Molecular and genetic dissection of neuronal necrotic-like death in Caenorhabditis elegans:

Neuronal necrosis is a major contributor to the devastating consequences of spinal cord injury, stroke, ischemia and neurodegenerative diseases. Detailed elaboration of the molecular mechanisms of neuronal necrosis will be essential for development of efficacious therapies. I studied neuronal necrosis in C. elegans, which involves death initiation by hyperactivated ion channels (MEC-4(d) and MEC10-(d)) in six touch neurons, elevation of intracellular Ca2+ via ER release, and activation of calpain and cathepsin proteases. I conducted the first genetic screen for enhancers of the mild necrosis-inducing stimulus conferred by MEC-10(d) to identify 18 medium-strong necrosis enhancer alleles (nen). The normal function of these genes should be to protect against necrosis in a native physiological context. One mec-10(d) necrosis enhancer is MEC-4 variant MEC-4(A149V). MEC-4(A149V) executes normal MEC-4 function in touch sensation and does not induce necrosis on its own, but rather combines with MEC-10(d) to create a strongly neurotoxic channel. The MEC-4(A149V) + MEC-10(d) channel conducts elevated Na+ and Ca2+ currents (with a disproportionate increase in Ca2+ current) in the Xenopus oocyte heterologous expression system. These data document the first example of synergistically toxic inter-subunit interactions in the DEG/ENaC channel class and provides evidence that Ca2+ current levels may be a decisive factor in necrosis. I also characterized another strong recessive necrosis enhancer, bz300, that appears to act downstream of channel-hyperactivation and upstream of ER-dependent cytoplasmic Ca2+ rise. nen(bz300) appears tightly linked to the unc-101 locus. Using a genome-inclusive RNAi testing of Ca2+-binding EF hand proteins, I identified potential key players (7 suppressors and 14 enhancers) in the necrosis pathway. I further confirmed that T05F1.1, which encodes a homologue of mammalian Nicalin, is a death enhancer by analysis of a deletion allele. Testing another model in the field, I used RNAi knockdown methods to demonstrate that Ca2+-induced calcium release proteins STIM-1 and Orai-1 are not required for MEC channel hyperactivation necrosis. I described the neuroprotective roles of heat shock response (HSR) and unfolded protein response (UPR) in mec-10(d)-induced necrosis. Decreasing the activities of HSF-1 and IRE-1/XBP-1 enhance cell death in parallel and non-redundant pathways, and their effects are slow and gradual. Epistasis analysis shows that cell death enhancement by these factors does not depend on the activity of calreticulin, a dramatic distinction from mec-4(d)-induced necrosis. My data indicate there might be additional stress factors that play key roles in the MEC-10(d) channel hyperactivation, in parallel with the calcium disturbance by ER release. Overall, my data advance understanding of physiological necrosis modulation.Ph.D.Includes bibliographical references (p. 224-234)by Wenying Zhan