ROLE OF C-TERMINUS OF HSC70 INTERACTING PROTEIN IN DETERMINING NEURONAL FATE IN ACUTE INJURY
JEANNETTE N. STANKOWSKI
Dissertation under the direction of Professor BethAnn McLaughlin
The decision to remove or refold oxidized, denatured or misfolded proteins by heat shock protein 70 (HSP70) and its binding partners is critical to determine cell fate. Acute overexpression of the ubiquitin ligase C-terminus of HSC70 interacting protein (CHIP) can compensate for failure of other ubiquitin ligases and enhance protein turnover and survival under chronic neurological stress. CHIPs ability to alter cell fate following acute neurological injury has however, not been assessed. Using post-mortem human tissue samples, we provide first evidence that cortical CHIP expression is increased following ischemic stroke. Oxygen glucose deprivation in vitro led to rapid protein oxidation, antioxidant depletion, proteasome dysfunction and a significant increase in CHIP. To determine if CHIP upregulation enhances neural survival, we overexpressed CHIP in vitro and evaluated cell fate 24hr following oxidative stress. Surprisingly, we observed that CHIP overexpressing cell lines fared worse against acute oxidative injury, accumulated more ubiquitinated and oxidized proteins and experienced decreased baseline proteasome activity suggesting that long-term upregulation of CHIP can be maladaptive. Conversely, decreasing CHIP expression in primary neuronal cultures using siRNA improved survival following oxidative stress, suggesting that the observed increase in CHIP following stroke-like injuries may negatively impact the neuroprotective potential of HSP70. To determine if cellular outcome could be further increased in an acute injury setting, we moved to a CHIP knockout model system and found increased levels of total oxidized proteins in brain tissue and accelerated calcium-induced mitochondrial permeability transition activities in these mice. Using the biotin-avidin-capture methodology to identify specific protein targets of oxidative stress, we found that known modulators of mitochondrial homeostasis or dynamics were not oxidatively modified. These results support previous findings of decreased lifespan and impaired survival upon injury observed in CHIP deficient animals. Together, these data suggest that CHIP expression must be tightly regulated in an acute injury setting as CHIP plays an essential role in regulating neuronal redox tone, and that strategies aimed at increasing CHIP expression levels may have previously unappreciated deleterious effects.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-01242011-140331 |
| Date | 24 January 2011 |
| Creators | Stankowski, Jeannette Nicole |
| Contributors | Dr. Pat Levitt, Dr. BethAnn McLaughlin, Dr. Deborah Murdock, Dr. Brian Wadzinski |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-01242011-140331/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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