Parkinson disease (PD) is the second most common neurodegenerative disorder1,2. Growing evidence suggests a causative role of misfolded forms of the protein, α-synuclein (αSyn), in the pathogenesis of PD3,4. Intraneuronal aggregates of αSyn occur in Lewy bodies and Lewy neurites5, the cytopathological hallmarks of PD and the related disorders called synucleinopathies. αSyn has long been defined as a “natively unfolded” monomer of ∼14 kDa6 that is believed to acquire α-helical secondary structure only upon binding to lipid vesicles7. This concept derives from the widespread use of recombinant bacterial expression protocols for in vitro studies, and of overexpression, sample heating and/or denaturing gels for cell culture and tissue studies. In contrast, we report that endogenous αSyn isolated and analyzed under non-denaturing conditions from neuronal and non-neuronal cell lines, brain tissue and living human cells occurs in large part as a folded tetramer of ∼58 kDa. Multiple methods, including analytical ultracentrifugation, scanning transmission electron microscopy and in vivo cell crosslinking, confirmed the occurrence of the tetramer. Native, cell-derived αSyn showed α-helical structure without lipid addition and had much greater lipid binding capacity than the recombinant αSyn studied heretofore. Whereas recombinantly expressed monomers readily aggregated into amyloid-like fibrils in vitro, native human tetramers underwent little or no amyloid-like aggregation. Based on these findings, we propose that destabilization of the helically folded tetramer precedes αSyn misfolding and aggregation in PD and other human synucleinopathies and that small molecules which stabilize the physiological tetramer could reduce αSyn pathogenicity.

Aggregation of a-synuclein (aS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of aS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that aS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, aS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age-dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous aS. In contrast, siRNA-mediated downregulation of aS results in elongated mitochondria in cell culture. aS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, aS prevents fusion of differently labelled mitochondrial populations. Thus, aS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of aS is rescued by coexpression of PINK1, parkin or DJ-1 but not the PD-associated mutations PINK1 G309D and parkin D1-79 or by DJ-1 C106A.

β-Sheet-rich α-synuclein (αS) aggregates characterize Parkinson's disease (PD). αS was long believed to be a natively unfolded monomer, but recent work suggests it also occurs in α-helix-rich tetramers. Crosslinking traps principally tetrameric αS in intact normal neurons, but not after cell lysis, suggesting a dynamic equilibrium. Here we show that freshly biopsied normal human brain contains abundant αS tetramers. The PD-causing mutation A53T decreases tetramers in mouse brain. Neurons derived from an A53T patient have decreased tetramers. Neurons expressing E46K do also, and adding 1-2 E46K-like mutations into the canonical αS repeat motifs (KTKEGV) further reduces tetramers, decreases αS solubility and induces neurotoxicity and round inclusions. The other three fPD missense mutations likewise decrease tetramer:monomer ratios. The destabilization of physiological tetramers by PD-causing missense mutations and the neurotoxicity and inclusions induced by markedly decreasing tetramers suggest that decreased α-helical tetramers and increased unfolded monomers initiate pathogenesis. Tetramer-stabilizing compounds should prevent this.

Background: ␣Syn is central to Parkinsonism, but its native state is unsettled. Results: A new, facile method for cross-linking ␣Syn in living cells, including neurons, reveals a major 60-kDa form consistent with a tetramer. Cell lysis destabilizes it, yielding mostly monomers. Conclusion: ␣Syn exists principally as a metastable tetramer in vivo. Significance: Models of native ␣Syn as an unfolded monomer should be reconsidered.

Copy number mutations implicate excess production of α-synuclein as a possibly causative factor in Parkinson’s disease (PD). Using an unbiased screen targeting endogenous gene expression, we discovered that the β2-adrenoreceptor (β2AR) is a regulator of the α-synuclein gene (SNCA). β2AR ligands modulate SNCA transcription through histone 3 lysine 27 acetylation of its promoter and enhancers. Over 11 years of follow-up in 4 million Norwegians, the β2AR agonist salbutamol, a brain-penetrant asthma medication, was associated with reduced risk of developing PD (rate ratio, 0.66; 95% confidence interval, 0.58 to 0.76). Conversely, a β2AR antagonist correlated with increased risk. β2AR activation protected model mice and patient-derived cells. Thus, β2AR is linked to transcription of α-synuclein and risk of PD in a ligand-specific fashion and constitutes a potential target for therapies.

Alpha-synuclein (aS) is a 140-amino-acid protein that is involved in a number of neurodegenerative diseases. In Parkinson's disease, the protein is typically encountered in intracellular, high-molecular-weight aggregates. Although aS is abundant in the presynaptic terminals of the central nervous system, its physiological function is still unknown. There is strong evidence for the membrane affinity of the protein. One hypothesis is that lipid-induced binding and helix folding may modulate the fusion of synaptic vesicles with the presynaptic membrane and the ensuing transmitter release. Here we show that membrane recognition of the N-terminus is essential for the cooperative formation of helical domains in the protein. We used circular dichroism spectroscopy and isothermal titration calorimetry to investigate synthetic peptide fragments from different domains of the full-length aS protein. Site-specific truncation and partial cleavage of the full-length protein were employed to further characterize the structural motifs responsible for helix formation and lipid-protein interaction. Unilamellar vesicles of varying net charge and lipid compositions undergoing lateral phase separation or chain melting phase transitions in the vicinity of physiological temperatures served as model membranes. The results suggest that the membrane-induced helical folding of the first 25 residues may be driven simultaneously by electrostatic attraction and by a change in lipid ordering. Our findings highlight the significance of the aS N-terminus for folding nucleation, and provide a framework for elucidating the role of lipid-induced conformational transitions of the protein within its intracellular milieu.

α-Synuclein (αS) is a highly abundant neuronal protein that aggregates into β-sheet-rich inclusions in Parkinson's disease (PD). αS was long thought to occur as a natively unfolded monomer, but recent work suggests it also occurs normally in α-helix-rich tetramers and related multimers. To elucidate the fundamental relationship between αS multimers and monomers in living neurons, we performed systematic mutagenesis to abolish self-interactions and learn which structural determinants underlie native multimerization. Unexpectedly, tetramers/multimers still formed in cells expressing each of 14 sequential 10-residue deletions across the 140-residue polypeptide. We postulated compensatory effects among the six highly conserved and one to three additional αS repeat motifs (consensus: KTKEGV), consistent with αS and its homologs β-and γ-synuclein all forming tetramers while sharing only the repeats. Upon inserting in-register missense mutations into six or more αS repeats, certain mutations abolished tetramer formation, shown by intact-cell cross-linking and independently by fluorescentprotein complementation. For example, altered repeat motifs KLKEGV, KTKKGV, KTKEIV, or KTKEGW did not support tetramerization, indicating the importance of charged or small residues. When we expressed numerous different in-register repeat mutants in human neural cells, all multimer-abolishing but no multimer-neutral mutants caused frank neurotoxicity akin to the proapoptotic protein Bax. The multimer-abolishing variants became enriched in bufferinsoluble cell fractions and formed round cytoplasmic inclusions in primary cortical neurons. We conclude that the αS repeat motifs mediate physiological tetramerization, and perturbing them causes PD-like neurotoxicity. Moreover, the mutants we describe are valuable tools for studying normal and pathological properties of αS and screening for tetramer-stabilizing therapeutics.alpha-synuclein | multimer | tetramer | Parkinson's disease | neurotoxicity

Background: Mitochondrial dysfunction and aggregation of ␣-synuclein both contribute to Parkinson disease. Results: Prefibrillar ␣-synuclein oligomers reduce the Ca 2ϩ retention time of isolated mitochondria respiring with complex I but not II substrates. Conclusion: Oligomeric ␣-synuclein promotes mitochondrial dysfunction in a Ca