The hypothesis driving this proposal is that presynaptic dysfunction is a common causative factor leading to cell death in multiple inherited neurodegenerative diseases. This hypothesis is based on the observations that 1) synaptic function mediates neuronal survival during development, 2) mutations which strongly impair presynaptic function result in massive, progressive neuronal degeneration, 3) a number of presynaptic proteins have been directly implicated in neurodegenerative diseases and 4) neuronal dysfunction/synapse loss is known to precede by a substantial period the manifestation of cell death in these diseases. To date, however, there is no established direct evidence of synaptic dysfunction mediating neuronal death during neurodegenerative disease states. The goal of this proposal is to assay synaptic maintenance in two genetic models of neurodegenerative diseases: Drosophila models of Parkinson's Disease (PD), a classic """"""""protein storage"""""""" disease, and Niemann-Pick Type C (NP-C), a classic """"""""lipid storage"""""""" disease. Drosophila was selected for its attractive properties as a new molecular genetic model of neurodegeneration, and its long history as the foremost genetic model for synaptic studies. PD and NP-C were selected as representative of a large number of related neurodegenerative disorders. The Drosophila PD model has been recently established through transgenic over-expression of human alpha-synuclein (a presynaptic protein) and shown to accurately recapitulate the diagnostic features of human PD. A Drosophila model of NP-C is being established through mutation (loss-of-function) of the endogenous NPC I gene, the known cause of human NP-C disease. Specifically, this proposal is to conduct age-progressive studies of synaptic mechanisms in Drosophila PD and NP-C models to correlate synaptic maintenance with the onset, progression and prevalence of neurodegeneration.
The first aim i s to improve Drosophila models by generating fluorescently tagged alpha-synuclein and NPCI proteins whose levels can be reversibly regulated through a temperature-dependent ubiquination strategy. Secondly, to confirm gross features of neurodegeneration in these models with behavioral assays and examination of nervous system/neuronal architecture. Third, and most importantly, to assay synaptic development, function and maintenance in these models. Assays will include electrophysiological measurements of neurotransmission, quantitative fluorescent optical imaging of protein and lipid dynamics in the presynaptic terminal and ultrastructural studies of presynaptic architecture. Together, these studies will allow a conclusive determination of whether synaptic maintenance is compromised in PD and NP-C, and is the causative factor that leads to neuronal cell death and neurodegeneration in these disease states.
Showing the most recent 10 out of 11 publications
