Several independent lines of evidence have linked the nerve terminal- enriched synuclein family of proteins to Parkinson's disease. Genetic studies have linked mutations in the alpha-synuclein gene to the disease in several families. Synucleins are a major constituent of Lewy bodies, a hallmark histopathology of Parkinson's disease. Over-expression of synucleins are a major constituent of Lewy bodies, a hallmark histopathology of Parkinson's disease. Over-expression of synucleins in mice causes Lewy body-like neuropathology and motor deficits. While there is a wealth of information on the genetic and histological features of synucleins, very little information exists on the function of the proteins in normally functioning nerve terminals. We propose to study the function of these proteins in nerve terminals using classic neurophysiologic experiments in an isolated nerve terminals using classic neurophysiologic experiments in an isolated nerve terminal. Pre-synaptic injection of agents that will modify synuclein function will be made into the giant pre-terminal of a primitive vertebrate (lamprey). The effects of pre-terminal injections of several synuclein affecting agents will be analyzed with electrophysiologic and ultrastructural methods. Inj3ected agents will include: full length and fragments of lamprey synuclein, recombinant human alpha-synuclein containing Parkinson's disease associated mutations (A53T and A30P). Mock casein kinase I and src kinase phosphorylated (S129E/S87E) human alpha-synuclein and antibodies against lamprey synuclein. Using paired intracellular recordings between the pre- and post-synaptic elements we will examine the effects of injections on the ESPS generated by intraoxonal stimulation. Many parameters of synaptic vesicle release kinetics will be examined. In addition, giant pre-synaptic elements receiving injections of agents followed by varying levels of stimulation will be examined under the electron microscope. Effects of injections on the synaptic vesicle cycle can be established by characteristic changes in nerve terminal morphology. Establishing the function of the synucleins in normal neurotransmitter release will shed light on the function and dysfunction of the protein in Parkinson's disease.
