C-Terminus of Heat Shock Cognate 70 Interacting Protein Increases Following Stroke and Impairs Survival Against Acute Oxidative Stress

Abstract

The decision to remove or refold oxidized, denatured, or misfolded proteins by heat shock protein 70 and its binding partners is critical to determine cell fate under pathophysiological conditions. 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. The ability of CHIP to alter cell fate after acute neurological injury has not been assessed. Using postmortem human tissue samples, we provide the first evidence that cortical CHIP expression is increased after ischemic stroke. Oxygen glucose deprivation in vitro led to rapid protein oxidation, antioxidant depletion, proteasome dysfunction, and a significant increase in CHIP expression. To determine if CHIP upregulation enhances neural survival, we overexpressed CHIP in vitro and evaluated cell fate 24 h after acute oxidative stress. Surprisingly, CHIP overexpressing cells fared worse against oxidative injury, accumulated more ubiquitinated and oxidized proteins, and experienced decreased proteasome activity. Conversely, using small interfering RNA to decrease CHIP expression in primary neuronal cultures improved survival after oxidative stress, suggesting that increases in CHIP observed after stroke like injuries are likely correlated with diminished survival and may negatively impact the neuroprotective potential of heat shock protein 70.

rAAV8-733-Mediated Gene Transfer of CHIP/Stub-1 Prevents Hippocampal Neuronal Death in Experimental Brain Ischemia

“…16 Our results contrast those of Stankowski et al, 21 who reported that primary neuronal cultures transfected with a small interfering RNA (siRNA) against CHIP resulted in improved survival following oxidative stress. Such a discrepancy is likely due to their use of dissociated cell cultures, whereas we examined both in vitro and in vivo models of hypoxic responses in structured brain tissue.…”

CHIP Is an Essential Determinant of Neuronal Mitochondrial Stress Signaling

“…In contrast to the benefits associated with transiently increased CHIP expression in blocking cell death induced by PD mutant genes (53), prolonged overexpression of CHIP worsens outcome following acute injury and causes proteasomal uncoupling (51), suggesting that a fine balance exists in the temporal expression of CHIP that affords neuroprotection.…”

“…We previously demonstrated that CHIP expression is increased in postmortem human tissue samples in patients following ischemic stroke as well as in an in vitro model of neuronal ischemia (51). In contrast to the benefits associated with transiently increased CHIP expression in blocking cell death induced by PD mutant genes (53), prolonged overexpression of CHIP worsens outcome following acute injury and causes proteasomal uncoupling (51), suggesting that a fine balance exists in the temporal expression of CHIP that affords neuroprotection.…”

“…Commercial vendors of chaperone antibodies as well as reagents and supplies used for immunoblotting, immunofluorescence, and cell culture experiments are the same as previously described (3,51). Additional primary antibodies used for immunoblotting in this study include p66 shc (566807; EMD), VDAC, COX IV, and Parkin (4866, 4850, and 2132, respectively; Cell Signaling), PINK1, Mfn1, and Drp1 (BC100-494, NB110-58853, and NB110-55288, respectively; Novus Biologicals), polyclonal CHIP (PC711; Calbiochem), LC3 (PD014; MBL International), and Cytochrome c (556433; BD Pharmingen).…”

“…GSH and GSSG concentrations were measured by highperformance liquid chromatography (HPLC) as previously described (51). Briefly, PND35 WT, Het, and CHIP KO animals were anesthetized and cervically dislocated.…”