we observed that cytochrome c was retained in the mitochondria of Puma inferior neurons suggesting that Puma is necessary for Bax induced mitochondrial membrane permeabilization. Differences Chk1 inhibitor between groups were considered statistically significant when p,0 and post hoc Tukeys test and were dependant on ANOVA. 05. The specific BH3 only genes involved with apoptotic signaling as well as the mechanisms by which they are regulated varies with respect to the cell-type and death stimulus. In CGNs apoptosis induced by potassium withdrawal could be avoided by actinomycin D or cycloheximide suggesting that de novo transcription is prone to the activation of BH3 only proteins and critical to the initiation of apoptosis. We therefore examined expression of BH3 only genes following potassium withdrawal in CGNs using quantitative RT PCR. We found no change in transcript levels of a few BH3 only household members including Noxa, Bid and Bad, however in keeping with previous reports we observed an increase in Hrk/DP5 and Bim mRNA levels. Apparently, we also noticed a marked escalation in Puma mRNA, a comparatively unstudied BH3 only member in this context. Consistent with the increase in Puma, Bim and mRNA protein amounts were also found to be improved following potassium Lymph node withdrawal. . Several studies including those from our research group have demonstrated that Puma plays a key part in regulating neuronal apoptosis in diverse injury paradigms. Consequently, we wanted to ascertain whether Puma is required for potassium deprivation induced apoptotic cell death in cerebellar granule neurons. To address this we compared the degree of apoptotic cells in CGNs based on wild-type littermates and Puma deficient put through potassium withdrawal. We found that neurons missing Puma exhibited a marked reduction in the number of apoptotic Dabrafenib GSK2118436A nuclei compared with wild type cells following potassium withdrawal. . One of the critical steps in the intrinsic apoptotic pathway is Bax mediated mitochondrial depolarization and mitochondrial outer membrane permeabilization. Thus we examined the position of Puma in controlling these Baxmediated apoptotic functions. To determine mitochondrial membrane potential we stained wild type and Puma inferior neurons with the mitochondria potentiometric dye Mitotracker Red. In contrast to wild-type neurons the great majority of Puma deficient neurons maintained the ability to uptake Mitotracker Red under low potassium circumstances showing that Puma is needed for mitochondrial membrane depolarization. Moreover, while potassium starvation triggered a robust induction of caspase 3 like activity in wild type neurons it was markedly decreased in Puma deficient neurons. As Bim has also been implicated in neuronal apoptosis induced by trophic component deprivation, we also examined the level of apoptosis in CGNs produced from Bim null mice following potassium deprivation.