomarkers,554,556 as their release is part of the process that recycles or eliminates nonfunctional mitochondrial fragments.184 Changes in ncRNAs, which regulate mitochondrial protein expression‎ and mitochondrial function-related signaling pathways, can reflect metabolic and functional alterations in mitochondria.557 MicroRNAs, a specific type of ncRNA, play a role in the pathogenesis of mitochondrial diseases and could aid in diagnosis. For example, oxidative stress-induced microRNA-9/9* has been implicated in the MELAS phenotype, while downregulation of microRNA-181a/b, which promotes MQC through the activation of mitochondrial biogenesis and mitophagy, has shown protective effects on RGCs in an LHON mouse model.558 Further studies on microRNA-181a/b downregulation in the treatment of inherited retinal diseases underscore the importance of microRNAs.559 Additionally, microRNA-27b-3p has demonstrated significant diagnostic value in patients with MELAS.560 However, these biomarker changes are not exclusive to mitochondrial diseases and can also be observed in other conditions,554,561 necessitating further research into their sensitivity and specificity.

Furthermore, under conditions of stress, apoptosis, or necrosis, damaged mtDNA can be released from cells as cell-free circulating mtDNA (ccf-mtDNA).562 The accumulation of damaged mtDNA contributes to the increase of ccf-mtDNA in plasma, making it a potential biomarker for mtDNA genetic disorders, particularly MELAS, with a sensitivity of 44% and specificity of 94%.554

Given the profound impact of metabolic and functional disorders on genetic mitochondrial diseases, the fields of proteomics, lipidomics, and metabolomics are being explored for their potential applications.563,564,565,566,567 While their diagnostic value is not yet fully established, these approaches hold great promise for the future.

DNA and RNA sequencing

Given that mtDNA mutations are the primary cause of genetic mitochondrial diseases, mtDNA testing should be prioritized in the diagnostic process. The advent of NGS has significantly shifted the diagnostic approach from a biopsy-first strategy to a genetic-first strategy.568,569 With the development and widespread application of NGS, the genetic diagnostic yield for these disorders has increased from 10–20% in the pre-NGS era to 40–60% today.41,568 However, the diagnostic yield of NGS in suspected mitochondrial disease cases varies, with reports indicating a range of 7% to 70%.227 Recently, the United Kingdom introduced new practice guidelines for the genetic diagnosis of mitochondrial diseases, aiming to standardize and guide the use of DNA sequencing.569

Comprehensive mtDNA testing should include sequencing the entire mtDNA genome, analyzing mtDNA deletions, assessing mtDNA copy number, and determining the heteroplasmy levels of mtDNA mutations.7,547,551 NGS has become the first-line methodology for mtDNA testing.547 Various laboratory techniques, such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), allele-specific oligonucleotide polymerase chain reaction (ASO-PCR), single-strand conformation polymorphism (SSCP), long-range PCR, and Southern blot, have traditionally been used to screen for point mutations and deletions in mtDNA.551 For analyzing mtDNA copy number, which often reflects mtDNA depletion, real-time quantitative PCR (RT-qPCR) is commonly used.551 Assessing the heteroplasmy level of mutant mtDNA is also necessary, as it may correlate with disease severity and progression; this is typically done using methods like pyrosequencing or PCR,546 though these approaches are increasingly being replaced by NGS.570 A promising approach combines long-range PCR-based enrichment with NGS, enabling both quantitative and qualitative detection of every base in the entire mitochondrial genome.570 This method can detect heteroplasmy levels as low as 1–10%, with improved sensitivity due to specific mtDNA enrichment strategies and NGS platforms.7 However, it is essential to distinguish these findings from normal tissue states, which may carry low-level heteroplasmy.228 Although blood is the most commonly used sample for mtDNA testing, it is also advisable to assess mtDNA in urine and other affected tissues due to the possibility of tissue-specific mutations.547

As discussed, multiple mtDNA deletion and depletion are often caused by nDNA mutations. Consequently, 75–90% of pediatric mitochondrial diseases are attributed to nDNA mutations, making nDNA sequencing particularly important for diagnosing childhood-onset mitochondrial diseases.551 NGS of nuclear genes involved in mtDNA maintenance, mitochondrial function, and metabolism using targeted gene panels and whole exome sequencing is a preferred approach.571 Other sequencing methods also play vital roles; for example, Sanger sequencing remains a valuable first-line test for identifying common mutant genes in certain populations.569

Emerging RNA sequencing, or transcriptomics, has recently shown promise for diagnosing mitochondrial diseases.571 This technique serves as an essential complement to genome sequencing and can help diagnose suspected mitochondrial diseases that remain genetically undetermined after genome sequencing.572 RNA sequencing methods can be divided into short-read cDNA sequencing, long-read cDNA sequencing, and direct RNA sequencing.573 RNA sequencing provides insights into aberrant splicing and altered transcript levels due to abnormal gene expression‎ or mono-allelic expression‎.571 For instance, RNA sequencing has detected a splicing variant in the CLPP gene associated with Perrault syndrome and mono-allelic expressed variants in the ALDH18A1 gene, which encodes an enzyme involved in mitochondrial proline metabolism, linked to cutis laxa III.572 RNA sequencing also has identified splice site mutations in mitochondrial diseases.574,575 Furthermore, tRNA sequencing can uncover defective tRNAs and reduced tRNA levels, which are implicated in many mitochondrial diseases.571 For example, defective N1-methyladenosine (m1A) modification in mt-tRNALys and mtDNA mutation-caused tRNAAla reduction, discovered through tRNA sequencing, have provided valuable insights into the pathogenesis of mitochondrial diseases.69,280

Biopsy

A tissue biopsy is widely regarded as the diagnostic gold standard for mitochondrial diseases, particularly in cases where genetic testing has not provided a definitive diagnosis.547 Detecting OXPHOS or mtDNA defects in biopsy specimens provides compelling evidence for the presence of mitochondrial disease. Muscle tissue is often preferred for biopsy due to its high energy demand, making it particularly susceptible to OXPHOS dysfunction.549 Skin biopsies, which allow for the analysis of fibroblasts, are also a viable alternative.576 Genetic, histopathological, and biochemical eval‎uations of biopsy specimens are crucial in this diagnostic process.

Testing mtDNA in biopsy specimens offers greater sensitivity in detecting low-level mtDNA heteroplasmy and assessing mtDNA copy number compared to blood samples.547 Histological stains such as Haematoxylin and eosin (H&E) and modified Gomori trichrome are used to examine mitochondrial structure and morphology. The modified Gomori trichrome stain, in particular, can reveal ragged-red fibers, which indicate abnormal mitochondrial proliferation in response to energy failure, and can also highlight cytochrome c oxidase (COX, respiratory complex IV) deficiency.577,578 Sequential staining with succinate dehydrogenase (SDH, respiratory chain complex II) and COX provides additional insights into the activity of these complexes.549 Since SDH is entirely encoded by nuclear DNA, while mtDNA encodes three subunits of COX, COX activity is a more direct reflection of mtDNA maintenance.579 In normal tissue, SDH staining appears as ragged-blue fibers.580,581 Due to the uneven distribution of mtDNA mutations along muscle fibers, COX staining often shows a mosaic pattern of COX-negative and COX-positive fibers in cross-sections.579 In combined COX and SDH staining, the COX activity is typically indicated by a brown stain that overshadows the blue SDH stain. However, reduced COX activity allows the SDH stain to become more visible, presenting as blue fibers.579 This staining combination provides two key insights: a mosaic pattern of SDH-positive but COX-negative fibers indicates an mtDNA defect involving COX, while a mosaic pattern of both SDH-positive and COX-positive fibers suggests an mtDNA defect involving NADH dehydrogenase (respiratory complex I) or cytochrome c reductase (respiratory complex III).579 To specifically detect NADH dehydrogenase activity, nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) staining should be employed.582 A reduction in NADH-dehydrogenase activity results in decreased or absent blue-purple color in NADH-TR staining, with an increase in stained ragged blue fibers in the subsarcolemmal region.549

In addition to assessing OXPHOS function in blood, as discussed earlier, the biochemical analysis of mitochondrial respiratory chain enzymes, isolated from muscle tissue or cultured fibroblasts, can directly reflect OXPHOS defects.583,584 CoQ10 assessment is also important, given its critical role in electron transport within OXPHOS.585 Spectrophotometric or colorimetric assays are commonly used to measure enzymatic activity.584,586 Successful enzymological analysis requires the use of internal controls, typically normalized to SDH activity or mitochondrial citrate synthase levels.547,586

Electron microscopy offers detailed visualization of mitochondrial quantity, inclusions, and ultrastructural abnormalities, demonstrating its diagnostic potential for mitochondrial nephropathy, cardiomyopathy, and hepatopathy.587,588,589

Respirometry

High-resolution respirometry is a valuable methodology for assessing mitochondrial respiration, specifically OXPHOS function.590 This technique can be applied to various subjects, including isolated mitochondria, intact cells, and three-dimensional systems such as tissue slices, and it is typically measured using two primary setups: chamber-based platinum electrodes and microplate-based fluorescent readings.591 The substrate-uncoupler-inhibitor titration (SUIT) protocol is particularly effective in this context, as it allows for the measurement of oxygen consumption (flux) by specific respiratory complexes, thereby reflecting enzyme activity.590,592,593 By calculating flux control ratios—ratios of oxygen flux under different respiratory control conditions—the SUIT protocol enables internal normalization, facilitating comparisons across different studies.593 This method has shown promise in the diagnosis of mitochondrial diseases in several studies.594,595,596

However, there are no perfect diagnostic methodologies. Diagnosing atypical and novel genetic mitochondrial disorders requires a thoughtful combination of these various diagnostic approaches. Moreover, the importance of integrating clinical observations with laboratory examinations cannot be overstated, as this connection is crucial for accurate diagnosis.

Potential therapeutic strategies of mitochondria in genetic disorders

Effective treatments for most mitochondrial diseases remain elusive. However, mitochondrial replacement therapy (MRT) and gene therapy represent promising foundational approaches for treating mitochondrial genetic disorders and preventing their transmission to future generations. Additionally, ongoing research is exploring the restoration of post-transcriptional tRNA modifications in mitochondria and the development of targeted therapies for specific conditions. Below, we provide a detailed summary of potential therapeutic strategies and discuss their possible clinical applications. (Fig. 7, Fig. 8, Table 3).

Fig. 7

Procedure for Mitochondrial Replacement Therapy. a The procedure for pronuclear transfer (PNT) involves extracting the pronucleus from a zygote of a healthy donor with wild-type mtDNA and from a patient with defective mtDNA. The cytoplast from the patient and the pronucleus from the healthy donor are then removed. Finally, the pronucleus from the patient and the cytoplast containing wild-type mtDNA from the healthy donor are fused, reconstructing a zygote with the patient’s pronucleus and wild-type mtDNA. b The procedure for spindle-chromosome complex transfer (ST) involves extracting the spindle-chromosome complex from a metaphase II oocyte of a healthy donor with wild-type mtDNA and from a patient with defective mtDNA. The cytoplast from the patient and the spindle-chromosome complex from the healthy donor are removed. Finally, the spindle-chromosome complex from the patient and the cytoplast containing wild-type mtDNA from the healthy donor are fused, reconstructing an oocyte with the patient’s spindle-chromosome complex and wild-type mtDNA. c The first polar body transfer (PB1T) procedure involves extracting the spindle-chromosome complex from a metaphase II oocyte of a healthy donor with wild-type mtDNA and the first polar body from a metaphase II oocyte of a patient with defective mtDNA. The cytoplast from the patient and the spindle-chromosome complex from the healthy donor are then removed. Finally, the first polar body from the patient and the cytoplast containing wild-type mtDNA from the healthy donor are fused, reconstructing an oocyte with the patient’s first polar body and wild-type mtDNA. d The second polar body transfer (PB2T) procedure involves extracting the female pronucleus from a zygote of a healthy donor with wild-type mtDNA and the second polar body from a zygote of a patient with defective mtDNA. The zygote from the patient and the female pronucleus from the healthy donor are then removed. Finally, the second polar body from the patient and the zygote containing wild-type mtDNA and the male pronucleus from the healthy donor are fused, reconstructing a zygote with the patient’s second polar body and wild-type mtDNA

Full size image

Fig. 8

Gene Therapy and Post-Transcriptional Modification Strategies for Mitochondrial Genetic Disorders. a MitoTALENs consist of TALE fused with FokI nucleases, while mtZFNs are composed of ZFP linked to FokI nucleases. MTS guides mtZFNs and mitoTALENs to the mitochondria. ZFP and TALE selectively bind to predetermined defective mtDNA target sequences, after which FokI dimerizes and cleaves the mtDNA adjacent to these binding sites, causing double-strand breaks that lead to the elimination of defective mtDNA. The remaining wild-type mtDNA can then replicate, altering the heteroplasmy ratio. b The targeted gene sequence, along with transcriptional regulatory elements and MTS, is packaged into an AAV vector, which is then delivered to the nucleus of the recipient cell. Inside the nucleus, the AAV uncoats and releases single-stranded DNA, which replicates to form double-stranded DNA. RNA polymerase then transcribes this DNA into mRNA. The mRNA exits the nucleus and is translated into the corresponding protein at the ribosomes in the cytoplasm. MTS guides these proteins to the mitochondria, where they undergo further processing and perform their respective functions. c DdCBEs are engineered by fusing MTS, split-DddAtox halves, UGIs, and either ZFPs or TALEs. DddAtox catalyzes the deamination of cytosine to uracil, while UGIs prevent uracil-DNA glycosylase from excising uracil, resulting in C-to-T editing during replication. Additionally, by linking MTS, split-DddAtox halves, TadA8e (an engineered adenine deaminase), and TALEs, TadA8e catalyzes the deamination of adenine to inosine, which pairs with cytosine during replication, thereby achieving targeted A-to-G editing. d Modifying the tRNA binding domain of nuclear-encoded aminoacyl tRNA synthetase or overexpressing aminoacyl tRNA synthetase can enhance aminoacylation efficiency and stabilize translation products. The expression‎ of post-transcriptional negative regulators like microRNAs can inhibit the expression‎ of mitochondrial RNA-modifying enzymes, thereby affecting mt-tRNA modifications. Using microRNA antagonists could potentially reverse disease phenotypes. Furthermore, overexpressing mt-tRNA-modifying enzymes can correct anticodon first nucleotide modification defects in mt-tRNA, improving ribosomal translation within mitochondria. Overexpression‎ of mitochondrial translation elongation factors EFTu and EFG2 can also partially suppress amino acid misincorporation caused by mtDNA mutations during the translation elongation process. mtDNA mitochondrial DNA; mitoTALENs mitochondria-targeted transcription activator-like effector nucleases; mtZFNs mitochondria-targeted zinc-finger nucleases; MTS mitochondrial targeting sequence; NES nuclear export signal; DdCBEs DddAtox-derived cytosine base editors; ZFP zinc finger proteins; TALE transcription activator-like effector; UGIs uracil glycosylase inhibitors; TadA8e deoxyadenosine deaminase; AAV adeno-associated virus

Full size image

Table 3 Clinical trials for mitochondrial diseases

Full size table

Mitochondrial replacement therapy

Pronuclear transfer

Pronuclear transfer (PNT) is a technique that replaces the mitochondrial genome by transferring the parental pronucleus from zygotes with mutated mtDNA into enucleated zygotes containing healthy mitochondria.597 Initial studies show that less than 2% of the donor’s mtDNA persists in early embryos, and it becomes undetectable after development to the blastocyst stage in vitro.598 In mouse models with large-scale mtDNA deletions, PNT has successfully corrected mtDNA-related phenotypes in offspring,599 demonstrating its potential in treating mitochondrial genetic diseases. In 2015, the United Kingdom Parliament approved regulations allowing the use of PNT and ST, with other countries also exploring these techniques.600 However, the long-term effects on live-born offspring remain uncertain. Technical challenges, such as cytoplasmic leakage or incomplete separation of the nucleoplasm, may result in the transfer of a portion of the donor’s cytoplasm during PNT.601 Over time, the proportion of donor mtDNA may increase,599,601 potentially affecting the efficacy of MRT due to factors like enrichment, genetic drift, and mitochondrial bottleneck effects.602,603,604,605 This highlights the importance of minimizing or even eliminating mtDNA carryover during embryo transfer procedures.605 Vitrification of patient oocytes could help reduce mtDNA carryover and provide the option for oocyte storage.601,606 Additionally, female pronuclei, being smaller and containing fewer mitochondria than those used in PNT and ST, are easier to isolate and position, reducing the need for cytoskeletal inhibitors and avoiding premature oocyte activation.607 In a notable advancement, researchers isolated the female pronucleus from the second polar body and transferred it to another zygote, resulting in the live birth of four healthy cynomolgus monkeys.608 Moreover, techniques that enforce mitochondrial autophagy in reconstructed embryos produced by PNT have shown promise in reducing or eliminating mtDNA heterogeneity, enhancing safety.605

Despite these advancements, several potential issues require careful consideration. PNT results in the loss of approximately half of the embryos.609 While some ethical frameworks define pronuclear fusion as the beginning of embryonic life, PNT does not involve such fusion, which may reduce ethical concerns.605,610 Nevertheless, the loss of embryos remains a significant consideration.605,610 Furthermore, interactions between nDNA and mtDNA are extensive, with normal mitochondrial function relying on the coevolution of these two genomes.611,612 Disruption of these interactions post-PNT could potentially alter gene expression‎ and phenotypic traits in offspring.600 This functional incompatibility between nuclear and mitochondrial genomes could even lead to reproductive isolation in mammals.613 To mitigate the risks associated with mito-nuclear interactions after mitochondrial replacement, it is recommended to match mitochondrial genotypes between donors and recipients and to determine acceptable variation levels between donor and patient mtDNA haplotypes.600 Until the implications of these interactions are better understood, combining mitochondrial replacement therapy with prenatal screening is advised.601 Ongoing follow-up and research will be essential to fully understand and address these issues.

Spindle-chromosome complex transfer

A study comparing mtDNA carryover and embryo development outcomes using germinal vesicle nuclear transfer, ST, and PNT in a mouse model614 found that germinal vesicle nuclear transfer did not result in blastocyst formation, while PNT and ST performed comparably well.614 ST involves replacing mutated mtDNA in patient oocytes by isolating the spindle-chromosome complex from the patient’s unfertilized mature MII oocytes and transferring it into the cytoplasm of enucleated healthy oocytes.597 Notably, Tachibana et al. successfully employed ST to produce healthy rhesus monkey offspring, which contained nDNA from the spindle donor and nearly homogenous mtDNA from the cytoplasm donor.44 In human studies, ST performed on unfertilized mature MII oocytes resulted in zygotes and derived embryonic stem cell lines with normal euploid karyotypes and donor-only mtDNA.615 Furthermore, the transfer of vitrified spindles into fresh cytoplasm led to the birth of four healthy rhesus monkey infants, with no significant changes in mtDNA heterogeneity.615 The use of cryopreserved human oocytes for ST has also been shown to eliminate mtDNA heteroplasmy while maintaining normal mitochondrial activity.616 Cryopreservation primarily affects the oocyte cytoplasm rather than the spindle, making it suitable for storing patient oocytes.615

There has been a reported case of a female patient with Leigh syndrome, carrying the m.8993 T > G mutation, giving birth to a healthy child using ST technology.46 However, the live birth following ST involved electrofusion,46 a technique that can prematurely activate the oocyte to enter late anaphase II, potentially leading to incomplete meiotic recovery post-fertilization and increasing the risk of abnormal pronuclear formation and aneuploidy.615 To mitigate the risk of premature oocyte activation, researchers have explored alternative approaches such as using chemical or mechanical methods,617 lowering temperatures to induce partial depolymerization of the spindle-chromosome complex in mature MII oocytes,616 and adjusting the sequence of intracytoplasmic sperm injection and ST.618 Despite the potential of ST, the procedure can result in donor mtDNA carryover.44,609 Although low-level mtDNA heteroplasmy often diminishes during MRT, genetic drift can sometimes cause mtDNA to revert to its original genotype, compromising the effectiveness of MRT.602,619 This reversion may be linked to the preferential replication of specific D-loop conservative sequence box II region polymorphisms.619 To address this, ongoing research is being conducted to reduce mitochondrial carryover and enhance the efficiency and safety of MRT.620 Strategies such as lowering cytochalasin B concentration before ST and PNT,621 generating aggregated chromosomes using the phosphodiesterase inhibitor 3-isobutyl 1-methylxanthine followed by transfer,622 and implementing ST with maximal residue removal in MII oocytes618 have shown promise in significantly reducing residual mtDNA levels. While ST is considered safe and effective44,618 and ethically acceptable,623 the optimal methodology has yet to be determined, and the risks of genetic drift necessitate further research.623

Polar body transfer

Wang et al. demonstrated that germline genomes derived from ST, PNT, and polar body transfer (PBT) in mice could lead to embryos capable of normal fertilization and producing viable offspring.609 The F1 generation from PBT exhibited minimal donor mtDNA carryover, which remained stable into the F2 generation.609 Notably, blastocysts resulting from first polar body transfer (PB1T) displayed lower average levels of mtDNA carryover compared to those from second polar body transfer (PB2T).624 Embryonic stem cells derived from PB1T blastocysts maintained low and stable mtDNA carryover during extended proliferation and differentiation, both in vitro and in vivo.624 Due to the mitochondrial inheritance bias during meiosis, polar bodies harbor minimal to undetectable mitochondria.609 PBT proceeds without the need for cytoskeletal disruptors,624 thereby further reducing donor mtDNA carryover. Additionally, the membrane encasing polar bodies protects the genome and facilitates their isolation.609,624 Combining PBT with ST or PNT in individual donor oocytes could potentially halve the number of donor oocytes needed, thereby improving MRT efficiency.609 Given that polar bodies and oocytes share the same genome, they might be more suitable as nuclear transfer donors than pronucleus or spindle-chromosome complexes.609 Wang et al. also demonstrated that PB1T could effectively substitute ST, yielding healthy macaque monkeys with stable mtDNA heteroplasmy below 5% and no mtDNA drift.625 However, due to the higher mitochondrial concentration in dense clusters within the first polar body in intact oocytes—compared to the uniform mitochondrial distribution in the ooplasm in ST—and the slightly larger volume of the first polar body relative to ST, the isolated first polar body contained more mtDNA than the isolated spindle complex,625 contrary to findings in mice.609 Nonetheless, polar bodies are distinct byproducts of meiosis, tasked with extruding the surplus genome, which might affect their quality.626 Further refinements in PBT techniques and additional preclinical studies on human oocytes and zygotes are necessary to assess the safety, efficacy, and feasibility of PBT.620

Gene therapy

Allotopic expression‎

Mitochondrial proteins, encoded by nuclear genes, are synthesized in the cytoplasm before being imported into mitochondria.627 This process has propelled advances in treating mitochondrial genetic diseases through the allotopic expression‎ approach. This method re-encodes defective mtDNA gene sequences into nuclear-compatible sequences, aligned with the “universal” genetic code.628,629 These sequences are combined with appropriate transcriptional regulatory elements and mitochondrial targeting peptides, then delivered to the cell nucleus via suitable vectors.628,629 The resultant mRNA is translated in the cytosol,628,629 after which the mitochondrial targeting peptides facilitate the import of these proteins into mitochondria, achieving the allotopic expression‎ of functional mtDNA genes to compensate for genetic defects.628,629 Specifically, re-engineered and stabilized allotopic expression‎ of MT-ATP6 and MT-ATP8 allows their processing, import, and integration into complex V,630,631,632 while MT-ND3,633 MT-ND4,628 and MT-ND6634 are successfully imported into mitochondria and incorporated into complex I, rescuing mitochondrial dysfunction caused by corresponding mtDNA mutations. These mutations can lead to the loss of mitochondrial proteins, defects in respiratory chain complexes, and impaired oxidative phosphorylation.

Although human tRNA is typically not imported into mitochondria,635 Kolesnikova et al. demonstrated partial mitochondrial import and proper aminoacylation of nDNA-encoded yeast tRNALys derivatives in human cells, partially restoring mitochondrial function in MERRF cells.636 Furthermore, the Leishmania RNA import complex, entering human cells via a caveolin-1-dependent pathway, can induce the import of endogenous cytosolic tRNA, encoded by the nucleus, into mitochondria, rescuing dysfunction caused by mutated mt-tRNA genes.637

In LHON mice or rats, intravitreal injection of adeno-associated virus (AAV) vectors carrying nuclear-encoded human MT-ND4 genes resulted in the allotopic expression‎ of the wild-type MT-ND4 gene, with subsequent accumulation of mRNA and protein in RGCs and optic nerve axons, successfully importing them into mitochondria.629,638 The ND4 protein assembles with three complex I subunits, integrating into the respiratory chain complex I without disrupting its activity.629 This integration preserves complex I function, preventing vision loss, RGC apoptosis, and degeneration, as well as optic nerve atrophy induced by mtDNA mutations.629,639 AAV-mediated allotopic expression‎ of the nuclear-encoded MT-ND4 gene appears to be a feasible and safe treatment for LHON with mutated mtDNA, with clinical trials underway.47 However, the timing of gene therapy is crucial, as oxidative damage, RGC apoptosis, and axonal loss may be partly irreversible.639 Therefore, administering gene therapy after vision loss but before optic nerve atrophy, or targeting the asymptomatic contralateral eye in patients with acute unilateral vision loss, may offer benefits.628,639

Despite the promise of allotopic expression‎, challenges remain. Only a small fraction of allotopically expressed mitochondrial proteins localize to mitochondria632,640 and functionally integrate,641 likely due to the high hydrophobicity of these proteins, which hinders efficient mitochondrial import.640 This necessitates further refinement in processing and import efficiency.632,642 Combining recoded mtDNA sequences with optimized 3’ untranslated regions and 5’ mitochondrial targeting sequences (MTS) can enhance protein localization and import.629,642 The importability of hydrophobic peptides may also be improved by enhancing the expression‎ of molecular chaperones.640 Additionally, codon optimization of mitochondrial genes can improve the efficiency and stability of recoded mtDNA gene expression‎ in the nucleus.632 LHON is characterized by a 100% m.11778 G > A mutation. Although LHON mammalian models exhibit symptoms of acute vision loss similar to human LHON, the LHON model retains a normal MT-ND4 gene.638 Continued research is needed to explore the therapeutic potential of allotopic expression‎ for other mitochondrial diseases.

Gene replacement therapy

Currently, the application of gene replacement therapy for various mitochondrial genetic diseases is the subject of extensive research and development. Normal genes can be delivered via AAV transduction to replace defective nuclear genes in mitochondrial genetic disorders, achieving therapeutic effects. Frataxin plays a key role in Fe-S cluster biosynthesis.643 In Friedreich’s ataxia, Frataxin deficiency leads to primary Fe-S cluster defects, reducing enzyme activity associated with these clusters, resulting in mitochondrial iron accumulation, dysfunction, and cellular damage.644 An AAV vector containing the Frataxin gene facilitates the expression‎ of human Frataxin in Friedreich’s ataxia mouse models, restoring Fe-S cluster-associated protein levels and enzyme activity in cardiomyocytes.644,645 This restoration normalizes Fe-S biosynthesis, corrects iron accumulation, improves mitochondrial ultrastructure and abnormal cardiac myofibrils, and thus prevents and reverses the cardiomyopathy phenotype.644,645 However, therapeutic outcomes are highly dependent on the cardiac biodistribution of the vector.646 Furthermore, the therapeutic window for AAV-mediated Frataxin gene therapy is narrow; overexpression‎ of Frataxin may induce oxidative stress and significantly increase labile iron pool levels, leading to hepatotoxicity and cardiotoxicity.647

A single intrathecal administration of AAV9/hSURF1 partially restores complex IV levels and activity, showing potential for treating SURF1-related Leigh syndrome.648 Additionally, intravenous and intracerebroventricular injections of AAV2/9-hNDUFS4 enhance complex I activity, improving weight, motor function, and lifespan in NDUFS4 knockout Leigh syndrome mouse models.649 However, neither intravenous nor intracerebroventricular administration alone fully improved the clinical phenotype, indicating limitations in standard AAV vector transduction.649 In contrast, intravenous injection of the brain-penetrating AAV.PHP.B-NDUFS4 vector restored mitochondrial complex I activity and function, improved behavior, corrected brain, retina, and heart pathologies, restored weight, and extended lifespan in mice.650,651 However, the AAV.PHP.B vector’s efficacy is limited to certain mouse strains and is not applicable to primates.652,653 The use of self-complementary AAV9 vectors, effective across mammals and enhancing the transcription rate of recombinant human NDUFS4, restored complex I activity and assembly in Leigh syndrome mice, significantly extending their lifespan.654

Delivering CRISPR/Cas9 and TYMP cDNA via lipid nanoparticles, polymeric nanoparticles, or AAV2/8 viral vector can efficiently integrate TYMP into the TYMP and Alb loci of hepatocytes in the MNGIE mouse model.655 This approach increases TP activity in plasma, reduces nucleoside levels, and shows promise for treating MNGIE.655 AAV-TYMP has been shown to elevate hepatic TP activity in MNGIE mouse models, normalize nucleoside and mitochondrial nucleotide metabolism, enhance mtDNA replication, correct mitochondrial dysfunction, and alleviate functional phenotypes.656 The alpha-1-antitrypsin promoter, demonstrating optimal efficacy,497 could also minimize the dosage required for clinical effectiveness.656

Moreover, the AAV2/8 vector can mediate the expression‎ of the human ETHE1 gene in the liver of ethylmalonic encephalopathy mouse models, effectively clearing circulating hydrogen sulfide, correcting plasma thiosulfate levels, restoring sulfur dioxygenase activity, significantly improving disease phenotypes, and extending lifespan—highlighting its potential for future clinical applications in treating ethylmalonic encephalopathy.657

In Barth syndrome cells, AAV-TAZ transduction increases mtDNA copy number and enhances mitochondrial structure and function.658 Similarly, AAV9-TAZ ameliorates mitochondrial structural defects in a Barth syndrome mouse model, improving cardiac and skeletal muscle function659,660 and rescuing neonatal mice from mortality, cardiac dysfunction, and fibrosis. However, its efficacy and duration depend on the number of cardiomyocytes transduced.660 Notably, administration of AAV9 following low-intensity aerobic exercise can enhance AAV transduction efficiency in the heart and skeletal muscles.661

RNA-based therapy

Delivering double-stranded RNA, single-stranded silencing RNA, or antisense oligonucleotides (ASOs) to Friedreich’s ataxia cells can specifically target or excise the intronic GAA trinucleotide repeat sequence, thereby reducing R-loop formation between the expanded repeat RNA and complementary genomic DNA.662,663,664 This intervention reverses the transcriptional silencing of Frataxin, decreases the production of aberrant early-terminated Frataxin transcripts, and increases both Frataxin mRNA and protein levels, presenting a promising therapeutic strategy for Friedreich’s ataxia.662,663,664 Gapmer oligonucleotides complementary to the adenine-adenine-guanine repeat sequence within the Frataxin gene have shown a higher efficacy in activating Frataxin RNA and protein expression‎.665 Research by Li et al. indicates that co-delivering oligonucleotides targeting the 5’ or 3’ untranslated regions of Frataxin can extend the mRNA half-life, leading to increased steady-state levels of Frataxin mRNA and protein, suggesting a novel approach to upregulating mRNA levels in any transcriptionally downregulated disorder.666 Despite the successful delivery of ASOs in Frataxin mouse models, the anticipated increase in Frataxin expression‎ was not observed, possibly due to the limited potency of the compounds, differences in the regulatory mechanisms of the Frataxin gene,667 or off-target effects.668 Additionally, two phosphorothioate-based ASOs with G-rich motifs were identified668 that indirectly activate Frataxin expression‎ in Friedreich’s ataxia cells but similarly failed to induce Frataxin expression‎ in Friedreich’s ataxia mouse models.668 Thus, determining the efficacy of double-stranded RNA, single-stranded silencing RNA, and ASOs in animal models is essential, necessitating further investigation.

Peptide nucleic acid oligomers

PNAs were the pioneering tool for achieving mitochondrial heteroplasmy shift.669 These molecules can selectively bind to complementary DNA or RNA sequences, effectively inhibiting replication and translation.670 Researchers have synthesized PNAs that are complementary to human mtDNA templates containing deletion breakpoints or single-base mutations, which specifically inhibit the replication of mutant human mtDNA templates in vitro.42 To address the challenge of delivering PNAs to mitochondria within cells, Muratovska et al. conjugated an 11-mer PNA to a lipophilic phosphonium cation.670 This phosphonium-PNA conjugate is non-cytotoxic, remains stable within cells, and can selectively inhibit the in vitro replication of mtDNA carrying the human m.8344 A > G mutation associated with MERRF.670 Despite the promising results PNAs have shown in inhibiting mutant mtDNA replication, their broader application is constrained. A PNA must be at least 7-mer in length to target a unique site within mtDNA.669 Additionally, during mtDNA replication, nucleic acid derivatives may fail to bind to their complementary sequences, and it remains uncertain whether single-stranded mtDNA at the replication fork can effectively interact with PNAs.669,670 Consequently, the widespread use of PNAs remains limited.

Mitochondria-targeted restriction endonucleases

Pathogenic mtDNA variations can introduce unique restriction endonuclease cleavage sites.671 MTS directs the restriction endonucleases into mitochondria, which cleaves pathogenic mtDNA at specific recognition sites, resulting in double-strand breaks (DSBs) that lead to the elimination of defective mtDNA.671,672,673 This process allows the remaining wild-type mtDNA to replicate, thereby altering the degree of heteroplasmy and offering a potential treatment for mitochondrial genetic disorders.671,672,673 Early research in 2002 demonstrated that mitochondria-targeted SmaI restriction endonuclease could selectively eliminate mutated mtDNA, allowing the replication of wild-type mtDNA and restoring normal cellular ATP levels and Δψm.674 Subsequent in vivo and in vitro studies have shown that mitochondria-targeted restriction endonucleases, such as PstI,675 ApaLI,676 Scal,677 and R.XmaI,678 can effectively eliminate mutant mtDNA at corresponding sites, thereby facilitating a shift in heteroplasmy. These interventions resulted in notable improvements in ATP synthase function and alleviated mitochondrial dysfunction, demonstrating the potential of mitochondria-targeted restriction endonucleases (mtREs) to prevent disease onset or reverse clinical symptoms in patients with specific pathogenic heteroplasmic mtDNA mutations. Additionally, they may inhibit the transgenerational transmission of human mitochondrial diseases.673,676,678,679,680 Compared to other gene-editing technologies, mtREs offer a significantly higher specificity, minimizing off-target activity and preventing mtDNA copy number depletion.681 However, two major challenges may limit the application of this method. First, the cellular delivery of mtREs could raise safety concerns.680 Second, the utility of mtREs is restricted to targeting specific heterogeneous mtDNA mutations.680 Many mtDNA mutations do not create new restriction enzyme sites, rendering mtREs ineffective against these mutations,42 which hinders their clinical application.669 To extend this approach to other pathogenic mtDNA mutations, the development of nucleases with novel specificities is essential.679

Mitochondria-targeted zinc-finger nucleases

Zinc finger nucleases (ZFNs) consist of zinc finger proteins (ZFPs) linked to FokI nucleases, with ZFPs further connected to MTS and nuclear export signals (NES). These modifications guide mitochondria-targeted zinc-finger nucleases (mtZFNs) to the mitochondria, where ZFPs can selectively bind to specific DNA target sequences. Upon binding, FokI nucleases dimerize and cleave the DNA adjacent to the ZFP binding sites.669,682 While traditional ZFNs can target mtDNA, identifying suitable ZFN pairs for certain mutations poses a challenge. To address this, researchers developed heterodimeric ZFNs that bind both mutant and adjacent wild-type mtDNA sequences. However, this approach often results in the degradation of wild-type mtDNA and rapid mtDNA depletion. To overcome this, researchers created single-chain ZFNs by conjugating two FokI nuclease catalytic domains to ZFPs. These single-chain ZFNs demonstrated greater selectivity for pathogenic point mutations in mtDNA682 and proved more effective than their single-domain counterparts.682 Despite this improvement, single-chain ZFNs are ineffective against large mtDNA deletions and present potential safety concerns.683 To address these limitations, researchers redesigned conventional dimeric mtZFNs, ensuring that monomers did not affect mtDNA.683 The improved mtZFNs effectively eliminated point mutations and large-scale mtDNA deletions, reducing the mutant mtDNA haplotype load below the pathogenic threshold, thereby restoring OXPHOS function and improving mitochondrial respiration.683 Both in vitro and in vivo experiments with mtZFN-AAV targeting the m.5024 C > T tRNAAla mutation demonstrated a partial shift in heteroplasmy, leading to improved steady-state levels of mt-tRNAAla, as well as enhanced mitochondrial respiration and metabolic function.672 Additionally, Gammage et al. achieved near-complete correction of mtDNA mutations and rescued mitochondrial respiratory function and metabolic defects through either consecutive short-term mtZFN treatments or finely controlled, optimized mtZFN expression‎.681 This approach minimized off-target effects and unwanted depletion of mtDNA copy numbers, proving more efficient than mtREs and mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs).681 Despite these advancements, ZFN expression‎ is associated with cytotoxicity due to off-target site cleavage. This issue might be mitigated by equipping mtZFNs with tightly regulated expression‎ systems or by optimizing NES and ZFP sequences to reduce cytotoxicity.682,684

Mitochondria-targeted transcription activator-like effector nucleases

MitoTALENs are composed of targeted transcription activator-like effectors (TALEs) that bind to specific DNA sequences, coupled with FokI nucleases that dimerize to cleave mtDNA. Numerous in vitro and in vivo studies have demonstrated that mitoTALENs can reduce the load of pathogenic mtDNA and rescue associated functional phenotypes.685,686 Additionally, mitoTALENs have shown effectiveness in reducing the human m.3243 A > G mtDNA mutation in porcine oocytes,685 as well as the NZB mtDNA in MII oocytes of the NZB/BALB heterozygous mouse model.680 Targeting human m.14459 G > A and m.9176 T > C mutant mtDNA using mitoTALENs has led to the specific elimination of these mutant mitochondrial genomes.680 These results highlight the potential of mitoTALENs for selectively eliminating mutant mtDNAs and preventing their germline transmission. Furthermore, mitoTALEN nickases, which are derivatives of mitoTALEN with an inactive FokI domain on one monomer, can induce single-strand breaks at specific sites in human mtDNA. This process leads to mtDNA deletions687,688 and facilitates the creation of new animal models for studying single large-scale mtDNA deletion diseases.689

Although mitoTALENs may not be as potent as mtREs in preventing the spread of germline mitochondrial diseases,680 they offer greater design flexibility compared to mtREs and mtZFNs.690 However, mitoTALENs come with several limitations. Unlike more precise gene-editing technologies, ZFN and TALEN are incapable of performing precise single-base editing, making them unsuitable for correcting homogenous mtDNA mutations.691 Additionally, most mitoTALENs require a thymidine base at position 0 of the target DNA binding site,692 and the mutation’s sequence context, along with methylation or other epigenetic modifications of the mtDNA target sequence, can impact the TALE’s efficiency.693,694 The large size of mitoTALENs also complicates their encapsulation in many vector systems.695,696 Some researchers have attempted to overcome this by fusing a monomeric nuclease domain derived from the I-TevI homologous endonuclease to the TALE DNA-binding domain697 or by designing shorter, more specific mitoTALENs.698 However, these approaches often limit DNA sequence recognition.698 Another significant challenge is the potential for non-specific cleavage by mitoTALENs, which can lead to substantial depletion of mtDNA copies and induce cytotoxicity,314,692 necessitating precise dose control of the constructs.681 Moreover, while mitoTALENs exhibit minimal cleavage activity against wild-type mtDNA,699 further development is needed to create more effective, safer, and easier-to-deliver mitoTALENs in the future.

CRISPR/Cas9

The design of single-guide RNA (sgRNA) for targeting mtDNA mutations involves adding MTS upstream and downstream of the Cas9 gene and the 3’ untranslated region of the target gene.700,701 This configuration enables mitochondria-targeted Cas9 (mito-Cas9) to cleave mtDNA at specific sites dictated by the sgRNA.700,701 It has been shown that the mito-Cas9 system can be successfully translocated into mitochondria, where it can introduce exogenous single-stranded DNA oligonucleotides into mtDNA, thereby facilitating the creation of cellular models of disease-causing mtDNA mutations.701 However, the efficiency of mtDNA editing using mito-Cas9 systems can be limited by several factors, including the sequence characteristics of the target region and the variable targeting efficiencies of different sgRNAs.701 Optimizing the mito-Cas9 system might involve enhancing mitochondrial RNA transport702 or employing engineered Cas proteins with higher editing efficiency.703 Nevertheless, the lack of an RNA transporter system within the double-membrane structure of mammalian mitochondria31,704 results in inefficient or defective nucleic acid import into mitochondria.691,700 Additionally, the absence of homologous recombination and non-homologous end-joining pathways for repairing DSBs in mitochondria further complicates the manipulation of mtDNA.689,705 In summary, any attempt to import synthetic RNA molecules into mitochondria based on naturally occurring mechanisms in human cells is likely to be sporadic and inefficient.681 Consequently, the application of CRISPR/Cas9 technology to mtDNA manipulation remains highly challenging and will require further extensive research to overcome these obstacles.

Mitochondria-targeted meganucleases

Mitochondria-targeted meganucleases (mitoARCUS), derived from the naturally occurring I-CreI nucleic acid endonucleases, are small, highly specific single-component proteins.706 These nucleases possess the ability to recognize DNA sequences with single-base pair differences, generating DSBs. They can be efficiently packaged into individual viral vectors, requiring only minimal AAV titers, and do not show signs of vector-associated or transient mtDNA depletion toxicity.707,708 In heterozygous mice with the m.5024 C > T mutation, AAV9-mitoARCUS significantly altered heteroplasmy and restored mt-tRNAAla levels.708,709 Additionally, AAV9-mitoARCUS modulated heterogeneity and enhanced mitochondrial-encoded protein homeostasis and respiratory function in m.3243 A > G cell lines and mouse models.707 However, the need to redesign target-specific mitoARCUS for each mutation and the complexity involved in reengineering I-CreI to recognize new targets remain substantial challenges that must be addressed.705,707,710

DddA-derived cytosine base editors

DddA-derived cytosine base editors (DdCBEs) are engineered by fusing MTS, split-DddAtox halves, TALE, and uracil glycosylase inhibitors.710 The DddAtox enzyme catalyzes the conversion of cytosine to uracil,710 while the uracil glycosylase inhibitors prevent uracil-DNA glycosylase from removing uracil, leading to C-to-T mutations during subsequent DNA replication without introducing DSBs. This mechanism ensures high specificity and product purity in targeted edits 715. DdCBEs have facilitated mitochondrial base editing in human embryos711,712 and enabled precise, heritable C-to-T base editing at specific mtDNA sites in zebrafish, mice, and rats. These models replicate phenotypes akin to human mitochondrial diseases, thereby advancing the precise modeling of these conditions.710,713,714,715,716 Additionally, Mok et al. developed monomeric DdCBEs derived from non-toxic, full-length DddAtox variants, achieving C-to-T editing with reduced off-target effects.717 Zinc finger deaminases, which combine zinc finger DNA-binding proteins, DddAtox, and uracil glycosylase inhibitors, also catalyze targeted C-to-T transitions in mtDNA.718 Optimizing the architecture of zinc finger DdCBEs has improved editing efficiency, reduced off-target effects, facilitated packaging in AAV vectors, and potentially decreased immunogenicity.719 These advances enable the correction of pathogenic mtDNA mutations and the modeling of mitochondrial diseases.

Ongoing research focuses on enhancing the base editing efficiency of DdCBEs and expanding their applications. For example, co-injection of mitoTALEN has been shown to enhance DdCBE-NES-mediated mtDNA editing.720 Variants of DddA with increased base editing efficiency and a broader target range have been developed using phage-assisted continuous evolution and phage-assisted discontinuous evolution.720,721 The DddA11 variant, in particular, has expanded HC (H = A, C, or T) sequence compatibility, although it remains less effective for GC targets.722 CRISPR-mediated nuclear and TALE-based mitochondrial DdCBEs, utilizing dsDNA deaminase derived from Roseburia intestinalis interbacterial toxin (riDddAtox), have successfully achieved C-to-T editing of HC and GC targets.723 Fusion of transactivators to DddAtox or riDddAtox has significantly increased editing efficiency in both nDNA and mtDNA.723 Moreover, combining the DddA11 variant with activation-inducible cytidine deaminase has further improved C-to-T editing efficiencies across various targets.724 In another approach, TALE-linked deaminases designed by Cho et al. catalyze the hydrolytic deamination of adenine to produce inosine, which pairs with cytosine during replication, enabling targeted A-to-G editing in mitochondria with high efficiency.725 The A-to-G editing efficiency was further enhanced by fusing the DddA6 variant with TALE-linked deoxyadenosine deaminase.724

However, DdCBEs may induce off-target activities in mtDNA,712,726 likely due to non-specific interactions between TALE and DNA or spontaneous assembly of split DddAtox deaminases.727 To mitigate these issues, Lee et al. developed high-fidelity DdCBEs that are both efficient and precise, avoiding off-target mutations.727 Adding NES sequences also helps reduce off-target editing while improving on-target efficiency.724 DdCBEs offer precise mtDNA base editing both in vitro and in vivo, with the ability for germline transmission.728 This method is particularly valuable for creating mitochondrial disease-associated cell lines and animal models, deepening our understanding of mitochondrial disorders and providing potential avenues for correcting both homoplasmic and heteroplasmic pathogenic variants.

Post-transcriptional modifications

Post-transcriptional modifications of mitochondrial RNA play a critical role in finely regulating the synthesis and stability of the 13 mitochondrial proteins.280 These modifications stabilize tRNA and introduce wobble modifications, which can ameliorate mitochondrial translation defects and present potential therapeutic avenues for mitochondrial genetic diseases.729 In vitro studies have demonstrated that the m.3290 T > C mutation can restore the hypomodified 5-taurinomethyluridine in mt-tRNALeu(UUR) with the m.3243 A > G mutation, thereby improving mitochondrial translation in MELAS, facilitating respiratory chain complex formation, and enhancing oxygen consumption rates.730 Additionally, acquiring wobble modifications in mt-tRNALeu(CUN) with the m.12300 G > A mutation can also alleviate respiratory defects associated with the m.3243 A > G mutation.731 Introducing wobble modifications in other isoacceptor tRNAs may also yield similar benefits.731

In addition, the defects in mt-tRNA modification and subsequent mitochondrial protein translation can be restored by regulating the expression‎ of mt-tRNA-modifying enzymes. Mitochondrial translation optimization 1 (MTO1) and GTP-binding protein 3 (GTPBP3) are responsible for catalyzing the biosynthesis of 5-taurinomethyluridine.732 High-dose oral taurine has been shown to increase MTO1 expression‎,733 effectively preventing stroke-like episodes in MELAS by correcting the first anticodon nucleotide modification defect in mt-tRNALeu(UUR).734 Moreover, MTO1 overexpression‎ restores 5-taurinomethyluridine in mutant mt-tRNALeu(UUR) in MELAS and the 2-thiouridine derivative in mutant mt-tRNALys in MERRF, enabling efficient decoding of homologous codons independently of taurine supplementation.729 MTO1 may also function as an RNA chaperone, stabilizing pathogenic mt-tRNA mutations, enhancing tRNA aminoacylation efficiency, and supporting mitochondrial protein synthesis.280,729 Furthermore, ketogenic diet can improve OXPHOS defects independently of MTO1-mediated tRNA modifications, suggesting an alternative therapeutic approach.735 Overexpression‎ of TRMT61B can restore the N1-methyladenosine modification at position 58 in mt-tRNALys with the m.8344 A > G mutation in MERRF, thereby finely regulating mitochondrial protein synthesis and stability.280 Cysteine is essential for the 2-thiomodification of mt-tRNA,736 and L-cysteine has been shown to partially rescue mitochondrial translation defects in cells with m.3243 A > G and m.8344 A > G mutations.737 Additionally, N-acetyl-cysteine has demonstrated benefits for mitochondrial translation in cells deficient in tRNA 5-methylaminomethyl-2-thiouridylate methyltransferase (TRMU) and MTO1,736 indicating that restoring specific tRNA modifications could mitigate mitochondrial disease symptoms.

Further research by Meseguer et al. has revealed that retrograde signals from mitochondria to the nucleus, such as ROS or Ca2+, can increase microRNA expression‎ in cells with various mtDNA mutations. These microRNAs, acting as post-transcriptional negative regulators, also influence mt-tRNA modifications by modulating the expression‎ of mt-tRNA-modifying enzymes, thereby exacerbating disease phenotypes. Notably, microRNA-335/335* regulates GTPBP3 and MTO1 expression‎.738 High ROS levels induce microRNA-9/9* expression‎ via the NF-κB pathway, directly targeting and reducing TRMU, GTPBP3, and MTO1 mRNA and protein levels, impairing non-mutant mt-tRNA modifications and worsening the MELAS phenotype.264 Consequently, microRNA antagonists may offer potential strategies to counteract these deleterious effects.264,738

Additional approaches to improve mitochondrial protein synthesis function include the use of the mTOR inhibitor rapamycin, which can enhance erythroid differentiation by inhibiting mTOR signaling and protein synthesis, effectively alleviating anemia symptoms in MLASA.502,739 Modifying the tRNA-binding domain of nuclear-encoded human mitochondrial phenylalanyl-tRNA synthetase can increase the aminoacylation efficiency of mt-tRNAPhe with the G34A mutation in MERRF.740 Overexpressing human mitochondrial leucyl-tRNA synthetase can stabilize tRNALeu(UUR) and mitochondrial translation products, thereby rescuing respiratory chain defects in cells with the m.3243 A > G mutation.741 Furthermore, overexpressing mitochondrial translation elongation factors EFTu and EFG2 can improve quality control during translational elongation, partially suppressing amino acid misincorporation in complex III, complex II, and ATP6 caused by the m.3243 A > G mutation, thus ameliorating respiratory chain assembly defects.742

Drug therapy

Advancements in pharmaceutical technology have led to the development of numerous drugs that are now being applied to treat mitochondrial disorders, with many having entered the clinical trial stage. These include CoQ10, idebenone, EPI-743, N-acetylcysteine, elamipretide, RT001, KH176, omaveloxolone, bezafibrate, pioglitazone, deferiprone, sodium dichloroacetate, L-arginine, interferon-gamma (IFN-γ), recombinant human erythropoietin, deoxynucleotide monophosphate and deoxynucleoside, among others. Each of these drugs operates through distinct mechanisms, targeting various aspects of mitochondrial dysfunction to potentially alleviate the symptoms or slow the progression of these disorders.

Antioxidants

CoQ10 plays a critical role in the mitochondrial respiratory chain,743 enhancing electron transport and ATP production, regulating redox signaling, stabilizing the mPTP, and preventing autophagy and apoptosis.744 Consequently, CoQ10 supplementation can reverse pathophysiological alterations and significantly improve clinical outcomes in conditions such as Leigh syndrome and MELAS.263,745,746 Furthermore, combining Idebenone with CoQ10 therapy may enhance therapeutic efficacy.746 CoQ10 offers tangible benefits for patients with mitochondrial diseases.

Idebenone, a CoQ10 analog, is the first drug approved in Europe for the treatment of LHON.45 It is reduced by cytosolic NAD(P)H oxidoreductase I (NQO1)747 and bypasses LHON-associated complex I dysfunction, restoring mitochondrial function by shuttling electrons directly from the cytoplasm to complex III. This mechanism maintains cellular energy production, restores ATP levels, and reduces ROS production.747,748 Both in vitro and in vivo studies have demonstrated Idebenone’s protective effects on RGCs and retinal integrity, preserving visual function.749,750 Idebenone has been shown to restore or maintain visual function in LHON, prevent color vision loss, and improve extraocular nerve dysfunction.751,752 Its long-term efficacy for LHON has been documented,753 although the therapeutic effect varies depending on the disease stage and specific pathogenic mtDNA mutation.754 Innovative delivery methods, such as PCL intravitreal implants loaded with Idebenone755 and biodegradable poly microspheres,756 offer controlled and prolonged intraocular administration, providing a new strategy for sustained LHON treatment. Idebenone is also used in the treatment of MELAS,757 Leigh syndrome,758 and Friedreich’s ataxia.759 However, Idebenone’s effect on mitochondrial respiratory chain function is dose-dependent; it can inhibit complex I activity while increasing complex II activity,760 potentially transforming from an antioxidant to a prooxidant and inducing mitochondrial dysfunction depending on its concentration and NQO1 expression‎ levels.761,762 Due to its narrow therapeutic range, high doses of Idebenone can be cytotoxic, particularly in the ganglion cell layer.761 Genetic variations in NQO1 protein levels significantly influence Idebenone’s efficacy and toxicity, especially in NQO1-deficient cell lines,747,761 necessitating consideration of the patient’s NQO1 genotype and mtDNA mutation before treatment.747 Additionally, Idebenone is ineffective in correcting mitochondrial energy metabolism defects in conditions of CoQ deficiency.763

EPI-743, a novel p-benzoquinone therapeutic agent, enhances endogenous glutathione biosynthesis and improves oxidative status by modulating oxidoreductase enzyme activity, leading to clinical improvements in some hereditary mitochondrial disorders.764,765 EPI-743 has been shown to delay disease progression in Leigh syndrome765,766 and positively impact the recovery of visual function in LHON.767 Some evidence suggests that EPI-743 may be more potent than Idebenone.768

N-acetylcysteine, a precursor to glutathione, plays a critical role in restoring glutathione balance, improving mitochondrial complex IV dysfunction, reducing cellular oxidative damage, and ameliorating neuromuscular dysfunctions in Leigh syndrome models.769 When combined with cysteamine bitartrate, these benefits are further enhanced. Additionally, a combination therapy involving glucose, niacin, and N-acetylcysteine has been shown to synergistically improve respiratory chain complex I dysfunction, reduce mitochondrial stress, and boost metabolic and glutathione levels, thereby increasing resilience and preventing acute neurological and biochemical decompensation.770

Elamipretide, a mitochondria-targeted aromatic-cationic tetrapeptide, efficiently penetrates the OMM and rapidly localizes to the inner membrane, where it binds to cardiolipin or monolysocardiolipin.771 By improving the function of specific proteins involved in mitochondrial dynamics and mitophagy, Elamipretide can restore mitochondrial morphology and function,772 thereby enhancing skeletal muscle and cardiovascular performance in Barth syndrome and alleviating related clinical symptoms and disease progression.773,774 Additionally, Elamipretide may improve visual function in LHON775 and increase exercise capacity in patients with primary mitochondrial myopathy.776

RT001, a deuterated ethyl linoleate, is an orally bioavailable synthetic deuterated polyunsaturated fatty acid designed to inhibit lipid autoxidation and protect cells from oxidative stress.777 It has shown potential therapeutic effects in Friedreich’s ataxia, where it has been demonstrated to improve peak work capacity and oxygen consumption.778 However, clinical trials have yielded mixed results, with one trial indicating that RT001 may not be beneficial for treating Friedreich’s ataxia.779

KH176 exhibits dual antioxidant and redox-modulating properties.780 As a ROS-redox modulator, KH176 preserves the microstructure of the NDUFS4 mouse brain, reduces lipid peroxidation, and mitigates RGC degeneration, leading to improved rotational and gait performance in NDUFS4 mice.781 By targeting the thioredoxin/peroxiredoxin system, KH176 effectively lowers ROS levels and offers protection to cells with OXPHOS deficiencies.780 It also ameliorates neuronal network dysfunction and transcriptomic changes linked to m.3243 A > G heteroplasmy in neurons derived from human iPSCs.782 However, clinical studies by Janssen et al. suggest that KH176 may not significantly enhance clinical outcomes in patients with mitochondrial m.3243 A > G spectrum disorders.783

Metabolic modifiers

Omaveloxolone, an Nrf2 activator and NF-kB inhibitor, targets inflammatory and metabolic pathways.784,785 It enhances substrate availability and complex I activity, reduces endogenous lipid peroxidation and mitochondrial ROS levels, and elevates glutathione levels, thereby protecting cells from oxidative stress, maintaining Δψm, promoting mitochondrial respiration, and preventing cell death.786 Omaveloxolone has shown significant improvements in neurological function in Friedreich’s ataxia and can markedly slow disease progression with a favorable safety and tolerability profile.787 It is the first drug approved in the United States and Europe for treating Friedreich’s ataxia in patients aged 16 and older.48 However, given the multi-system nature of Friedreich’s ataxia, a cure may ultimately require combination therapy. Omaveloxolone may also enhance mitochondrial function and submaximal exercise tolerance, reducing heart rate and lactate production during exercise, which could benefit those with mitochondrial myopathy.788 Other compounds, such as (+)-Epicatechin,789 A0001,790 and Nomlacofusp,791 have also shown potential in treating Friedreich’s ataxia, though further research is necessary.

Bezafibrate, a PPAR activator, upregulates downstream PPAR target genes, increases mitochondrial biogenesis, and prevents cardiac dysfunction in mouse models of Barth syndrome792,793 and exercise intolerance.793 It also improves disturbances in the antioxidant system, mitochondrial quality control proteins, and mitochondrial function, offering potential treatment for Barth syndrome and dilated cardiomyopathy with ataxia syndrome.794 Additionally, bezafibrate enhances metabolic programming in Leigh syndrome neural progenitor cells by promoting SURF1 gene expression‎ and inducing PGC-1α, thereby restoring neuronal morphogenesis.795 It increases survival rates and mitigates disease progression in Leigh syndrome mouse models.796 In carriers of the m.3243 A > G mutation, bezafibrate induces mitochondrial biogenesis, improves cardiac function, and alters metabolomic profiles while increasing mitochondrial disease biomarkers in serum.797

Pioglitazone, a PPAR-γ agonist, increases levels of the human insulin-degrading enzyme and PITRM1 protein through PPAR-γ activation.798 This mechanism restores mitochondrial targeting sequence pre-processing and alleviates feedback inhibition of mitochondrial processing peptidase activity in fibroblasts from PITRM1-deficient patients, improving Frataxin maturation and mitochondrial function.798 In combination with deoxynucleosides, pioglitazone may increase mtDNA copy number and mitochondrial mass, reduce mtDNA-encoded transcripts, and improve mitochondrial respiration in MELAS cells with the m.3243 A > G mutation.799 Similarly, rosiglitazone, another PPAR-γ agonist, enhances Frataxin levels in Frataxin-deficient dorsal root ganglia neurons, boosting mitochondrial biogenesis, function, calcium homeostasis, and cell survival.800 Rosiglitazone also improves energy metabolism by increasing fatty acid β-oxidation in Frataxin-deficient cardiomyocytes and enhances motor function in Friedreich’s ataxia mouse models,800 indicating its potential as a treatment for this condition both in vitro and in vivo.800

Sodium dichloroacetate inhibits accelerated lactate production in mtDNA deletion mice, ameliorates chronic lactic acidosis, improves mitochondrial biogenesis, restores respiratory function, and extends lifespan.801 It may offer certain benefits for patients with mitochondrial diseases.802 Oral sodium dichloroacetate stimulates cellular energy metabolism by activating residual enzyme activity, effectively reducing blood and cerebrospinal fluid lactate levels in children with congenital lactic acidosis due to pyruvate dehydrogenase complex mutations.803

L-arginine, a precursor for nitric oxide synthesis, enhances nitric oxide formation in patients with impaired endothelial function.804 It has been shown to improve aerobic capacity and muscle metabolism in MELAS,805 and reverse endothelial dysfunction.806 L-arginine can also reduce the frequency and severity of stroke-like episodes and slow the progression of MELAS.807 Long-term L-arginine supplementation appears promising for MELAS therapy,808 though the existing evidence is of poor methodological quality, and both intravenous and oral L-arginine have shown limited clinical benefits in the acute or preventive treatment of MELAS.809 L-arginine may primarily serve to prevent the onset of stroke-like episodes.810 Citrulline, which increases de novo arginine synthesis and enhances nitric oxide production, could potentially offer better therapeutic effects than arginine.811 More rigorous trials are needed to fully eval‎uate the efficacy and safety of L-arginine and citrulline therapies.

Chelator

Deferiprone is known to redistribute iron and stimulate the expression‎ of Frataxin, a mitochondrial iron chaperone.459,812 This action facilitates the chelation of mitochondrial labile iron, which plays a role in oxidative stress, and leads to the reactivation of iron-deficient aconitase, reduction of iron accumulation, and mitigation of iron-induced ROS synthesis and mitochondrial stress in Friedreich’s ataxia cardiomyocytes.459,812 As a result, deferiprone helps restore respiratory chain protein levels, salvages mitochondrial function, inhibits TSFR gene expression‎, and improves calcium handling dynamics, thereby enhancing cardiac function.459,812 However, the use of high doses of deferiprone can negatively impact cellular Fe-S enzyme activity and reduce Frataxin levels.459,813 Additionally, Lim et al. identified a novel lipophilic iron chelator, 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone, which rapidly penetrates cells to induce iron efflux and protect Friedreich’s ataxia fibroblasts from hydrogen peroxide-induced cytotoxicity, showing potential as a treatment for iron overload.814

Others

IFN-γ has emerged as a potential treatment for Friedreich’s ataxia. Both in vitro and in vivo studies have demonstrated that IFN-γ can upregulate Frataxin levels by modulating Frataxin gene transcription, which helps improve sensory and motor deficits in Friedreich’s ataxia mouse models.815 IFN-γ enhances the expression‎ of Nrf2 and manganese-dependent superoxide dismutase in Friedreich’s ataxia cells, activating the non-canonical Nrf2 pathway via p21. This activation reduces the cells’ sensitivity to hydrogen peroxide-induced cell death, thereby offering protective effects.816 IFN-γ treatment may also reduce cardiomyocyte damage and improve cardiac function in Friedreich’s ataxia cardiomyopathy.817

A single high dose of erythropoietin has been shown to sustainably elevate Frataxin levels in Friedreich’s ataxia, reduce oxidative stress markers, and improve clinical symptoms.818 However, this treatment can lead to an increase in hematocrit levels.818 In vitro studies have demonstrated that carbamylated erythropoietin, an erythropoietin derivative, can increase Frataxin levels independently of erythropoietin receptor activity and without inducing erythropoiesis.819 Despite these promising results, a Phase II clinical trial indicated that carbamylated erythropoietin might not have significant therapeutic effects on Friedreich’s ataxia820

In TK2 deficiency, deoxythymidine triphosphate levels are significantly diminished, resulting in an imbalance in the mitochondrial deoxyribonucleoside triphosphate pool.821 Administering oral TK2 products such as deoxycytidine and deoxythymidine monophosphates, or rapidly degradable deoxypyrimidine monophosphate products like deoxythymidine and deoxycytidine, can effectively increase deoxythymidine triphosphate concentrations in TK2-deficient mouse models.822,823,824 This treatment has been shown to restore mtDNA copy numbers and improve the activity and levels of mitochondrial respiratory chain enzymes, thereby delaying disease onset, alleviating symptoms, and extending lifespan in these models.822,823,824 Clinical studies have further demonstrated that deoxynucleotide monophosphate and deoxynucleoside therapies significantly enhance survival, swallowing, respiratory, and motor functions in patients with TK2-deficient myopathy, showcasing both efficacy and a favorable safety profile.825,826 However, because deoxythymidine and deoxycytidine therapy alone cannot completely halt or reverse the progression of TK2 deficiency, a combined therapeutic approach using AAV-TK2 gene therapy alongside deoxynucleosides may provide a more comprehensive and effective treatment for TK2 deficiency.827

Cell therapy

Unlike skeletal muscles, satellite cells—dormant myoblasts that can be activated to re-enter the cell cycle and fuse with existing muscle fibers—rarely or never harbor mutant mtDNA.828,829 This unique characteristic enables muscle fiber regeneration through satellite cells, which can be stimulated by resistance training or injury-induced muscle regeneration,828,829,830 promoting hypertrophy and the incorporation of satellite cells into muscle fibers.828,829,830 This process facilitates the transfer of wild-type mtDNA from satellite cells to mature muscle fibers, altering the heteroplasmy and ultimately enhancing muscle strength and oxidative capacity.828,829,830 Although satellite cells and myoblasts are primarily suited for local muscle administration,831 mesoangioblasts—stem cells capable of fusing with damaged muscles—can either directly repair muscle tissue or augment the satellite cell pool to promote muscle regeneration.832,833 Mesoangioblasts can adhere to and traverse vascular endothelial cells, allowing for systemic arterial administration.834 In vitro studies have shown that the fusion of wild-type mesoangioblasts with myotubes carrying the m.3271 T > C or m.3291 T > C mutations can reduce the mtDNA mutation load and improve mitochondrial function.831 Moreover, mesoangioblasts from patients with mtDNA mutations or large-scale deletions show negligible corresponding mtDNA mutations or deletions and demonstrate mitochondrial function, proliferation, and myogenic differentiation abilities comparable to those of wild-type mesoangioblasts.835 Thus, mesoangioblasts present a viable option for autologous myogenic cell therapy, enabling the regeneration of new muscle fibers without mtDNA mutations or deletions following muscle injury.835 Additionally, eccentric exercise can induce skeletal muscle inflammation, prompting monocyte migration, which may enhance the efficacy of such treatments.835,836

In MNGIE, the infusion of healthy donor platelets or the use of hemodialysis and peritoneal dialysis can transiently restore circulating TP and temporarily reduce plasma levels of thymidine and deoxyuridine.837,838 However, these effects are short-lived and do not address neurological functions.837 HSCT offers a more definitive solution by permanently restoring TP function and thereby curing MNGIE.839 Both in vitro and in vivo studies have shown that HSCT can restore TP activity, correct the imbalance in the mitochondrial deoxyribonucleoside triphosphate pool in the liver, and safely halt disease progression.840,841 HSCT has been shown to correct the biochemical abnormalities and clinical manifestations of MNGIE,489,842 with a standardized protocol now in place.843 Recent research has also explored haploidentical transplantation as a potential treatment option.844 However, the timing of treatment, the preconditioning regimen, and donor selection are critical factors in determining therapeutic outcomes.839 HSCT is recommended for patients with the best donor match, younger age, and milder symptoms.489 Due to the increased risk of morbidity and mortality associated with advanced disease progression and complications, HSCT is not advised for patients with advanced MNGIE.489,845

Enzyme replacement therapy

The high mortality risk associated with HSCT and the scarcity of matched donors limit the accessibility of this treatment for many patients.844,845 An alternative approach involves lentiviral transduction of hematopoietic stem and erythroid cell lines to produce reticulocytes containing active TP.846 Erythrocyte-encapsulated thymidine phosphorylase (EE-TP) can catalyze the metabolism of thymidine and deoxyuridine, which freely diffuse across the red blood cell membrane, converting them into normal products.847 This process reduces plasma nucleosides, improves mitochondrial dysfunction, alleviates clinical symptoms, and has shown good safety and tolerability, making EE-TP a potential enzyme replacement therapy for MNGIE.848 However, the therapeutic effects are temporary, as metabolite levels return to abnormal values once treatment is discontinued.848 Regular intravenous injections of EE-TP can help manage intracellular nucleotide imbalance in patients with MNGIE,847 positioning EE-TP as a viable treatment option for those without suitable HSCT donors or for patients with irreversible end-stage disease.848 Nonetheless, preclinical toxicity assessments have indicated that EE-TP might pose severe toxicity risks in MNGIE, necessitating careful management of immune reactions.847 To address these concerns, Vocht et al. developed active TP-encapsulating nanoreactors, which, due to their stability and lack of cytotoxicity and inflammatory response, could offer a more effective and safer enzyme replacement therapy option.849

Organ transplantation therapy

Liver transplantation has emerged as a novel therapeutic option for mitochondrial genetic diseases involving the liver. The liver is a major source of TP,850 and in MNGIE, liver transplantation has been shown to restore TP activity, rapidly normalize nucleoside levels, and maintain them at stable low levels, leading to the improvement and stabilization of various clinical symptoms.851,852 While liver transplantation may not achieve complete clinical recovery, it offers a potentially safer alternative to allogeneic HSCT, especially in patients with underlying liver disease.851 Additionally, liver transplantation has been explored as a treatment for ethylmalonic encephalopathy.853 However, in an 18-month-old patient with ethylmalonic encephalopathy, liver transplantation only partially improved symptoms and did not result in a complete cure,854 suggesting that the procedure is most effective when performed before irreversible neurological damage occurs. In Wolcott-Rallison syndrome, which is caused by EIF2AK3 mutations, single or combined transplantation of the liver, pancreas, and kidneys has been associated with improved overall health, although further clinical follow-up is necessary to confirm‎ these outcomes.855,856 The decision to perform liver transplantation in patients with mtDNA depletion syndromes and deoxyguanosine kinase deficiency remains controversial, as post-transplant outcomes for these conditions are generally poor.857

Exercise therapy

Aerobic training

Exercise intolerance is a preval‎ent symptom in patients with mitochondrial disease, directly correlating with the severity of impaired muscle OXPHOS.858,859 Reduced physical activity significantly contributes to decreased exercise capacity in individuals with mitochondrial myopathies.860 Several studies have shown that moderate-intensity aerobic training can enhance OXPHOS in skeletal muscle, promote mitochondrial proliferation, and increase the levels of respiratory chain enzymes.861 However, the impact of exercise on muscle mutational load remains unclear, with some studies suggesting that mutational load may increase with training,861 while others report no significant change in muscle mtDNA amounts or mutational load levels.859,860 Consequently, long-term studies are necessary to eval‎uate the safety and efficacy of exercise as a treatment for patients with mitochondrial myopathy. Exercise limitations in mitochondrial myopathy may also be compounded by chronic conditions such as cytochrome oxidase deficiency, in addition to primary mitochondrial dysfunction.862 Combining aerobic training with oral sodium dichloroacetate therapy could potentially improve aerobic capacity and motor function.862 Moreover, low-intensity aerobic exercise has been shown to facilitate targeted transgene delivery to specific organs, potentially enhancing the safety of gene therapy in human patients.661 Further research is needed to optimize exercise training regimens to maximize their benefits for patients with mitochondrial disease, ensuring both safety and therapeutic efficacy.

Resistance training

Endurance exercise promotes mitochondrial turnover, biogenesis, and angiogenesis by activating PGC-1α expression‎ in skeletal muscle, thereby enhancing mitochondrial function.863,864 It also improves mitochondrial morphology and boosts antioxidant capacity.864 Training interventions that combine aerobic and resistance exercise have been shown to enhance mTOR-activated signaling pathways, PGC-1α signaling related to muscle mitochondrial biogenesis and anabolism, as well as OXPHOS complex activity and redox balance in muscle tissues.865 These combined improvements lead to increased aerobic fitness and muscle strength, as demonstrated in mice.865

Induced pluripotent stem cells and organoid

Recent advancements in generating patient-specific human iPSCs and their derived cells and organoids have established robust models for investigating the pathophysiological mechanisms of various mitochondrial genetic diseases, including MELAS,866 MERRF,283 Leigh syndrome,795 Friedreich’s ataxia,867 and LHON868 These iPSCs, along with their derived cells and organoids, address the challenges of sample acquisition while preserving patient-specific genetic backgrounds and accurately replicating disease characteristics.869 Notably, these technologies have become powerful tools for screening potential therapeutic drugs and developing novel treatment strategies. For example, Guo et al. reprogrammed skin fibroblasts from a patient with DGUOK-mutated mtDNA depletion syndrome into iPSCs, which were then differentiated into hepatocyte-like cells and liver organoids.869 This study not only uncovered the mechanism linking iron overload to hepatocyte death but also identified N-acetylcysteine as a potential therapeutic intervention to inhibit ferroptosis in patients with mtDNA depletion syndrome.869 Similarly, iPSCs derived from patients with Alpers’ syndrome, as well as their differentiated neural stem cells and cortical organoids, revealed that nicotinamide riboside could ameliorate mitochondrial defects and exert neuroprotective effects.870 Furthermore, researchers are also utilizing iPSCs derived from patients with mitochondrial diseases to investigate the role and efficacy of gene-editing tools.685 Extensive research highlights the potential of iPSCs and their derived cells and organoids in modeling mitochondrial genetic diseases and exploring therapeutic strategies, offering renewed hope to patients. However, mtDNA alterations may occur during iPSC reprogramming, potentially affecting the reprogramming and differentiation processes.871 These changes could lead to the emergence of immunogenic neo-epitopes.872 Therefore, monitoring and dynamic analysis of mtDNA integrity should be integral to the quality control processes in iPSC production to ensure both safety and efficacy.871

Overall, interventions such as MRT, gene therapy, pharmacological treatments, and cell therapy have demonstrated significant therapeutic potential in clinical trials for mitochondrial genetic disorders. These approaches primarily function through mechanisms including the replacement of defective mtDNA/nDNA, antioxidation, metabolic regulation, and mitochondrial protection. Although the widespread clinical application of these technologies continues to face numerous challenges, efforts can be made to overcome these issues. For example, reducing the possibility of genetic drift in MRT might be achievable through gene editing technologies or the development of new biomaterials, such as the generation of in vitro oocytes.873 Additionally, the timing of gene therapy, tailored approaches for different mtDNA/nDNA defects, and more efficient delivery systems will further enhance the efficacy and application of gene therapy. Research on personalized therapeutic strategies and the synergistic effects of multiple drugs could mitigate the variability in drug efficacy caused by factors such as administration methods, individual differences, and varying disease stages. Numerous clinically non-translational but potentially effective therapeutic approaches remain. Mesenchymal stem cells can transfer functionally normal mitochondria to cells with hereditary mtDNA defects via TNT and mitoEVs, thereby increasing the proportion of normal mtDNA, improving and sustaining mitochondrial function in the long term.874 Further in vivo studies on the long-term effects and mechanisms of mitochondrial transfer are essential to ensure the safety and efficacy of these therapies, ultimately optimizing cell therapy for clinical application.874 In mitochondrial diseases, oxidative stress due to the disruption of oxidative phosphorylation leads to an imbalance between oxygen delivery and utilization, resulting in oxygen toxicity.12 Hypoxia can reduce the production of free radicals or abnormal signaling substrates and activate the vHL-PHD-HIF hypoxic transcriptional program, which in turn activates glycolysis to generate ATP and reduces ROS production caused by impaired electron transfer in ETC, thereby rescuing the ETC-inhibited cellular phenotype.12 Reducing oxygen delivery and consumption may offer therapeutic or preventive benefits for mitochondrial diseases.12,875 Further research is required to optimize and investigate the long-term effects of hypoxia, as well as its applicability in humans.875 The advent of induced pluripotent stem cells and organoids also provides powerful models for exploring the pathogenesis and therapeutic strategies of mitochondrial genetic disorders, potentially leading to the discovery of more effective treatments.

유도 만능 줄기세포와 오가노이드

환자 특이적 인간 유도 만능 줄기세포(iPSCs) 및 그 유래 세포와 오가노이드의 생성 기술의 최근 발전은 MELAS,866 MERRF,283 Leigh 증후군,795 Friedreich의 운동실조증,867 및 LHON868 등 다양한 미토콘드리아 유전 질환의 병리생리학적 메커니즘을 연구하기 위한 강력한 모델을 확립했습니다. 이러한 iPSCs, 그 파생 세포 및 오가노이드와 함께, 환자 특이적 유전적 배경을 유지하면서 샘플 획득의 어려움을 극복하고 질병 특성을 정확히 재현하는 데 기여합니다.869 특히, 이러한 기술은 잠재적 치료 약물 스크리닝 및 새로운 치료 전략 개발을 위한 강력한 도구로 부상했습니다.

예를 들어, Guo 등 연구진은 DGUOK 돌연변이 미토콘드리아 DNA 고갈 증후군 환자의 피부 섬유아세포를 iPSC로 재프로그래밍한 후 간세포 유사 세포와 간 장기유사체로 분화시켰습니다.869 이 연구는 철 과부하와 간세포 사멸 간의 메커니즘을 규명했을 뿐만 아니라, 미토콘드리아 DNA 고갈 증후군 환자의 철사멸을 억제하는 잠재적 치료제로 N-아세틸시스테인을 식별했습니다.869

마찬가지로, 알퍼스 증후군 환자로부터 유래한 iPSC 및 그 분화 신경 줄기세포와 피질 장기체는 니코틴아미드 리보사이드가 미토콘드리아 결함을 개선하고 신경 보호 효과를 발휘할 수 있음을 보여주었습니다.870

또한 연구자들은 미토콘드리아 질환 환자로부터 유도된 iPSC를 활용해 유전자 편집 도구의 역할과 효능을 조사하고 있습니다.685 광범위한 연구는 iPSC 및 그 유래 세포와 오가노이드가 미토콘드리아 유전 질환 모델링과 치료 전략 탐색에 잠재력을 지니며, 환자에게 새로운 희망을 제시하고 있음을 강조합니다. 그러나 iPSC 재프로그래밍 과정에서 mtDNA 변이가 발생할 수 있으며, 이는 재프로그래밍 및 분화 과정에 영향을 미칠 수 있습니다.871 이러한 변화는 면역원성 신에피토프의 출현을 유발할 수 있습니다.872 따라서 iPSC 생산 과정의 품질 관리에 mtDNA 무결성의 모니터링 및 동적 분석이 필수적입니다.871

전체적으로

MRT, 유전자 치료, 약물 치료, 세포 치료와 같은 개입 방법은

미토콘드리아 유전 질환의 임상 시험에서 상당한 치료 잠재력을 보여주었습니다.

이러한 접근 방식은 결함 있는 mtDNA/nDNA 교체, 항산화, 대사 조절, 미토콘드리아 보호 등 다양한 메커니즘을 통해 작용합니다. 이러한 기술의 광범위한 임상 적용은 여전히 수많은 도전 과제에 직면해 있지만, 이러한 문제를 극복하기 위한 노력이 가능합니다. 예를 들어, MRT에서의 유전적 변이 가능성을 줄이는 것은 유전자 편집 기술이나 새로운 생체 재료 개발, 예를 들어 체외에서 생성된 난자 등을 통해 달성될 수 있습니다.873 또한, 유전자 치료의 시점, 다양한 mtDNA/nDNA 결함에 맞는 맞춤형 접근법, 더 효율적인 전달 시스템은 유전자 치료의 효능과 적용 범위를 더욱 향상시킬 것입니다. 개인 맞춤형 치료 전략과 다중 약물의 시너지 효과에 대한 연구는 투여 방법, 개인 차이, 질병 단계의 다양성 등으로 인한 약물 효능의 변이를 완화할 수 있습니다. 임상적으로 전환되지 않았지만 잠재적으로 효과적인 치료 접근법이 여전히 존재합니다. 중간엽 줄기 세포는 TNT와 mitoEVs를 통해 유전적 mtDNA 결함이 있는 세포에 기능적으로 정상적인 미토콘드리아를 전달하여 정상 mtDNA의 비율을 증가시키고 장기적으로 미토콘드리아 기능을 개선 및 유지할 수 있습니다.874 미토콘드리아 전달의 장기적 효과와 메커니즘을 평가하기 위한 추가적인 in vivo 연구는 이러한 치료법의 안전성과 효능을 확보하고 궁극적으로 임상 적용을 위한 세포 치료법을 최적화하는 데 필수적입니다.874 미토콘드리아 질환에서 산화적 인산화 장애로 인한 산화 스트레스는 산소 공급과 이용의 불균형을 초래하여 산소 독성을 유발합니다.12 저산소증은 자유 라디칼이나 이상 신호 전달 물질의 생성을 감소시키고 vHL-PHD-HIF 저산소 전사 프로그램을 활성화합니다. 이는 다시 글리코lysis를 활성화하여 ATP를 생성하고 전자 전달 체계(ETC)의 손상으로 인한 ROS 생성을 감소시켜 ETC 억제 세포 형질을 회복시킵니다.12 산소 공급과 소비를 감소시키는 것은 미토콘드리아 질환에 대한 치료적 또는 예방적 혜택을 제공할 수 있습니다.12,875 저산소증의 장기적 효과와 인간에서의 적용 가능성을 최적화하고 조사하기 위해 추가 연구가 필요합니다.875 유도 만능 줄기세포와 오가노이드의 등장도 미토콘드리아 유전 질환의 병리 메커니즘과 치료 전략을 탐구하는 강력한 모델을 제공하며, 더 효과적인 치료법 발견으로 이어질 수 있습니다.

Conclusion and Future perspectives

Since the first description of LHON in 1871,19 and the subsequent discoveries of the roles of mtDNA and nDNA in mitochondrial genetic disorders in 198832,33 and 1995,37 the field has transitioned from the pre-molecular era to the molecular era. This deeper understanding of mitochondrial biology has significantly advanced our ability to elucidate pathogenesis and explore effective therapies.

Significant progress has been made in understanding the pathogenesis, improving diagnosis, and developing treatments for mitochondrial diseases. Novel mechanisms of MQC, such as migrasome-mediated exocytosis,190 mitolysosome exocytosis,188 and mitopherogenesis,194 have been identified, highlighting the role of a robust MQC network in enhancing mitochondrial resilience to stress and damage. Beyond OXPHOS disturbances, the broader implications of mitochondrial dysfunction and its downstream effects are receiving increasing attention. Abnormalities induced by mtDNA or nDNA mutations—such as impaired mito-nuclear communication, mitochondrial dynamics, and mitophagy—are now recognized as key factors in mitochondrial diseases.126,227 Notably, mutation-specific and cell-specific mito-nuclear communication has been shown to significantly influence the tissue heterogeneity observed in these disorders.541 Advances in NGS and other technologies have markedly improved diagnostic accuracy. Promising therapeutic strategies are also advancing, with MRT showing significant potential in treating and preventing mitochondrial genetic diseases and their germline transmission.609 The success of ST technology in particular is encouraging.46 Gene therapy, especially AAV-mediated allogeneic expression‎ and gene replacement, is another highly promising approach, with several therapies now in clinical trials.47 Additional interventions such as exercise,876 pharmacological treatments,48,754,826 cell therapy,843 enzyme replacement therapy,848 and organ transplantation877 have demonstrated beneficial effects on mitochondrial diseases.

However, several urgent challenges remain. The genotype-phenotype correlation in mitochondrial diseases is complex and not fully understood.4 Despite advancements in iPSC and organoid technology, as well as gene editing, developing accurate disease models continues to be a significant challenge, limiting research progress. Existing cell (cybrid) and animal models are insufficient, particularly due to issues such as tissue specificity and heteroplasmy shift.226 Additionally, not all mutations can be modeled, as some homozygous mutations result in embryonic lethality.369 In terms of diagnosis, DNA sequencing remains the primary method for detecting mitochondrial diseases, but it faces significant limitations. Secondary mtDNA mutations caused by nDNA mutations, tissue heteroplasmy, and overlapping phenotypes complicate sequencing efforts, leading to a low overall diagnostic yield.549,571 This often necessitates repeated and combined testing methods, increasing the economic burden. Regarding treatment, a complete cure for mitochondrial diseases remains elusive. The application of MRT faces potential limitations due to functional incompatibilities between nuclear and mitochondrial genomes,613 residual donor mtDNA,609 and ethical concerns.878 Current mtDNA editing approaches are limited to correcting a few specific point mutations,879 and concerns about off-target effects and safety undermine confidence in gene therapy.680,719 Additionally, allogeneically expressed mitochondrial proteins are highly hydrophobic, and only a small portion successfully localizes to mitochondria, limiting the effectiveness of allogeneic expression‎ therapy.880 Significant challenges remain in overcoming these obstacles.

Future research should increasingly focus on the relationship between genetic defects and mitochondrial dysfunction, along with the subsequent consequences. Novel MQC mechanisms offer promising avenues for investigation in mitochondrial diseases. Delving into tissue-specific and cell-specific mito-nuclear communication pathways is crucial to understanding these conditions better. Epigenetics, particularly lactylation, may play a significant role in mitochondrial diseases and warrant further exploration. A deeper understanding of mito-nuclear communication could also provide solutions to mito-nuclear incompatibility issues encountered after MRT. Given the challenges of unstable diagnostic yield and variants of uncertain significance in genome sequencing, integrating multi-omics approaches as complementary diagnostic tools could enhance diagnostic accuracy. To address the delivery limitations of gene therapy, future efforts should concentrate on developing methods to efficiently introduce nuclear-encoded products into mitochondria to ensure they function correctly. Prioritizing the development of gene therapy tools that are more precise, efficient, minimally off-target, safer, and easier to deliver is essential. Additionally, the combination of gene therapy with MRT could yield unexpected therapeutic benefits. Therapies such as drugs and exercise also require clinical evidence-based guidance for their application. Notably, combining different therapeutic approaches may offer synergistic benefits. The use of iPSCs and organoid technologies should be expanded to create models for specific mutations associated with mitochondrial genetic disorders. These models would facilitate the study of disease phenotypes and post-treatment changes, advancing our understanding of disease mechanisms and aiding in the development of new treatment strategies. Exploring the differentiation of pluripotent stem cells into oocytes for MRT could reduce the need for egg retrieval‎ from healthy donors and minimize the waste of oocytes. The application of iPSCs and organoids as models for gene therapy also holds significant potential.

This review systematically summarizes the physiological aspects of mitochondrial metabolism, the intracellular and intercellular MQC network, and mitochondrial inflammation and apoptosis. It highlights potential molecular mechanisms, recent diagnostic advancements, and therapeutic developments in mitochondrial diseases, contributing to a deeper understanding of these conditions and guiding future research design and clinical translation of precise diagnostics and effective therapies. The future directions for research, diagnosis, and treatment outlined here aim to inspire and inform subsequent studies.

결론 및 미래 전망

1871년 LHON의 첫 번째 보고 이후,19 1988년32,33 및 1995년37 미토콘드리아 유전 질환에서 mtDNA와 nDNA의 역할이 발견되면서, 이 분야는 분자 전 시대에서 분자 시대로 전환되었습니다. 미토콘드리아 생물학에 대한 이 깊은 이해는 병리 메커니즘을 규명하고 효과적인 치료법을 탐구하는 능력을 크게 향상시켰습니다.

미토콘드리아 질환의 병인 메커니즘 이해, 진단 개선, 치료법 개발 분야에서 중요한 진전이 이루어졌습니다. 미토콘드리아 품질 관리(MQC)의 새로운 메커니즘, 예를 들어 미그라소ーム 매개 분비190, 미토리소좀 분비188 및 미토포게네시스194가 식별되어, 스트레스와 손상에 대한 미토콘드리아의 회복력을 강화하는 강력한 MQC 네트워크의 역할을 강조하고 있습니다. OXPHOS 장애를 넘어, 미토콘드리아 기능 장애와 그 하류 효과의 광범위한 영향이 점점 더 주목받고 있습니다.

mtDNA 또는 nDNA 돌연변이에 의해 유발된 이상—예를 들어 미토-핵 통신 장애, 미토콘드리아 동역학 장애, 미토파지 장애—는 이제 미토콘드리아 질환의 핵심 요인으로 인정받고 있습니다.126,227 특히, 돌연변이 특이적 및 세포 특이적 미토-핵 통신이 이러한 질환에서 관찰되는 조직 이질성에 크게 영향을 미친다는 것이 밝혀졌습니다.541 NGS 및 기타 기술의 발전은 진단 정확도를 크게 향상시켰습니다. 유망한 치료 전략도 발전 중이며, MRT는 미토콘드리아 유전성 질환 및 그 유전적 전파를 치료하고 예방하는 데 상당한 잠재력을 보여주고 있습니다.609 특히 ST 기술의 성공은 격려할 만합니다.46 유전자 치료, 특히 AAV 매개 이종 세포 유전자 발현 및 유전자 교체는 또 다른 매우 유망한 접근법으로, 현재 여러 치료법이 임상 시험 중입니다.47 운동,876 약물 치료,48,754,826 세포 치료,843 효소 교체 치료,848 및 장기 이식877과 같은 추가적 개입은 미토콘드리아 질환에 유익한 효과를 보여주었습니다.

그러나 여러 긴급한 과제가 남아 있습니다. 미토콘드리아 질환의 유전자형-표현형 상관관계는 복잡하고 완전히 이해되지 않았습니다.4 iPSC 및 오가노이드 기술, 유전자 편집 기술의 발전에도 불구하고 정확한 질환 모델 개발은 여전히 주요 도전 과제이며, 이는 연구 진전을 제한하고 있습니다. 기존의 세포(사이브리드) 및 동물 모델은 조직 특이성과 헤테로플라스미 변화와 같은 문제로 인해 불충분합니다.226 또한 모든 돌연변이를 모델링할 수 없으며, 일부 동형접합 돌연변이는 배아 치사율을 유발합니다.369 진단 측면에서는 DNA 시퀀싱이 미토콘드리아 질환을 탐지하는 주요 방법이지만, 심각한 한계를 가지고 있습니다. nDNA 변이로 인한 2차 mtDNA 변이, 조직 이형성, 중복된 증상 등은 시퀀싱 노력을 복잡하게 만들어 전체 진단 수율을 낮춥니다.549,571 이로 인해 반복적이고 결합된 검사 방법이 필요하게 되어 경제적 부담이 증가합니다. 치료 측면에서 미토콘드리아 질환의 완전한 치료는 여전히 달성되지 않았습니다. MRT의 적용은 핵과 미토콘드리아 유전체 간의 기능적 불호환성,613 잔여 기증자 mtDNA,609 및 윤리적 문제로 인해 잠재적 한계를 가지고 있습니다.878 현재의 mtDNA 편집 접근법은 몇 가지 특정 점 돌연변이를 교정하는 데 제한되어 있으며,879 표적 외 효과와 안전성에 대한 우려는 유전자 치료에 대한 신뢰를 약화시키고 있습니다.680,719 또한, 이종 유래로 발현된 미토콘드리아 단백질은 고도로 친수성이 낮아 미토콘드리아에 성공적으로 국소화되는 비율이 낮아 이종 유래 발현 치료의 효과를 제한합니다.880 이러한 장애물을 극복하는 데는 여전히 상당한 도전 과제가 남아 있습니다.

미래 연구는 유전적 결함과 미토콘드리아 기능 장애 간의 관계 및 그 후속 영향에 대한 이해를 심화하는 데 집중해야 합니다. 새로운 MQC 메커니즘은 미토콘드리아 질환 연구에서 유망한 접근 방식으로 주목받고 있습니다. 조직 특이적 및 세포 특이적 미토-핵 통신 경로를 탐구하는 것은 이러한 질환을 더 잘 이해하는 데 필수적입니다. 에피제네틱스, 특히 락틸레이션은 미토콘드리아 질환에서 중요한 역할을 할 수 있으며 추가 연구가 필요합니다. 미토-핵 통신에 대한 깊은 이해는 MRT 후 발생하는 미토-핵 불호환성 문제에 대한 해결책을 제공할 수 있습니다. 유전체 염기서열 분석에서 진단 정확도가 불안정하고 의미 불명의 변이체가 존재하는 문제를 고려할 때, 보완적인 진단 도구로 다중 오믹스 접근법을 통합하면 진단 정확도를 높일 수 있습니다. 유전자 치료의 전달 한계를 해결하기 위해, 향후에는 핵에서 암호화된 제품을 미토콘드리아에 효율적으로 도입하여 제대로 기능하도록 하는 방법 개발에 집중해야 합니다. 더 정확하고, 효율적이며, 오프 타겟이 적고, 안전하며, 전달이 쉬운 유전자 치료 도구의 개발을 우선시하는 것이 중요합니다. 또한 유전자 치료와 MRT의 결합은 예상치 못한 치료적 혜택을 가져올 수 있습니다. 약물 및 운동과 같은 치료법은 적용을 위해 임상적 근거 기반 지침이 필요합니다. 특히 다양한 치료 접근법의 결합은 시너지 효과를 제공할 수 있습니다. 미토콘드리아 유전 질환과 관련된 특정 돌연변이를 모델링하기 위해 iPSC 및 오가노이드 기술의 활용을 확대해야 합니다. 이러한 모델은 질병 표현형과 치료 후 변화를 연구하는 데 도움을 주어 질병 메커니즘에 대한 이해를 심화시키고 새로운 치료 전략 개발을 지원할 것입니다. MRT를 위해 다능성 줄기세포를 난자로 분화시키는 것을 탐구하면 건강한 기부자로부터 난자를 채취하는 필요성을 줄이고 난자 낭비를 최소화할 수 있습니다. iPSC와 오가노이드를 유전자 치료 모델로 적용하는 것도 큰 잠재력을 가지고 있습니다.

이 리뷰는 미토콘드리아 대사, 세포 내외 미토콘드리아 품질 관리(MQC) 네트워크, 미토콘드리아 염증 및 아포토시스 등 미토콘드리아 질환의 생리학적 측면을 체계적으로 요약합니다. 잠재적 분자 메커니즘, 최근 진단 기술의 발전, 치료법 개발을 강조함으로써 이러한 질환에 대한 이해를 심화시키고 정밀 진단 및 효과적인 치료법의 임상 적용을 위한 연구 설계에 방향을 제시합니다. 본 논문에서 제시된 연구, 진단, 치료의 미래 방향은 후속 연구에 영감을 주고 지침을 제공하기 위해 마련되었습니다.

References

1.