Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

Aggregation of alpha-synuclein (ASYN) in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T) exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.

[1]  T. Golde,et al.  Divergent effects of the H50Q and G51D SNCA mutations on the aggregation of α‐synuclein , 2014, Journal of neurochemistry.

[2]  A. Gitler,et al.  The novel Parkinson's disease linked mutation G51D attenuates in vitro aggregation and membrane binding of α-synuclein, and enhances its secretion and nuclear localization in cells. , 2014, Human molecular genetics.

[3]  H. Lashuel,et al.  The H50Q Mutation Enhances α-Synuclein Aggregation, Secretion, and Toxicity* , 2014, The Journal of Biological Chemistry.

[4]  Sandra Tenreiro,et al.  Harnessing the power of yeast to unravel the molecular basis of neurodegeneration , 2013, Journal of neurochemistry.

[5]  Ronald Melki,et al.  G51D α‐synuclein mutation causes a novel Parkinsonian–pyramidal syndrome , 2013, Annals of neurology.

[6]  Christos Proukakis,et al.  A novel α-synuclein missense mutation in Parkinson disease , 2013, Neurology.

[7]  J. Hardy,et al.  α-Synucleinopathy associated with G51D SNCA mutation: a link between Parkinson’s disease and multiple system atrophy? , 2013, Acta Neuropathologica.

[8]  T. Outeiro,et al.  Assessing the Subcellular Dynamics of Alpha-synuclein Using Photoactivation Microscopy , 2013, Molecular Neurobiology.

[9]  E. Masliah,et al.  The many faces of α-synuclein: from structure and toxicity to therapeutic target , 2012, Nature Reviews Neuroscience.

[10]  P. Aebischer,et al.  Use of viral vectors to create animal models for Parkinson's disease , 2012, Neurobiology of Disease.

[11]  T. Outeiro,et al.  Alpha-synuclein: from secretion to dysfunction and death , 2012, Cell Death and Disease.

[12]  G. Braus,et al.  Aggregate Clearance of α-Synuclein in Saccharomyces cerevisiae Depends More on Autophagosome and Vacuole Function Than on the Proteasome* , 2012, The Journal of Biological Chemistry.

[13]  M. Chesselet,et al.  Animal models of the non-motor features of Parkinson's disease , 2012, Neurobiology of Disease.

[14]  B. Hyman,et al.  Alpha-synuclein aggregation involves a bafilomycin A1-sensitive autophagy pathway , 2012, Autophagy.

[15]  B. Mollenhauer,et al.  Development of electrochemiluminescence-based singleplex and multiplex assays for the quantification of α-synuclein and other proteins in cerebrospinal fluid. , 2012, Methods.

[16]  H. Lashuel,et al.  Elucidating the role of C-terminal post-translational modifications using protein semisynthesis strategies: α-synuclein phosphorylation at tyrosine 125. , 2012, Journal of the American Chemical Society.

[17]  P. Aebischer,et al.  Mimicking Phosphorylation at Serine 87 Inhibits the Aggregation of Human α-Synuclein and Protects against Its Toxicity in a Rat Model of Parkinson's Disease , 2012, The Journal of Neuroscience.

[18]  C. Hetz The unfolded protein response: controlling cell fate decisions under ER stress and beyond , 2012, Nature Reviews Molecular Cell Biology.

[19]  S. Kügler,et al.  Aggregation of αSynuclein promotes progressive in vivo neurotoxicity in adult rat dopaminergic neurons , 2011, Acta Neuropathologica.

[20]  Kostas Vekrellis,et al.  Pathological roles of α-synuclein in neurological disorders , 2011, The Lancet Neurology.

[21]  S. Kügler,et al.  Sumoylation inhibits α-synuclein aggregation and toxicity , 2011, The Journal of cell biology.

[22]  E. Bézard,et al.  A tale on animal models of Parkinson's disease , 2011, Movement disorders : official journal of the Movement Disorder Society.

[23]  M. Zurita,et al.  Synphilin suppresses α‐synuclein neurotoxicity in a Parkinson's disease Drosophila model , 2011, Genesis.

[24]  Mohamad Saad,et al.  Imputation of sequence variants for identification of genetic risks for Parkinson's disease: a meta-analysis of genome-wide association studies , 2011, The Lancet.

[25]  Fred H. Gage,et al.  In vivo demonstration that α-synuclein oligomers are toxic , 2011, Proceedings of the National Academy of Sciences.

[26]  B. Hyman,et al.  Heat‐shock protein 70 modulates toxic extracellular α‐synuclein oligomers and rescues trans‐synaptic toxicity , 2011, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[27]  Adam J. Trexler,et al.  Single molecule characterization of α-synuclein in aggregation-prone states. , 2010, Biophysical journal.

[28]  T. Südhof,et al.  α-Synuclein Promotes SNARE-Complex Assembly in Vivo and in Vitro , 2010, Science.

[29]  E. Masliah,et al.  Phosphorylation at S87 Is Enhanced in Synucleinopathies, Inhibits α-Synuclein Oligomerization, and Influences Synuclein-Membrane Interactions , 2010, The Journal of Neuroscience.

[30]  B. Hyman,et al.  Tyrosine and serine phosphorylation of alpha-synuclein have opposing effects on neurotoxicity and soluble oligomer formation. , 2009, The Journal of clinical investigation.

[31]  Sonja W. Scholz,et al.  Genome-Wide Association Study reveals genetic risk underlying Parkinson’s disease , 2009, Nature Genetics.

[32]  H. Jäckle,et al.  Pre‐fibrillar α‐synuclein variants with impaired β‐structure increase neurotoxicity in Parkinson's disease models , 2009, The EMBO journal.

[33]  Zhiping Hu,et al.  Golgi apparatus and neurodegenerative diseases , 2008, International Journal of Developmental Neuroscience.

[34]  P. Lansbury,et al.  Phosphorylation at Ser-129 but Not the Phosphomimics S129E/D Inhibits the Fibrillation of α-Synuclein* , 2008, Journal of Biological Chemistry.

[35]  Bradley T. Hyman,et al.  Formation of Toxic Oligomeric α-Synuclein Species in Living Cells , 2008, PloS one.

[36]  P. Lansbury,et al.  The impact of the E46K mutation on the properties of alpha-synuclein in its monomeric and oligomeric states. , 2007, Biochemistry.

[37]  H. Cai,et al.  Coats, tethers, Rabs, and SNAREs work together to mediate the intracellular destination of a transport vesicle. , 2007, Developmental cell.

[38]  S. Michnick,et al.  A highly sensitive protein-protein interaction assay based on Gaussia luciferase , 2006, Nature Methods.

[39]  Yukio Fujita,et al.  Fragmentation of Golgi apparatus of nigral neurons with α-synuclein-positive inclusions in patients with Parkinson’s disease , 2006, Acta Neuropathologica.

[40]  N. Gonatas,et al.  Fragmentation of the Golgi apparatus in neurodegenerative diseases and cell death , 2006, Journal of the Neurological Sciences.

[41]  P. Fraser,et al.  Small Ubiquitin-like Modifier (SUMO) Modification of Natively Unfolded Proteins Tau and α-Synuclein* , 2006, Journal of Biological Chemistry.

[42]  C. Shults Lewy bodies. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Robert E. Schmidt,et al.  The alpha-synuclein mutation E46K promotes aggregation in cultured cells , 2006, Experimental Neurology.

[44]  N. Bonini,et al.  Snaring the Function of α-Synuclein , 2005, Cell.

[45]  M. Feany,et al.  α-Synuclein phosphorylation controls neurotoxicity and inclusion formation in a Drosophila model of Parkinson disease , 2005, Nature Neuroscience.

[46]  A Dürr,et al.  Causal relation between α-synuclein locus duplication as a cause of familial Parkinson's disease , 2004, The Lancet.

[47]  Philippe Amouyel,et al.  α-synuclein locus duplication as a cause of familial Parkinson's disease , 2004, The Lancet.

[48]  J. Hoenicka,et al.  The new mutation, E46K, of α‐synuclein causes parkinson and Lewy body dementia , 2004, Annals of neurology.

[49]  Matthew J. Farrer,et al.  Comparison of kindreds with parkinsonism and α‐synuclein genomic multiplications , 2004 .

[50]  S. Lindquist,et al.  Yeast Cells Provide Insight into Alpha-Synuclein Biology and Pathobiology , 2003, Science.

[51]  Janel O. Johnson,et al.  α-Synuclein Locus Triplication Causes Parkinson's Disease , 2003, Science.

[52]  M. Cookson,et al.  α‐Synuclein implicated in Parkinson's disease is present in extracellular biological fluids, including human plasma , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[53]  K. O’Malley,et al.  Parkinsonian Mimetics Induce Aspects of Unfolded Protein Response in Death of Dopaminergic Neurons* , 2003, Journal of Biological Chemistry.

[54]  Smita Patel,et al.  Golgi Fragmentation Occurs in the Cells with Prefibrillar α-Synuclein Aggregates and Precedes the Formation of Fibrillar Inclusion* , 2002, The Journal of Biological Chemistry.

[55]  Seung-Jae Lee,et al.  Characterization of cytoplasmic alpha-synuclein aggregates. Fibril formation is tightly linked to the inclusion-forming process in cells. , 2002, The Journal of biological chemistry.

[56]  E. Masliah,et al.  α-Synuclein is phosphorylated in synucleinopathy lesions , 2002, Nature Cell Biology.

[57]  B. Hyman,et al.  α-Synuclein–enhanced green fluorescent protein fusion proteins form proteasome sensitive inclusions in primary neurons , 2001, Neuroscience.

[58]  M. Tabaton,et al.  Full length α-synuclein is present in cerebrospinal fluid from Parkinson's disease and normal subjects , 2000, Neuroscience Letters.

[59]  J. Trojanowski,et al.  Synucleins Are Developmentally Expressed, and α-Synuclein Regulates the Size of the Presynaptic Vesicular Pool in Primary Hippocampal Neurons , 2000, The Journal of Neuroscience.

[60]  P. Lansbury,et al.  Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson's disease: implications for pathogenesis and therapy. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[61]  L. Meijer,et al.  Constitutive Phosphorylation of the Parkinson's Disease Associated α-Synuclein* , 2000, The Journal of Biological Chemistry.

[62]  D. Neill,et al.  Aggregates from mutant and wild‐type α‐synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of β‐sheet and amyloid‐like filaments , 1998, FEBS letters.

[63]  Robert L. Nussbaum,et al.  Mutation in the α-Synuclein Gene Identified in Families with Parkinson's Disease , 1997 .

[64]  J. Dartigues,et al.  Prevalence of parkinsonism and Parkinson's disease in Europe: the EUROPARKINSON Collaborative Study. European Community Concerted Action on the Epidemiology of Parkinson's disease. , 1997, Journal of neurology, neurosurgery, and psychiatry.

[65]  H. Braak,et al.  100 years of Lewy pathology , 2013, Nature Reviews Neurology.

[66]  A. Schapira,et al.  A Novel Alpha-Synuclein Missense Mutation in Parkinson's Disease , 2012 .

[67]  M. Feany,et al.  Alpha-synuclein phosphorylation controls neurotoxicity and inclusion formation in a Drosophila model of Parkinson disease. , 2005, Nature neuroscience.

[68]  N. Bonini,et al.  Snaring the function of alpha-synuclein. , 2005, Cell.

[69]  A Dürr,et al.  Causal relation between alpha-synuclein gene duplication and familial Parkinson's disease. , 2004, Lancet.

[70]  M. Farrer,et al.  Comparison of kindreds with parkinsonism and alpha-synuclein genomic multiplications. , 2004, Annals of neurology.

[71]  Philippe Amouyel,et al.  Alpha-synuclein locus duplication as a cause of familial Parkinson's disease. , 2004, Lancet.

[72]  E. Masliah,et al.  alpha-Synuclein is phosphorylated in synucleinopathy lesions. , 2002, Nature cell biology.

[73]  L. Meijer,et al.  Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein. , 2000, The Journal of biological chemistry.

[74]  M. Tabaton,et al.  Full length alpha-synuclein is present in cerebrospinal fluid from Parkinson's disease and normal subjects. , 2000, Neuroscience letters.

[75]  R. Krüger,et al.  Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease. , 1998, Nature genetics.

[76]  M G Spillantini,et al.  Alpha-synuclein in Lewy bodies. , 1997, Nature.

[77]  S E Ide,et al.  Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. , 1997, Science.