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The Process of Lewy Body Formation, Rather Than Simply Α-Synuclein Fibrillization, Is One of the Major Drivers of Neurodegeneration

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The Process of Lewy Body Formation, Rather Than Simply Α-Synuclein Fibrillization, Is One of the Major Drivers of Neurodegeneration The process of Lewy body formation, rather than simply α-synuclein fibrillization, is one of the major drivers of neurodegeneration Anne-Laure Mahul-Melliera, Johannes Burtschera, Niran Maharjana, Laura Weerensa, Marie Croisierb, Fabien Kuttlerc, Marion Leleud,e, Graham W. Knottb, and Hilal A. Lashuela,1 aLaboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland; bBioEM Core Facility and Technology Platform, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland; cBiomolecular Screening Core Facility and Technology Platform, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland; dGene Expression Core Facility and Technology Platform, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland; and eSwiss Institute of Bioinformatics, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland Edited by Pietro De Camilli, Yale University, New Haven, CT, and approved December 31, 2019 (received for review August 27, 2019) Parkinson’s disease (PD) is characterized by the accumulation of mis- and LB formation and maturation and to correlate these events with folded and aggregated α-synuclein (α-syn) into intraneuronal inclu- alteration in cellular pathways and functions/dysfunctions. sions named Lewy bodies (LBs). Although it is widely believed that Until recently, the great majority of cellular models of synucleino- α-syn plays a central role in the pathogenesis of PD, the processes pathies were based on overexpression of WT or mutant α-syn alone, that govern α-syn fibrillization and LB formation remain poorly un- with other proteins, or coupled to treatment with other stress inducers derstood. In this work, we sought to dissect the spatiotemporal (8–10). However, very few studies investigated the extent to which events involved in the biogenesis of the LBs at the genetic, molecular, these aggregates reproduce the cardinal structural and organizational biochemical, structural, and cellular levels. Toward this goal, we fur- features of the bona fide LB (11): 1) Dense core with irradiating ther developed a seeding-based model of α-syn fibrillization to gen- filaments (classical brainstem type LB) or fibrillary structure without a erate a neuronal model that reproduces the key events leading to LB central core (cortical LB); 2) p62, ubiquitin (ub), and phosphorylated formation, including seeding, fibrillization, and the formation of in- α-syn (pS129) immunoreactivity; and 3) the presence of membranous NEUROSCIENCE clusions that recapitulate many of the biochemical, structural, and organelles (4, 5, 12–16). In these studies, the aggregation properties of organizational features of bona fide LBs. Using an integrative omics, α-syn were indirectly assessed (8, 17, 18) while characterization of the biochemical and imaging approach, we dissected the molecular LB-like properties was mostly limited to assessing the immunoreac- events associated with the different stages of LB formation and their tivity for selected LB markers (8–10). When electron microscopy contribution to neuronal dysfunction and degeneration. In addition, (EM) was used, it clearly demonstrated that these α-syn inclusions do we demonstrate that LB formation involves a complex interplay be- not share the compositional complexity and morphological features of tween α-syn fibrillization, posttranslational modifications, and inter- actions between α-syn aggregates and membranous organelles, including mitochondria, the autophagosome, and endolysosome. Fi- Significance nally, we show that the process of LB formation, rather than simply fibril formation, is one of the major drivers of neurodegeneration Although converging evidence point to α-synuclein aggrega- through disruption of cellular functions and inducing mitochondria tion and Lewy body (LB) formation as central events in Par- damage and deficits, and synaptic dysfunctions. We believe that this kinson’s disease, the molecular mechanisms that regulate these model represents a powerful platform to further investigate the processes and their role in disease pathogenesis remain elu- mechanisms of LB formation and clearance and to screen and evalu- sive. Herein, we describe a neuronal model that reproduces the ate therapeutics targeting α-syn aggregation and LB formation. key events leading to the formation of inclusions that re- capitulate the biochemical, structural, and organizational fea- α‐synuclein | Parkinson’s disease | aggregation | Lewy body | seeding tures of bona fide LBs. This model allowed us to dissect the molecular events associated with the different stages of LB n-depth postmortem neuropathological examinations of hu- formation and how they contribute to neuronal dysfunctions man brains from patients with Parkinson’s disease (PD) and and degeneration, thus providing a powerful platform for I α related synucleinopathies have revealed the existence of different evaluating therapeutics targeting -synuclein aggregation and subtypes of pathological inclusions that are enriched in aggregated LB formation and to identify and validate therapeutic targets for the treatment of Parkinson’s disease. forms of α-synuclein (α-syn), including fibrils (1–6). This has led to α the hypothesis that aggregation of -syn has a primary role in the Author contributions: A.-L.M.-M., J.B., N.M., L.W., G.W.K., and H.A.L. designed research; formation of the Lewy bodies (LBs) and other α-syn pathological A.-L.M.-M., J.B., N.M., L.W., and M.C. performed research; A.-L.M.-M., J.B., N.M., L.W., F.K., aggregates and therefore in the pathogenesis of synucleinopathies M.L., and G.W.K. analyzed data; and A.-L.M.-M., J.B., N.M., and H.A.L. wrote the paper. (7). However, the molecular and cellular processes that trigger The authors declare no competing interest. and govern the misfolding, fibrillization, LB formation, and spread This article is a PNAS Direct Submission. of α-syn in the brain remain poorly understood. Published under the PNAS license. The absence of experimental models that reproduce all of the Data deposition: The proteomic dataset has been deposited in the ProteomeXchange via the stages of LB formation and maturation has limited our ability to PRIDE database, Project Name: Temporal proteomic analyses of the protein contents found decipher the different processes involved in LB formation and the in the insoluble fraction of the a-synuclein PFF-treated neurons (accession no. PXD016850). The transcriptomic dataset has been deposited in the Gene Expression Omnibus (GEO) da- contribution of these processes to the pathogenesis of PD and tabase, https://www.ncbi.nlm.nih.gov/geo (accession no. GSE142416). Higher-quality figures synucleinopathies. To address this knowledge gap, it is crucial to are available at Figshare (https://doi.org/10.6084/m9.figshare.11842389.v2). develop cellular and animal models that not only produce α-syn 1To whom correspondence may be addressed. Email: [email protected]. aggregates but also recapitulate the process of LB formation at the This article contains supporting information online at https://www.pnas.org/lookup/suppl/ biochemical, structural, and organizational levels. This is funda- doi:10.1073/pnas.1913904117/-/DCSupplemental. mental to elucidate the key events associated with α-syn aggregation www.pnas.org/cgi/doi/10.1073/pnas.1913904117 PNAS Latest Articles | 1of12 Downloaded by guest on September 28, 2021 the LBs (8, 19, 20), suggesting that the majority of α-syn cellular α-syn aggregates in seeding models, our ICC data revealed the models reproduce some aspects of α-syn aggregation or fibril for- formation of aggregates that are heterogeneous in size, shape, mation, but do not recapitulate all of the key events leading to LB and subcellular distribution in the same population of PFF- formation and maturation. treated neurons (Fig. 1 B–I and SI Appendix, Fig. S3 B and C). Recently, it was shown that exogenously added preformed fibrils Biochemical analysis of the insoluble fraction of the PFF-treated (PFFs) of α-syn can act as seeds to initiate the misfolding and ag- neurons confirmed the accumulation of both truncated and high gregation of endogenous α-syn,inbothcellular(21)andanimal molecular weight (HMWs) α-syn species that were positively models (22), in the absence of α-syn overexpression. Although long stained with a pan-synuclein antibody and with an antibody filamentous aggregates, immunoreactive for the standard LB mak- against pS129 (Fig. 1J and SI Appendix, Fig. S7), consistent with ers, were observed, the transition from fibrils to LB-like structures previous reports from our group (25). These insoluble fractions has not been reported. This suggests that the neuronal seeding displayed a potent seeding activity in neuronal primary culture model, as initially developed, is a suitable model for investigating the (SI Appendix, Fig. S4) and induced the formation of seeded ag- processes involved in fibrils formations but not LB formation. gregates with a morphology and a subcellular distribution similar Given that LBs have been consistently shown to have complex to those formed in PFFs-treated neurons (SI Appendix, Figs. S3C composition and organization and usually contain other non- and S4B). These findings demonstrate that the newly formed + proteinaceous material (lipids) (23, 24) and membranous or- pS129 α-syn filamentous aggregates contain α-syn seeding- ganelles (4, 5, 12–16), we hypothesized that the transition from fibrils competent
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