Molecular mechanisms of collybistin-dependent gephyrin clustering at inhibitory synapses

• Applicant: Dr. Theofilos Papadopoulos
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2011 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 203252528

Collybistin (Cb) is a brain-specific guanine nucleotide exchange factor that interacts with gephyrin, the major scaffolding protein at inhibitory postsynapses. Cb and gephyrin mutations in humans have been reported to cause diverse neurological and psychiatric disorders. My work is devoted to the elucidation of the mechanisms that regulate Cb-mediated clustering of gephyrin at GABAergic postsynapses, a key step of neuronal network formation in the hippocampus. The project proposed here has three major objectives that are intimately interconnected.i) Previous work indicates an essential role of phosphatidylinositol-3-phosphate (PI3P) in Cb-dependent gephyrin clustering. Here, the enzymes and signaling mechanisms that regulate the generation of PI3P on endomembranes involved in the intracellular trafficking of Cb during synaptogenesis will be identified. ii) Recently, a point mutation in human Cb (R290H) was shown to underlie a heritable form of epilepsy and mental retardation. Our preliminary characterization of the R290H-protein indicates that the mutation perturbs PI3P binding. In this project, the changes in the biochemical, structural and functional properties of the R290H mutant that underlie its dysfunction at inhibitory synapses will be defined.iii) Our work led to the identification of the Rho-GTPase TC10 as a key regulator of Cb-mediated gephyrin clustering. Furthermore, preliminary results indicate that TC10 binds to phosphoinositides. Here, these findings will be extended by examining the physiological role of TC10 during synaptogenesis in vivo using knockout mice. In addition, the involvement of TC10 in the regulation of Cb binding to PI3P-rich endomembranes will be further investigated.Together, the experiments proposed are expected to unravel how localized PI3P generation and GTPase-induced Cb-activation regulate the recruitment of Cb, and thereby contribute to the formation of gephyrin clusters at developing postsynaptic sites. In addition, they should help to clarify the pathomechanism that causes epilepsy and mental retardation in humans carrying the Cb R290H mutation.

• DFG Programme: Research Grants