Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) produce profound motor and cognitive impairment associated with aggregation of ?-synuclein (?Syn). In these diseases, ?Syn undergoes complex post-translational modifications and fragmentation. In particular, many reports show increasing levels of C- terminally truncated (?C) ?Syn in brains of familial PD ?Syn mutation carriers and as a function of disease severity. Notably, truncated ?C-?Syn can induce soluble full-length ?Syn to co-assemble and thus accelerate, or even initiate, its aggregation. Limited research has focused on the proteases mediating ?C-?Syn truncation, their potential activation pathways, and the specific forms of the ?Syn substrate. Known caspase and calpain recognition sites occur in the ?Syn C-terminus, and inhibiting these proteases lowers ?C-?Syn and subsequent ?Syn aggregation. Moreover, the PI's recent studies of ?Syn in rodents exposed to paraquat, a herbicide linked to sporadic PD risk, showed elevated ?C-?Syn due to activation of calpain-1. However, these and other studies examining ?Syn in neurons or brain tissue have used denaturing conditions that preclude discriminating between monomers and the physiological ?Syn tetramer/multimers that our and other labs recently discovered. Using an intact-cell crosslinking assay that preserves the lysis-sensitive tetramers/multimers, we have shown that all fPD missense mutants shift normal tetramers to monomers. Further, we reported that mutating the N-terminal KTKEGV repeat motif, namely expressing E35K+E46K (?2K?) and E35K+E46K+E61K (?3K?) that mimic a doubling or tripling of the fPD E46K mutation, decreases the propensity of ?Syn to form normal tetramers/multimers. The resultant excess monomers associate with membranes, form ?Syn-rich vesicular inclusions, cause neurotoxicity, and could be subject to ?C truncation. Hence our central hypothesis is that excess ?Syn monomers arising under both familial and `sporadic' PD conditions provide the substrate for adverse proteolytic truncation. To pursue this hypothesis, we propose (Aim 1) to phenotype in detail our novel ?2K? and ?3K? tetramer-abrogating mice that develop neuronal aggregates in nigra and cortex containing ?C-?Syn and show progressive motor deficits with prominent tremor. As a distinct but complementary approach (Aim 2), we will apply paraquat to activate calpain-1 and to see if the resultant increase ?C-?Syn alters the multimer-to-monomer ratio by sequestering monomers into aggregates. Mechanistically, cholesterol-rich caveolar membranes regulate Ca2+ entry and calpain-1 activity, so we will ask if the calpain-mediated ?C truncation induced by paraquat or the fPD mutations is associated with changes of caveolar structure/structural proteins. In sum, we will generate new tools including novel tetramer-abrogating ?Syn tg mice and paraquat-treated neurons to identify ?Syn forms and examine proteases underlying the adverse ?C truncation that contributes to the progressive aggregation centrally implicated in PD and DLB.

Public Health Relevance

Our research grant addresses a critical public health concern by filling gaps in our understanding of the mechanisms of ?Syn truncation and aggregation and their relationship to neuronal death in PD and related human diseases. The overall goal is (1) to exploit our recent finding that an excess of aSyn monomers arising from abrogation of physiological ?Syn multimers provides substrates for adverse proteolytic cleavage; and (2) to use tetramer-abolishing mutants as well as models of toxin-induced protease activity to guide the future generation of protease inhibitors for treating PD and DLB.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1)
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Sutherland, Margaret L
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Brigham and Women's Hospital
United States
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