Abstract/Session Information for Program Number 758A
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Session Information
Session Title: Cell Death and Neurodegeneration   Session Type: Poster
Session Location: Pauley Pavilion   Session Time: Sunday - Tuesday
Abstract Information
Poster Board Number: 758A   Presentation Time: SUN, June 26, 2005, 1:30-3:00PM
Keywords: KW09 - Cell death
Abstract Content
A non-biased, in vivo genetic screen for genes that protect against necrosis. Wenying Zhang, Monica Driscoll. Dept Mol Biol & Biochem, Rutgers Univ, Piscataway, NJ.

   Necrosis, a non-apoptotic cell death often initiated by ion channel hyperactivation, plays a major role in the initial and prolonged death of neurons consequent to neuronal injury and diseases. Defining necrotic death mechanisms should suggest plausible molecular intervention strategies.
   The goal of our lab is to elaborate molecular mechanisms of necrosis by identifying the genes critical for necrosis regulation and execution. In C. elegans, a large sidechain AA substitution for a small residue near the pore in the touch channel subunit MEC-4 causes channel hyperactivation and excess ion influx, which elicits an intracellular Ca2+ elevation via ER release; consequently calpain and cathepsin proteases are activated (Xu et al., Neuron. 2001 957; Syntichaki et al., Nature. 2002 939.). We have conducted extensive genetic screens for suppressors of mec-4(d)-induced necrosis (see abstract by Gerstbrein et al.). However, we have not yet screened for genetic mutations that enhance necrosis. Necrosis enhancer mutations should identify genes that normally exert a protective function against necrosis in a physiological context.
   We conducted a classic forward screen of the C. elegans genome to identify mutations that enhance necrosis. Our screen strategy is based on mec-10(d)-induced death of the touch neurons (Huang, et al., Nature 1994 467). mec-10(d) encodes a large sidechain AA near the channel pore that weakly hyperactivates the touch channel when transgenically introduced. We constructed with a strain that is Is5[pmec4GFP];Is[mec-10(d)] in which nearly all 6 touch receptor neurons survive at 20oC. We then mutagenized and screened for rare animals in which many touch neurons die, as evidenced by the loss of touch neuron fluorescence. We found that the Union Biometrica BIOSORT, an instrument that functions like FACS sorter for nematodes and allows automatic fluorescence-based screening, was adequately sensitive to help sort out even modest necrosis enhancer mutants. We screened 18,500 EMS-mutagenized genomes for mutations that enhance necrosis. After eliminating candidates that simply lost the GFP reporter signal, we are left with 18 strong/moderate cell death enhancer alleles and 44 weak cell death enhancers. We will report on phenotypic and mapping characterization of the necrosis enhancers that will underlie cloning efforts. This work was supported in part by a Predoctoral Fellowship from the New Jersey Commission on Spinal Cord Research to Wenying Zhang.
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