Role of C-terminus of HSC70 interacting protein in determining neuronal fate in acute injury

Abstract

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. CHIP’s 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.