Molecular mechanisms of collybistin-dependent gephyrin clustering at inhibitory synapses
Final Report Abstract
Collybistin (CB) is a brain-specific guanine nucleotide exchange factor that interacts with gephyrin, the major scaffolding protein at inhibitory postsynaptic sites. 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. Our work led to the identification of the Rho-GTPase TC10 as a key regulator of CB-mediated gephyrin clustering. Recently, we have shown that TC10 binds to monophosphorylated phosphoinositides and that TC10 acts as a specificity-regulator of the binding of CB to certain phosphoinositides. Our data define a protein-lipid interaction network that controls the clustering of gephyrin and gamma-aminobutyric acid type-A receptors (GABAARs) at synapses. Within this network, TC10 and monophosphorylated phosphoinositides, particularly phosphatidylinositol-3-phosphate (PI3P), provide a coincidence detection platform that allows the accumulation and activation of CB in endomembranes. Upon activation, TC10 induces a phospholipid affinity switch in CB, which allows CB to specifically interact with phosphoinositide species present at the plasma membrane, such as PI(4,5)P2 and PI(3,4,5)P3. Our findings explain the hitherto enigmatic facts that native CB is present in both endosomal and plasma membrane compartments and that CB can operate at postsynaptic membranes to recruit gephyrin despite its intrinsic preference for endosomal-based phosphoinositides. GABAergic interneurons in the hippocampus play a role in shaping neuronal network patterns and oscillations that are involved in higher brain functions like memory, attention and perception. CB deficiency in humans leads to epilepsy, autism, mental retardation, aggressive behaviour and anxiety. Whether CB-deficiency affects the postsynaptic clustering of GABAARs in the different subtypes of hippocampal GABAergic interneurons has not been yet investigated. Recently, we indicated that in the brains of CB-deficient mice, the clustering of gephyrin is unaltered in neuronal subpopulations endogenously expressing the α3 subunit of GABAARs. GABAAR-α3 is abundantly expressed in the reticular thalamic nuclei (nRT), an area rich in GABAergic interneurons, which indicates an important role of α3-containing GABAARs in localizing gephyrin at postsynaptic sites of interneuronal synapses. Moreover, exogenous expression of GABAAR-α3 partially rescues the impaired clustering of gephyrin in cultured hippocampal neurons derived from CB KO animals. These results indicated an important role of GABAAR-α3 in priming gephyrin-mediated and CB-independent formation of inhibitory postsynapses. In addition to the aforementioned published work of the last funding period, we established a unbiased cell-based screening in order to search for novel proteins involved in the formation of inhibitory synapses in the mammalian forebrain. Our screen led to the identification of a scaffolding protein which interacts directly with the SH3 domain of CB and plays an important role in the regulation of the gephyrin clustering in parvalbumin-positive interneurons of the hippocampus.
Publications
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Collybistin activation by GTP-TC10 enhances postsynaptic gephyrin clustering and hippocampal GABAergic neurotransmission. Proc. Natl. Acad. Sci. U.S.A. 110: 20795-20800 (2013)
Mayer, S., Kumar, R., Jaiswal, M., Soykan, T., Ahmadian, M.R., Brose, N., Betz, H., Rhee J.S., Papadopoulos, T.
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Lipid binding defects and perturbed synaptogenic activity of a collybistinR290H mutant that causes epilepsy and intellectual disability. J. Biol. Chem. 290: 8256-8270 (2015)
Papadopoulos, T., Schemm, R., Grubmüller, H., Brose, N.
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IQ Motif and SEC7 Domain-Containing Protein 3 (IQSEC3) Interacts with Gephyrin to Promote Inhibitory Synapse Formation. J. Biol. Chem. 291: 10119-10130 (2016)
Um, J.W, Choii, G., Park, D., Kim, D., Jeon, S., Kang H., Mori, T., Papadopoulos, T., Yoo, T., Lee, Y., Kim, E., Tabuchi, K., Ko, J.
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Specificity of Collybistin-Phosphoinositide Interactions - Impact of the individual protein domains. J. Biol. Chem. 291: 244-254 (2016)
Ludolphs, M., Schneeberger, D., Soykan, T., Schäfer, J., Papadopoulos, T., Brose, N., Schindelin, H., Steinem, C.
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Endosomal Phosphatidylinositol 3-Phosphate Promotes Gephyrin Clustering and GABAergic Neurotransmission at Inhibitory Postsynapses. J. Biol. Chem. 292: 1160-1177 (2017)
Papadopoulos, T.§, Rhee, H.J., Subramanian, D., Paraskevopoulou, F., Mueller, R., Schultz, C., Brose, N., Rhee, J.-S., Betz, H.
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A GTPase-induced switch in phospholipid affinity of collybistin contributes to synaptic gephyrin clustering. J. Cell Sci. 133 (2020): jcs232835
Kilisch M., Mayer S., Mitkovski M., Roehse, H, Hentrich, J., Schwappach, B., Papadopoulos, T.
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Neuroligin-2 dependent conformational activation of collybistin reconstituted in supported hybrid membranes. J. Biol. Chem. (2020): jbc.RA120.015347
Schäfer, J. Förster, L., Mey, I. Papadopoulos, T. Brose, N. Steinem, C.
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(2021). Collybistin SH3- protein isoforms are expressed in the rat brain promoting gephyrin and GABA-A receptor clustering at GABAergic synapses. J. Neurochem. 157: 1032-1051
George, S., Bear, J. Jr., Taylor, M.J., Kanamalla, K., Fekete, C.D., Chiou, T.T., Miralles, C.P., Papadopoulos, T. De Blas, A.L.
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(2021). The α3 subunit of GABAA receptors promotes formation of inhibitory synapses in the absence of collybistin. J. Biol. Chem. 296: 100709
Wagner, S., Lee C.-K., Rojas, L., Specht, C.G., Rhee, J.-S., Brose N., Papadopoulos, T.
