Decrements in nuclear glucocorticoid receptor (GR) protein levels and DNA binding in aged rat hippocampus.

Hippocampal glucocorticoid receptors (GRs) are believed to play a major role in age-related cognitive decline and cellular vulnerability. It has been proposed that these receptors mediate damaging effects of elevated glucocorticoid secretion on cellular function. In the present report we present evidence that intracellular trafficking of the GR is impaired with hippocampal aging, manifest as decreased nuclear translocation and deficient DNA binding. We also present evidence that chaperone proteins responsible for GR nuclear translocation are decreased with hippocampal aging. Age-related nuclear GR decreases are not observed in hypothalamus, indicating regional specificity of trafficking deficits. Aging did not affect nuclear or cytosolic MR levels. These data suggest that GR signaling is diminished, rather than enhanced, during hippocampal aging. Diminished GR signaling capacity may attenuate the beneficial effects of glucocorticoids on hippocampal regulation of memory and stress integration.

Glutamate mediates cell death and increases the Bax to Bcl-2 ratio in a differentiated neuronal cell line.

Excessive stimulation of the NMDA receptor by glutamate induces cell death and has been implicated in the development of several neurodegenerative diseases. While apoptosis plays a role in glutamate-mediated toxicity, the mechanisms underlying this process have yet to be completely determined. Recent evidence has shown that exposure to excitatory amino acids regulates the expression of the antiapoptotic protein, Bcl-2, and the proapoptotic protein, Bax, in neurons. Since it has been suggested that the ratio of Bax to Bcl-2 is an important determinant of neuronal survival, the reciprocal regulation of these Bcl-2 family proteins may play a role in the neurotoxicity mediated by glutamate. Here, we have used a differentiable neuronal cell line, N1E-115, to investigate the molecular properties of glutamate-induced cell death. Annexin V staining was used to determine apoptotic cell death between 0 and 5 days differentiation with DMSO/low serum. Immunoblot analysis was used to determine whether the expression of Bcl-2 or Bax was modulated during the differentiation process. Bcl-2 protein levels were increased during maturation while Bax expression remained unchanged. Maximum Bcl-2 expression was observed following 5 days of differentiation. Examination of Bcl-2 and Bax following glutamate treatment revealed that the expression of these proteins was inversely regulated. Exposure to glutamate (0.001-10 mM) for 20+/-2 h resulted in a dose-dependent decrease in cell survival (as measured by MTT analysis) that was maximal at 10 mM. These results further support the role of apoptosis in glutamate-mediated cell death. Furthermore, a significant decrease in Bcl-2 levels was observed at 1 mM and 10 mM glutamate (32.1%+/-4.8 and 33.7+/-12.8%, respectively) while a significant upregulation of Bax expression (88.2+/-17.9%) was observed at 10 mM glutamate. Interestingly, Bcl-2 and Bax levels in cells treated with glutamate from 12-24 h were not significantly different from those of control. Taken together, these findings provide additional evidence for the reciprocal regulation of Bcl-2 and Bax expression by glutamate and suggest that neuronal excitotoxicity may, in part, result from the inverse regulation of these proteins.