A Dissertation Submitted For The Degree Of Doctor Of Philosophy

Part Ⅰ Metformin regulates the membrane trafficking of GAB A A receptor in cultured hippocampal neuronsAim:Metformin is a safe and effective hypoglycemic drug, so far still widely used in clinical treatment of type2diabetes. Recent studies suggest that metformin can promote the neurogenesis in hippocampus, and can also improve spatial learning and memory in mice. Numerous studies found that, GABAergic neurotransmission plays a crucial role in neuronal differentiation, growth of dendritic spines and synapse formation. Given the close relationship between the nerve regeneration and GABAergic neurotransmission, we hypothesized that GABAergic neurotransmission may be involved in metformin-induced neurogenesis, and the membrane trafficking of γ-aminobutyric acid type A receptor (GABAAR) was a key factor of inhibitory synaptic transmission. Thus, our experiment investigated whether metformin regulated the membrane trafficking of GABAAR in hippocampal neurons.Methods:Immunofluorescence and protein crosslinking techniques were used to test whether metformin had an effect on membrane trafficking GABAAR in the primary cultured hippocampal neurons. Whole-cell patch-clamp technique was employed to test whether metformin had an effect on GABAergic synaptic transmission in the primary cultured hippocampal neurons.Results:(1) Metformin promoted the surface expression of y2subunit-containing GABAAR in primary cultured hippocampal neurons.(2) Western blotting experiment confirmed that metformin had no effect on protein expression of y2subunit-containing GABAAR, but promoted the membrane expression of y2subunit-containing GABAAR in the primary cultured hippocampal neurons.(3) Metformin increased the amplitude of miniature IPSCs (mlPSCs) and frequency in the primary cultured hippocampal neurons.Conclusion:These results suggest that the metformin, the first-line drugs used to treat diabetes can promote membrane expression of y2subunit-containing GABAAR in primary hippocampal neurons. It also enhanced the GABAAR-mediated inhibitory postsynaptic neurotransmission. Part Ⅱ The mechanisms underlying the regulation of metformin in membrane trafficking of GABAA receptor in hippocampal neuronsAim:The previous work has demonstrated that metformin could promote the membrane trafficking of GABAAR in primary hippocampal neurons, the underlying mechanism needs to be further explored. More and more studies demonstrate that GABA receptor-associated protein (GABARAP) plays an important role in the trafficking of GABAAR, mainly through the interaction of GABARAP and GABAAR y2subunit. GABARAP belongs to autophagy-related genes8(autophagy-related protein8, Atg8) family member, which are critical for autophagosome biogenesis and maturation, whereas transcriptional regulation of autophagy-related genes is depended on the nuclear transcription factor FoxO3a. Meanwhile, a lot of evidence shows that the phosphorylation of FoxO3a caused by AMPK, a key kinase that regulates energy balance, can lead to increased transcriptional activity into the nucleus. Therefore, we hypothesized that metformin may activate AMPK-FoxO3a signaling pathway to increase the expression of GABARAP, thus enhance interaction with GABARAP and y2subunit of GABAAR, resulting in the enhancing GABAergic neurotransmission in the primary cultured hippocampal neurons. Therefore, this study investigated the role of AMPK-FoxO3a-GABARAP in membrane trafficking of GABAAR during metformin treatment.Methods:Co-immunoprecipitation was used to detect the effcects of metformin on the interaction of GABARAP with the y2subunit of the GABAAR in the primary cultured hippocampal neurons. qRT-PCR and western blotting were used to detect the effcects of metformin on the mRNA and protein levels of GABARAP and FoxO3a in primary cultured hippocampal neurons. Protein cross-linking method and immunofluorescence technology were employed to detect whether lentivirus silencing FoxO3a could prevent the metformin-induced expression of GABARAP and membrane trafficking of GABAAR. Chromatin immunoprecipitation was employed to detect the interaction of FoxO3a promoter with GABARAP. Pharmacological AMPK inhibitor compound C and lentivirus silencing AMPK expression were used to detect whether downregulated AMPK activation could inhibit the metformin-induced expression of GABARAP and membrane trafficking of GABAAR. Immunofluorescence experiment was performed to detect whether metformin has an effect on the translocation of FoxO3a in the primary cultured hippocampal neurons.Results:(1) Metformin-induced GABAaR insertion was mediated by increased expression of GABARAP and interactions with γ2subunit.(2) FoxO3a was implicated in metformin-induced upregulation of GABARAP expression and surface expression of GABAAR.(3) AMPK activation was required for metformin-induced activation of FoxO3a signaling in cultured rat hippocampal neurons.Conclusion:In the primary cultured hippocampal neurons, the activation of AMPK-FoxO3a-GABARAP signaling pathway mediated metformin-induced membrane trafficking of GABAAR. Part Ⅲ The effect and mechanisms of metformin on anxiety-like behavior of ratsAim:In vitro studies have shown that metformin can activate AMPK-FoxO3a-GABARAP signaling pathway, thus contributing to membrane trafficking of GABAAR. Thus, we asked whether metformin can produce anxiolytic effects. Therefore, this study investigated the impact of metformin on anxiety-like behavior.Methods:Immunofluorescence and protein crosslinking techniques were used to test whether metformin had an effect on membrane trafficking of GABAAR on the pyramidal neurons of hippocampal CA1region in rats. Open field test (OFT) and elevated plus maze (EPM) were employed to assay the effecs of metformin and TAT-GABARAP on anxiety-like behavior and locomotor activity. Western blotting was used to test the effecs of metformin on the activation AMPK-FoxO3a-GABARAP signaling pathway in hippocampal CA1region, and the interaction between GABARAP and GABAAR y2subunit. The glucose test strips was used to detect whether metformin had an effect on basal blood sugar level. Results:(1) Metformin increased the surface expression of GABAAR in hippocampus CA1region.(2) Metformin-treated rats displayed anxiolytic-like behavior in open field test and elevated plus maze experiments.(3) Metformin activated AMPK-FoxO3a-GABARAP signaling pathway in the hippocampal CA1region and enhanced the interaction between the y2subunit of the GABAAR and GABARAP.(4) TAT-GABARAP was able to block the anxiolytic-like behavior induced by metformin.(5) Metformin did not affect basal blood sugar levels.Conclusion:Our results suggest that the metformin produces anxiolytic effect by increase the surface trafficking of GABAAR, providing a candidate pharmacological approach for treatment of anxiety disorder.