Monitoring α-synuclein ubiquitination dynamics reveals key endosomal effectors mediating its trafficking and degradation

While defective α-synuclein homeostasis is central to Parkinson’s pathogenesis, fundamental questions about its degradation remain unresolved. We have developed a bimolecular fluorescence complementation assay in living cells to monitor de novo ubiquitination of α-synuclein and identified lysine residues 45, 58, and 60 as critical ubiquitination sites for its degradation. This is mediated by NBR1 binding and entry into endosomes in a process that involves ESCRT I-III for subsequent lysosomal degradation. Autophagy or the autophagic chaperone Hsc70 is dispensable for this pathway. Antibodies against diglycine-modified α-synuclein peptides confirmed that endogenous α-synuclein is similarly ubiquitinated in the brain and targeted to lysosomes in primary and iPSC-derived neurons. Ubiquitinated α-synuclein was detected in Lewy bodies and cellular models of aggregation, suggesting that it may be entrapped with endo/lysosomes in inclusions. Our data elucidate the intracellular trafficking of de novo ubiquitinated α-synuclein and provide tools for investigating the rapidly turned-over fraction of this disease-causing protein.

[1]  G. Tofaris.  Initiation and progression of α-synuclein pathology in Parkinson’s disease , 2022, Cellular and Molecular Life Sciences.

[2]  T. Golde,et al.  Optical pulse labeling studies reveal exogenous seeding slows α-synuclein clearance , 2022, NPJ Parkinson's disease.

[3]  R. Fischer,et al.  Phenotypic manifestation of α-synuclein strains derived from Parkinson’s disease and multiple system atrophy in human dopaminergic neurons , 2021, Nature Communications.

[4]  Jason J. Davis,et al.  Validation of α‐Synuclein in L1CAM‐Immunocaptured Exosomes as a Biomarker for the Stratification of Parkinsonian Syndromes , 2021, Movement disorders : official journal of the Movement Disorder Society.

[5]  C. Behrends,et al.  Systematically defining selective autophagy receptor-specific cargo using autophagosome content profiling. , 2021, Molecular cell.

[6]  Jason J. Davis,et al.  Serum neuronal exosomes predict and differentiate Parkinson’s disease from atypical parkinsonism , 2020, Journal of Neurology, Neurosurgery, and Psychiatry.

[7]  G. Knott,et al.  The process of Lewy body formation, rather than simply α-synuclein fibrillization, is one of the major drivers of neurodegeneration , 2020, Proceedings of the National Academy of Sciences.

[8]  A. Murzin,et al.  Structures of α-synuclein filaments from multiple system atrophy , 2020, Nature.

[9]  W. V. van IJcken,et al.  Lewy pathology in Parkinson’s disease consists of crowded organelles and lipid membranes , 2019, Nature Neuroscience.

[10]  R. Riek,et al.  A cullin-RING ubiquitin ligase targets exogenous α-synuclein and inhibits Lewy body–like pathology , 2019, Science Translational Medicine.

[11]  P. De Camilli,et al.  VPS13A and VPS13C are lipid transport proteins differentially localized at ER contact sites , 2018, The Journal of cell biology.

[12]  I. Dikic,et al.  Mechanism and medical implications of mammalian autophagy , 2018, Nature reviews. Molecular cell biology.

[13]  P. Chinnery,et al.  Stem cell modeling of mitochondrial parkinsonism reveals key functions of OPA1 , 2018, Annals of neurology.

[14]  D. Selkoe,et al.  Loss of native &agr;-synuclein multimerization by strategically mutating its amphipathic helix causes abnormal vesicle interactions in neuronal cells , 2017, Human molecular genetics.

[15]  Jüergen Cox,et al.  The MaxQuant computational platform for mass spectrometry-based shotgun proteomics , 2016, Nature Protocols.

[16]  A. Bax,et al.  Nuclear Magnetic Resonance Observation of α-Synuclein Membrane Interaction by Monitoring the Acetylation Reactivity of Its Lysine Side Chains , 2016, Biochemistry.

[17]  A. Goldberg,et al.  Deubiquitinase Usp8 regulates α-synuclein clearance and modifies its toxicity in Lewy body disease , 2016, Proceedings of the National Academy of Sciences.

[18]  Samantha J. Stehbens,et al.  NBR1 enables autophagy-dependent focal adhesion turnover , 2016, The Journal of cell biology.

[19]  Thomas J. Jentsch,et al.  Optogenetic Acidification of Synaptic Vesicles and Lysosomes , 2015, Nature Neuroscience.

[20]  J. Hurley,et al.  ESCRTs are everywhere , 2015, The EMBO journal.

[21]  M. Dong,et al.  ESCRTs Cooperate with a Selective Autophagy Receptor to Mediate Vacuolar Targeting of Soluble Cargos. , 2015, Molecular cell.

[22]  T. Südhof,et al.  α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation , 2014, Proceedings of the National Academy of Sciences.

[23]  S. Gygi,et al.  Why do cellular proteins linked to K63‐polyubiquitin chains not associate with proteasomes? , 2013, The EMBO journal.

[24]  Li Yu,et al.  Synthesis and screening of 3-MA derivatives for autophagy inhibitors , 2013, Autophagy.

[25]  E. Klann,et al.  Macroautophagy deficiency mediates age-dependent neurodegeneration through a phospho-tau pathway , 2012, Molecular Neurodegeneration.

[26]  G. Tofaris.  Lysosome‐dependent pathways as a unifying theme in Parkinson's disease , 2012, Movement disorders : official journal of the Movement Disorder Society.

[27]  Yanji Xu,et al.  Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis. , 2012, Journal of proteome research.

[28]  A. Kakita,et al.  Autophagic adapter protein NBR1 is localized in Lewy bodies and glial cytoplasmic inclusions and is involved in aggregate formation in α-synucleinopathy , 2012, Acta Neuropathologica.

[29]  B. Hyman,et al.  Distinct Roles In Vivo for the Ubiquitin–Proteasome System and the Autophagy–Lysosomal Pathway in the Degradation of α-Synuclein , 2011, The Journal of Neuroscience.

[30]  A. Goldberg,et al.  Ubiquitin ligase Nedd4 promotes α-synuclein degradation by the endosomal–lysosomal pathway , 2011, Proceedings of the National Academy of Sciences.

[31]  L. Santambrogio,et al.  Microautophagy of cytosolic proteins by late endosomes. , 2011, Developmental cell.

[32]  M. Komatsu,et al.  A role for NBR1 in autophagosomal degradation of ubiquitinated substrates. , 2009, Molecular cell.

[33]  Kostas Vekrellis,et al.  Wild Type α-Synuclein Is Degraded by Chaperone-mediated Autophagy and Macroautophagy in Neuronal Cells* , 2008, Journal of Biological Chemistry.

[34]  B. Hyman,et al.  CHIP Targets Toxic α-Synuclein Oligomers for Degradation* , 2008, Journal of Biological Chemistry.

[35]  M. Farrer,et al.  Genomic investigation of α‐synuclein multiplication and parkinsonism , 2008, Annals of neurology.

[36]  R. Barbour,et al.  Phosphorylation of Ser-129 Is the Dominant Pathological Modification of α-Synuclein in Familial and Sporadic Lewy Body Disease* , 2006, Journal of Biological Chemistry.

[37]  A. Helenius,et al.  Rab7 Associates with Early Endosomes to Mediate Sorting and Transport of Semliki Forest Virus to Late Endosomes , 2005, PLoS biology.

[38]  C. Ross,et al.  Ubiquitylation of synphilin-1 and alpha-synuclein by SIAH and its presence in cellular inclusions and Lewy bodies imply a role in Parkinson's disease. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[39]  G. Forloni,et al.  Proteasome inhibition and aggregation in Parkinson's disease: a comparative study in untransfected and transfected cells , 2003, Journal of neurochemistry.

[40]  B. Ghetti,et al.  Ubiquitination of α-Synuclein in Lewy Bodies Is a Pathological Event Not Associated with Impairment of Proteasome Function* , 2003, Journal of Biological Chemistry.

[41]  Jeremy N. Skepper,et al.  α-Synuclein Is Degraded by Both Autophagy and the Proteasome* , 2003, Journal of Biological Chemistry.

[42]  L. Stefanis,et al.  Proteasomal Inhibition-Induced Inclusion Formation and Death in Cortical Neurons Require Transcription and Ubiquitination , 2002, Molecular and Cellular Neuroscience.

[43]  L. Serpell,et al.  Proteasomal degradation of tau protein , 2002, Journal of neurochemistry.

[44]  M. Spillantini,et al.  α‐Synuclein metabolism and aggregation is linked to ubiquitin‐independent degradation by the proteasome , 2001, FEBS letters.

[45]  John Q. Trojanowski,et al.  Induction of α-Synuclein Aggregation by Intracellular Nitrative Insult , 2001, The Journal of Neuroscience.

[46]  T. Chase,et al.  Degradation of α-Synuclein by Proteasome* , 1999, The Journal of Biological Chemistry.

[47]  R. Kopito,et al.  Aggresomes: A Cellular Response to Misfolded Proteins , 1998, The Journal of cell biology.

[48]  A. Jonas,et al.  Stabilization of α-Synuclein Secondary Structure upon Binding to Synthetic Membranes* , 1998, The Journal of Biological Chemistry.

[49]  E. Yeh,et al.  Characterization of NEDD8, a Developmentally Down-regulated Ubiquitin-like Protein* , 1997, The Journal of Biological Chemistry.

[50]  C. Overly,et al.  Quantitative measurement of intraorganelle pH in the endosomal-lysosomal pathway in neurons by using ratiometric imaging with pyranine. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[51]  R. Scheller,et al.  The rat brain synucleins; family of proteins transiently associated with neuronal membrane. , 1991, Brain research. Molecular brain research.

[52]  V. Quaranta.  Inhibitors , 1991, Encyclopedia of Molecular Pharmacology.

[53]  V. Tennyson,et al.  Phase and Electron Microscopic Observations of Lewy Bodies and Melanin Granules in the Substantia Nigra and Locus Caeruleus in Parkinson's Disease , 1965 .