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Molecular and genetic dissection of neuronal necrotic-like death in Caenorhabditis elegans
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Title
Molecular and genetic dissection of neuronal necrotic-like death in Caenorhabditis elegans
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ETD_1579
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http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.000051430
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eng
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theses
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Neuroscience
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Topic
Neurons--Necrosis
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Caenorhabditis elegans
Abstract
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.
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.
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electronic resource
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xvi, 236 p. : ill.
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Ph.D.
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Includes bibliographical references (p. 224-234)
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by Wenying Zhang
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Zhang
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Wenying
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1976
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Wenying Zhang
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Christopher
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Christopher Rongo
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Driscoll
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Monica
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Monica Driscoll
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Konsolaki
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Mary
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Mary Konsolaki
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Rutgers University
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Graduate School - New Brunswick
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2009
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2009-05
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xx
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Rutgers University Electronic Theses and Dissertations
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Graduate School - New Brunswick Electronic Theses and Dissertations
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doi:10.7282/T3JD4X1B
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ETD doctoral
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The author owns the copyright to this work.
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Copyright protected
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Open
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Zhang
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Wenying
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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