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.

Public Health Relevance

Parkinson's disease (PD) is second only to Alzheimer's as the most common neurodegenerative illness, causing the loss of dopamine producing neurons in the substantia nigra and devastating motor impairment.(1) PD pathology is characterized by the aggregation of the protein ?-synuclein (?-syn),(7) and intermediate-sized ?-syn aggregates known as oligomers are thought to cause disease.(28) This study aims to provide the first direct demonstration of ?-syn oligomer toxicity in an animal model system in vivo, and an investigation into the role of dopamine itself in disease mechanisms, bearing significant implications for treatment design in PD.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31NS087779-02
Application #
8979451
Study Section
Special Emphasis Panel (ZRG1-F03A-N (20))
Program Officer
Sutherland, Margaret L
Project Start
2014-12-01
Project End
2016-08-31
Budget Start
2015-12-01
Budget End
2016-08-31
Support Year
2
Fiscal Year
2016
Total Cost
$29,869
Indirect Cost
Name
University of Pennsylvania
Department
Pediatrics
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Mor, Danielle E; Tsika, Elpida; Mazzulli, Joseph R et al. (2017) Dopamine induces soluble ?-synuclein oligomers and nigrostriatal degeneration. Nat Neurosci 20:1560-1568
Mor, Danielle E; Ugras, Scott E; Daniels, Malcolm J et al. (2016) Dynamic structural flexibility of ?-synuclein. Neurobiol Dis 88:66-74