This project's overall goal is to identify genes that undergo abnormal levels of expression in neurons affected by Parkinson Disease (PD) and that contribute to their death. The rationale is that neuron death in models of PD and other neurodegenerations requires transcription-dependent gene regulation. Once such genes are recognized and their causal roles in neuron death established, they will become potential targets for preventive/ameliorative therapy in PD. During the last period, we used SAGE to detect approximately 1,200 transcripts (out of 14,000) that are significantly up-regulated in a cell culture PD model. Among other findings, the SAGE profile revealed that PD mimetics induce an endoplasmic reticulum stress response that may contribute to death. For the next period, we propose the following specific aims: 1 To continue to """"""""mine"""""""" our SAGE data of 6-OHDA responsive genes. Bioinformatics will be used to match additional SAGE tags to known transcripts and to provide updated assessments of the potential functional roles of the regulated genes in neuronal death and/or PD. 2. To continue studies of the functional roles of select 6-OHDA-responsive genes identified by SAGE. Gene selection will reflect deduced potential relevance to PD and neuron death. Initial emphasis will be on ER stress genes and in particular on the pro-apoptotic transcription factor CHOP. Eight additional regulated genes have been chosen for initial emphasis. Studies will include loss- and gain-of-function experiments with PC12 cells and sympathetic neurons cultured from wild-type and mutant mice. When suitable animal models (null or transgenic) become available, these will be exploited with our collaborators Drs. Burke and Przedborski. 3. To continue examining whether responsive genes detected in our SAGE study are also up-regulated in animals models of PD and in PD tissue. Animal models will be examined collaboratively with Drs. Burke and Przedborski. PD and control tissue will be examined by a) immmunohistochemistry of SNpc and sympathetic neurons (SCG) and b) by quantitative PCR using RNA from such neurons harvested by laser capture microdissection. 4. To use data generated by SAGE or MPSS of control and PD neurons to detect additional regulated transcripts for studies of causal roles in neuronal death in PD. This longer-range aim that will seek to exploit gene profiling of control and PD neurons. Comparison of profiles from SCG, SNpc and PC12 cells will provide a powerful filter for identification of genes for functional in vitro and in vivo analyses as described above.
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