Autophagy ISSN: 1554-8627 (Print) 1554-8635 (Online) Journal homepage: https://www.tandfonline.com/loi/kaup20 Intracellular crowding by age-dependent neuromelanin accumulation disrupts neuronal proteostasis and triggers Parkinson disease pathology Miquel Vila, Ariadna Laguna & Iria Carballo-Carbajal To cite this article: Miquel Vila, Ariadna Laguna & Iria Carballo-Carbajal (2019) Intracellular crowding by age-dependent neuromelanin accumulation disrupts neuronal proteostasis and triggers Parkinson disease pathology, Autophagy, 15:11, 2028-2030, DOI: 10.1080/15548627.2019.1659621 To link to this article: https://doi.org/10.1080/15548627.2019.1659621 © 2019 The Author(s). Published by Informa Published online: 03 Sep 2019. UK Limited, trading as Taylor & Francis Group. Submit your article to this journal Article views: 1658 View related articles View Crossmark data Citing articles: 12 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=kaup20 AUTOPHAGY 2019, VOL. 15, NO. 11, 2028–2030 https://doi.org/10.1080/15548627.2019.1659621 AUTOPHAGIC PUNCTUM Intracellular crowding by age-dependent neuromelanin accumulation disrupts neuronal proteostasis and triggers Parkinson disease pathology a,b,c a Miquel Vila , Ariadna Laguna , and Iria Carballo-Carbajala a Neurodegenerative Diseases Research Group, Vall d’Hebron Research Institute (VHIR)-Center for Networked Biomedical Research on Neurodegenerative Diseases (CIBERNED), Barcelona, Spain; bDepartment of Biochemistry and Molecular Biology, Autonomous University of Barcelona, Barcelona, Spain; cCatalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain ABSTRACT ARTICLE HISTORY In Parkinson disease (PD), there is a preferential degeneration of neurons that contain the dark-brown Received 19 July 2019 cytoplasmic pigment neuromelanin, in particular dopaminergic neurons of the substantia nigra (SN), the Revised 4 August 2019 loss of which leads to the typical motor symptoms of the disease and constitutes the cardinal Accepted 5 August 2019 pathological diagnostic criterion for PD. Neuromelanin is generally considered a byproduct of dopamine KEYWORDS oxidative metabolism and, in humans, it is first detected in early childhood and accumulates progres- Alpha-synuclein; autophagy; sively with age until occupying most of the neuronal cytoplasm, as neurons apparently lack the means cellular trafficking; to degrade or eliminate this pigment. Aging is the main risk factor for developing PD, but the molecular dopamine; Lewy bodies; substrate underlying this link remains unknown. Despite the close and long-established association neurodegeneration; between neuromelanin and PD, the potential contribution of neuromelanin to PD pathogenesis has neuromelanin; Parkinson’s remained elusive because, in contrast to humans, common laboratory animal species, such as rodents, disease lack neuromelanin. To overcome this major limitation, we have recently generated the first experimental in vivo rodent model exhibiting age-dependent production and accumulation of human-like neurome- lanin within PD-vulnerable dopaminergic nigral neurons, at levels up to those reached in elderly humans. Our model is based on the unilateral viral vector-mediated membrane-delimited mature lysosomes/autolysosomes or inside expression in the rat SN of human (Hs) TYR (tyrosinase), the double-membrane-delimited autophagosomes that accumulated rate-limiting enzyme for the synthesis of peripheral melanins with age until occupying most of the neuronal cytoplasm. (e.g. skin and hair) that is also present at very low levels in the Consistent with the autophagic nature of neuromelanin granules, human SN, where it might play a putative role in neuromelanin lysosomal and autophagosomal markers increase in parallel with synthesis. Using this unique neuromelanin-producing rat neuromelanin production and colocalize with neuromelanin gran- model, we found that progressive intracellular neuromelanin ules in HsTYR-expressing neurons. The continuous buildup of accumulation above a specific threshold ultimately compromises neuromelanin within autophagic compartments in these cells is neuronal function and triggers, in an age-dependent manner, associated with a parallel decrease in lysosomal-mediated proteo- pathological features typical of PD, including impaired dopa- lysis and a marked reduction in ubiquitin-proteasome system mine release, motor deficits, Lewy body (LB)-like inclusion for- (UPS) activity. Relevant to humans, both autophagy and the mation and nigrostriatal neurodegeneration [1]. Relevant to UPS have been reported to be impaired in neuromelanin-laden humans, intracellular neuromelanin levels reach this pathogenic but not in non-melanized regions from post-mortem PD brains. threshold in both PD patients and pre-symptomatic PD subjects Consistent with a failure in autophagy and UPS-mediated proteo- (i.e. incidental Lewy body disease) but not in aged-matched lysis, neuromelanin-laden cells exhibit a marked accumulation of elderly control brains. In parallel to dopaminergic cell death, SQSTM1/p62, a common component of PD-associated neuro- HsTYR-overexpressing rodents also exhibit other neuropatho- pathological inclusions, and an extensive deposition of SNCA/α- logical features typical of PD, such as extracellular neuromela- synuclein oligomers, as directly visualized by an SNCA proximity nin, released from dying neurons, surrounded by, or within, ligation assay. In HsTYR-overexpressing rodents, cytoplasmic activated microglia (a process known as neuronophagia, which inclusions emerge within neuromelanin-filled areas, either over- is indicative of ongoing neurodegeneration). lapping with or displacing neuromelanin granules, and often In humans, intracellular neuromelanin is known to continu- appear as early multiple punctate small aggregates immunoposi- ously accumulate over a lifetime within undegraded or partly tive for SQSTM1 that seem to progressively coalesce into more degraded macroautophagic/autophagic structures, probably in compacted LB-like spherical structures, as similarly reported in an (unsuccessful) attempt by the cell to degrade this insoluble post-mortem PD brains. PD-type inclusion formation in HsTYR- pigment. In agreement with this, neuromelanin from HsTYR- overexpressing rodents peaks at the time of early neuronal dys- overexpressing rodents appears enclosed within single- function and is substantially reduced once neurodegeneration is CONTACT Miquel Vila miquel.vila@vhir.org Neurodegenerative Diseases Research Group, Vall d’Hebron Research Institute (VHIR)-Center for Networked Biomedical Research on Neurodegenerative Diseases (CIBERNED), Barcelona 08035, Spain © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. AUTOPHAGY 2029 established, suggesting that inclusion-containing neurons are failure of cellular proteostasis, well beyond SNCA, in neu- those that become dysfunctional and preferentially degenerate in romelanin-laden neurons. Consistent with a late-stage pro- these animals. In line with this, the number of neuronal inclusions teostasis failure in which cell function and survival may in PD brains at advanced stages of the disease is much lower than become compromised, neuromelanin-producing cells also that observed in early PD cases. Remarkably, while some of the exhibit accumulation of dysfunctional mitochondria and PD-type inclusions from HsTYR-overexpressing rodents are increased production of reactive oxygen species, 2 patho- immunopositive for SNCA, the latter is dispensable for inclusion genic events that have been consistently linked to PD. formation and nigrostriatal degeneration in these animals, as Confirming a pivotal pathogenic role for impaired proteos- attested by experiments combining HsTYR-induced neuromela- tasis in neuromelanin-laden neurons, enhancement of lyso- nin production with SNCA genetic ablation. This observation somal-mediated proteolysis in HsTYR-overexpressing suggests that SNCA, which is currently regarded as a major patho- rodents by overexpression of the master autophagy regula- genic factor in PD, might not be in fact required for LB formation tor TFEB (transcription factor EB) reduces intracellular and PD-linked neurodegeneration. neuromelanin to levels below the pathogenic threshold, The accumulation of SQSTM1-immunopositive inclu- attenuates PD-like inclusion formation, prevents nigrostria- sions in HsTYR-overexpressing rodents suggests a general tal neurodegeneration and reverses motor deficits in these Figure 1. Proposed pathogenic effects of age-dependent intracellular neuromelanin accumulation in humans (top) and HsTYR-overexpressing rats (bottom). The continuous, age-dependent intracellular buildup of neuromelanin within undegraded autophagic compartments above a specific pathogenic threshold of accumulation (dotted red line) is associated with a general failure of cellular proteostasis concomitant with lysosomal/autophagy dysfunction, UPS impairment, Lewy body-type inclusion formation, reduced mitochondrial respiration, increased production of reactive oxygen species, impaired neurotransmission, nigrostriatal neurodegeneration and neuroinflammation, all of which are major pathological features of PD. Enhancement of lysosomal-mediated proteolysis with TFEB reduces intracellular neuromelanin to levels below the pathogenic threshold, attenuates PD-like inclusion formation, prevents nigrostriatal neurodegeneration and reverses motor impairment in HsTYR-overexpressing rodents. Part of TFEB’s effects are attributed to enhanced exocytosis of neuromelanin-filled lysosomes outside the cell, thus supporting the feasibility and therapeutic potential of modulating intracellular neuromelanin levels in vivo. 2030 M. VILA ET AL. animals. Part of TFEB’s effects at reducing intracellular Disclosure statement neuromelanin levels are attributed to TFEB-induced exocy- No potential conflict of interest was reported by the authors. tosis of neuromelanin-filled lysosomes outside the cell, thereby demonstrating the feasibility and therapeutic poten- tial of modulating intracellular neuromelanin levels in vivo. Funding Overall, our results reveal a novel pathogenic scenario in This work was supported by the Michael J. Fox Foundation for Parkinson’s PD in which the continuous, age-dependent buildup of Research [TAP 15291]; Ministerio de Economía y Competitividad neuromelanin within autophagic compartments may ulti- [SAF2015-73997-JIN]; Ministerio de Economía y Competitividad mately exhaust the vesicular storage capacity of the cell, [SAF2016-77541-R]; “la Caixa” Foundation [LCF/PR/HR17/52150003]. interfere with lysosomal proteases and other degradative pathways, impair intracellular vesicular trafficking and ORCID alter endocytic/secretory tasks, creating a cellular traffic jam that interferes with intracellular communication and Miquel Vila http://orcid.org/0000-0002-1352-989X Ariadna Laguna http://orcid.org/0000-0002-9732-6677 protein synthesis/degradation, eventually leading to Lewy pathology and PD-linked neuronal dysfunction/degenera- tion (Figure 1). Accordingly, strategies to modulate intra- Reference cellular neuromelanin levels may provide unprecedented [1] Carballo-Carbajal I, Laguna A, Romero-Gimenez J, et al. Brain therapeutic opportunities to prevent, halt or delay neuronal tyrosinase overexpression implicates age-dependent neuromelanin dysfunction and degeneration linked to PD and brain production in Parkinson’s disease pathogenesis. Nat Commun. aging. 2019;10(1):973. PMID: 30846695.