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Biochem J. Author manuscript; available in PMC 2022 Mar 21.
Published in final edited form as:
Biochem J. 2021 May 28; 478(10): 1959–1976.
doi: 10.1042/BCJ20200917
PMCID: PMC8935502
NIHMSID: NIHMS1786328
PMID: 34047789
Understanding amphisomes
Dhasarathan Ganesan and Qian Cai
Author information Copyright and License information PMC Disclaimer
The publisher's final edited version of this article is available at Biochem J
Abstract
Amphisomes are intermediate/hybrid organelles produced through the fusion of endosomes with autophagosomes within cells. Amphisome formation is an essential step during a sequential maturation process of autophagosomes before their ultimate fusion with lysosomes for cargo degradation. This process is highly regulated with multiple protein machineries, such as SNAREs, Rab GTPases, tethering complexes, and ESCRTs, are involved to facilitate autophagic flux to proceed. In neurons, autophagosomes are robustly generated in axonal terminals and then rapidly fuse with late endosomes to form amphisomes. This fusion event allows newly generated autophagosomes to gain retrograde transport motility and move toward the soma, where proteolytically active lysosomes are predominantly located. Amphisomes are not only the products of autophagosome maturation but also the intersection of the autophagy and endo-lysosomal pathways. Importantly, amphisomes can also participate in non-canonical functions, such as retrograde neurotrophic signaling or autophagy-based unconventional secretion by fusion with the plasma membrane. In this review, we provide an updated overview of the recent discoveries and advancements on the molecular and cellular mechanisms underlying amphisome biogenesis and the emerging roles of amphisomes. We discuss recent developments towards the understanding of amphisome regulation as well as the implications in the context of major neurodegenerative diseases, with a comparative focus on Alzheimer’s disease and Parkinson’s disease.
초록
암피솜은
세포 내에서 엔도솜과 오토파지솜의 융합을 통해 생성되는
중간/하이브리드 소기관입니다.
암피솜 형성은
화물 분해를 위해 리소좀과 최종적으로 융합하기 전에
오토파지솜이 순차적으로 성숙하는 과정에서 필수적인 단계입니다.
이 과정은
고도로 조절되며,
자가포식 플럭스가 원활하게 진행될 수 있도록
SNARE, Rab GTPase, 테더링 복합체 및 ESCRT와 같은
여러 단백질 기계가 관여합니다.
뉴런에서 오토파지솜은
축삭 말단에서 강력하게 생성된 후
후기 엔도솜과 빠르게 융합하여 암피좀을 형성합니다.
이러한 융합을 통해 새로 생성된 오토파지솜은
역행 수송 운동성을 획득하고
단백질 분해 활성 리소좀이 주로 위치한
소마 쪽으로 이동할 수 있습니다.
앰피솜은
오토파지솜 성숙의 산물일 뿐만 아니라
오토파지와 엔도리소좀 경로의 교차점이기도 합니다.
중요한 것은
암피솜이 원형질막과의 융합을 통해
역행성 신경 영양 신호 또는
오토파지 기반의 비전통적 분비와 같은 비표준 기능에도 참여할 수 있다는 점입니다.
이 리뷰에서는
암피솜 생성의 기초가 되는 분자 및 세포 메커니즘과
암피솜의 새로운 역할에 대한 최근 발견과 발전에 대한
업데이트된 개요를 제공합니다.
알츠하이머병과 파킨슨병을 중심으로
암피솜 조절에 대한 이해를 향한
최근의 발전과 주요 신경 퇴행성 질환의 맥락에서
암피솜이 갖는 의미에 대해 논의합니다
Introduction
Macroautophagy, hereafter referred to as autophagy, is a major cytosolic degradative system involving sequestration of damaged cellular components and dysfunctional organelles within autophagosomes for subsequent lysosomal clearance [1]. This mechanism relies on dedicated autophagy regulators — the autophagy-related proteins — and can be induced by various stress stimuli such as nutrient deprivation, energy loss, redox condition, hypoxia, as well as the presence of protein aggregates and intracellular pathogens. Upon autophagy activation, an initial step is to form a pre-autophagosomal membrane called phagophore or isolation membrane that is elongated to facilitate cargo engulfment and finally enclosed to form autophagosomes. The outer membrane of autophagosomes thus fuses with lysosomes to generate autolysosomes so that the sequestered cargoes are degraded within autolysosomes through the activity of lysosomal hydrolases. In the past twenty years, numerous studies have demonstrated that the maturation of autophagosomes is a more complex process, which requires successive fusion events with the endo-lysosomal compartment [2]. Before ultimately fusing with lysosomes, autophagosomes can fuse with late endosomes (LEs)/multivesicular bodies (MVBs) to generate an intermediate/hybrid organelle — amphisome. The formation of amphisomes facilitates autophagosomes to further mature, which supplies autophagosomes with several key molecules such as SNAREs, Rab GTPases, and tethering complexes that are necessary for subsequent fusion with lysosomes to produce autolysosomes.
Although the autophagy system has been extensively studied in non-neuronal cells, the function of this system in neurons is still far from being clear. Autophagy failure has been indicated as a major concern, relevant to the development of the pathologies in major neurodegenerative diseases [3]. Neurons are highly polarized cells and characterized by a complex dendritic arbor and a very long axon that emerges from the soma and bridges vast distances that can extend more than a meter in the human body. Given the post-mitotic nature of neurons, sophisticated mechanisms that assure the timely disposal of damaged proteins and organelles are essential for neuronal health and function. Autophagy serves as a key quality control mechanism that ensures the physical and functional integrity of proteins and organelles and thus plays a pivotal role in the maintenance of neuronal homeostasis. Autophagosomes are continuously generated in the distal axons of neurons and thus neurons face unique challenges to remove these autophagosomes from axonal terminals for their clearance in the soma, where mature lysosomes are predominantly located. Fusion with LEs/MVBs to form amphisomes is necessary for nascent autophagosomes to gain retrograde transport motility so that amphisomes function as a transporter to mediate the delivery of autophagic cargoes to the soma for lysosomal degradation, which is critical for autophagy function in neurons.
In addition, amphisomes provide a crossroad for the intersection of the autophagic and endocytic pathways. In recent years, while significant efforts have been taken to advance our detailed understanding of the autophagy mechanism in non-neuronal and neuronal cells, the latest studies have unveiled potential roles of amphisomes beyond autophagy-mediated cargo elimination within cells. In this review, we summarize the progress that has been made in the elucidation of the molecular and cellular mechanisms underlying the autophagosome to LE fusion events during autophagosome maturation, and discuss the non-canonical functions of amphisomes implicated in multiple physiological processes. Finally, we review recent findings of the potential roles of amphisomes under pathophysiological conditions, with a focus on neurodegenerative diseases.
소개
거대 오토파지(이하 오토파지라고 함)는
손상된 세포 성분과
기능 장애 소기관을
오토파지솜 내에서 격리하여
이후 리소좀을 제거하는 주요 세포질 분해 시스템입니다 [1].
이 메커니즘은
자가포식 관련 단백질인 자가포식 조절 인자에 의존하며
영양소 결핍,
에너지 손실,
산화 환원 상태,
저산소증,
단백질 응집체 및 세포 내 병원균의 존재와 같은
다양한 스트레스 자극에 의해 유도될 수 있습니다.
오토파지가 활성화되면
초기 단계는 화물 포집을 용이하게 하기 위해
길쭉하게 늘어난 식세포 또는 분리막이라고 하는 오토파지 전막을 형성하고
최종적으로 둘러싸서 오토파지를 형성하는 것입니다.
이렇게 만들어진
오토파지좀의 외막은
리소좀과 융합하여 오토리소좀을 생성하고,
격리된 화물들은 리소좀 가수분해효소의 활성을 통해
오토리소좀 내에서 분해됩니다.
지난 20년 동안 수많은 연구를 통해
오토파지좀의 성숙은
보다 복잡한 과정으로,
소포체 내 구획과의 연속적인 융합 이벤트가 필요하다는 것이 입증되었습니다[2].
궁극적으로
리소좀과 융합하기 전에 오토파지솜은
후기 엔도솜(LE)/다소체(MVB)와 융합하여
중간/혼합 소기관인 암피좀을 생성할 수 있습니다.
암피솜의 형성은
오토파지좀이 더욱 성숙할 수 있도록 도와주며,
오토파지좀은 이후 리소좀과 융합하여
오토리소좀을 생성하는 데 필요한 S
NARE, Rab GTPase 및 테더링 복합체와 같은 여러 핵심 분자를 오토파지좀에 공급합니다.
자가포식 시스템은
비신경세포에서 광범위하게 연구되어 왔지만,
신경세포에서 이 시스템의 기능은 아직 명확하게 밝혀지지 않았습니다.
주요 신경 퇴행성 질환의 병리 발달과 관련하여
자가포식 장애가 주요 관심사로 지적되어 왔습니다[3].
뉴런은
고도로 분극화된 세포로,
복잡한 수상돌기와 체세포에서 나와
인체에서 1미터 이상 뻗어 있는 광대한 거리를 연결하는
매우 긴 축삭이 특징입니다.
뉴런의 유사 분열 후 특성을 고려할 때,
손상된 단백질과 소기관을 적시에 처리하는 정교한 메커니즘은
뉴런의 건강과 기능에 필수적입니다.
오토파지는
단백질과 세포 소기관의 물리적, 기능적 무결성을 보장하는 핵심 품질 관리 메커니즘으로,
신경세포 항상성 유지에 중추적인 역할을 합니다.
오토파지좀은
뉴런의 원위 축삭에서 지속적으로 생성되므로
뉴런은 성숙한 리소좀이 주로 위치한 체세포질에서 제거하기 위해
축삭 말단에서 오토파지좀을 제거하는 독특한 과제에 직면합니다.
초기 오토파지가
역행 수송 운동성을 획득하여
암피좀을 형성하기 위해서는 LE/MVB와 융합하여
암피좀이 수송체로서 기능하여
리소좀 분해를 위한 자가포식화물의 체세포로의 전달을 매개하는 것이 필요한데,
이는 신경세포의 자가포식 기능에 매우 중요합니다.
또한
암피솜은
자가포식 경로와 세포 내 경로가 교차하는 교차로를 제공합니다.
최근 몇 년 동안
비신경세포와 신경세포의 자가포식 메커니즘에 대한 자세한 이해를 증진하기 위해
많은 노력을 기울여 왔으며,
최신 연구에서는
세포 내 자가포식 매개 화물 제거를 넘어서는
암피솜의 잠재적 역할이 밝혀졌습니다.
이 리뷰에서는
오토파지좀 성숙 과정에서
오토파지좀과 LE 융합 사건의 기초가 되는
분자 및 세포 메커니즘을 규명하는 과정에서 이루어진 진전을 요약하고,
여러 생리적 과정에 관여하는 암피좀의 비표준적 기능에 대해 논의합니다.
마지막으로
신경 퇴행성 질환에 초점을 맞춰
병리 생리학적 조건에서 암피솜의 잠재적 역할에 대한 최근 연구 결과를 검토합니다.
Molecular machinery in amphisome biogenesis
Amphisomes have been defined as degradative compartments within cells, which are produced through the fusion of autophagosomes with LEs/MVBs [4]. Achieved knowledge about the biogenesis of amphisomes is mostly from the studies conducted in non-neuronal cells [5, 6]. Here, we discuss several key molecules that have been indicated to drive amphisome generation, including soluble N-ethylmaleimide-sensitive factor activating protein receptors (SNAREs), Rab GTPases, tethering complexes, and endosomal sorting complex required for transport (ESCRT) proteins.
암피좀 생성의 분자 메커니즘
암피솜은
세포 내의 분해 구획으로 정의되어 왔으며,
이는 오토파지솜과 LE/MVB의 융합을 통해 생성됩니다 [4].
암피솜의 생물학적 생성에 대한 지식은
대부분 비신경세포에서 수행된 연구에서 얻은 것입니다 [5, 6].
여기에서는
가용성 N-에틸말레이미드 민감 인자 활성화 단백질 수용체(SNARE),
Rab GTPase,
테더링 복합체,
수송에 필요한 소포체 분류 복합체(ESCRT) 단백질 등
암피좀 생성을 촉진하는 것으로 알려진
몇 가지 주요 분자에 대해 설명합니다.
SNARE machinery
Like many other organelles in cells, membrane fusion events involve the SNARE proteins [7]. The vesicle-localized (v)- and target-membrane-bound (t)-SNARE proteins are localized respectively on two different vesicles and interact progressively to assemble a quadruple helix called the trans-SNARE. This trans-SNARE forms a zipper, which brings two opposing membranes closer and generates a pulling force on both membranes allowing them to fuse. SNARE proteins also participate in the key steps of autophagosome formation and subsequent maturation. Most v-SNAREs have an arginine residue in the center of the SNARE domain (R-SNAREs), whereas a glutamine (or aspartate) residue is found in syntaxins and SNAP-25-like proteins (Q-SNAREs) of the t-SNAREs [8, 9]. The R/v-SNARE protein VAMP7 and its partners — syntaxin 7 (Qa/t-SNARE) and syntaxin 8 (Qc/t-SNARE) were demonstrated to direct the formation of autophagosomes by mediating fusion of Atg16 vesicles with phagophores. Thus, the organelles can reach a proper size critical for their maturation into autophagosomes [10]. These SNAREs are also required for autophagosome biogenesis in yeast [11]. The maturation of autophagosomes into amphisomes requires syntaxin 17, an autophagosomal Q/t-SNARE, which exclusively localizes to the outer membrane of completed autophagosomes and is necessary for the autophagosome to LE fusion events [12, 13]. Importantly, syntaxin 17 interacts with synaptosomal-associated protein 29 (SNAP29) to form the syntaxin 17/SNAP29 complex, allowing their binding to VAMP8, the R-SNARE localized to the endosomal and lysosomal membranes. Syntaxin 17 is known to be recruited to autophagosomes through its LC3-interacting region (LIR) and its autophagosomal localization can be regulated by the lysosomal-associated membrane protein 2 (LAMP-2), LC3/GABA type A receptor-associated protein (GABARAP), and immunity-related GTPase M (IRGM) [13–15]. Loss of LAMP-2 or the silencing IRGM disrupts autophagic flux due to a mislocalization of syntaxin 17 [14, 15]. In neurons, we have provided direct evidence that syntaxin 17 mediates autophagosome fusion with LEs to create amphisome hybrid organelle [16, 17]. Syntaxin 17 RNAi in primary neurons significantly decreases the number of amphisomes coupled with abnormal accumulation of autophagosomes, suggesting that autophagosome maturation into amphisomes is halted as the result of the impaired fusion between autophagosomes and LEs [16, 17]. So far, syntaxin 17 is the only confirmed SNARE protein driving the formation of amphisomes in neurons.
In addition to syntaxin 17, other SNARE proteins have been identified to participate in the autophagosome/amphisome to lysosome fusion. The SNARE Vti1B, which localizes to LEs and lysosomes, and its interacting partners — syntaxin 6 and VAMP3/Cellubrevin — were reported to have a role in the fusion of autophagosomes with LEs or lysosomes [18, 19]. In contrast, data from another study concluded that Vti1B played no role in autophagic fusion [13]. The conflicting observations are likely attributed to differential regulation of the binding partners upon autophagy induction in response to distinct stimuli. Additionally, VAMP3/Cellubrevin has been indicated to play a role in membrane trafficking in non-neuronal cells. Studies in K562 reticulocytes, a cell line from human blood, have demonstrated that VAMP3/Cellubrevin and VAMP7 are another R-SNAREs that steer autophagosome-LE fusion to produce amphisomes [20]. VAMP3/Cellubrevin is highly abundant in the brain [21]. However, its role in amphisome biogenesis in neurons is poorly understood. In a recent study, YKT6 was proposed as a novel autophagosomal SNARE protein that forms a SNARE complex with SNAP29 and lysosomal syntaxin 7 in HeLa cells, which is required for autophagosomal fusion independent of syntaxin 17 [22]. Interestingly, in Drosophila, YKT6 was shown to localize to lysosomes and autolysosomes and form a SNARE complex with syntaxin 17 and SNAP29, which can be outcompeted from this SNARE complex by VAMP7 [23]. These findings suggest that YKT6 acts as a non-conventional, regulatory SNARE in this process. More investigations are needed to address whether YKT6 is also involved in the formation of amphisomes.
SNARE 기계
세포의 다른 많은 세포 소기관과 마찬가지로
막 융합 이벤트에는 SNARE 단백질이 관여합니다[7].
소포 국소화(v)- 및 표적 막 결합(t)-SNARE 단백질은 각
각 두 개의 다른 소포에 국한되어 점진적으로 상호 작용하여
트랜스-SNARE라고 하는 4중 나선을 조립합니다.
이 트랜스-SNARE는 지퍼를 형성하여
두 개의 반대편 막을 더 가깝게 만들고
두 막에 당기는 힘을 발생시켜 두 막이 융합할 수 있도록 합니다.
SNARE 단백질은 또한
오토파지솜 형성 및 후속 성숙의 주요 단계에도 관여합니다.
대부분의 v-SNARE는 SNARE 도메인의 중앙에 아르기닌 잔기가 있는 반면(R-SNARE), t-SNARE의 신택신과 SNAP-25 유사 단백질(Q-SNARE)에는 글루타민(또는 아스파르트산염) 잔기가 있습니다[8, 9]. R/v-SNARE 단백질 VAMP7과 그 파트너인 신택신 7(Qa/t-SNARE) 및 신택신 8(Qc/t-SNARE)은 Atg16 소포와 식세포의 융합을 매개하여 오토파지솜의 형성을 유도하는 것으로 입증되었습니다. 따라서 세포 소기관은 오토파지좀으로 성숙하는 데 중요한 적절한 크기에 도달할 수 있습니다 [10]. 이러한 SNARE는 효모에서 오토파지솜 생성을 위해서도 필요합니다 [11]. 오토파지솜이 암피솜으로 성숙하려면 완성된 오토파지솜의 외막에만 국한되어 있고 오토파지솜과 LE 융합 이벤트에 필요한 오토파지솜 Q/t-SNARE인 신택신 17이 필요합니다 [12, 13]. 중요한 것은 신택신 17이 시냅토솜 관련 단백질 29(SNAP29)와 상호작용하여 신택신 17/SNAP29 복합체를 형성함으로써 소포체 및 리소좀 막에 국한된 R-SNARE인 VAMP8에 결합할 수 있다는 점입니다. 신택신 17은 LC3 상호작용 영역(LIR)을 통해 오토파지로 모집되는 것으로 알려져 있으며, 오토파지의 위치는 리소좀 관련 막 단백질 2(LAMP-2), LC3/GABA A형 수용체 관련 단백질(GABARAP), 면역 관련 GTPase M(IRGM)에 의해 조절될 수 있습니다[13-15]. LAMP-2의 손실 또는 IRGM의 침묵은 신택신 17의 잘못된 위치로 인해 자가포식 플럭스를 방해합니다 [14, 15]. 뉴런에서 우리는 신택신 17이 암피솜 하이브리드 소기관을 생성하기 위해 LE와 오토파지솜 융합을 매개한다는 직접적인 증거를 제시했습니다 [16, 17]. 원시 뉴런에서 신택신 17 RNAi는 오토파지솜의 비정상적인 축적과 함께 암피솜의 수를 현저히 감소시켜 오토파지솜과 LE 간의 융합 장애의 결과로 암피솜으로의 성숙이 중단됨을 시사합니다 [16, 17]. 지금까지 신택신 17은 뉴런에서 암피솜 형성을 주도하는 SNARE 단백질로 확인된 유일한 단백질입니다.
신택신 17 외에도 오토파지좀/암피좀과 리소좀의 융합에 관여하는 다른 SNARE 단백질이 확인되었습니다. LE와 리소좀에 국한되는 SNARE Vti1B와 상호 작용하는 파트너인 신택신 6 및 VAMP3/Cellubrevin은 오토파지좀과 LE 또는 리소좀의 융합에 중요한 역할을 하는 것으로 보고되었습니다 [18, 19]. 이와는 대조적으로 다른 연구 데이터에서는 Vti1B가 오토파지 융합에 아무런 역할을 하지 않는다고 결론지었습니다[13]. 이러한 상반된 관찰 결과는 서로 다른 자극에 대한 반응으로 오토파지가 유도될 때 결합 파트너의 차별적 조절에 기인하는 것으로 보입니다. 또한, VAMP3/셀루브레빈은 비신경세포에서 막 이동에 중요한 역할을 하는 것으로 나타났습니다. 인간 혈액에서 추출한 세포주인 K562 망상 적혈구를 대상으로 한 연구에 따르면 VAMP3/Cellubrevin과 VAMP7은 오토파지솜-LE 융합을 유도하여 앰피솜을 생성하는 또 다른 R-SNARE입니다 [20]. VAMP3/셀루브레빈은 뇌에 매우 풍부합니다[21]. 그러나 뉴런에서 암피솜 생성과정에서의 역할은 잘 알려져 있지 않습니다. 최근 연구에서 YKT6는 신택신 17과 무관하게 자가포식체 융합에 필요한 HeLa 세포에서 SNAP29 및 리소좀 신택신 7과 SNARE 복합체를 형성하는 새로운 자가포식체 SNARE 단백질로 제안되었습니다 [22]. 흥미롭게도 초파리에서 YKT6는 리소좀과 자가 리소좀에 국한되어 신택신 17 및 SNAP29와 SNARE 복합체를 형성하는 것으로 나타났으며, 이 SNARE 복합체는 VAMP7에 의해 경쟁할 수 있습니다 [23]. 이러한 연구 결과는 YKT6가 이 과정에서 비전통적인 조절 SNARE로 작용한다는 것을 시사합니다. YKT6가 암피솜 형성에도 관여하는지 여부는 더 많은 연구가 필요합니다.
Rab GTPases and tethering complexes
While SNAREs are essential for the fusion of membranes, these SNARE proteins alone are not sufficient and require other factors to complete the process, particularly Rab GTPases and two major tethering complexes — the homotypic fusion and protein sorting complex (HOPS) and class C core vacuole/endosome tethering complex (CORVET). CORVET serves as a Rab5 GTPase effector complex, whereas HOPS is an evolutionarily conserved membrane tethering complex for membranes containing Rab7 GTPase and is critical for the autophagosome to LE/lysosome fusion events [24–26]. The interaction of Rab proteins with these tethering complexes brings membranes into contact, appearing to be a key event in endosomal fusion, which is further enhanced by SNAREs present on both membranes. Rab5 is the predominant GTPase on early endosomes (EEs), whereas Rab7 is abundant on LEs/MVBs [27, 28]. Most cargo sorting takes place in EEs that need to mature into LEs/MVBs, a process characterized by increased luminal acidification and the switch from Rab5 to Rab7, so-called ‘Rab conversion’. Such a mechanism is critical for the cargoes sorted into intraluminal vesicles to be degraded upon fusion with catalytically active lysosomes [29]. Interestingly, both Rab5 and Rab7 were reported to be present on autophagic membranes [30, 31]. A role of Rab5 in the early steps of the autophagy pathway is to regulate the removal of defined targets by inducing autophagy [32]. However, Rab5 is dispensable for autophagosome maturation [33], and indeed the exchange of Rab5 to Rab7 is essential for the formation of amphisomes/autolysosomes [19, 31]. Rab7 is associated with autophagosomes prior to autophagosomal fusion with lysosomes, which is activated by the guanine nucleotide exchange factor (GEF) complex, Mon1-Ccz1 [30, 34]. By binding to Atg8 in yeast and Atg8a in Drosophila, the GEF complex is recruited to autophagosomes and enhances Atg8-dependent activation of Rab7, thereby promoting autophagosome-endolysosome fusion. The HOPS complex was proposed to play a role in autophagy maturation by directly interacting with syntaxin 17 to facilitate the proper assembly of the SNARE complex [35, 36]. Moreover, UV radiation resistance-associated gene (UVRAG) can interact with the HOPS core complex and facilitate the fusion of autophagosomes with LEs/lysosomes by stimulating Rab7 activity [37]. Some studies have suggested that biogenesis of lysosome-related organelles complex 1 (BLOC-1)-related complex (BORC), Atg14, and BIRC6 function as tethers and thereby regulate autophagosome maturation [38–40]. In particular, BORC recruits the HOPS tethering complex to lysosomal membranes and facilitates the assembly of the syntaxin 17/VAMP8/SNAP29 complex, which is involved in amphisome formation. Atg14 binds to the syntaxin 17/SNAP29 complex on autophagosomes and primes this complex for VAMP8 interaction to promote autophagosome-endolysosome fusion. BIRC6 is an E2/E3 ubiquitin-conjugating enzyme and interacts with two Atg8 members GABARAP and GABARAPL1 as well as with syntaxin 17, acting as tethering components that regulate autophagosome-LE/lysosome fusion. Therefore, these observations support the notion that Rab GTPases along with the tethering complexes play a crucial role in the regulation of progressive maturation of autophagosomes. However, whether these key molecules regulate amphisome biogenesis in neurons remains largely unknown.
ESCRT machinery
Endosomal sorting complexes required for transport (ESCRT) machinery are known to mediate the maturation of LEs/MVBs. Several reports have established that ESCRTs are also involved in the regulation of autophagic flux whereas defects in ESCRTs interfere with autophagosome maturation [41–44]. The ESCRT machinery is connected to autophagy by driving autophagosome-LE/MVB fusion [45]. Studies have provided strong evidence that mutations in ESCRTs disrupt the formation of amphisomes and impact autophagy function. For example, ESCRT-associated AAA-ATPase Vps4/SKD1 is found to dissociate the ESCRTs from the endosomal membrane, a critical step for endosomal maturation. Interestingly, overexpression of the dominant-negative form Vps4/SKD1E235Q leads to an increase in autophagosomes but a decrease in autolysosomes [46]. In line with this data, lysobisphosphatidic acid (LBPA), a lipid found in LEs, fails to be delivered to autophagosomes in Vps4/SKD1E235Q cells under starvation conditions. These findings consistently suggest that impaired fusion of autophagosomes with LEs/MVBs halts the delivery of endocytic cargoes to autophagosomes in the mutant cells [46]. Similar observations were obtained in D. melanogaster ovarian follicular cells and fat bodies, in which expressing vps4 dominant-negative mutants increased autophagosomal accumulation [44]. Mutant cells for vps28 (ESCRT-I), vps25 (ESCRT-II), or vps32 (ESCRT-III) exhibit an increase in autophagosomes while amphisomes or autolysosomes are reduced. Defects in autophagosome maturation and fusion with lysosomes were also reported in ESCRT-0 VPS27/HRS depleted HeLa cells and MEFs [47]. Loss of VPS32/SNF7 (ESCRT-III) or expression of a mutant form CHMP2Bintron5 in neurons led to an increase in the number of autophagosomes [42]. Moreover, the ESCRT-1 subunit TSG101 was found to participate in the regulation of the amphisome-lysosome fusion event [48]. The ESCRT-0 Tom1 protein was reported to promote the formation of amphisomes through its interaction with myosin VI and the autophagy adaptors NDP52, T6BF, and optineurin [49]. Unlike almost all other myosins, myosin VI moves toward the minus end of actin filaments and functions in a variety of intracellular processes including sorting in the endocytic pathway [50]. Myosin VI localizes to autophagosomes and the interaction of myosin VI with the ESCRT-0 Tom1 in endocytic compartments facilitates the trafficking of endocytic cargo to autophagosomes. In the cells lacking myosin VI or the ESCRT-0 TOM1, abnormal accumulation of immature autophagosomes and impaired fusion with lysosomes were observed, accompanied by a significant reduction in the clearance of protein aggregates. This study has suggested that myosin VI likely docks or tethers the ESCRT-0 TOM1-associated endosomes to autophagosomes, thereby enhancing the autophagosome to endosome fusion events during autophagosome maturation [49]. Collectively, these findings suggest that ESCRTs are critical for autophagosome maturation, which can promote the fusion of endosomes and autophagosomes to form amphisomes and act as a hub-like system driving the final maturation of both LEs/MVBs and autophagosomes. Thus, alteration of LEs/MVBs can cause defects in autophagosome maturation and then the fusion with lysosomes.
The intersection of the autophagy and endocytosis pathways at amphisomes
Mounting evidence indicates that the autophagy and endocytic pathways are strongly interconnected in non-neuronal cells and neurons and share multiple common machineries. Amphisomes are formed upon the fusion of LEs/MVBs with autophagosomes, thus providing a crossroad for these two pathways (Figure 1).
Defects in the biogenesis and maturation of endosomes can have an impact on amphisome generation and thereby compromise autophagy function. Several endocytic regulators, such as Rab7 and ESCRTs, essential for the proper function of the endocytic pathway, have been shown to directly regulate autophagy or the fusion of autophagosomes with LEs/lysosomes as discussed above [6, 51]. Atg9 is known to contribute the membrane to the phagophore assembly site during the biogenesis of autophagosomes. Interestingly, the endocytic pathway was reported to control the proper function of Atg9. Studies have revealed that this transmembrane protein colocalizes with transferrin-, Rab11-, Rab7-, and Rab9-associated endosomes [52, 53]. The trafficking of Atg9 was disrupted in cells in the absence of the components of clathrin-mediated endocytosis, including clathrin and the assembly protein (AP) complex 2 (AP-2) or AP-4 [54, 55]. One work has uncovered that the clathrin and AP-2 complex are also relevant to Atg16 trafficking and the formation of pre-autophagosomal structures [56]. In addition, studies from C. elegans, Drosophila, and mammals have demonstrated that the ESCRT machinery is involved in autophagy regulation [57, 58]. Genetic disruptions of ESCRTs cause autophagosome accumulation and trigger neurodegeneration [42–44]. On the other hand, the autophagy machinery was reported to play a role in the function of the endocytic pathway. A recent study showed that cells lacking Atg7 or Atg16L1 displayed abnormal early endosomes, which resulted in disturbed trafficking of epidermal growth factor receptors [59]. Vps34, a mammalian phosphatidylinositol (PI) 3-kinase, drives the biogenesis of phosphatidylinositol 3-phosphate (PI3P), a lipid required for autophagosome formation and the proper function of the autophagy system [60]. Vps34 is widely distributed in the brain and deletion of Vps34 in neurons leads to impaired axonal pruning, synapse loss, and progressive neurodegeneration [61–63]. Importantly, these defects are attributed to a disruption of the endocytic pathway rather than autophagy dysfunction [62, 63]. However, whether Vps34 plays a role in the biogenesis of amphisomes is currently clear. Collectively, these studies have established the direct involvement of the endocytic system and its players in the regulation of autophagosome biogenesis and maturation, but the knowledge on the role of autophagy in endocytosis is still limited. Also, whether these mechanisms have an impact on amphisome formation needs to be addressed.
암피솜에서 자가포식 경로와 엔도사이토시스 경로의 교차점
자가포식 경로와 소포체 경로가
비신경세포와 신경세포에서 강하게 상호 연결되어 있으며
여러 가지 공통된 메커니즘을 공유한다는 증거가 늘어나고 있습니다.
암피솜은
LE/MVB와 오토파지솜이 융합하여 형성되므로
이 두 경로의 교차로를 제공합니다(그림 1).
엔도솜의 생성과 성숙에 결함이 있으면
암피좀 생성에 영향을 미쳐
오토파지 기능이 손상될 수 있습니다.
내포세포 경로의 적절한 기능에 필수적인
Rab7 및 ESCRT와 같은 여러 내포세포 조절 인자는
위에서 설명한 대로 오토파지 또는
오토파지와 LE/리소좀의 융합을 직접 조절하는 것으로 나타났습니다 [6, 51].
Atg9은 오토파지솜의 생체 생성 과정에서 세포막을 식세포 조립 부위에 기여하는 것으로 알려져 있습니다. 흥미롭게도 세포 내 경로가 Atg9의 적절한 기능을 제어하는 것으로 보고되었습니다. 연구에 따르면 이 막 통과 단백질은 트랜스페린, Rab11, Rab7 및 Rab9 관련 엔도솜과 공동 위치하는 것으로 밝혀졌습니다 [52, 53]. 클라트린과 조립 단백질(AP) 복합체 2(AP-2) 또는 AP-4를 포함한 클라트린 매개 세포 내 이동의 구성 요소가 없는 경우 세포에서 Atg9의 이동이 중단되었습니다[54, 55]. 한 연구에서는 클라트린과 AP-2 복합체가 Atg16 트래피킹 및 자가포식 전 구조의 형성과도 관련이 있다는 사실이 밝혀졌습니다 [56]. 또한 초파리, 초파리, 포유류에 대한 연구를 통해 ESCRT 메커니즘이 오토파지 조절에 관여한다는 사실이 입증되었습니다 [57, 58]. ESCRT의 유전적 장애는 자가포식소체 축적을 유발하고 신경 퇴화를 유발합니다 [42-44]. 한편, 자가포식 기계는 세포 내 경로의 기능에 중요한 역할을 하는 것으로 보고되었습니다.
최근 연구에 따르면 Atg7 또는 Atg16L1이 결핍된 세포는 비정상적인 초기 엔도솜을 나타내며, 그 결과 표피 성장 인자 수용체의 트래픽이 방해받는 것으로 나타났습니다 [59]. 포유류 포스파티딜이노시톨(PI) 3-키나아제인 Vps34는 자가포식체 형성과 자가포식 시스템의 적절한 기능에 필요한 지질인 포스파티딜이노시톨 3-포스페이트(PI3P)의 생성을 주도합니다[60]. Vps34는 뇌에 널리 분포하며 뉴런에서 Vps34를 결손하면 축삭 가지치기, 시냅스 손실 및 진행성 신경 퇴행이 발생합니다[61-63]. 중요한 것은 이러한 결함이 자가포식 기능 장애가 아닌 세포 내 경로의 파괴에 기인한다는 점입니다 [62, 63]. 그러나 Vps34가 암피솜의 생체 생성에 중요한 역할을 하는지는 현재 명확하지 않습니다.
이러한 연구들을 종합해 볼 때,
오토파지솜 생성과 성숙의 조절에 있어
세포 내 시스템과 그 플레이어가
직접적으로 관여한다는 사실은 밝혀졌지만,
세포 내에서의
오토파지의 역할에 대한 지식은
여전히 제한적입니다.
또한 이러한 메커니즘이
앰피솜 형성에 영향을 미치는지 여부도
규명해야 합니다.
Amphisomes in autophagosome maturation.
Autophagosomes undergo a progressive maturation process by interacting with multi-vesicular bodies (MVBs)/late endosomes (LEs) to generate intermediate/hybrid compartments — amphisomes, which are the intersection of the autophagy and endocytic pathways. Several key molecules such as SNAREs, Rab GTPases, tethering complexes, and ESCRTs are involved in the autophagosome-MVB/LE fusion event.
오토파지솜 성숙의 앰피솜.
오토파지솜은
다중 소포체(MVB)/후기 엔도솜(LE)과 상호 작용하여
중간/혼성 구획인 앰피솜을 생성함으로써
점진적인 성숙 과정을 거치며,
이는 오토파지와 세포 내 경로가 교차하는 교차점입니다.
오토파지솜-MVB/LE 융합 이벤트에는 SNARE, Rab GTPase, 테더링 복합체 및 ESCRT와 같은 여러 주요 분자가 관여합니다.
Amphisome retrograde transport in the axons of neurons
In non-neuronal cells, the autophagosome-LE fusion that produces amphisomes has a transient nature and is quickly transformed into autolysosomes for degradation. In neurons, autophagosomes are continuously generated in axonal terminals and need to move back to the soma for the clearance of autophagic cargoes within autolysosomes [64–71]. Retrograde axonal transport, driven by microtubule-dependent minus-end-directed dynein motors, is a common feature of both endosomes and autophagosomes through which targeted materials and autophagic cargoes from distal axons can be delivered to the soma for lysosomal degradation. Mutation or inhibition of the motor activity in dynein motors limits autophagic clearance [72, 73]. In addition to dynein motors, this mechanism involves the adaptors that attach dynein motors to targeted cargoes and thereby enable cargo retrograde movement. Multiple motor adaptors for retrograde axonal transport of endosomes have been described. Intriguingly, the function of these motor adaptors is not restricted to the transport of endosomes, and they also drive the transport of autophagosomes [74–79], suggesting a common mechanism for retrograde transport of both endosomes and autophagosomes.
Our previous studies have established that Snapin functions as a dynein motor adaptor to mediate retrograde transport of LEs in the axons of neurons [64, 80]. Through its direct interaction with the dynein intermediate chain (DIC), Snapin recruits dynein motors to the membrane of LEs, enabling LE retrograde movement toward the soma. Moreover, we have demonstrated that newly generated autophagosomes in distal axons rapidly fuse with LEs to form amphisomes through which autophagosomes are loaded with the dynein-Snapin motor-adaptor transport machinery and thus gain retrograde transport motility [16, 17]. Such a mechanism is required for the removal of autophagic cargoes from axonal terminals to facilitate their clearance within autolysosomes after fusion with lysosomes in the soma, which reduces autophagic stress in distal axons (Figure 2). Our recent studies have further uncovered that the dynein-Snapin-mediated retrograde transport coordinates with Ras homolog enriched in brain (RHEB)-dependent mitophagy to promote the elimination of damaged mitochondria at synaptic terminals, a mechanism critical for the maintenance of mitochondrial homeostasis and synaptic integrity [81, 82]. Apart from the dynein-Snapin transport complex, AP-2 was also reported to regulate axonal transport of autophagosomes/amphisomes, which involves the association of AP-2αA, a large brain-specific subunit of AP-2 with LC3 and of AP-2β with the p150Glued subunit of the dynein motor cofactor — the dynactin complex [76]. Neurons in the absence of AP-2 exhibit impaired retrograde transport of autophagosomes/amphisomes and autophagy defects. Interestingly, the involvement of AP-2 in autophagosome transport in axons could be independent of its established role in endocytosis. However, it is unclear how the dynein-Snapin and AP-2-dynactin transport machineries are coordinated to regulate retrograde transport of amphisomes in the axons of neurons.
뉴런 축삭에서의 암피좀 역행 수송
비신경세포에서 암피좀을 생성하는 오토파지솜-LE 융합은
일시적인 성격을 가지며,
오토리소좀으로 빠르게 변환되어 분해됩니다.
뉴런에서 오토파지솜은
축삭 말단에서 지속적으로 생성되며
오토리소좀 내에서 오토파지화물을 제거하기 위해
소마로 다시 이동해야 합니다 [64-71].
미세소관 의존성 마이너스 말단 방향 다이네인 모터에 의해 구동되는
역행 축삭 수송은
원위 축삭의 표적 물질과 자가포식화물을 리소좀 분해를 위해
체세포로 전달할 수 있는 엔도솜과 오토파지솜의 공통적인 특징입니다.
다이네인 모터의 운동 활동을
돌연변이하거나 억제하면
이 메커니즘에는
다이네인 모터 외에도 다이네인 모터를 표적화물에 부착하여
화물의 역행 이동을 가능하게 하는
어댑터가 포함됩니다.
엔도솜의 역행성 축삭 수송을 위한
여러 모터 어댑터가 설명되어 있습니다.
흥미롭게도 이러한
모터 어댑터의 기능은
엔도솜의 수송에만 국한되지 않고
오토파지솜의 수송도 주도하며[74-79],
이는 엔도솜과 오토파지솜 모두의 역행 수송에 대한 공통된 메커니즘을 시사합니다.
이전 연구에서는 스냅인이 뉴런 축삭에서 LE의 역행 수송을 매개하는 다이네인 모터 어댑터로 기능한다는 사실이 밝혀졌습니다 [64, 80]. 스냅인은 다이네인 중간 사슬(DIC)과의 직접적인 상호작용을 통해 다이네인 모터를 LE의 막에 모집하여 LE가 소체를 향해 역행할 수 있게 합니다.
또한,
원위 축삭에서 새로 생성된 오토파지솜이
LE와 빠르게 융합하여 암피솜을 형성하고,
이를 통해 오토파지솜에 다이네인-스냅인 모터-적응체 수송 기계가 탑재되어
역행 수송 운동성을 얻는다는 사실을 입증했습니다 [16, 17].
이러한 메커니즘은
축삭 말단에서 자가포식화물을 제거하여
체내 리소좀과 융합한 후
자가리소좀 내에서 제거를 용이하게 하여
원위 축삭의 자가포식 스트레스를 줄이는 데 필요합니다(그림 2).
최근 연구에서는
다이네인-스나핀 매개 역행 수송이
뇌에 풍부한 Ras 상동체(RHEB)와 협력하여
시냅스 말단에서 손상된 미토콘드리아의 제거를 촉진함으로써
미토콘드리아의 항상성 및 시냅스 완전성 유지에
중요한 메커니즘임을 추가로 발견했습니다[81, 82].
다이나인-스내핀 수송 복합체 외에도
AP-2는 오토파지솜/암피솜의 축삭 수송을 조절하는 것으로 보고되었는데,
이는 AP-2의 큰 뇌 특이적 하위 단위인 AP-2αA와 LC3 및 AP-2β와
다이나인 운동 보조 인자인 다이낙틴 복합체의 p150Glued 하위 단위의 연관성을 포함합니다 [76].
AP-2가 없는 뉴런은
오토파지좀/암피좀의 역행 수송 장애와 오토파지 결함을 나타냅니다.
흥미롭게도,
축삭에서 오토파지솜 수송에 AP-2가 관여하는 것은
세포 내에서의 기존 역할과는 무관할 수 있습니다.
그러나
신경세포 축삭에서 암피좀의 역행 수송을 조절하기 위해
다이네인-스내핀과 AP-2-다이낙틴 수송 메커니즘이 어떻게 조정되는지는 불분명합니다.
Amphisome retrograde transport in the axons of neurons.
Formation of amphisomes through nascent autophagosome fusion with LEs in distal axons enables the dynein-Snapin motor-adaptor complex to drive retrograde transport of amphisomes for lysosomal degradation in the soma of neurons. In addition, signaling amphisomes, comprising a subset of amphisomes, mediate retrograde movement of p75NTR or BDNF-activated TrkB receptors from axonal terminals toward the soma, participating in the long-range retrograde neurotrophic signaling in neurons.
뉴런의 축삭에서 암피솜 역행 수송.
원위 축삭에서 초기 자가포식체와 LE의 융합을 통한 암피솜의 형성은
다이나인-스내핀 모터-적응체 복합체가
뉴런의 체세포에서 리소좀 분해를 위한
암피솜의 역행 수송을 유도할 수 있게 합니다.
또한 암피솜의 하위 집합으로 구성된 신호 암피솜은
뉴런의 장거리 역행성 신경 영양 신호에 관여하여
축삭 말단에서 체질을 향한 p75NTR 또는 BDNF 활성화 TrkB 수용체의 역행성 이동을 매개합니다.
Amphisomes in retrograde neurotrophic signaling
In neurons, autophagy also plays a non-degradative role in the regulation of neurotrophic signaling. Neurotrophins are a family of growth factors including the brain-derived neurotrophic factor (BDNF) with an essential role in the retrograde control of critical neuronal functions such as axonal outgrowth, synaptogenesis, and synaptic plasticity [83]. The signaling process is initiated by neurotrophin binding to and activating the tyrosine receptor kinase (Trk) receptor. The ligand-receptor complex is then internalized into intracellular signaling compartments called signaling endosomes that undergo long-distance retrograde transport in axons, which allows the retrograde propagation of the signal [83]. Through the isolation of these carriers, earlier studies uncovered the presence of activated Trk receptors and the dynein motor transport machinery [84]. Importantly, the endosomal markers Rab5 and Rab7 were also found to be associated with these signaling compartments, thus termed signaling endosomes, which represent a subset of endosomes [74, 85]. Using Botulinum neurotoxin (BoNT) that travels in signaling endosomes containing the neurotrophin receptors p75NTR, studies have revealed that autophagosomes carry BoNT as cargo and mediate BoNT retrograde movement in axons [86, 87]. Importantly, cargoes sequestered within these autophagosomes are partially provided by the endocytic pathway, suggesting that these compartments represent amphisomes in nature following fusion with LEs/MVBs.
It is known that retrograde trafficking of BDNF-activated TrkB receptors in signaling endosomes constitutes an important long-distance signaling mechanism that conveys information from nerve terminals to the soma [88]. Recent studies suggest that BDNF/TrkB receptors are transported by amphisomes in which the incorporation of activated TrkB receptors from LEs confers signaling capabilities [75, 76]. Our earlier studies provided evidence that the dynein-Snapin-mediated retrograde transport participates in the regulation of BDNF/TrkB signaling in neurons [74]. Interestingly, SIPA1L2, a RapGAP protein, was proposed to function as a motor adaptor of TrkB-amphisomes to dynein motors by directly interacting with TrkB and Snapin, thereby temporally and spatially modulating long-range and local TrkB signaling [75]. Disruption of amphisome retrograde transport leads to defects in BDNF/TrkB signaling, as evidenced by a severe impairment of neuronal arborization and a reduction in BDNF levels, a major target of TrkB [76]. This work has also shown that prevention of autophagosome biogenesis mimics defective neuronal arborization, indicating that long-distance retrograde transport of TrkB receptors in amphisomes is required for BDNF-TrkB-mediated regulation of gene expression. Another study has further demonstrated that TrkB-amphisomes not only are essential for long-range BDNF/TrkB signaling but also regulate TrkB signaling at single presynaptic terminals [75]. This temporal and spatial control of TrkB signaling is achieved by modulating retrograde transport of TrkB-amphisomes. Interestingly, stopover of the transport complex at single synaptic terminals results in local activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) signaling at synaptic boutons along with enhancement of synaptic transmission. Defects in this mechanism trigger impairment in presynaptic long-term plasticity at hippocampal mossy fiber terminals and defective spatial separation, suggesting its relevance to presynaptic plasticity. Collectively, neurons have utilized the spatial segregation of autophagosomes/amphisomes and lysosomes to integrate retrograde neurotrophic signaling and ensure the long-range signaling capabilities of receptors (Figure 2).
However, it remains unclear what the molecular and biochemical properties of signaling amphisomes are, whether signaling amphisomes contain engulfed cargoes targeted for clearance, and how the distally activated neurotrophin receptors escape from lysosomal degradation upon their arrival to the soma. Further investigations including ultrastructural analysis are required to advance our understanding of the biogenesis of signaling amphisomes.
역행성 신경 영양 신호의 암피좀
뉴런에서 오토파지는
신경 영양 신호의 조절에 비분해적인 역할도 합니다.
뉴로트로핀은
뇌유래신경영양인자(BDNF)를 포함한 성장 인자 계열로
축삭 돌기 성장,
시냅스 형성 및
시냅스 가소성과 같은
중요한 신경세포 기능의 역행 조절에 필수적인 역할을 합니다[83].
신호 전달 과정은
뉴로트로핀이
티로신 수용체 키나아제(Trk) 수용체와 결합하여
활성화함으로써 시작됩니다.
그런 다음
리간드-수용체 복합체는
축삭에서 장거리 역행 수송을 거치는 신호 엔도솜이라는
세포 내 신호 구획으로 내재화되어
신호의 역행 전파를 가능하게 합니다 [83].
이러한 운반체의 분리를 통해
초기 연구에서는 활성화된 Trk 수용체와
다이닌 운동 수송 기계의 존재를 밝혀냈습니다 [84].
중요한 것은
엔도솜 마커인 Rab5와 Rab7도
이러한 신호 구획과 연관되어 있는 것으로 밝혀져
엔도솜의 하위 집합을 나타내는 신호 엔도솜이라고 불렀습니다 [74, 85].
뉴로트로핀 수용체 p75NTR을 포함하는
신호 전달 엔도솜에서 이동하는 보툴리눔 신경독소(BoNT)를 사용한 연구에 따르면
오토파지솜은 화물로서 BoNT를 운반하고
축삭에서 BoNT 역행 운동을 매개하는 것으로 밝혀졌습니다[86, 87].
중요한 것은
이러한 오토파지솜 내에 격리된 화물은
부분적으로 세포 내 경로에 의해 제공되며,
이는 이러한 구획이 LE/MVB와 융합된 후 자연에서 암피솜을 나타낸다는 것을 시사합니다.
신호 전달 엔도솜에서
BDNF 활성화 TrkB 수용체의 역행 이동은
신경 말단에서 체세포로 정보를 전달하는
중요한 장거리 신호 전달 메커니즘을 구성하는 것으로 알려져 있습니다 [88].
최근 연구에 따르면
BDNF/TrkB 수용체는
암피솜에 의해 운반되며,
LE에서 활성화된 TrkB 수용체를 통합하면
우리의 이전 연구에서는
다이닌-스나핀 매개 역행 수송이
뉴런에서 BDNF/TrkB 신호의 조절에 참여한다는 증거를 제공했습니다 [74].
흥미롭게도,
RapGAP 단백질인 SIPA1L2는 TrkB 및 Snapin과
직접 상호 작용하여 장거리 및 국소 TrkB 신호를 시간적, 공간적으로 조절함으로써
TrkB-암피솜의 모터 어댑터로서
다이닌 모터에 작용하는 것으로 제안되었습니다 [75].
암피솜 역행 수송의 중단은
신경 세포의 심각한 손상과 T
rkB의 주요 표적인 BDNF 수준의 감소로 입증된 것처럼
BDNF/TrkB 신호의 결함으로 이어집니다 [76].
이 연구는 또한
오토파지솜 생성을 방지하면
결함이 있는 신경세포 수목화가 모방되며,
이는 암피솜에서 TrkB 수용체의 장거리 역행 수송이
BDNF-TrkB 매개 유전자 발현 조절에 필요하다는 것을 보여줍니다.
또 다른 연구에서는
TrkB-암피솜이
장거리 BDNF/TrkB 신호에 필수적일 뿐만 아니라
단일 시냅스 전 단자에서 TrkB 신호도 조절한다는 사실이 추가로 입증되었습니다 [75].
이러한
TrkB 신호의 시간적 및 공간적 제어는
TrkB-앰피솜의 역행 수송을 조절함으로써 이루어집니다.
흥미롭게도 단
일 시냅스 말단에서 수송 복합체의 중간 기착은
시냅스 전달의 향상과 함께 시냅스 부톤에서
세포 외 신호 조절 단백질 키나제 1/2(ERK1/2) 신호의 국소적 활성화를 초래합니다.
이 메커니즘의 결함은
해마 이끼 섬유 단자에서 시냅스 전 장기 가소성의 손상과
공간 분리 결함을 유발하여 시
냅스 전 가소성과의 관련성을 시사합니다.
종합적으로
뉴런은
오토파지좀/암피좀과 리소좀의 공간적 분리를 활용하여
역행성 신경 영양 신호를 통합하고
수용체의 장거리 신호 기능을 보장합니다(그림 2).
그러나
신호 전달 앰피솜의 분자적 및 생화학적 특성이 무엇인지,
신호 전달 앰피솜에 제거 대상이 되는 화물이 포함되어 있는지,
원위에서 활성화된 뉴로트로핀 수용체가 체세포에 도착한 후
어떻게 리소좀 분해에서 벗어나는지는 아직 명확하지 않습니다.
신호 암피솜의 생물학적 발생에 대한 이해를 증진하기 위해서는
초구조 분석을 포함한 추가 조사가 필요합니다.
Secretory amphisomes
Amphisomes have been defined as degradative organelles in cells. However, several studies have suggested that an amphisome population with non-degradative function — secretory amphisomes — constitutes an unconventional secretion mechanism through autophagy-based fusion with the plasma membrane. In goblet cells of the intestinal epithelium, amphisomes were shown to promote the secretion of mucus granules, which is required for providing the barrier of the mucus that protects against intestinal pathogens [89]. Another study reported secretory functions of amphisomes in interferon (IFN)-γ-stimulated lung epithelial cells [90]. IFN-γ treatment activates autophagy in lung epithelial cells, which triggers the extracellular secretion of annexin A2 (ANXA2), a phospholipid-binding protein. This unconventional secretion takes place likely through ANXA2 containing amphisomes that are positive for LC3B and CD63, and also depends on Atg5, Rab11, Rab8A, and Rab27A. These observations suggest that the formation of LEs/MVBs and autophagosomes and the subsequent fusion with the plasma membrane are relevant processes for ANXA2 secretion. However, further studies are needed to distinguish autophagy-mediated unconventional secretion from the release of exosomes. Several lines of studies have demonstrated that LE/MVB formation is also needed for autophagy-dependent secretion of interleukin (IL)-1β, a pro-inflammatory cytokine [91]. Importantly, IL-1β secretion is unaffected by downregulation of autophagosome/amphisome fusion with lysosomes as well as cargo degradation [92], suggesting that IL-1β carriers can fuse directly with the plasma membrane. These findings consistently indicate that the extensive crosstalk between the autophagy and endocytic systems promotes autophagy-based unconventional secretion. Of note, Rab8A plays a role in both IFN-γ-stimulated ANXA2 secretion [90] and autophagy-mediated IL-1β secretion [93]. Amphisome fusion with the plasma membrane was also proposed to be relevant to membrane remodeling essential for the final stage of reticulocyte maturation [94]. In particular, large glycophorin A-containing vesicles formed at the cytosolic face of the plasma membrane can be internalized and fuse with autophagosomes before their exocytosis to expel the contents within autophagosomes. The mechanism of cargo selection and the common molecular machinery that drives the fusion of secretory amphisomes with the plasma membrane remain to be elucidated, and further studies are also needed to delineate the possible connections between autophagy-associated conventional secretion and exosome release.
분비 암피솜
암피솜은
세포의 분해성 소기관으로 정의되어 왔습니다.
그러나
여러 연구에 따르면
비분해 기능을 가진 암피솜 집단인 분비성 암피솜은
자가포식 기반의 원형질막과의 융합을 통해
비전통적인 분비 메커니즘을 구성한다고 합니다.
장 상피의 잔 세포에서
암피솜은 장내 병원균으로부터 보호하는 점액 장벽을 제공하는 데 필요한
점액 과립의 분비를 촉진하는 것으로 나타났습니다 [89].
또 다른 연구에서는
인터페론(IFN)-γ 자극 폐 상피 세포에서
암피솜의 분비 기능이 보고되었습니다 [90].
IFN-γ 치료는
폐 상피 세포에서 자가포식을 활성화하여
인지질 결합 단백질인 아넥신 A2(ANXA2)의 세포 외 분비를 촉발합니다.
이러한 비정상적인 분비는 아마도 LC3B 및 CD63에 양성인 암피좀을 포함하는 ANXA2를 통해 일어나며 Atg5, Rab11, Rab8A 및 Rab27A에도 의존합니다. 이러한 관찰은 LE/MVB와 오토파지솜의 형성과 그 후의 원형질막과의 융합이 ANXA2 분비와 관련된 과정임을 시사합니다. 그러나 오토파지를 매개로 한 비전형적 분비와 엑소좀의 방출을 구별하기 위해서는 추가 연구가 필요합니다. 여러 연구에 따르면 LE/MVB 형성은 전 염증성 사이토카인인 인터루킨(IL)-1β의 오토파지 의존적 분비에도 필요하다는 것이 입증되었습니다 [91]. 중요한 것은 IL-1β 분비가 리소좀과의 오토파지/암피좀 융합 및 화물 분해의 하향 조절에 영향을 받지 않는다는 점입니다[92]. 이는 IL-1β 운반체가 혈장막과 직접 융합할 수 있음을 시사합니다. 이러한 연구 결과는 자가포식과 세포 내 시스템 간의 광범위한 누화가 자가포식 기반의 비전통적 분비를 촉진한다는 것을 일관되게 나타냅니다. 주목할 만한 점은 Rab8A가 IFN-γ에 의해 자극되는 ANXA2 분비 [90]와 오토파지 매개 IL-1β 분비 [93] 모두에서 역할을 한다는 점입니다. 또한 암피솜과 원형질막의 융합은 망상 적혈구 성숙의 마지막 단계에 필수적인 막 리모델링과 관련이 있는 것으로 제안되었습니다 [94]. 특히, 혈장막의 세포질 표면에 형성된 글리코포린 A를 함유한 대형 소포체는 세포외로 배출되기 전에 내재화되어 오토파지솜과 융합되어 오토파지솜 내의 내용물을 배출할 수 있습니다. 화물 선택 메커니즘과 분비 앰피솜과 원형질막의 융합을 유도하는 공통 분자 메커니즘은 아직 밝혀지지 않았으며, 오토파지와 관련된 기존 분비와 엑소좀 방출 사이의 가능한 연관성을 설명하기 위한 추가 연구가 필요합니다.
Amphisomes in neurodegenerative diseases
Neurodegenerative diseases are characterized by progressive degeneration and loss of neurons, structures, and functions of the nervous system. The common feature of these diseases is a progressive accumulation of misfolded proteins, protein aggregates, or fibrils in neurons. Autophagy constituents the key quality control mechanism for the elimination of misfolded and aggregation-prone proteins and dysfunctional organelles within neurons. However, current data suggest that alterations in the autophagy and endocytic pathways underlie the earliest events in neurodegenerative diseases preceding the appearance of neuropathology [3]. Abnormal accumulation of autophagosomes is a prominent feature in the brains of patients with Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic Lateral Sclerosis (ALS), or Huntington’s disease (HD), but the underlying mechanisms remain largely unknown. Importantly, the misfolded proteins or aggregates formed by these proteins can be transmitted from affected neurons to healthy neurons. This neuron-to-neuron transmission of secreted pathologically misfolded proteins or amyloid has been proposed as the molecular basis of propagation of protein malconformation cytopathology and disease progression in neurogenerative diseases. While amphisomes and LEs normally fuse with lysosomes for degradation, these prelysosomal compartments may fuse with the plasma membrane to release any contained amyloid under the conditions of perturbed membrane trafficking and/or lysosomal deficiency. Enormous interest in the unconventional secretion of amyloids from neurons has led to the rapid growth of new findings in this field. Here we discuss the potential roles of amphisomes in the pathogenesis of major neurodegenerative diseases (Figure 3).
신경 퇴행성 질환의 암피좀
신경 퇴행성 질환은
신경계의 신경세포, 구조 및 기능이
점진적으로 퇴화되고 상실되는 것이 특징입니다.
이러한 질환의 공통적인 특징은
뉴런에 잘못 접힌 단백질, 단백질 응집체 또는
원섬유가 점진적으로 축적된다는 점입니다.
오토파지는
뉴런 내에서 잘못 접히거나 응집되기 쉬운 단백질과
기능 장애를 일으키는 소기관을 제거하는
핵심적인 품질 관리 메커니즘을 구성합니다.
그러나 현재 데이터에 따르면
자가포식 및 세포 내 경로의 변화는
신경 병리가 나타나기 전 신경 퇴행성 질환에서
가장 먼저 발생하는 사건의 근간을 이루고 있습니다[3].
오토파지솜의 비정상적인 축적은
알츠하이머병(AD), 파킨슨병(PD), 근위축성 측색 경화증(ALS), 헌팅턴병(HD) 환자의 뇌에서
두드러진 특징이지만
그 근본적인 메커니즘은 아직까지 거의 알려지지 않은 상태입니다.
중요한 것은
잘못 접힌 단백질 또는
이러한 단백질에 의해 형성된 응집체가 영향을 받은 뉴런에서
건강한 뉴런으로 전달될 수 있다는 점입니다.
분비된 병리적으로 잘못 접힌 단백질 또는
아밀로이드의 신경세포 간 전달은
신경 퇴행성 질환에서 단백질 기형 세포 병리 및
질병 진행의 분자적 근거로 제안되었습니다.
암피좀과 LE는
일반적으로 리소좀과 융합하여 분해되지만,
이러한 전리소좀 구획은 막 이동이 교란되거나 리소좀이 결핍된 조건에서
혈장막과 융합하여 함유된 아밀로이드를 방출할 수 있습니다.
뉴런에서 아밀로이드가 비정상적으로 분비되는 것에 대한 관심이 높아지면서
이 분야에서 새로운 발견이 빠르게 증가하고 있습니다.
여기에서는 주요 신경 퇴행성 질환의 발병 기전에서 암피솜의 잠재적 역할에 대해 논의합니다(그림 3).
Amphisomes in neurodegenerative diseases.
Abnormal accumulation of amphisomes is a feature of autophagic stress in major neurodegenerative diseases. In Alzheimer’s disease (AD), defective amphisome retrograde transport leads to amphisome retention in axons as a consequence of the dynein-Snapin uncoupling due to the interaction of oligomeric amyloid β (Aβ)42 with dynein motors. Such a defect impairs autophagic clearance and augments Aβ production by promoting amphisome-enriched β-site APP cleaving enzyme 1 (BACE1) cleavage of amyloid precursor protein (APP). AP-2 reduction also contributes to impeded retrograde movement of amphisomes in AD axons. Autophagic stress might trigger the amphisome-mediated extracellular secretion of Aβ and tau in AD and other tauopathy diseases. In Parkinson’s disease (PD), autophagy-based unconventional secretion through amphisomes enhances the extracellular release of α-synuclein and participates in the transmission of α-synucleinopathy. In Amyotrophic Lateral Sclerosis (ALS) and Huntington’s disease (HD), aberrant accumulation of enlarged amphisomes is attributed to lysosomal deficiency in ALS motor neurons or the altered endocytic system in HD neurons. In contrast, amphisome formation is disrupted in Niemann-Pick type C1 (NPC1) disease.
신경 퇴행성 질환의 암피좀.
암피솜의 비정상적인 축적은
주요 신경 퇴행성 질환에서
자가포식 스트레스의 특징입니다.
알츠하이머병(AD)에서는 올리고머 아밀로이드 β(Aβ)42와 다이네인 모터의 상호작용으로 인한 다이네인-스냅인 결합 해체의 결과로 암피좀 역행 수송 결함이 축삭에 암피좀을 보유하게 됩니다. 이러한 결함은 아밀로이드 전구체 단백질(APP)의 암피솜 농축 β-부위 APP 절단 효소 1(BACE1) 분해를 촉진하여 자가포식 제거를 손상시키고 Aβ 생성을 증가시킵니다. AP-2 감소는 또한 AD 축삭에서 암피솜의 역행 이동을 방해하는 데 기여합니다. 자가포식 스트레스는 AD 및 기타 타우 병증 질환에서 암피좀 매개 Aβ 및 타우의 세포 외 분비를 유발할 수 있습니다. 파킨슨병(PD)에서는 암피좀을 통한 자가포식 기반의 비전형적 분비가 α-시누클린의 세포 외 방출을 증가시키고 α-시누클린병의 전파에 관여합니다. 근위축성 측색 경화증(ALS)과 헌팅턴병(HD)에서 비대해진 암피솜의 비정상적인 축적은 ALS 운동 신경세포의 리소좀 결핍 또는 HD 신경세포의 변화된 세포 내 시스템으로 인해 발생합니다. 이와는 대조적으로, 니만-픽 C1형(NPC1) 질환에서는 암피솜 형성이 중단됩니다.
Alzheimer’s disease
AD is the most common form of neurodegenerative disease and a leading cause of dementia in the aging populations [95]. The disease progression involves cognitive decline, memory loss, and neuronal death in the cerebral cortex and subcortical regions. AD patient brains are characterized by extracellular amyloid plaque deposits, composed of agglomerated amyloid β (Aβ) peptides, as well as intracellular accumulation of neurofibrillary tangles (NFTs), consisting of hyperphosphorylated tau protein. The increase in amyloidogenic processing of amyloid precursor protein (APP), which leads to Aβ overproduction, is a key feature underlying the pathogenesis of AD [96–98]. Strong evidence indicates a link between alterations in the autophagy and endolysosomal systems and early AD pathophysiology. Massive accumulation of autophagic vacuoles (AVs) along with endosomes were observed in induced pluripotent stem cells (iPSCs)-derived neurons and post-mortem brains of both familial and sporadic AD patients [65, 99–105]. However, the cause of such autophagosomal and endosomal proliferations in AD brains remains poorly understood.
We and others have demonstrated that AVs drastically accumulate within the dystrophic neurites and synaptic terminals of AD brains [3, 69]. This raises a fundamental question as to whether defects in the removal of newly generated autophagosomes from distal axons through retrograde transport disrupt autophagic clearance and thus trigger autophagic stress in AD neurons. Our work has revealed aberrant accumulation of amphisomes at presynaptic terminals in the brains of AD patients and AD-related mutant human APP (hAPP) transgenic (Tg) mice. Importantly, such a defect is attributed to impaired retrograde transport of amphisomes [69]. Furthermore, soluble Aβ42 oligomers enriched in axons interact with dynein motors. This interaction interferes with the coupling of the dynein motor with its adaptor Snapin, interrupting the attachment of dynein motors to amphisomes. As a result, dynein-Snapin-driven retrograde transport of amphisomes is hampered, thereby trapping amphisomes in distal axons and impairing their degradation within lysosomes in the soma of AD neurons [69, 106]. In agreement with these findings, deletion of snapin in mice phenocopies AD-linked synaptic autophagic stress, whereas overexpression of Snapin in mutant hAPP neurons decreases autophagic accumulation at presynaptic terminals by enhancing retrograde transport of amphisomes. Our studies have further shown that β-site APP cleaving enzyme 1 (BACE1), a rate-limiting enzyme for APP amyloidogenic processing and Aβ generation, is concentrated within LEs/amphisomes and the dynein-Snapin transport machinery-loaded LEs/amphisomes promote BACE1 retrograde trafficking to lysosomes for degradation in the soma [107–109]. Such a mechanism is critical for the modulation of BACE1 turnover and its β secretase activity, thus controlling BACE1 cleavage of APP and Aβ production. In addition to the dynein-Snapin-driven retrograde transport-mediated control of BACE1 trafficking and turnover, a recent study reported that AP-2, originally proposed to mediate the reformation of synaptic vesicles and the retrograde transport of amphisomes containing BDNF/TrkB receptors [76, 110], was also involved in the regulation of BACE1 trafficking and degradation [111]. iPSC-derived neurons from patients with late-onset AD displayed a decrease in AP-2 levels. Moreover, deletion of AP-2 in mouse brains increases BACE1 accumulation within LEs/amphisomes coupled with elevated Aβ generation. Therefore, these studies consistently suggest that defects in retrograde transport of amphisomes exacerbate autophagy failure and halt BACE1 trafficking toward lysosomes for proper degradation, aggravating amyloid pathology in AD.
In AD brains, a significant amount of APP is processed at the plasma membrane and then Aβ is released to the extracellular space, contributing to the formation of amyloid plaques. In addition to its direct release from the plasma membrane, Aβ can be generated from the inside of neurons and secreted through unconventional mechanisms. Of note, several studies indicate that intracellular APP and Aβ localize to LEs/MVBs and exosomes in the vulnerable neurons of AD brains, and the prevention of LE/MVB fusion with lysosomes can enhance the release of Aβ [112–115]. These observations suggest that Aβ can be produced and deposited into the lumen of LEs followed by the release through the fusion of LEs/MVBs with the plasma membrane. It is known that pathogenic forms of misfolded proteins and protein aggregates are the substrates of the autophagy pathway [3]. Studies using mouse models and cell culture have shown that AVs are enriched in Aβ-generating machinery where Aβ can be produced [101, 116–118]. The cellular levels of APP and Aβ were reported to be partially regulated by autophagy [119]. Interestingly, Atg7 deficiency in AD mouse brains leads to intracellular accumulation of Aβ, accompanied by a significant reduction in extracellular amyloid plaque burden [117]. Thus, these results support an important role for autophagy in the regulation of Aβ secretion and amyloidogenesis. It is conceivable that retrograde transport impairment-induced massive accumulation of AVs, especially amphisomes, may promote the release of Aβ at the axonal terminals of AD neurons. However, direct evidence of autophagy-based Aβ secretion in AD neurons is still lacking and whether Aβ is released through autophagosomes or amphisomes remains undefined.
Tauopathies are characterized by abnormal accumulation of hyperphosphorylated tau proteins in the cytoplasm that results in the formation of tau aggregates and fibrils along with NFTs, a pathogenic hallmark of tauopathy diseases, including AD. A growing body of evidence suggests that amphisomes are involved in autophagy-dependent tau secretion. Studies have shown that membranous organelle-based unconventional secretion leads to the extracellular release of tau either in a free form or bound to a vesicle. Autophagy stress and the presence of tau within AVs are characteristics in the brains of AD and other tauopathies [120]. These tau-enriched AVs could be redirected for secretion by directly fusing with the plasma membrane. It was reported that the release of tau is enhanced upon autophagy induction by starvation or pharmacological agents or upon lysosomal deficiency, but is decreased under the condition of autophagy inhibition [121–124]. Through electron microscopy analysis at the ultrastructural level, a study from neuroblastoma cells has provided a piece of direct evidence showing that AVs possibly containing tau were approaching the plasma membrane and perhaps releasing tau [125]. Moreover, autophagy induction upon the oxygen-glucose deprivation (OGD) was shown to promote the release of free tau along with tau within microvesicles (MVs) positive for LC3, suggesting the possibility of tau secretion through amphisomes [123]. Given the fact that autophagy induction directs the fusion of LEs/MVBs with AVs to form amphisomes [126], tau targeted for the extracellular release likely arrives in amphisomes through LEs/MVBs rather than autophagosomes. A close link has been established between autophagy and other unconventional secretion pathways [127]. Thus, more studies are required to advance our understanding of whether and how tau utilizes AVs as a carrier in the process of secretion, and whether autophagy-mediated tau release plays a key role in driving the propagation of tau pathologies in tauopathy diseases.
Parkinson’s disease
PD is the second most common neurodegenerative disease and is characterized by the formation of Lewy bodies and progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta [128, 129]. α-synuclein, the most abundant protein in Lewy body inclusions of PD [130], is the substrate of the autophagy pathway and can be removed and degraded through autophagy activity. On the other hand, α-synuclein aggregates have also been indicated to inhibit autophagy function [131–133]. Aberrant autophagy function and autophagosome accumulation are also observed in PD patient brains and have been implicated in the pathogenesis of PD [3]. Also, α-synuclein has been documented to undergo unconventional secretion from cultured nerve cells [134–140]. Interneuronal transmission of endogenously produced and secreted α-synuclein has been demonstrated both in vitro and in vivo [134, 141–146]. These results highlight a critical role of α-synuclein secretion and uptake in the transmission of α-synucleinopathy with Lewy body diseases. Earlier studies have suggested endosomes as the secretory organelles for the dissemination of α-synuclein by exocytosis [137, 138]. Prior to exocytosis, cytosolic α-synuclein is imported into intraluminal vesicles of LEs/MVBs dependent and independent of the ESCRT complex [147, 148]. Amphisomes were shown to undergo physiological exocytosis, which can be enhanced by stressful or pathological conditions [89]. In nerve growth factor (NGF)-differentiated PC12 catecholaminergic nerve cells, expressing PD-related protein TPPP/p25α induces lysosomal deficiency by blocking fusion of amphisomes with lysosomes. In line with this finding, these cells exhibit an increase in the amphisome-mediated release of α-synuclein monomer and aggregates to the extracellular surroundings [136, 149]. The secretion of α-synuclein was found to be upregulated by Rab8, which was previously reported to be involved in vesicle docking to the acceptor membrane upstream of the SNARE machinery in membrane fusion events [150]. Recent work provided additional evidence that α-synuclein was released and transferred via amphisome-like structures upon inhibition of the autophagy-lysosomal pathway, suggesting that amphisomes could be an alternative candidate for α-synuclein secretion besides LEs/MVBs [151]. More work is needed to elucidate detailed mechanisms underlying α-synuclein secretion through autophagy-based unconventional secretion and its relevance to the development of PD pathologies.
Other neurodegenerative diseases
The mechanisms underlying autophagy dysregulation in other neurodegenerative diseases have been understudied. ALS is characterized by progressive degeneration of motor neurons in the brain and the spinal cord, leading to muscle weakness, atrophy, and paralysis [152]. While 5%–10% of ALS patients suffer from familial ALS, mutations in superoxide dismutase 1 (SOD1) account for 2% of total disease cases. Similar to Aβ, tau, and α-synuclein, several lines of evidence have demonstrated prion-like propagation of mutant SOD1 misfolding via exosome-dependent and exosome-independent mechanisms in neuronal cells [153–155]. Defective autophagy has been indicated in ALS, but distinct underlying mechanisms were proposed [156–160]. A mutant form of ALS2, an activator of Rab5, was proposed to disrupt ALS2-dependent activation of Rab5, leading to defects in the formation of amphisomes in this familial form of ALS [157]. In a mouse model of ALS expressing mutant human SOD1, lysosomal defects have been found to result in abnormal accumulation of amphisome-like structures [66]. However, whether amphisomes are directly involved in SOD1 secretion and interneuron transmission in ALS remains largely unknown. Niemann-Pick type C1 (NPC1) disease is a lipid-storage disorder associated with neurodegeneration and liver function and is characterized by cholesterol accumulation within LEs/lysosomes resulting from disease-causing mutations in the NPC1 protein [161, 162]. A study reported that defects in amphisome formation caused by a failure in SNARE machinery hinder the maturation of autophagosomes and thus impair autophagy function in NPC1 disease-linked cells [163]. Interestingly, stimulation of autophagy or expression of functional NPC1 protein restores the function of this pathway. HD is a fatal neurodegenerative disorder caused by an expansion of a polyglutamine tract in the huntingtin (htt) protein that mediates the formation of intracellular protein aggregates. Accumulation of enlarged amphisomes was reported in cultured neurons expressing a mutant htt fragment, which is attributed to a disruption of the endocytic system that triggers neurodegeneration [164].
Conclusions and perspectives
By interacting with endosomes to form amphisomes, autophagosomes undergo a sequential maturation process before ultimate fusion with lysosomes, eventually resulting in the degradation of sequestrated autophagic cargoes [45, 165–167]. Many membrane fusion machineries have been identified to be involved in the fusion between autophagosomes and endosomes. These proteins need to be delivered to the surface of autophagosomes to avoid premature fusion with lysosomes. Thus, autophagosome-endosome fusion to generate amphisomes could be a critical step in controlling the progressive maturation of autophagosomes. Despite evidence of the importance of this intermediate step in the autophagy pathway, it is largely overlooked. Many studies in the field have been focusing on autophagosome-lysosome fusion but with very limited data describing amphisome formation and the fusion of amphisomes to lysosomes. Importantly, the mechanism underlying amphisome biogenesis in neurons remains poorly understood. Amphisomes also play a non-degradative role in the regulation of retrograde neurotrophic signaling in neurons. However, how the biogenesis of signaling amphisomes occurs and whether cargoes sequestered within signaling amphisomes are also targets for autophagic clearance are still largely unknown. Autophagy is crucial for memory formation and age-related cognitive decline has been linked to autophagy dysfunction. Thus, whether autophagy deficits impair neurotrophic signaling and whether the absence of BDNF/TrkB signaling as a result of the dysregulation of signaling amphisomes contributes to age-associated cognitive impairment are critical questions for future studies. Moreover, autophagy failure has been implicated in the pathogenesis of neurodegenerative diseases [3]. Detailed elucidation of amphisome biogenesis is necessary to advance our understanding of this middle step in autophagosome maturation and to clarify whether and how the amphisome stage is affected as well as its impact on disease pathologies. A better understanding of this pathway would highlight a potential avenue for biomarkers. Given the rapid development in this field in recent years, these important questions need to be addressed in a timely manner.
The autophagy and endolysosomal pathways directly intersect at amphisomes, the contents of which have multiple fates, including lysosomal degradation or extracellular release. Physiological exocytosis of amphisomes through autophagy-based unconventional secretion has been demonstrated to participate in multiple biological processes and can be facilitated by the crosstalk between autophagy and the endolysosomal system. Under the conditions of disturbed membrane trafficking and/or lysosomal deficiency, amphisomes accumulate within cells, which either leads to cell death or promotes the exocytosis of the accumulating amphisomes, as a last resort, to release contents to the surroundings. In neurodegenerative diseases, some studies indicate that enhanced exocytosis of amphisomes can mitigate the toxicity induced by the aberrant accumulation of disease-causing protein aggregates/amyloid [136]. However, direct evidence of autophagy-based secretion in diseased neurons is still lacking and it is also unclear whether amphisome-dependent amyloid release cooperates with the exosomal pathway. More work is required to elucidate detailed mechanisms and their relevance to the development of neurodegeneration. On the other hand, amphisomes-mediated autophagy-dependent amyloid secretion has been proposed as a mechanism underlying the propagation of pathologies associated with neurodegenerative diseases. Thus, there is a suspicion that the manipulations of amphisome secretion through membrane trafficking pathways would be a promising strategy for therapeutic developments to enhance amyloid clearance and thus combat disease pathologies. However, from the propagation perspective, not all secreted amyloids are the same. Amphisomes likely release both monomeric amyloid and soluble amyloid in the form of proteotoxic oligomers and aggregates [168]. Therefore, it is important to determine which of these species are most relevant for disease propagation to develop focused therapeutic strategies in the future.
Acknowledgements
The authors thank Y. Zuo and J. Yoon for editing and other members of the Cai laboratory for their assistance and discussion.
Funding
This work was supported by National Institutes of Health grants R01NS089737 and R01GM135326 (to Q.C.)
Abbreviations
AD | Alzheimer’s disease |
ALS | Amyotrophic Lateral Sclerosis |
ANXA2 | annexin A2 |
AP | assembly protein (AP) |
APP | Amyloid precursor protein |
AVs | autophagic vacuoles |
Aβ | amyloid β |
BACE-1 | β-site APP cleaving enzyme 1 |
BDNF | brain-derived neurotrophic factor |
BoNT | Botulinum neurotoxin |
BROC | biogenesis of lysosome-related organelles complex 1 (BLOC-1)-related complex |
CORVET | class C core vacuole/endosome tethering complex |
DIC | dynein intermediate chain |
EE | early endosome |
ERK1/2 | extracellular signal-regulated protein kinase 1/2 |
ESCRT | endosomal sorting complexes required for transport |
GABARAP | GABA type A receptor-associated protein |
GEF | guanine nucleotide exchange factor |
HD | Huntington’s disease |
HOPS | homotypic fusion and protein sorting complex |
htt | Huntingtin protein |
IFN | interferon |
IL | interleukin |
iPSCs | induced pluripotent stem cells |
IRGM | immunity-related GTPase M |
LAMP-2 | lysosomal-associated membrane protein 2 |
LBPA | lysobisphosphatidic acid |
LC3 | microtubule-associated proteins 1A/1B light chain 3 |
LE | late endosomes |
LIR | LC3-interacting region |
MVB | multivesicular body |
MVs | microvesicles |
NFTs | neurofibrillary tangles |
NGF | nerve growth factor |
NPC1 | Niemann-Pick type C1 |
OGD | oxygen-glucose deprivation |
PD | Parkinson’s diseases |
PI3P | phosphatidylinositol 3-phosphate |
RHEB | Ras homolog enriched in brain |
SNAP29 | synaptosomal-associated protein 29 |
SNAREs | soluble N-ethylmaleimide-sensitive factor activating protein receptors |
SOD1 | superoxide dismutase 1 |
Tg | transgenic |
Trk | tyrosine receptor kinase |
UVRAG | UV radiation resistance-associated gene |
VAMP | vesicle associated membrane proteins |
Footnotes
Competing Interests
The authors declare that there are no competing interests associated with the manuscript.
References