Title

Gephyrin and the postsynaptic GABAergic complex

Date of Completion

January 2007

Keywords

Biology, Neuroscience|Biology, Cell

Degree

Ph.D.

Abstract

Although gephyrin is an important postsynaptic scaffolding protein at GABAergic synapses, the role of gephyrin for GABAergic synapse formation and/or maintenance is still under debate. In order to address this question, I manipulated the gephyrin expression levels by using small hairpin RNAs (shRNAs) to decrease gephyrin expression, by overexpressing a gephyrin-EGFP fusion protein to decrease the gephyrin clustering and by overexpression of wild type gephyrin in cultured hippocampal pyramidal cells to detect their effects on synapses. I found that decreased postsynaptic gephyrin clustering led to not only decreased postsynaptic GABA(A) receptor clusters but also decreased GABAergic innervation. It is concluded that gephyrin plays a critical role for the stability of GABAergic synapses. ^ Further more, I showed that altering the levels of expression/clustering of gephyrin also affects glutamatergic synapses. Decreasing gephyrin clusters led to increased size but not density of both the postsynaptic glutamatergic PSD-95 clusters and the presynaptic glutamatergic terminals contacting the pyramidal cells. Overexpression of gephyrin led to a slight decrease in size but not in density of both PSD-95 clusters and presynaptic glutamatergic terminals. However, the size or density of the glutamate GluR1-AMPA, or NR1-NMDA receptor clusters was not affected. It is concluded that the expression and clustering of gephyrin, a postsynaptic GABAergic scaffold protein, also plays a role in the control of the size of glutamatergic contacts. ^ I also showed that GRIP1c4-7 is a short splice form of GRIP1a, present in rat genome. The N-terminal sequence specifies its membrane localization. GRIP1c4-7 and GRIP1a/b interact with gephyrin by co-precipitation from rat brain extracts and from extracts of HEK293 cells. Moreover, purified gephyrin binds to purified GRIP1c4-7 or GRIP1a/b as shown by an affinity pull down assay, indicating that these molecules directly interact with each other. Postsynaptic gephyrin clusters colocalized with GRIP1c4-7 and GRIP1a/b clusters in cultured hippocampal neurons. These results indicate that GRIP1 proteins could be involved in the trafficking and recycling of postsynaptic gephyrin at GABAergic synapses. ^ By using gamma2-GABAAR shRNAs, an approach which is different from the knockout mice, we have shown that gamma2 subunit is important for the clustering of beta2/3-GABAARs and gephyrin. More importantly, we have shown that disrupting normal postsynaptic gamma2-GABAAR clustering leads to a significant reduction in the GABAergic innervation. The studies were done in cultured hippocampal pyramidal cells. I further extended the research to in vivo studies by transfecting neurons with in utero electroporation where I found that decreased GABA(A)R clustering and GABAergic innervation of pyramidal neurons in the post-natal rat cerebral cortex after in utero transfection of these neurons with the gamma2 shRNAs. All these results support the hypothesis that γ2 subunit is essential for the postsynaptic clustering of the GABAAR and the postsynaptic clustering of GABAAR is essential for the stability of the GABAergic synapses. ^ All these studies put together show: (I) gephyrin is critical for the postsynaptic clustering of many GABAARs; (II) γ2-GABA AR clustering is critical for the postsynaptic clustering of gephyrin and (III) the postsynaptic clustering of GABAAR and gephryin is cirtical for the stability of the presynaptic GABAergic innervation. ^