Re: 파킨슨병 Neuromelanin 색소에 대하여

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뉴로멜라닌(뇌 특히 흑질에 많이 존재하는 멜라닌 색소)이 나이가 들면서 과도한 축적

--> 임계치를 넘어서면 파킨슨 증상...

Scientific Perspective

Open Access

Neuromelanin, aging, and neuronal vulnerability in Parkinson's disease

Miquel Vila MD, PhD

First published: 28 June 2019

https://doi.org/10.1002/mds.27776

Citations: 78

Relevant conflicts of interests/financial disclosures: Vall d'Hebron Research Institute has applied for a use patent for the modulation of neuromelanin levels in the treatment of Parkinson's disease and/or brain aging.

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Abstract

Neuromelanin, a dark brown intracellular pigment, has long been associated with Parkinson's disease (PD). In PD, neuromelanin-containing neurons preferentially degenerate, tell-tale neuropathological inclusions form in close association with this pigment, and neuroinflammation is restricted to neuromelanin-containing areas. In humans, neuromelanin accumulates with age, which in turn is the main risk factor for PD. The potential contribution of neuromelanin to PD pathogenesis remains unknown because, in contrast to humans, common laboratory animals lack neuromelanin. The recent introduction of a rodent model exhibiting an age-dependent production of human-like neuromelanin has allowed, for the first time, for the consequences of progressive neuromelanin accumulation—up to levels reached in elderly human brains—to be assessed in vivo. In these animals, intracellular neuromelanin accumulation above a specific threshold compromises neuronal function and triggers a PD-like pathology. As neuromelanin levels reach this threshold in PD patients and presymptomatic PD patients, the modulation of neuromelanin accumulation could provide a therapeutic benefit for PD patients and delay brain aging.

짙은 갈색의 세포 내 색소인 뉴로멜라닌은 

오랫동안 파킨슨병(PD)과 연관되어 왔습니다. 

PD에서는 

뉴로멜라닌을 함유한 뉴런이 우선적으로 퇴화하며, 

이 색소와 밀접하게 연관된 신경 병리학적인 내포물이 형성되고 

신경염증이 뉴로멜라닌 함유 영역으로 제한됩니다. 

사람의 경우 뉴로멜라닌은 

나이가 들면서 축적되며, 

이는 결국 파킨슨병의 주요 위험 요인입니다. 

파킨슨병(PD)에서는 뉴로멜라닌 함유 뉴런, 특히 흑질 도파민 신경세포의 선택적 퇴화가 일어납니다. 인간의 경우 뉴로멜라닌은 나이가 들면서 축적되며, 후자는 PD의 주요 위험 인자입니다. 인간과 달리 일반적인 실험동물은 뉴로멜라닌이 부족하기 때문에 뉴로멜라닌이 PD 발병에 기여하는 바는 아직 알려지지 않았습니다. 말초 멜라닌의 합성은 뇌에도 낮은 수준으로 존재하는 효소인 티로시나아제에 의해 매개됩니다. 여기서 우리는 쥐 흑질에서 인간 티로시나아제를 과발현하면 흑질 도파민 신경세포 내에서 인간과 유사한 뉴로멜라닌이 나이에 따라 노인에게 도달하는 수준까지 생산된다는 사실을 보고합니다. 이러한 동물에서 특정 역치 이상의 세포 내 뉴로멜라닌 축적은 운동 저하증, 루이체 유사 형성 및 흑질 신경 퇴화를 포함한 연령 의존적 PD 표현형과 관련이 있습니다. 리소좀 단백질 정체성을 강화하면 세포 내 뉴로멜라닌이 감소하고 티로시나아제 과발현 동물의 신경 퇴화를 예방할 수 있습니다. 우리의 연구 결과는 세포 내 뉴로멜라닌 수치가 PD의 시작을 위한 임계치를 설정할 수 있음을 시사합니다.

인간과 달리 일반적인 실험동물에는 

뉴로멜라닌이 부족하기 때문에 

뉴로멜라닌이 PD 발병에 미치는 잠재적 기여는 아직 알려지지 않았습니다. 

최근 인간과 유사한 뉴로멜라닌의 연령 의존적 생성을 보이는 설치류 모델이 도입되면서 처음으로 노인의 뇌에서 도달하는 수준까지 뉴로멜라닌 축적의 결과를 생체 내에서 평가할 수 있게 되었습니다. 이 동물에서 세포 내 뉴로멜라닌이 특정 임계치 이상으로 축적되면 신경세포 기능이 손상되고 PD와 유사한 병리가 유발됩니다. 

뉴로멜라닌 수치가 

PD 환자 및 전증상 PD 환자에서 이 역치에 도달하면 

뉴로멜라닌 축적을 조절하면 

PD 환자에게 치료 효과를 제공하고 뇌 노화를 지연시킬 수 있습니다.

© 2019 The Author. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Neuromelanin and PD: A 100-Year-Old Relationship

A century ago, in 1919, Konstantin Tretiakoff reported for the first time in his remarkable doctorate thesis the presence of a marked loss of pigmented neurons in the substantia nigra (SN), visible with the naked eye, in the brains of Parkinson's disease (PD) patients.1 Although this finding failed to gain him any significant recognition during his lifetime, his observation remains to this day the cardinal pathologic diagnostic criterion for PD.1 The loss of nigral pigmented neurons, which we now know produce the neurotransmitter dopamine, leads to the classical motor symptoms of PD and constitutes the only robust clinico-pathological correlation associated with the disease.1 The pigment contained within these neurons, termed neuromelanin because of its similar appearance to cutaneous melanin, is so abundant in the SN of the human brain that this structure can be seen macroscopically as a darkened area (hence the origin of the name given to this brain region).2 Later, other neuromelanin-containing neurons in different brain regions were also found to consistently degenerate in PD; these include the noradrenergic neurons of the locus coeruleus and dorsal motor nucleus of the vagus, leading to characteristic nonmotor symptoms of the disease.3, 4 In contrast, neuronal loss in nonmelanized brain regions appears either inconsistent, not specific to PD, or secondary to the loss of interconnected neuromelanin-containing neurons.5-7

In the normal human midbrain, dopamine-producing cell groups markedly differ from each other in terms of the percentage of neuromelanin-pigmented neurons they contain.8-10 In PD, the estimated cell loss in these groups correlates directly with the percentage of neuromelanin-pigmented neurons normally present in them.8-10 Likewise, within each neuromelanin-containing cell group, there is greater relative sparing of weakly pigmented than of strongly melanized neurons.8 For instance, the loss of catecholaminergic neurons in PD is severe in the SN pars compacta (SNpc), in which virtually all neurons are pigmented, and almost undetectable in the central gray substance, in which most catecholaminergic neurons are not pigmented.9 In the peri- and retro-rubral regions of the midbrain, which contain pigmented and nonpigmented catecholaminergic neurons in similar proportions, the population of melanized catecholaminergic neurons is significantly decreased, whereas the total population of catecholaminergic neurons devoid of neuromelanin is not affected.9 Similarly, dopaminergic neurons in the ventral tegmental area, which are largely spared in PD, produce minimal neuromelanin over a lifetime.8, 11

Classical Lewy bodies (LB), that is, α-synuclein-containing intracytoplasmic inclusions that constitute the pathological hallmark of PD besides cell loss, and their presumed precursor structures, termed pale bodies, typically appear within the intracellular areas of the cytoplasm in which neuromelanin accumulates and form in close physical association with this pigment.12 Consistent with these observations, studies on human brains have shown that α-synuclein redistributes to the neuromelanin pigment in early stages of PD and becomes entrapped within neuromelanin granules.13-15 Further linking the PD neuropathology with neuromelanin, neuroinflammatory changes occurring in PD brains, such as activation of innate (ie, microgliosis) and adaptive (ie, lymphocyte infiltration) immune responses, are highly localized within neuromelanin-containing areas and only sparingly observed in nonmelanized regions.16 For instance, extracellular neuromelanin released from dying neurons activates microglia in PD brains to phagocytize and degrade/eliminate this pigment.17 Also, neuromelanin-containing SNpc neurons from PD brains exhibit Immunoglobulin G accumulation and antigenic major histocompatibility complex class I expression‎ in a manner directly correlated to neuronal loss.18 In addition, antimelanin antibodies are increased in the sera of PD patients.19

The PD pathogenesis thus appears to be inextricably linked to the presence of neuromelanin. However, despite the close and long-established association between neuromelanin and PD, the physiological significance of neuromelanin and its potential contribution to PD pathogenesis remain unknown because, in contrast to humans, laboratory animal species commonly used in experimental research, such as rodents, lack neuromelanin. Although neuromelanin is actually present in some other species as varied as monkeys,20, 21 dolphins,22 and frogs,23 the highly abundant quantity of neuromelanin in the brain stem seems unique to humans, as a dark pigmentation of this brain area is not apparently observed in other animal species at the macroscopic level.20 In fact, in the mammalian brain, neuromelanin accumulation increases progressively as the evolutionary relation to man becomes closer,20 and humans are the only species that naturally develops PD.24 Consequently, a factor so intimately linked to PD such as neuromelanin has been surprisingly neglected to date in experimental in vivo paradigms of the disease.

뉴로멜라닌과 PD: 100년 역사의 관계

한 세기 전인 1919년, 콘스탄틴 트레티아코프는

그의 놀라운 박사 학위 논문에서 파킨슨병(PD) 환자의 뇌에서

육안으로 볼 수 있는 흑질(SN)의 색소 신경세포가

현저하게 손실되어 있음을 처음으로 보고했습니다.1

이 발견은

그의 생전에 큰 인정을 받지 못했지만,

그의 관찰은 오늘날까지

PD의 주요 병리학적 진단 기준으로 남아 있습니다.1

현재

신경전달물질 도파민을 생성하는 것으로 알려진

흑질 색소 신경세포의 손실은

PD의 전형적인 운동 증상을 유발하며

이 질환과 관련된 유일한 강력한 임상 병리학적 상관관계를 구성하고 있습니다.1

이 뉴런에 포함된 색소는

피부 멜라닌과 비슷한 모양으로 인해

뉴로멜라닌이라고 불리며,

인간 뇌의 SN에 매우 풍부하여

이 구조를 거시적으로 어두운 영역으로 볼 수 있습니다(따라서 이 뇌 영역에 주어진 이름의 유래). 2

인간의 뇌에서 색소 뉴런이 소실되는 것은 오랫동안 파킨슨병(PD)의 특징이었습니다. 도파민 뉴런의 시냅스 전 말단에 있는 뉴로멜라닌(NM)은 PD를 비롯한 신경 퇴행성 질환의 주요 원인으로 떠오르고 있습니다. 이 미니 리뷰에서는 뉴로멜라닌과 시냅스 말단의 여러 분자 간의 상호작용에 대해 설명하고 이러한 상호작용이 PD를 포함한 신경 퇴행성 질환에 어떤 영향을 미치는지 설명합니다. 뉴로멜라닌은 가역적으로 아민을 함유한 신경독소( 예: MPTP)와 결합하고 상호작용하여 말단에서 그 작용을 강화함으로써 결국 산화 스트레스와 미토콘드리아 기능 장애로 인해 멜라닌 함유 뉴런의 불안정성과 퇴행을 유발할 수 있습니다. 특히 뉴로멜라닌은 파이 접합 시스템에서 철과 결합된 헴 유사 구조의 큰 싱크대를 제공함으로써 화학 독성에 대한 감수성을 부여하는 것으로 보이며, 이 시스템은 파이 스택과 철에 대한 리간드 결합을 포함한 상호작용을 안정화하기 위한 것으로 보입니다. 나이가 들면서 도파민 합성 경로의 명백한 감소와 함께 NM이 점진적으로 축적되는 것을 고려할 때, NM이 이 세포에서 사용되는 주요 기능성 모노아민인 도파민과도 결합할 수 있는지에 대한 즉각적인 의문이 제기되어야 합니다. 이 발견이 시사하는 바가 매우 큼에도 불구하고 이 아이디어는 적절히 다루어지지 않은 것으로 보입니다. 따라서 우리는 도파민이 뉴로멜라닌에서 분리될 수 있는 잠재적 메커니즘과 이러한 가역적 관계의 의미에 대해 가정합니다. 흥미롭게도 뉴로멜라닌이 막 결합 소포에서 도파민을 격리하고 방출할 수 있다면, 이 세포 내 시냅스 전 메커니즘은 도파민 뉴런의 화학적 기억의 한 형태가 될 수 있습니다.

나중에

다른 뇌 영역의 다른 뉴로멜라닌 함유 뉴런도

PD에서 지속적으로 퇴화되는 것으로 밝혀졌으며,

여기에는 미주체의 흑질핵과 등쪽 운동핵의 노르아드레날린성 뉴런이 포함되어

질병의 특징적인 비운동 증상을 유발합니다.3, 4

대조적으로,

비 멜라닌 화 된 뇌 영역의 신경 세포 손실은

일관성이 없거나 PD에 특이적이지 않거나

상호 연결된 뉴로 멜라닌 함유 뉴런의 손실에 이차적으로 나타납니다 .5-7

정상적인 인간 중뇌에서 도파민을 생성하는 세포 그룹은

뉴로멜라닌 색소 뉴런의 비율 측면에서

서로 현저하게 다릅니다.8-10

PD에서 이러한 그룹의 추정 세포 손실은

일반적으로 존재하는 뉴로멜라닌 색소 뉴런의 비율과 직접적인 상관관계가 있습니다.8-10

마찬가지로,

각 뉴로멜라닌 함유 세포 그룹 내에서

강하게 멜라닌화된 뉴런보다

약하게 색소화된 뉴런이 상대적으로 더 많이 남아 있습니다.8

예를 들어,

PD에서 카테콜아민성 뉴런의 손실은

거의 모든 뉴런이 색소화된 SN 파스 콤팩타(SNpc)에서 심각하고

대부분의 카테콜아민성 뉴런이 색소가 없는 중앙 회색 물질에서는 거의 감지할 수 없습니다.9

색소성 및 비색소성 카테콜아민성 뉴런을 비슷한 비율로 포함하는

중뇌의 주변 및 후배엽 영역에서

멜라닌화 된 카테콜아민성 뉴런의 인구는 크게 감소하는 반면

뉴로멜라닌이 없는 카테콜아민성 뉴런의 전체 인구는 영향을받지 않습니다 .9

마찬가지로,

PD에서 대부분 보존되는 복부 분절 영역의 도파민성 뉴런은

일생 동안 최소한의 뉴로멜라닌을 생성합니다 .8, 11

세포 손실 외에 PD의 병리학 적 특징을 구성하는 고전적인 루이체 (LB),

즉 α- 시누클레인 함유 세포질 내 내포물 및 창백한 몸이라고하는 추정 된 전구체 구조는

일반적으로 뉴로 멜라닌이 축적되는 세포질의 세포 내 영역 내에 나타나고

이 색소와 밀접한 물리적 연관성을 형성합니다 .12

이러한 관찰과 일치하는 인간의 뇌에 대한 연구에 따르면

α-시누클레인은 PD의 초기 단계에서

뉴로멜라닌 색소로 재분배되고

뉴로멜라닌 과립 내에 포획됩니다.13-15.

PD 신경병리와 뉴로멜라닌을 더욱 연결시켜 보면,

선천성(즉, 미세아교증) 및 적응성(즉, 림프구 침윤) 면역 반응의 활성화와 같은

PD 뇌에서 발생하는 신경 염증성 변화는

뉴로멜라닌 함유 영역 내에서 매우 국소화되고 비멜라닌화 영역에서는 드물게 관찰됩니다.16

예를 들어,

죽어가는 뉴런에서 방출된 세포 외 뉴로멜라닌은

PD 뇌의 미세아교세포를 활성화하여

이 색소를 포식하고 분해/제거합니다.17

또한

PD 뇌의 뉴로멜라닌 함유 SNpc 뉴런은

신경세포 손실과 직접적으로 연관된 방식으로

면역글로불린 G 축적 및 항원성 주요 조직 적합성 복합체 클래스 I 발현이 나타납니다.18

또한

PD 환자의 혈청에서 항멜라닌 항체가 증가합니다.19

따라서

PD 발병 기전은

뉴로멜라닌의 존재와 불가분의 관계에 있는 것으로 보입니다.

파킨슨병(PD)은 노화와 관련된 질환으로 알츠하이머병 다음으로 두 번째로 흔한 신경 퇴행성 질환입니다. PD의 주요 증상은 흑질질 DA 뉴런의 세포 사멸로 인한 선조체의 신경전달물질 도파민(DA) 결핍과 함께 운동 장애를 동반합니다. PD에는 두 가지 주요 조직 병리학적인 특징이 있습니다. 주로 α-시누클레인 단백질로 구성된 루이체라고 하는 세포질 내포체와, 이 올리고머가 잘못 접혀 생성되는 신경 독성이 있어 DA 세포 사멸을 유발하는 것으로 알려진 뉴로멜라닌(NM)이라고 하는 검은 색소가 DA 신경세포에 포함되어 있고 PD에서 현저하게 감소하는 것입니다. 피부의 멜라닌 합성은 티로시나아제에 의한 도파퀴논(DOPAquinone, DQ)을 통해 이루어지는 반면, DA 뉴런에서의 NM 합성은 티로신 하이드 록실라제(TH)와 방향족 L- 아미노산 탈 카르 복실 라제(AADC)에 의한 DA퀴논(DAQ)을 통해 이루어지는 것으로 간주되어 멜라노 세포에서의 멜라닌 합성과 유사한 것으로 알려져 있습니다. 세포질의 DA는 반응성이 매우 높으며 자연적으로 또는 미확인된 티로시나아제에 의해 DAQ로 산화되어 NM으로 합성되는 것으로 추정됩니다. 특정 임계값 이상의 세포 내 NM 축적은 DA 뉴런 사멸 및 PD 표현형과 관련이 있는 것으로 보고되었습니다. 이 리뷰에서는 PD에서 NM의 생합성 및 병리 생리학에 대한 최근의 진전을 보고합니다.

그러나

뉴로멜라닌과 PD 사이의 긴밀하고 오랜 연관성에도 불구하고

뉴로멜라닌의 생리적 중요성과 PD 발병에 대한 잠재적 기여도는

인간과 달리 설치류와 같이 실험 연구에 일반적으로 사용되는 실험동물 종에는 뉴로멜라닌이 없기 때문에

아직 알려지지 않았습니다.

뉴로멜라닌은

실제로 원숭이,20, 21 돌고래,22 개구리 등 다양한 종에 존재하지만,23

뇌간에서 매우 풍부한 양의 뉴로멜라닌은

거시적 수준에서 이 뇌 영역의 어두운 색소 침착이 다른 동물 종에서는 명백하게 관찰되지 않기 때문에

인간에게만 고유한 것으로 보입니다.20

실제로 포유류의 뇌에서 뉴로멜라닌 축적은 인간과의 진화적 관계가 가까워질수록 점진적으로 증가하며,20 인간은 자연적으로 PD가 발생하는 유일한 종입니다.24

결과적으로

뉴로멜라닌과 같이 PD와 밀접한 관련이 있는 인자는

지금까지 질병의 생체 내 실험 패러다임에서 놀랍게도 소홀히 여겨져 왔습니다.

Synthesis of Neuromelanin: Mechanisms and Significance

In contrast to the widespread distribution of other brain pigments such as lipofuscin, neuromelanin is restricted to catecholamine-producing regions of the brain and forms only in neurons. Neuromelanin first becomes observable in the human SNpc at approximately 3 years of age and progressively accumulates over time within the cells in which it has been produced, as neurons apparently lack the mechanisms for degrading or eliminating this pigment.25, 26 As a consequence, intracellular neuromelanin builds up with age until occupying most of the neuronal cytoplasm.25 Although aging is the main risk factor for developing PD,27 the molecular substrate linking PD with aging is currently unknown.

Melanins (from the Greek word melanos [“black”]) are a group of complex, brown-black pigmented biopolymers that include eumelanin (the pigment associated with dark hair and skin), pheomelanin (characteristic of red and blond hair), and neuromelanin (ie, brain melanin, which is thought to contain a pheomelanin core and eumelanin surface28). Melanins derive from a complex biosynthetic pathway initiated with the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (l-dopa). After this common step, l-dopa serves as a precursor to both melanins and catecholamines, acting along separate pathways. Although the synthesis of peripheral melanin pigments (eg, in skin and hair) is relatively well understood, the mechanism leading to neuromelanogenesis is still a matter of speculation. Both neuromelanin and peripheral melanins are produced as oxidative products downstream from l-dopa. In fact, neuromelanin synthesis is regarded as a protective antioxidant mechanism to remove potentially toxic oxidized dopamine species, such as quinones and semiquinones, by their conversion into neuromelanin.29 However, although peripheral melanogenesis occurring within specialized cells (ie, melanocytes) is known to result from an enzymatically driven biosynthetic pathway in which tyrosinase is the key, rate-limiting enzyme,30 it is widely assumed that neuromelanin is instead produced by spontaneous nonenzymatic dopamine auto-oxidation.31 Several observations argue, however, against the latter concept. For instance, the distribution of neuromelanin does not fully match that of the dopamine-producing enzyme tyrosine hydroxylase (TH), with many dopamine and noradrenergic neurons completely lacking neuromelanin.8, 32-34 In addition, neuromelanin is not observed in most animal species despite the presence of dopamine and other catecholamines in these animals.20 Although this could be related to differences in lifespan or rate of catecholamine synthesis between species, experimentally induced increases in dopamine and/or oxidized dopamine in mice and rats, achieved either with chronic l-dopa treatment35, 36 or by genetically enhancing TH activity,37 are not sufficient by themselves to produce neuromelanin in these animals, as might be expected if neuromelanin represents a mere process of auto-oxidized dopamine. Along this line, it is worth noting that George C. Cotzias's initial use of l-dopa in PD patients was apparently aimed at restoring neuromelanin levels, and not dopamine, as he thought that PD might result from the loss of the neuromelanin pigment in the SNpc.38 This approach, however, failed to reinstate the missing pigment in PD brains (but it provided instead a dramatic antiparkinsonian effect by replacing the depleted dopamine, thus establishing a revolutionary new treatment for PD, although apparently for the wrong reason).38

The identification of specific phases and changes in the rate of neuromelanin production over time in humans25, 26 suggests the regulation of neuromelanin production and turnover, possibly through enzymatic processes,26 as is the case for all other melanin pigments.39 Interestingly, tyrosinase expression‎ might not be restricted to melanocytes but could also be present, although at very low levels, in the brain, including the human SNpc.40-43 For instance, brain tyrosinase messenger ribonucleic acid (mRNA) expression‎ has been reported in human SN by reverse transcription polymerase chain reaction/real-time polymerase chain reaction, although at barely detectable levels.41-43 Similarly, 3 different microarray experiments in human brain tissue (Entrez_id: 7299; Allen Brain Institute Seattle, Washington), nonhuman primate brain tissue (Entrez_id: 705792; National Institutes of Health [NIH] Blueprint Non-human Primate [NHP] Atlas), and developing human prenatal tissue (Entrez_id: 7299; BrainSpan Atlas of the developing human brain) detected the presence of some tyrosinase transcripts in the brain. Consistent with the very low levels at which tyrosinase mRNA might be expressed, if at all, in the brain, the actual protein has not been detected in the human brain when using immunohistochemistry or Western blot.44, 45 Similarly, mass spectrometry techniques failed to detect significant amounts of typical enzymes and proteins involved in melanogenesis, including tyrosinase, in isolated neuromelanin-containing organelles from human SN.15, 46, 47 In contrast, tyrosinase enzymatic activity has been reported in brain extracts from human SN,41 although at 100,000 times lower than in melanocytes.48 These inconsistent results could be potentially attributed to the very low levels at which tyrosinase might be actually expressed, if at all, in the brain and further experiments are thus warranted to unambiguously demonstrate or refute the existence of tyrosinase in the human brain. Although it is currently unknown whether tyrosinase may contribute to neuromelanin synthesis, this possibility is theoretically conceivable because tyrosinase not only mediates the hydroxylation of tyrosine and the oxidation of l-dopa necessary for the formation of peripheral melanins but also is able to oxidize dopamine's catechol ring, which is an essential event required for neuromelanin synthesis.49 In addition, a rare loss-of-function mutation in tyrosinase linked to albinism has been recently associated with an increased risk for PD, which has been attributed to a possible inability of this tyrosinase variant to synthesize neuromelanin in these patients and the subsequent accumulation of potentially toxic dopamine-derived species that cannot be detoxified by conversion into neuromelanin.50 On the other hand, arguing against a potential role of tyrosinase in neuromelanin synthesis, the presence of neuromelanin was reported in the brains of 2 subjects with albinism,51 which are usually assumed to lack tyrosinase activity. However, that report was published in the pregenetics diagnostic era and, in absence of specific information about the genetic type of albinism suffered by these cases, it cannot be excluded that residual tyrosinase activity was actually present in these subjects.52 Indeed, because of the clinical overlap between oculocutaneous albinism (OCA) subtypes, molecular diagnosis is necessary to establish the specific gene defect and thus the OCA subtype. Among the most common types of albinism, only the OCA1 subtype is actually caused by mutations in the tyrosinase gene.53 In turn, within OCA1 subjects, tyrosinase activity is completely abolished only in the OCA1A subtype while in OCA1B some enzymatic activity still remains, allowing some accumulation of melanin pigment over time.53 In this context, at least one of the subjects examined by Foley and Baxter was diagnosed with “partial albinism,”51 implying that some residual tyrosinase activity could have been present in these brains. Therefore, whether tyrosinase or other related enzymes may contribute to neuromelanin synthesis remains an open question.

Modeling Neuromelanin Production In Vivo: Age-Dependent Intracellular Neuromelanin Accumulation Drives PD Pathology

To assess the potential role of neuromelanin in PD pathogenesis, it would first be necessary to develop an in vivo model of human-like neuromelanin production, up to the abundant levels reached in elder humans, as no such animal model currently exists, experimentally or naturally. Considering the failure by previous studies to produce neuromelanin in vivo solely by increasing the levels of dopamine and oxidized dopamine,35-37 we recently opted as a potential alternative strategy for overexpressing human tyrosinase (hTyr) via a viral vector in the SNpc of both rats and mice, which normally lack neuromelanin.43 Strikingly, hTyr-overexpressing rodents exhibited an age-dependent production and accumulation of a neuromelanin-like pigment within nigral dopaminergic neurons that was virtually analogous to human neuromelanin43 (henceforth referred to as neuromelanin; see Fig. 1). Similar to humans, neuromelanin from hTyr-overexpressing rodents (1) could be visualized with the naked eye as a darkened SNpc area43 (Fig. 1A); (2) was detected as an hyperintense area by neuromelanin-sensitive high-resolution T1-weighted magnetic resonance imaging (MRI)43 (Fig. 1B); (3) accumulated progressively over time within SNpc dopaminergic neurons until occupying most of their neuronal cytoplasm, reaching levels consistent with those seen in elderly humans43 (Fig. 1C); (4) stained prominently with the melanin marker Masson-Fontana, which reflects the ability of neuromelanin to chelate metals43 (Fig. 1D); and (5) was encapsulated within autophagic structures exhibiting, at the ultrastructural level, an electron-dense matrix associated with characteristic lipid droplets43 (Fig. 1E). The striking resemblance between human neuromelanin and neuromelanin from hTyr-overexpressing rodents, combined with the fact that hTyr overexpression‎ is sufficient in itself to produce neuromelanin in these animals (as opposed to the lack of neuromelanin by solely increasing dopamine/oxidized dopamine levels), supports the possibility that brain tyrosinase or other related enzymes might potentially contribute to neuromelanin formation in humans. Whatever the case may be, these animals represent the first experimental in vivo model of the age-dependent production and accumulation of human-like neuromelanin within PD-vulnerable nigral neurons at levels up to those reached in elderly humans. The model thus constitutes a unique experimental tool enabling assessment of the possible consequences of neuromelanin production/accumulation on neuronal function and viability.

뉴로멜라닌의 합성: 메커니즘과 중요성

리포푸신과 같은 다른 뇌 색소가 광범위하게 분포하는 것과 달리

뉴로멜라닌은

뇌의 카테콜아민 생성 영역으로 제한되어 있으며

뉴런에서만 형성됩니다.

뉴로멜라닌은

약 3세에 인간의 SNpc에서 처음 관찰되기 시작하여

시간이 지남에 따라 생성된 세포 내에서 점진적으로 축적되는데,

뉴런은 이 색소를 분해하거나 제거하는 메커니즘이 없는 것으로 보입니다.25, 26

결과적으로

세포 내 뉴로멜라닌은 나이가 들면서 축적되어

신경 세포질의 대부분을 차지합니다.25

노화가

PD 발병의 주요 위험 요인이지만,27

PD와 노화를 연결하는 분자 기질은 현재 밝혀져 있지 않습니다.

멜라닌(그리스어 멜라노스 ["검은색"]에서 유래)은

유멜라닌(어두운 모발과 피부와 관련된 색소),

페오멜라닌(붉은색과 금발 모발의 특징),

뉴로멜라닌(뇌 멜라닌, 즉 페오멜라닌 코어와 유멜라닌 표면으로 구성된 것으로 생각되는 뇌 멜라닌28)을 포함하는

복잡한 갈색-검정색 생체고분자 그룹입니다.

멜라닌은

L-티로신이 L-디하이드록시페닐알라닌(l-도파)으로 수산화되면서 시작되는

복잡한 생합성 경로에서 파생됩니다.

이 일반적인 단계를 거친 후, l-도파는 멜라닌과 카테콜아민의 전구체 역할을 하며 별도의 경로를 따라 작용합니다. 말초 멜라닌 색소(예: 피부와 모발)의 합성은 비교적 잘 알려져 있지만, 신경 멜라닌 생성으로 이어지는 메커니즘은 여전히 추측의 대상입니다.

뉴로멜라닌과 말초 멜라닌은

모두 l-도파의 하류에서 산화 산물로 생성됩니다.

실제로

뉴로멜라닌 합성은

퀴논 및 세미퀴논과 같이 잠재적으로 독성이 있는 산화된 도파민 종을

뉴로멜라닌으로 전환하여 제거하는

보호 항산화 메커니즘으로 간주됩니다.29

그러나

특수 세포(즉, 멜라닌 세포) 내에서 발생하는

말초 멜라닌 생성은 티로시나아제가

핵심적인 속도 제한 효소인 효소 주도 생합성 경로에서 발생하는 것으로 알려져 있지만,30

뉴로멜라닌은 대신 자발적인 비효소적 도파민 자가 산화에 의해 생성된다고

널리 추정됩니다.31

그러나 후자의 개념에 반대하는 여러 관찰 결과도 있습니다.

예를 들어,

뉴로멜라닌의 분포는

도파민 생성 효소인 티로신 하이드 록실 라제(TH)의 분포와 완전히 일치하지 않으며,

많은 도파민 및 노르아드레날린성 뉴런에는

뉴로멜라닌이 전혀 없습니다.8, 32-34

또한 대부분의 동물 종에서 도파민 및 기타 카테콜아민이 존재함에도 불구하고 뉴로멜라닌은 관찰되지 않습니다.20 이는 종간 수명 또는 카테콜아민 합성 속도의 차이와 관련이 있을 수 있지만, 실험적으로 유도된 생쥐와 쥐의 도파민 및/또는 산화 도파민의 증가는 만성 l-도파 치료35, 36 또는 유전적으로 TH 활동을 강화함으로써 달성되며,37 뉴로멜라닌이 단순히 도파민의 자가 산화 과정을 나타내는 경우 예상할 수 있는 것처럼 이들 동물에서 뉴로멜라닌을 생성하기에 충분하지 않습니다. 이러한 맥락에서, 조지 C. 코치아스가 PD 환자에게 처음에 l-dopa를 사용한 것은 도파민이 아닌 뉴로멜라닌 수치를 회복하기 위한 것이었음을 주목할 필요가 있습니다. 그는 PD가 SNpc에서 뉴로멜라닌 색소의 손실로 인해 발생할 수 있다고 생각했기 때문입니다.38 그러나 이 접근법은 PD 뇌에서 사라진 색소를 복원하는 데는 실패했지만, 대신 고갈된 도파민을 대체하여 극적인 항파킨슨 효과를 제공함으로써 비록 잘못된 이유에서 비롯된 것이지만 PD에 대한 혁신적인 새로운 치료법을 확립했습니다.38

인간에서 시간에 따른 뉴로멜라닌 생성 속도의 특정 단계와 변화를 확인한 결과25,26 다른 모든 멜라닌 색소의 경우와 마찬가지로 효소 과정을 통해 뉴로멜라닌 생성 및 전환이 조절될 수 있음을 시사합니다.39 흥미롭게도 티로시나제 발현은 멜라닌 세포에만 국한되지 않고 매우 낮은 수준이지만 인간 SNpc를 포함한 뇌에도 존재할 수 있습니다.40-43 예를 들어, 역전사 중합효소 연쇄 반응/실시간 중합효소 연쇄 반응에 의해 인간 SN에서 뇌 티로시나제 메신저 리보핵산(mRNA) 발현이 거의 검출할 수 없는 수준이지만 보고되었습니다.41-43 마찬가지로, 인간 뇌 조직(Entrez_id: 7299; 워싱턴주 시애틀의 Allen Brain Institute), 비인간 영장류 뇌 조직(Entrez_id: 705792; 국립보건원[NIH] 청사진 비인간 영장류[NHP] 아틀라스), 발달 중인 인간 태아 조직(Entrez_id: 7299; 발달 중인 인간 뇌의 BrainSpan Atlas)에서 뇌에 일부 티로시나제 전사체의 존재를 감지했습니다. 면역조직화학이나 웨스턴 블롯을 사용할 때 티로시나아제 mRNA가 뇌에서 발현될 수 있는 매우 낮은 수준과 일치하는 실제 단백질은 인간의 뇌에서 검출되지 않았습니다.44, 45 마찬가지로, 질량 분석 기법은 인간 SN.15에서 분리된 뉴로멜라닌 함유 소기관에서 티로시나아제를 포함하여 멜라닌 형성에 관여하는 전형적인 효소와 단백질의 상당량을 검출하지 못했습니다, 46, 47 대조적으로, 인간 SN의 뇌 추출물에서 티로시나아제 효소 활성이 보고되었는데,41 비록 멜라닌 세포보다 10만 배 낮은 수준이지만.48 이러한 일관되지 않은 결과는 티로시나아제가 실제로 뇌에서 발현될 수 있는 매우 낮은 수준 때문일 수 있으며, 따라서 인간 뇌에서 티로시나아제의 존재를 명확하게 입증하거나 반박하기 위해서는 추가 실험이 필요할 수 있습니다. 현재 티로시나아제가 뉴로멜라닌 합성에 기여할 수 있는지는 알려지지 않았지만, 티로시나아제는 말초 멜라닌 형성에 필요한 티로신의 수산화와 l-도파의 산화를 매개할 뿐만 아니라 뉴로멜라닌 합성에 필수적인 도파민의 카테콜 고리를 산화할 수 있기 때문에 이론적으로 이러한 가능성을 생각할 수 있습니다.49 또한, 최근 백색증과 관련된 티로시나아제의 드문 기능 상실 돌연변이가 PD 위험 증가와 관련이 있는데, 이는 이러한 환자에서 이 티로시나아제 변이가 뉴로멜라닌을 합성하지 못하고 뉴로멜라닌으로 전환하여 해독할 수 없는 잠재적 독성 도파민 유래 종의 축적이 원인으로 추정되고 있습니다.50. 한편, 뉴로멜라닌 합성에 있어 티로시나아제의 잠재적 역할에 대한 반론으로, 일반적으로 티로시나아제 활성이 결여된 것으로 추정되는 백색증 환자 2명의 뇌에서 뉴로멜라닌의 존재가 보고되었습니다,51. 그러나 이 보고는 유전학 진단 전 시대에 발표된 것으로, 이 사례들이 앓고 있는 백색증의 유전적 유형에 대한 구체적인 정보가 없는 상황에서 이들 피험자에게 실제로 잔류 티로시나아제 활성이 존재했을 가능성을 배제할 수 없습니다.52 실제로 안구 피부 백색증(OCA) 아형 간의 임상적 중첩으로 인해 특정 유전자 결함과 따라서 OCA 아형을 확인하려면 분자 진단이 필요합니다. 가장 흔한 유형의 백색증 중 OCA1 아형만이 실제로 티로시나아제 유전자의 돌연변이로 인해 발생합니다.53 OCA1 피험자 내에서는 OCA1A 아형에서만 티로시나아제 활동이 완전히 소멸되는 반면 OCA1B에서는 일부 효소 활동이 여전히 남아 있어 시간이 지나면서 멜라닌 색소가 일부 축적될 수 있습니다.53 이러한 맥락에서 폴리와 박스터가 검사한 피험자 중 적어도 한 명은 "부분 백색증"51 진단을 받았으며, 이는 이러한 뇌에 일부 잔류 티로시나아제 활동이 존재할 수 있음을 의미합니다. 따라서 티로시나아제 또는 기타 관련 효소가 뉴로멜라닌 합성에 기여할 수 있는지 여부는 아직 미해결 과제로 남아 있습니다.

생체 내 뉴로멜라닌 생성 모델링: 연령에 따른 세포 내 뉴로멜라닌 축적이 PD 병리를 유발합니다.
PD 발병에서 뉴로멜라닌의 잠재적 역할을 평가하려면 먼저 인간과 유사한 뉴로멜라닌 생성의 생체 내 모델을 개발해야 하는데, 현재 실험적으로나 자연적으로 그러한 동물 모델이 존재하지 않기 때문에 노인에서 풍부한 수준까지 뉴로멜라닌을 생성할 수 있는 생체 내 모델을 개발하는 것이 필요합니다. 이전 연구에서 도파민과 산화 도파민의 수준을 높이는 것만으로 생체 내에서 뉴로멜라닌을 생성하는 데 실패한 것을 고려하여35-37 최근에는 일반적으로 뉴로멜라닌이 부족한 쥐와 생쥐의 SNpc에서 바이러스 벡터를 통해 인간 티로시나아제(hTyr)를 과발현하는 전략을 대안으로 선택했습니다.43 놀랍게도, hTyr 과발현 설치류는 연령에 따라 흑색 도파민 신경세포 내에서 인간 뉴로멜라닌과 거의 유사한 뉴로멜라닌 유사 색소43 (이후 뉴로멜라닌이라고 함, 그림 1 참조)가 생성 및 축적되는 것으로 나타났습니다. 인간과 마찬가지로, hTyr 과발현 설치류의 뉴로멜라닌은 (1) 육안으로 어두운 SNpc 영역43으로 시각화할 수 있었고 (2) 뉴로멜라닌에 민감한 고해상도 T1 가중 자기공명영상(MRI)43에서 초강력 영역으로 검출되었습니다(그림 1A); (3) 뉴로멜라닌에 민감하게 반응하는 자기공명영상(MRI)43에서 뉴로멜라닌이 검출되었습니다(그림 1B). 1B); (3) SNpc 도파민 성 뉴런 내에서 시간이 지남에 따라 점진적으로 축적되어 대부분의 신경 세포질을 차지할 때까지 노인에서 볼 수있는 수준에 도달했습니다43 (그림. 1C); (4) 뉴로멜라닌이 금속을 킬레이트화하는 능력을 반영하는 멜라닌 마커 Masson-Fontana로 두드러지게 염색됨43 (그림 1D); (5) 초구조 수준에서 특징적인 지질 방울과 관련된 전자 밀도 매트릭스를 나타내는 자가포식 구조 내에 캡슐화됨43 (그림 1E). 인간의 뉴로멜라닌과 hTyr 과발현 설치류의 뉴로멜라닌 사이의 놀라운 유사성은 도파민/산화된 도파민 수치만 증가시켜 뉴로멜라닌이 부족한 경우와 달리, hTyr 과발현 자체만으로도 이러한 동물에서 뉴로멜라닌을 생성하기에 충분하다는 사실과 함께 뇌 티로시나제 또는 기타 관련 효소가 인간에서 뉴로멜라닌 생성에 기여할 가능성을 뒷받침해 줍니다. 어떤 경우이든, 이 동물은 PD에 취약한 흑질 뉴런 내에서 인간과 유사한 뉴로멜라닌이 노인의 수준까지 연령에 따라 생성 및 축적되는 최초의 생체 내 실험 모델입니다. 따라서 이 모델은 뉴로멜라닌 생성/축적이 신경세포 기능 및 생존력에 미칠 수 있는 결과를 평가할 수 있는 독특한 실험 도구가 됩니다.

Figure 1

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Human-like neuromelanin production in tyrosinase-overexpressing rats. Representative images from elderly human control brains (top) and brains from rats unilaterally injected with an adeno-associated viral (AAV) vector expressing human tyrosinase (hTyr) above the right substantia nigra pars compacta (SNpc; bottom). Humans and AAV-hTyr-injected rats exhibit analogous neuromelanin production from a macroscopic (A,B), microscopic (C,D), and ultrastructural (E) point of view. No neuromelanin is observed in the contralateral (noninjected) hemisphere of AAV-hTyr-injected rats, as rodents normally lack this pigment. (A) Unstained midbrain (dashed outline, melanized SNpc). (B) Neuromelanin-sensitive high-resolution T1-weighted magnetic resonance imaging (dashed outline, melanized SNpc visualized as an hyperintense area). (C) Hematoxylin-eosin stained 5-μm-thick SNpc sections (unstained neuromelanin is seen in brown). (D) Masson-Fontana melanin staining in 5-μm-thick SNpc sections (neuromelanin is seen in dark brown). (E) Electron microscopy (neuromelanin, electron dense matrix; asterisks, associated lipid droplets). Scale bars, 12.5 μm (C,D), 500 nm (E). Adapted from ref. 43.

In this context, one of the main questions to be addressed concerns how a neuron containing very high levels of intracellular neuromelanin, to the point where the neuromelanin occupies most of the neuron's cytoplasm, can function properly. The answer is, in fact, that it cannot. Indeed, it was found in hTyr-overexpressing rodents that the progressive intracellular build-up of neuromelanin ultimately compromised neuronal function when allowed to accumulate above a specific threshold, eventually triggering in an age-dependent manner the main pathological features of PD, including hypokinesia, LB-like inclusion formation and nigrostriatal neurodegeneration.43 By ~4 months, nigrostriatal denervation in hTyr-overexpressing rodents was equivalent to that seen in early PD patients (<10 years’ evolution), and by ~2 years the extent of denervation reached levels comparable to those seen in advanced PD (>20 years’ evolution).43, 54 In parallel to dopaminergic cell death, these animals exhibited other neuropathological features typical of aged and PD human brains, including (1) extracellular neuromelanin, released from dying neurons43; (2) neuronophagia (ie, extracellular neuromelanin surrounded by, or within, activated microglia), indicative of an active, ongoing neurodegenerative process43; and (3) perivascular neuromelanin, resulting from the migration of neuromelanin-filled microglia to blood vessels to remove extracellular neuromelanin from the brain.43

Prior to degeneration, neuromelanin-filled neurons from hTyr-overexpressing rodents exhibited early signs of neuronal dysfunction similar to those occurring in PD patients, including impaired dopamine release, axonal swelling, decreased striatal levels of dopamine transporter, and phenotypic loss of TH expression‎, all of which was accompanied by early motor deficits in these animals.43 The occurrence of neuronal dysfunction before overt cell death may have important therapeutic implications, as it provides a therapeutic window in which neuronal function could be potentially restored before the actual loss of the cell. Interestingly, PD-type inclusion body formation in hTyr-overexpressing rodents, comprising both pale bodies–like and LB-like structures exclusively observed within neuromelanin-filled neurons, peaked at the time of early neuronal dysfunction and was substantially reduced once neurodegeneration was established.43 This observation suggest that inclusion-containing neurons are those that become dysfunctional and preferentially degenerate in these animals. Consistent with this, inclusion-containing neurons in both humans55 and hTyr-overexpressing rodents43 often exhibit TH downregulation, which reflects neuronal dysfunction at early stages of neurodegeneration.8 In addition, similar to hTyr-overexpressing rodents, the number of neuronal inclusions in PD brains at advanced stages of the disease is much lower than that observed in cases involving early PD.16

Overall, the unprecedented use of neuromelanin-producing hTyr-overexpressing rodents revealed that age-dependent neuromelanin production within SNpc dopaminergic neurons is associated with neuronal dysfunction and progressive nigrostriatal neurodegeneration, equivalent to that occurring in PD patients, once a certain threshold of intracellular neuromelanin accumulation is reached (Fig. 2). Relevant to humans, intracellular neuromelanin levels reach this threshold in both PD patients and subjects with incidental LB disease (ie, clinically healthy individuals exhibiting LB pathology at autopsy who are considered to represent early, presymptomatic stages of PD).43 In contrast, in healthy elderly individuals, intracellular neuromelanin levels remain below this threshold.43 Although previous studies reported a decrease in neuromelanin levels in dopaminergic neurons from PD patients,10 this was observed only in those neurons in the final stages of degeneration.13 In contrast, in adjacent, vulnerable (but yet morphologically intact) dopaminergic neurons, the neuromelanin density was augmented,13 further supporting the notion that increased intracellular neuromelanin levels may precede dopaminergic cell death and predispose these neurons to degeneration in PD. Please note that in these studies intracellular neuromelanin levels were measured by optical densitometry within individual neuromelanin-containing neurons,10, 13, 43 as opposed to regional quantifications of total SNpc neuromelanin,56 the latter being dramatically decreased in PD brains because of the massive loss of neuromelanin-containing dopaminergic neurons.56 Supporting a pathogenic role for intracellular neuromelanin accumulation when allowed to accumulate above a specific threshold within individual neurons, the lowering of intracellular neuromelanin to levels below this pathogenic threshold in hTyr-overexpressing rodents, in part by promoting the release of intracellular neuromelanin to the outside of the cell (see the next section for details), diminished PD-linked inclusion formation, attenuated nigrostriatal neurodegeneration, and reversed hypokinesia in these animals.43 These results demonstrate the feasibility and therapeutic potential of modulating intracellular neuromelanin levels in vivo and indicate that strategies to maintain or decrease intracellular neuromelanin to levels below its pathogenic threshold may provide unprecedented therapeutic opportunities to prevent, halt, or delay neuronal dysfunction and degeneration linked to PD and brain aging (Fig. 2).

Figure 2

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Putative pathogenic threshold of intracellular neuromelanin accumulation. (Top) Age-dependent intracellular neuromelanin accumulation in human tyrosinase (hTyr)–overexpressing rodents is associated with Parkinson's disease–like neuronal dysfunction and degeneration when reaching a specific threshold of accumulation (dotted red line)43. In aged postmortem brains from healthy individuals intracellular neuromelanin levels are maintained below this pathogenic threshold while in age-matched brains from Parkinson's disease patients and pre–Parkinson's disease (ie, incidental Lewy Body disease) subjects intracellular neuromelanin levels appear above this pathogenic threshold.43 Please note that such a pathogenic threshold would be valid regardless of the actual mechanism of neuromelanin synthesis (whether enzymatic or nonenzymatic). (Bottom) Neuronal “rejuvenation” strategies aimed at reducing intracellular levels of neuromelanin back to younger states (ie, below the pathogenic threshold of neuromelanin accumulation) should prevent, halt, or delay neuronal dysfunction and degeneration linked to both Parkinson's disease and brain aging. Because of the putative protective role of neuromelanin synthesis (ie, to remove potentially toxic dopamine-derived oxidative species from the cytosol), such strategies should probably favor the elimination of neuromelanin once it has already been produced (for instance, by promoting the exocytosis of neuromelanin-filled lysosomal/autophagic structures)43 instead of inhibiting neuromelanin synthesis per se. Photomicrographs correspond to hematoxylin-eosin stained human brain sections illustrating dopaminergic nigral neurons with high or low levels of intracellular neuromelanin.

Mechanisms of Neuromelanin-Linked Neurodegeneration

The oxidation of dopamine to form neuromelanin generates many o-quinones, which can be potentially toxic to neurons31 (Fig. 3). Indeed, dopamine oxidation has long been proposed as a leading pathogenic factor in PD (for a review on this specific topic, see, for instance, ref. 31). Therefore, the pathological PD phenotype observed in neuromelanin-producing hTyr-overexpressing rodents could potentially result from the production of toxic dopamine-derived intermediate species by overexpressed hTyr, with neuromelanin accumulation in these animals simply representing an inert by-product of these reactions. However, hTyr-overexpressing rodents showed no significant accumulation of oxidized dopamine species, either preceding or concomitant with neuronal dysfunction/degeneration.43 In fact, the significant accumulation of such potentially toxic species in these animals was actually prevented by their continuous conversion into neuromelanin,43 this being consistent with a putative protective antioxidant role of neuromelanin synthesis.31 Further arguing against a major pathogenic contribution of dopamine-mediated toxicity in hTyr-overexpressing rodents, it has been recently reported by 2 independent groups that dopamine-induced toxicity in rodents, either by chronic l-dopa treatment35 or by enhancing TH activity,37 is only observed in animals displaying additional PD-related alterations, such as DJ-1 deficiency35 or overexpression‎ of PD-linked A53T mutant α-synuclein,37 but not in regular wild-type animals35, 37 (as opposed to the PD phenotype observed in wild-type rodents overexpressing hTyr43). Indeed, although high concentrations of dopamine can be acutely toxic, mostly in vitro,11 the chronic enhancement of dopamine levels does not cause dopaminergic nerve terminal or cell body loss in vivo,35-37 probably because of compensatory protective mechanisms.57 In addition, dopamine-induced toxicity has been shown to be dependent on α-synuclein expression‎,11, 35, 37, 57 in contrast to the nonessential role of α-synuclein for neuromelanin-linked pathology in hTyr-overexpressing animals43 (see later for details). Although a potential pathogenic contribution of oxidized dopamine species in neuromelanin-producing hTyr-overexpressing rodents cannot be completely ruled out, because neuromelanin production is indissociable from dopamine oxidation, other explanations for the observed pathogenicity in these animals could be envisaged.

Figure 3

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Molecular mechanisms of neuromelanin-linked neurotoxicity. (1) Dopamine oxidation generates many o-quinones, which can be potentially toxic to neurons. However, the continuous conversion of these oxidized dopamine species into neuromelanin prevents their pathological accumulation in the cytoplasm,43 which is consistent with a protective antioxidant role of neuromelanin synthesis. (2) The continuous intracellular build-up of neuromelanin within undegraded lysosomal-autophagic structures, until occupying most of the neuronal cytoplasm, may physically interfere with intracellular trafficking/communication and ultimately exhaust the vesicular storage capacity of the cell, leading to a general failure of cellular proteostasis coursing with lysosomal/autophagy dysfunction, ubiquitin-proteasome (UPS) impairment, intracellular inclusion body formation, reduced mitochondrial respiration, increased production of reactive oxygen species, impaired neurotransmission, and neurodegeneration. All of these pathological changes are observed in neuromelanin-producing human tyrosinase–overexpressing animals and Parkinson's disease patients.43 (3) Extracellular neuromelanin released from dying neurons activates microglia that phagocytize this pigment43 (ie, neuronophagia) and may trigger an antigenic response able to induce a selective T cell-mediated cytotoxic attack against neuromelanin-laden neurons,18 potentially contributing to the progression of the neurodegenerative process.

In both humans and hTyr-overexpressing rodents, intracellular neuromelanin accumulates within lysosomal-autophagic structures in an attempt by the cell to degrade this pigment15, 58 (Fig. 3). However, because of its insoluble nature, neuromelanin cannot be degraded by lysosomes and remains trapped within undegraded or partly degraded autophagic structures that accumulate with age.58 The continuous build-up of neuromelanin within autophagic compartments may ultimately exhaust the vesicular storage capacity of the cell, interfere with lysosomal proteases and other degradative pathways, impair intracellular vesicular trafficking, and alter endocytic/secretory tasks.58, 59 Supporting this concept, l-dopa–induced toxicity in vitro is only observed at doses associated with neuromelanin formation11 and has been attributed to an interference by neuromelanin with intracellular neurotrophin signaling, and not to an acute toxic effect of l-dopa per se.59 Neuromelanin could thus be toxic to aminergic neurons insofar as it physically interferes with intracellular communication,60, 61 causing a “macromolecular crowding” effect,62 thereby interfering with the synthesis and degradation of cellular proteins.63 Consistent with this concept, the progressive accumulation of neuromelanin-filled autophagic structures in hTyr-overexpressing cells was associated with parallel decreases in both autophagic and ubiquitin-proteasome (UPS) degradation system activities, leading to a general failure of cellular proteostasis and subsequent inclusion formation.43 Relevant to PD, both autophagy and UPS systems are impaired in neuromelanin-laden, but not in nonmelanized, regions from postmortem PD brains.64, 65 Impairment of autophagy and UPS is indicative of a general, late-stage proteostasis failure in which cell function and survival become compromised.66 For instance, mitochondrial quality control is tightly linked to proteolytic cytosolic systems; if the latter are blocked, damaged mitochondria gradually accumulate.66 Consistent with this, neuromelanin-producing cells also exhibit impaired mitochondrial respiration and increased production of reactive oxygen species, 2 pathogenic events that have been consistently linked to PD.43 Confirm‎ing a pivotal pathogenic role for impaired proteostasis in neuromelanin-laden cells, enhancement of lysosomal-mediated proteolysis in hTyr-overexpressing rodents by overexpression‎ of transcription factor EB (TFEB) reduced intracellular neuromelanin to levels below the pathogenic threshold, attenuated PD-like inclusion formation, prevented nigrostriatal neurodegeneration, and reversed motor impairment in these animals.43 TFEB is a master regulator of autophagy that induces the biogenesis of lysosomes and autophagosomes, boosts autophagic cellular clearance, and modulates general proteostasis67-70 as part of a pleiotropic range of effects.71 Interestingly, some of TFEB's effects at promoting cellular clearance involve the activation of lysosomal exocytosis, a process in which lysosomes fuse with the plasma membrane and empty their contents to the outside of the cell.69, 72 Because neuromelanin is trapped within lysosomal-autophagic structures, TFEB-induced reduction of intracellular neuromelanin levels occurred, at least in part, by the exocytosis of neuromelanin-filled lysosomes outside the cell.43 This approach may thus represent a potential therapeutic tool to reduce intracellular neuromelanin levels below their pathogenic threshold of accumulation without interfering with neuromelanin synthesis.

α-Synuclein in PD: A Key Factor or a Misleading Clue?

Given the formation of α-synuclein-positive inclusions and oligomers in neuromelanin-producing hTyr-overexpressing rodents and the pathogenic role attributed to α-synuclein aggregation in PD, it is feasible that the pathological phenotype of these animals could be potentially mediated by α-synuclein. Strikingly, α-synuclein was found to be dispensable for PD-like inclusion formation and nigrostriatal degeneration, as well as for neuromelanin production, in hTyr-overexpressing rodents.43 Indeed, the induction of neuromelanin production by hTyr overexpression‎ in α-synuclein-deficient mice resulted in levels of pale bodies/LB-like inclusion formation and nigrostriatal degeneration comparable to those seen in hTyr-overexpressing wild-type animals.43 However, although Lewy pathology in the latter animals displayed LB markers such as p62, ubiquitin, or α-synuclein, in hTyr-overexpressing α-synuclein-deficient mice Lewy pathology displayed p62 and ubiquitin but lacked α-synuclein.43 These results indicate that α-synuclein, which is currently regarded as a major component of LB, might not in fact be required for LB formation or for neurodegeneration (whether in the absence of α-synuclein these structures can still be called “LB” is a matter of semantics). Consistent with a dispensable role of α-synuclein in the pathogenesis of PD, α-synuclein accumulation correlates poorly with clinical symptoms and synaptic/neuronal loss in PD patients.73 Also, although α-synuclein-derived peptides have been proposed to act as antigenic epitopes capable of inducing a selective T-cell-mediated cytotoxic attack in PD patients,74 the activation of both innate and adaptive immune responses in postmortem PD brains is localized within neuromelanin-containing areas and absent from nonmelanized regions such as the cortex despite the latter exhibiting abundant α-synuclein depositions.16, 18 Furthermore, α-synuclein pathology is not mandatory for the development of PD, as shown by some genetic PD cases caused by Leucine-rich repeat kinase 2 and parkin mutations that course with pure nigrostriatal degeneration without Lewy pathology.75 Moreover, phosphorylation of α-synuclein, which is widely considered to be an index of PD pathology, (1) can also be seen in tissue from control subjects76; (2) may result from a nonspecific cross-reaction with other phospho-proteins, such as phosphorylated neurofilaments77; and (3) does not affect neuronal survival in in vitro PD models.78 In addition, peripheral α-synuclein aggregates from PD patients, which are believed to spread to the brain and initiate PD, lack the capacity to promote α-synuclein pathology, propagate between neuronal networks, or induce neurodegeneration when inoculated into experimental animals.79 Finally, in the original studies reporting an apparent transmission of LB pathology from host PD patients to embryonic mesencephalic neurons grafted into the striatum many years earlier,80, 81 which gave rise to the currently preval‎ent pathogenic hypothesis of cell-to-cell transmission of α-synuclein, LB formation occurred exclusively within grafted neurons that contained adult levels of neuromelanin and was not observed in nonmelanized grafted cells or intrinsic striatal neurons of the host. Therefore, the latter studies may not in fact reflect a host-to-graft propagation of α-synuclein as was widely interpreted but, rather, indicate that grafted cells had reached pathogenic levels of intracellular neuromelanin accumulation. Overall, it is possible that α-synuclein pathology in PD may merely represent an epiphenomenon within a more general PD-causing pathogenic process, such as the one linked to neuromelanin accumulation.43 Although this concept does not undermine the value of α-synuclein as a potential biomarker of PD, it raises the importance of reassessing the value of α-synuclein as a potential therapeutic target for the disease.

Implications of Neuromelanin-Driven Pathology for PD and Brain Aging

The recent development of a rodent model producing human-like neuromelanin has allowed for the consequences of age-dependent neuromelanin accumulation up to levels reached in elderly humans to be experimentally assessed for the first time in an in vivo setting. The use of this animal model has revealed that the progressive intracellular neuromelanin build-up that occurs with aging ultimately triggers PD-like neuronal dysfunction and neurodegeneration once a specific threshold of neuromelanin accumulation is reached. Therefore, although neuromelanin synthesis per se may be neuroprotective, its long-term accumulation may have deleterious consequences.

From a pathogenic point of view, these results could explain major features of PD, including (1) the selective vulnerability of specific neuronal groups (ie, neurons containing neuromelanin); (2) the correlation of PD with age (as neuromelanin accumulates progressively with age); (3) the progressive nature of the neurodegenerative process (as not all neurons accumulate neuromelanin at the same rate); (4) the known PD-linked cellular alterations, such as mitochondrial dysfunction, oxidative stress, autophagy/UPS deficits, inclusion body formation or neuroinflammation (all of which may occur secondarily to intracellular neuromelanin accumulation or extracellular neuromelanin release); (5) the uniqueness of PD to humans (as only humans accumulate such high levels of neuromelanin); or (6) the supposed transmission of LB pathology from host PD patients to grafted embryonic mesencephalic neurons (as LB are only observed within aged grafted neurons containing adult levels of neuromelanin).

In a broader context, these results imply that all humans would potentially develop PD if they were to live long enough to reach the pathogenic threshold of intracellular neuromelanin accumulation. Supporting this concept, although only some individuals actually develop PD (1 to up ~5% of the population older than 60, increasing with age),82 10% to 30% of apparently healthy people older than 60 years exhibit PD-type LB pathology in their melanized brain stem nuclei (ie, incidental LB disease),83 and these subjects display intracellular neuromelanin levels above the pathogenic threshold defined in neuromelanin-producing hTyr-overexpressing rodents.43 In addition, parkinsonian signs such as bradykinesia, stooped posture, and gait disturbance are common in elderly individuals, in the absence of overt PD, reaching a preval‎ence of up to 50% in individuals older than 80 years of age.84, 85 Also, multiple reports have documented an age-related loss of pigmented nigral neurons, estimated at about 10% per decade, in otherwise healthy individuals.17 Consistent with age-dependent alterations of neuromelanin-containing neurons, brains from aged individuals commonly exhibit a downregulation of dopaminergic phenotypic markers and α-synuclein accumulations within neuromelanin-laden SNpc neurons54, 86 as well as abundant extracellular neuromelanin associated with sustained microglial activation17 when compared with young adult brains. Also, in a recent retrospective study on healthy individuals of different ages (from 5-83 years), the neuromelanin-sensitive MRI SNpc signal peaked in middle age before declining in older ages, which was attributed to an age-related decrease of melanized nigral neurons.87 Overall, neuromelanin-filled neurons from apparently healthy aged individuals exhibit early signs of neuronal dysfunction/degeneration. Whether these changes precede clinical PD or are part of normal brain aging remains to be determined. In any case, based on the results discussed here, PD will appear in those subjects that have reached earlier the pathogenic threshold of intracellular neuromelanin accumulation.

From a therapeutic point of view, these results also imply that strategies to maintain or decrease intracellular neuromelanin to levels below the pathogenic threshold should be able to prevent, halt, or delay neuronal dysfunction and degeneration linked to both PD and brain aging (Fig. 2). Because of the putative protective role of neuromelanin synthesis itself (ie, to prevent the cytosolic accumulation of dopamine-derived toxic species), potential therapies aimed at maintaining or reducing intracellular neuromelanin to levels below the pathogenic threshold should probably favor the elimination of neuromelanin from the cell once neuromelanin has already been produced instead of inhibiting neuromelanin synthesis per se, which may instead prove detrimental. It would be thus important for any therapeutic strategy aimed at modulating neuromelanin levels to tightly regulate neuromelanin levels above its protective threshold and below its pathogenic threshold.

If PD were indeed triggered by an excessive accumulation of intracellular neuromelanin, above a certain pathogenic threshold, then neuromelanin levels might provide an indication of the risk of developing PD. For instance, neuromelanin's binding of iron provides a paramagnetic source for its detection in living brain tissue by MRI.88 Currently, neuromelanin-sensitive MRI is mainly used to differentiate between PD patients and age-matched healthy controls based on the loss of neuromelanin-sensitive MRI signal in PD subjects secondary to SNpc neurodegeneration.88 However, one could envisage the use of this technique in subjects at risk for PD (eg, nonmanifesting carriers of PD-linked genetic mutations or subjects with idiopathic rapid eye movement sleep behavior disorder) or even in the general aging population. This would allow to assess whether these subjects might be approaching or reaching the pathogenic threshold of neuromelanin accumulation and thus allow the prompt application of potential neuromelanin modulatory therapies in these subjects to prevent disease. This could also be envisaged with the use of [18F]-AV-1451 (Flortaucipir), a novel Positron Emission Tomography (PET) tracer that was initially developed for its high affinity to neurofibrillary tau in Alzheimer's disease but was subsequently found to bind strongly and specifically to neuromelanin and melanin.89, 90 Alternatively, it could be also interesting to determine whether brain neuromelanin levels might correlate with, and thus be inferred from, the production and/or type of pigmentation in the skin or hair. Supporting this concept: (1) PD patients have an increased risk of developing cutaneous melanoma and, reciprocally, patients with cutaneous melanoma have an increased risk of developing PD91; (2) similar to neurons, cutaneous melanocytes derive from pluripotent neural crest cells and their maturation is influenced by the same signaling molecules that play a role in central and peripheral nervous tissue92; (3) loss-of-function variants of the melanocortin 1 receptor gene, which are associated with red hair and fair skin, are linked to an increased risk of both cutaneous melanoma and PD93, 94; (4) genetically modified mice carrying an inactivating mutation of melanocortin 1 receptor mimicking the human redhead phenotype have compromised nigrostriatal dopaminergic neuronal integrity and are more susceptible to dopaminergic parkinsonian neurotoxins.95

Concluding Remarks and Future Directions

The development of the first rodent model producing human-like neuromelanin in PD-vulnerable dopaminergic nigral neurons has allowed for the first time to experimentally assess the consequences of age-dependent neuromelanin accumulation up to levels reached in elderly humans in an in vivo setting. The observation in this animal model that age-dependent intracellular neuromelanin build-up ultimately leads to PD-like neuronal dysfunction and neurodegeneration when reaching a certain pathogenic threshold of accumulation may have far-reaching implications for both PD and brain aging.

Among the outstanding questions that remain to be answered is whether neuromelanin synthesis in humans occurs enzymatically, nonenzymatically, or by a combination of both. Although the pathogenic effects linked to neuromelanin accumulation discussed here appear to be independent of the actual mechanisms of neuromelanin synthesis, as they occur downstream of neuromelanin production and subsequent accumulation, the elucidation of the mechanisms of neuromelanin synthesis could provide important clues about the significance of neuromelanin production or identify potential molecular targets to modulate neuromelanin levels. Understanding these mechanisms may also help unravel why PD patients would reach the pathogenic threshold of neuromelanin accumulation earlier than healthy subjects. This could be due, for instance, to an increased or accelerated production of neuromelanin in PD patients caused by an upregulation of hTyr or other related enzymes, such as tyrosinase-related protein-1 and tyrosinase-related protein-2. Consistent with this, the expression‎ of tyrosinase-related protein-2 (ie, dopachrome tautomerase) is increased in dopaminergic neurons derived from induced pluripotent stem cells from PD patients.96 Alternatively, increased neuromelanin production in PD could arise from increased levels of free cytosolic dopamine that oxidizes into neuromelanin, for instance, by defects in vesicular monoamine transporter 2–mediated encapsulation of dopamine within synaptic vesicles, as observed in early PD cases.97 Indeed, there is an inverse relationship between neuromelanin content and vesicular monoamine transporter 2 immunoreactivity in human midbrain dopaminergic neurons, with neurons exhibiting the highest vesicular monoamine transporter 2 levels and the lowest neuromelanin levels being the less vulnerable to PD-linked neurodegeneration.63

It would also be important to further uncover the exact molecular mechanisms driving neuromelanin-linked pathology. In addition of the potential detrimental effects of a physical cytosolic overcrowding by neuromelanin or the formation of potentially toxic dopamine-derived oxidized species during neuromelanin synthesis, other factors could also be considered. For instance, neuromelanin has the ability to chelate various transition metal ions98 and neuromelanin is in fact the main iron storage molecule in SNpc dopaminergic neurons.99, 100 The equilibrium between iron, dopamine, and neuromelanin seems to be crucial for cell homeostasis,99, 101 and PD brains exhibit increased levels of iron when compared with healthy control subjects.100 In this context, neuromelanin may represent a potential source of toxic iron if the iron-binding capacity of this pigment becomes saturated.101 Similarly, the capacity of neuromelanin to bind environmental parkinsonian neurotoxins, such as 1-methyl-4-phenylpyridinium (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MPTP's active metabolite) or the pesticide paraquat could also be considered.102 For instance, it has been observed in macaques that among dopaminergic neurons, those containing neuromelanin are more susceptible to MPTP toxicity than nonmelanized neurons.103

These and other questions could now be addressed in vivo for the first time by means of the newly available neuromelanin-producing rodent model.43 This model could be used for (1) the study of the synthesis, biology, and pathogenic mechanisms of neuromelanin in vivo in both aging and disease, (2) the screening of novel therapeutic strategies aimed at modulating neuromelanin levels within the aged or diseased brain, and (3) the search for potential neuromelanin-related biomarkers for the risk, early detection, diagnosis, and/or progression of PD. The introduction into experimental in vivo research of a factor such as neuromelanin that is so intimately linked to PD and that has thus far been neglected in PD animal modeling should open new research avenues and could lead to a paradigm shift in the field of PD and, in a broader sense, brain aging.

Acknowledgments

The work described was funded by grants from the Ministry of Economy and Competitiveness (Spain), Institute of Health Carlos IIII–European Regional Development Fund, Parkinson's U.K., The Michael J. Fox Foundation, “la Caixa” Foundation, and Center for Networked Biomedical Research on Neurodegenerative Diseases (CIBERNED). Figures 2 and 3 were drawn by Dr. Celine Perier.

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