Acetylation of the Pro-Apoptotic Factor, p53 in the Hippocampus following Cerebral Ischemia and Modulation by Estrogen

Recent studies demonstrate that acetylation of the transcription factor, p53 on lysine(373) leads to its enhanced stabilization/activity and increased susceptibility of cells to stress. However, it is not known whether acetylation of p53 is altered in the hippocampus following global cerebral ischemia (GCI) or is regulated by the hormone, 17β-estradiol (17β-E(2)), and thus, this study examined these issues.The study revealed that Acetyl p53-Lysine(373) levels were markedly increased in the hippocampal CA1 region after GCI at 3 h, 6 h and 24 h after reperfusion, an effect strongly attenuated by 17β-E(2). 17β-E(2) also enhanced interaction of p53 with the ubiquitin ligase, Mdm2, increased ubiquitination of p53, and induced its down-regulation, as well as attenuated elevation of the p53 transcriptional target, Puma. We also observed enhanced acetylation of p53 at a different lysine (Lys(382)) at 3 h after reperfusion, and 17β-E(2) also markedly attenuated this effect. Furthermore, administration of an inhibitor of CBP/p300 acetyltransferase, which acetylates p53, was strongly neuroprotective of the CA1 region following GCI. In long-term estrogen deprived (LTED) animals, the ability of 17β-E(2) to attenuate p53 acetylation was lost, and intriguingly, Acetyl p53-Lysine(373) levels were markedly elevated in sham (non-ischemic) LTED animals. Finally, intracerebroventricular injections of Gp91ds-Tat, a specific NADPH oxidase (NOX2) inhibitor, but not the scrambled tat peptide control (Sc-Tat), attenuated acetylation of p53 and reduced levels of Puma following GCI.The studies demonstrate that p53 undergoes enhanced acetylation in the hippocampal CA1 region following global cerebral ischemia, and that the neuroprotective agent, 17β-E(2), markedly attenuates the ischemia-induced p53 acetylation. Furthermore, following LTED, the suppressive effect of 17β-E(2) on p53 acetylation is lost, and p53 acetylation increases in the hippocampus, which may explain previous reports of increased sensitivity of the hippocampus to ischemic stress following LTED

Enhanced glutamatergic and decreased GABAergic synaptic appositions to GnRH neurons on proestrus in the rat: modulatory effect of aging.

Previous work by our lab and others has implicated glutamate as a major excitatory signal to gonadotropin hormone releasing hormone (GnRH) neurons, with gamma amino butyric acid (GABA) serving as a potential major inhibitory signal. However, it is unknown whether GABAergic and/or glutamatergic synaptic appositions to GnRH neurons changes on the day of the proestrous LH surge or is affected by aging.To examine this question, synaptic terminal appositions on GnRH neurons for VGAT (vesicular GABA transporter) and VGLUT2 (vesicular glutamate transporter-2), markers of GABAergic and glutamatergic synaptic terminals, respectively, was examined by immunohistochemistry and confocal microscopic analysis in young and middle-aged diestrous and proestrous rats. The results show that in young proestrous rats at the time of LH surge, we observed reciprocal changes in the VGAT and VGLUT2 positive terminals apposing GnRH neurons, where VGAT terminal appositions were decreased and VGLUT2 terminal appositions were significantly increased, as compared to young diestrus control animals. Interestingly, in middle-aged cycling animals this divergent modulation of VGAT and VGLUT2 terminal apposition was greatly impaired, as no significant differences were observed between VGAT and VGLUT2 terminals apposing GnRH neurons at proestrous. However, the density of VGAT and VGLUT2 terminals apposing GnRH neurons were both significantly increased in the middle-aged animals.In conclusion, there is an increase in glutamatergic and decrease in GABAergic synaptic terminal appositions on GnRH neurons on proestrus in young animals, which may serve to facilitate activation of GnRH neurons. In contrast, middle-aged diestrous and proestrous animals show a significant increase in both VGAT and VGLUT synaptic terminal appositions on GnRH neurons as compared to young animals, and the cycle-related change in these appositions between diestrus and proestrus that is observed in young animals is lost

Organization of VGAT and VGLUT2 synaptic terminals onto GnRH neurons.

<p>Figure 2A, a low magnification confocal image of the brain section, encompassing POA regions, stained for VGAT (red), GnRH (blue) and VGLUT2 (green; channel hidden for clarity). Figure 2B is a <i>3D</i> confocal image created using Z-stacks. Figure 2C shows stereo views of a single GnRH neuron in various projections (60 projections). Figures 2 D1 and D2 show magnified stereo images of a GnRH neuron and organization of VGAT (red) and VGLUT2 (green) synaptic terminals. A synaptic terminal apposing GnRH neuron was confirmed and counted only when it apposed the cell body and dendrite (white arrows) throughout the rotation of the cell in multiple fields around Z axis. Scale bars represent 50 µm in Figure 2A, and 10 µm in Figure 2B and D.</p

Expression of VGAT and VGLUT2 synaptic proteins in the rPOA of the proestrous female rat.

<p>Figure 1A shows a schematic diagram of rat brain in coronal plane highlighting the POA region. Figures 1 B and C show 3D confocal images of VGLUT2 (green) and VGAT (red) obtained using 63X objective lens with additional 3X digital zoom. Both VGLUT2 and VGAT show punctate staining characteristics of presynaptic terminals. Figure 1D shows a merged (colocalized) image which indicates that while both VGLUT2 and VGAT proteins are highly expressed in the same region, only a small percentage (∼10%) of them show colocalization (white arrows) with each other. Figure 1E shows an enlarged view of VGAT (red) and VGLUT 2 (green) colocalization (>90%) with synaptophysin (blue) in the POA region. Figure 1F shows that almost all ERα positive (green) neurons show colocalization/co-expression (white arrows) of VGAT (red), VGLUT2 (blue) in the AVPV region. Scale bars represent 5 µm in Figure 1 B-D and 10 µm in Figure 1 E-F.</p

Cycle and age-dependent changes in VGAT and VGLUT2 terminals apposing GnRH neurons.

<p>Figure 3A and B, confocal images showing expression of VGAT (red), VGLUT2 (green) and GnRH neuron (blue) from a young (Figure 3A) and middle-aged (Figure 3B) proestrus female rat, respectively. Figure 3C, shows statistical analysis of VGAT and VGLUT2 synaptic terminals apposing GnRH neurons. In young animals at diestrus, average VGAT terminals apposing GnRH neurons were significantly (<i>P</i><0.001) higher (Figure 3C; bar Y1) than VGLUT2 terminals (Figure 3C; bar Y2). However, at proestrus afternoon, VGAT terminals apposing GnRH neurons were significantly (<i>P</i><0.01) decreased (Figure 3C; bar Y3) whereas VGLUT2 terminals (Figure 3C; bar Y4) were significantly increased (<i>P</i><0.001). In middle-aged animals at diestrus, average VGAT terminals (Figure 3C; bar M1) apposing GnRH neurons were not statistically different (<i>P</i>>0.05) from the number of VGLUT2 terminals (Figure 3C; bar M2). Similarly, at proestrus, the number of VGAT terminals (Figure 3C; bar M3) apposing GnRH neurons were also not statistically different (<i>P</i>>0.05) from the number of VGLUT2 terminals (Figure 3C; bar M4). Furthermore, in either diestrus or proestrus middle-aged animals, average VGAT (Figure3B, and Figure 3C; bars M1 and M3) and VGLUT2 (Figure3B, and Figure 3C; bars M2 and M4) terminals apposing GnRH neurons were significantly higher (<i>P</i><0.0001) compared to young either diestrus or proestrus animals (Figure 3C; bars Y1 andY3 for VGAT and bars Y2 and Y4 for VGLUT2). A further analysis at proestrus showed that in middle-aged animal, density of both VGAT (Figure 3B, and Figure 3D; bar M1) and VGLUT2 (Figure 3B, and Figure 3D; bar M2) terminals in the area in close vicinity of GnRH neurons was significantly (<i>P</i><0.0001) increased compared to young animals (Figure 3A, and Figure 3D; bar Y1 for VGAT and bar Y2 for VGLUT2). Scale in Figure 3 A and B represents 10 µm. Various abbreviations used in Figure 3C are: YD (young diestrus), YP (young proestrus), MD (middle-age diestrus) and MP (middle-age proestrus) animal groups.</p