Activation of the 20S proteasome core particle prevents cell death induced by oxygen- and glucose deprivation in cultured cortical neurons

Neuronal damage in brain ischemia is characterized by a disassembly of the proteasome and a decrease in its proteolytic activity. However, to what extent these alterations are coupled to neuronal death is controversial since proteasome inhibitors were shown to provide protection in different models of stroke in rodents. This question was addressed in the present work using cultured rat cerebrocortical neurons subjected to transient oxygen- and glucose-deprivation (OGD) as a model for in vitro ischemia. Under the latter conditions there was a time-dependent loss in the proteasome activity, determined by cleavage of the Suc-LLVY-AMC fluorogenic substrate, and the disassembly of the proteasome, as assessed by native-polyacrylamide gel electrophoresis followed by western blot against Psma2 and Rpt6, which are components of the catalytic core and regulatory particle, respectively. Immunocytochemistry experiments against the two proteins also showed differential effects on their dendritic distribution. OGD also downregulated the protein levels of Rpt3 and Rpt10, two components of the regulatory particle, by a mechanism dependent on the activity of NMDA receptors and mediated by calpains. Activation of the proteasome activity, using an inhibitor of USP14, a deubiquitinase enzyme, inhibited OGD-induced cell death, and decreased calpain activity as determined by analysis of spectrin cleavage. Similar results were obtained in the presence of two oleic amide derivatives (B12 and D3) which directly activate the 20S proteasome core particle. Together, these results show that proteasome activation prevents neuronal death in cortical neurons subjected to in vitro ischemia, indicating that inhibition of the proteasome is a mediator of neuronal death in brain ischemia.