[P1-3-39] Elucidating the role of microglia in the molecular basis of sex differences in Alzheimer's disease
Keywords:中枢神経、加齢・老化、オートファジー
[Purpose] Epidemiological studies have shown that Alzheimer's disease (AD) is less prevalent in men than in women; however, the underlying mechanisms remain unclear. Microglia, the primary innate immune cells in the brain, play a crucial role in AD by releasing inflammatory cytokines and degrading aggregated amyloid β (Aβ) via autophagy. Recent studies have shown that microglial characteristics depend on sex, suggesting their involvement in sexually differential susceptibility to AD. Given the critical role of sex differences in AD, we focused on testosterone, the major sex hormone in males, implicates in the progression of amyloid pathology in AD. Low plasma testosterone levels have been reported to be associated with an increased risk of AD in men. GPRC6A, a G protein-coupled receptor, is a testosterone receptor responsible for its rapid non-genomic action. In this study, we investigated whether testosterone-GPRC6A signaling in microglia regulates Aβ-induced autophagy.
[Materials & Methods] Mouse microglial cell line MG6 was used. Autophagic flux was assessed by immunoblotting and fluorescence microscopy, using phosphatidylethanolamine-conjugated microtubule-associated protein 1 light chain 3 (LC3-II) or p62, which mediates the degradation of ubiquitinated proteins by selective autophagy, as indicators. Gprc6a was silenced using two independent shRNAs.
[Results & Conclusion] We confirmed that GPRC6A, but not the nuclear androgen receptor, was expressed in MG6 cells, indicating that GPRC6A mainly mediates testosterone signaling in MG6 cells. We found that testosterone stimulation suppressed the phosphorylation of extracellular signal-regulated kinase (ERK) in MG6 cells, suppressing mTOR activation, and promoting Aβ-induced autophagy, which may degrade Aβ. Extracellular Aβ was observed inside the cells and colocalized with the autophagosome marker LC3, indicating the uptake and degradation of Aβ in MG6 cells. Autophagic vacuoles increased upon co-stimulation with Aβ and testosterone. Furthermore, genetic knockdown of GPRC6A restored testosterone-stimulated repressed phosphorylation of ERK, which activates mTOR, an autophagic inhibitor. These results indicate that testosterone-GPRC6A signaling enhances Aβ-induced autophagy in microglia, and thus may play a crucial role in the low susceptibility to AD in men.
[Materials & Methods] Mouse microglial cell line MG6 was used. Autophagic flux was assessed by immunoblotting and fluorescence microscopy, using phosphatidylethanolamine-conjugated microtubule-associated protein 1 light chain 3 (LC3-II) or p62, which mediates the degradation of ubiquitinated proteins by selective autophagy, as indicators. Gprc6a was silenced using two independent shRNAs.
[Results & Conclusion] We confirmed that GPRC6A, but not the nuclear androgen receptor, was expressed in MG6 cells, indicating that GPRC6A mainly mediates testosterone signaling in MG6 cells. We found that testosterone stimulation suppressed the phosphorylation of extracellular signal-regulated kinase (ERK) in MG6 cells, suppressing mTOR activation, and promoting Aβ-induced autophagy, which may degrade Aβ. Extracellular Aβ was observed inside the cells and colocalized with the autophagosome marker LC3, indicating the uptake and degradation of Aβ in MG6 cells. Autophagic vacuoles increased upon co-stimulation with Aβ and testosterone. Furthermore, genetic knockdown of GPRC6A restored testosterone-stimulated repressed phosphorylation of ERK, which activates mTOR, an autophagic inhibitor. These results indicate that testosterone-GPRC6A signaling enhances Aβ-induced autophagy in microglia, and thus may play a crucial role in the low susceptibility to AD in men.