DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a late-onset neurodegenerative disorder characterized by the progressive loss of dopamine (DA) producing neurons in the substantia nigra (SN).(1) The neuronal protein ?-synuclein (?-syn) is an important contributor since mutations in the ?-syn gene cause familial PD and aggregated wild type (WT) ?-syn is present within hallmark pathological inclusions termed Lewy bodies in sporadic cases.(2-7) Recently, it was shown that injection of ?-syn aggregates into WT mouse brain is sufficient to induce widespread ?-syn aggregation, DA cell death, and motor impairment.(24) However, the identity of disease-causing aggregated species is unknown. Growing evidence suggests that soluble oligomer aggregates of ?-syn are pathogenic,(28) yet a direct link between oligomers and neurodegeneration remains to be demonstrated. Moreover, oxidized DA stabilizes ?-syn oligomers in cell culture and cell-free systems,(38-41) and the effects of this interaction on neuronal health in vivo remains unexplored. In this study, ?-syn oligomers or oligomers stabilized by DA will be directly tested for their ability to induce DA cell degeneration in vivo. In Aim 1, biochemically defined oligomer species of human ?-syn either extracted from the nervous system of transgenic mice(18) ex vivo or generated from purified protein in vitro(26) will be injected into the SN of adult [?-syn transgenic] mice. Control mice wil be injected with ex vivo material depleted of ?-syn or in vitro purified monomer in order to support the attribution of observed phenotypes specifically to oligomeric ?-syn. The effects on ?-syn aggregation will be examined by biochemical separation of oligomer species and detection of inclusions by histology. Analysis of nigrostriatal degeneration will include DA neuron count in SN, synaptic protein expression in striatum, and motor function testing. [Oligomer-induced defects in autophagy will be tested as a possible mechanism of toxicity using electron microscopy and degradation assays.] In Aim 2, a novel lentiviral approach will be used to increase DA production in the SN of ?-syn transgenic mice,(18) in order to test the effects on ?-syn oligomerization and DA cell viability. Transgenic mice injected with an empty vector [or inactive form of the vector] will serve as a control for viral transduction, and enhancement of DA levels in WT mice will control for DA-related toxicity that is independent of human ?-syn. Preliminary data suggest that viral-mediated increase in catecholamine levels is accompanied by both stabilization of ?-syn oligomers of larger size and a significant decrease in the number of SN neurons. DA-stabilized oligomers will be characterized in terms of size, stability, and other biochemical properties. As in Aim 1, DA cell number in SN, synaptic protein expression in striatum, [deficits in autophagy], as well as motor functioning will indicate if DA neurons have degenerated [due to impairments in protein degradation]. The findings from these Aims will provide the first investigation into the ability of ?-syn oligomers to directly initiate disease inan otherwise [unaffected] host, and the role of DA in potentiating oligomer toxicity in vivo, with significant implications for defining ?-syn disease mechanisms and designing novel PD therapies.
|Effective start/end date||12/1/14 → 10/30/16|