No abstract

The anti-diabetic drug and peroxisome proliferator-activated receptor-gamma (PPARc) agonist, rosiglitazone, alters astrocyte activation; however, its mechanism remains less-known. We hypothesized participation of epidermal growth factor receptor (EGFR), known to control astrocyte reactivity. We first detected that rosiglitazone promoted glial fibrillary acidic protein (GFAP) expression in primary astrocytes as well as the mouse cerebral cortex, associated with increased EGFR activation. Screening for EGFR ligands revealed a rosiglitazone-mediated increase of heparin-binding epidermal growth factor (HB-EGF) in astrocytes, resulting in HB-EGF release into culture medium and mouse cerebrospinal fluid too. Treatment with HB-EGF-siRNA and EGFR inhibitors showed that the rosiglitazone-induced HB-EGF and p-EFGR were interdependent, which participated in GFAP increase. Interestingly, we observed that rosiglitazone could induce cellular and secreted-HB-EGF in neurons also, contributing toward the activated EGFR-induced GFAP in astrocytes. Probing whether these effects of rosiglitazone were PPARc-linked, revealed potential PPARc-responsive elements within HB-EGF gene. Moreover, gel-shift, site-directed mutagenesis, chromatinimmunoprecipitation and luciferase-reporter assays demonstrated a PPARc-dependent HB-EGF transactivation. Subsequently, we examined effects of rosiglitazone in a high-fat diet-fed diabetes mouse model, and supporting observations in the normal cortical cells, identified a rosiglitazone-induced GFAP, astrocyte and neuronal HB-EGF and secreted-HB-EGF in the cerebral cortex of diabetic mice. Moreover, assessing relevance of increased HB-EGF and GFAP revealed an anti-apoptotic role of rosiglitazone in the cerebral cortex, supported by a GFAP-siRNA as well as HB-EGF-siRNA-mediated increase in cleaved-caspase 3 and 9 levels in the rosiglitazone-treated astrocyte-neuron coculture. Overall, our study indicates that rosiglitazone may protect the brain, via a PPARc-dependent HB-EGF/EGFR signaling and increased GFAP.

Estrogen deficiency reduces estrogen receptor-alpha (ERα) and promotes apoptosis in the hippocampus, inducing learning-memory deficits; however, underlying mechanisms remain less understood. Here, we explored the molecular mechanism in an ovariectomized (OVX) rat model, hypothesizing participation of autophagy and growth factor signaling that relate with apoptosis. We observed enhanced hippocampal autophagy in OVX rats, characterized by increased levels of autophagy proteins, presence of autophagosomes and inhibition of AKT-mTOR signaling. Investigating upstream effectors of reduced AKT-mTOR signaling revealed a decrease in hippocampal heparin-binding epidermal growth factor (HB-EGF) and p-EGFR. Moreover, 17β-estradiol and HB-EGF treatments restored hippocampal EGFR activation and alleviated downstream autophagy process and neuronal loss in OVX rats. In vitro studies using estrogen receptor (ERα)-silenced primary hippocampal neurons further corroborated the in vivo observations. Additionally, in vivo and in vitro studies suggested the participation of an attenuated hippocampal neuronal HB-EGF and enhanced autophagy in apoptosis of hippocampal neurons in estrogen- and ERα-deficient conditions. Subsequently, we found evidence of mitochondrial loss and mitophagy in hippocampal neurons of OVX rats and ERα-silenced cells. The ERα-silenced cells also showed a reduction in ATP production and an HB-EGF-mediated restoration. Finally in concordance with molecular studies, inhibition of autophagy and treatment with HB-EGF in OVX rats restored cognitive performances, assessed through Y-Maze and passive avoidance tasks. Overall, our study, for the first time, links neuronal HB-EGF/EGFR signaling and autophagy with ERα and memory performance, disrupted in estrogen-deficient condition.