Sui, Yusheng; (2022) The Interaction of GABAA Receptors with Synaptic Adhesion Proteins Regulates the Formation and Function of GABAergic Synapses. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

GABAA receptors are crucial for maintaining the balance between the excitation and inhibition in the brain. They mediate tonic inhibition at extrasynaptic locations or phasic inhibition by generating inhibitory postsynaptic currents at GABAergic synapses. Formation of GABAergic synapses during development involves complex protein-protein interactions between the presynaptic and postsynaptic elements. The focus of my thesis has been on interactions between GABAA receptors and Neuroginin-2 (NL2)/Neurexin, Dystroglycan/Pikachurin complexes and Diazepam Binding Inhibitor (DBI), using a co-culture approach. I used stable HEK293 cell lines expressing synaptic (α2β2γ2) or extrasynaptic (α4β3δ) types of GABAA receptors in co-culture with GABAergic neurons to study synaptic contact formation by immunolabelling and confocal and super-resolution imaging. My results show that synaptic GABAA receptors can induce synaptogenesis on their own but also enhance synaptogenic effects of NL2. Extrasynaptic GABAA receptors do not induce synaptic contacts but can enhance synaptogenesis induced by NL2, although significantly less than synaptic GABAA receptors. Synaptic GABAA receptors and NL2 cooperativity is mediated by direct interactions between the GABAA receptors γ2 subunit and NL2 via their intracellular domains. Functional assessment of synaptic contacts was done using electrophysiology and confocal imaging of the activity-dependent fluorescent labelling of presynaptic terminals forming contacts with HEK293 cells. Using the same approaches, I have also shown that Dystroglycan, a well-established synaptic adhesion protein, does not induce synaptic contacts but in combination with a synaptic matrix protein Pikachurin, can induce synaptogenesis in co-cultures. In a separate set of experiments, I have shown that expression of DBI, a multifunctional protein that inhibits allosteric modulation of GABAA receptors by benzodiazepines, can also promote synaptic contact formation in this system. In summary, my studies indicate that synaptic GABAA receptors play an important structural role during synaptogenesis and provide evidence for the key interaction between these receptors and NL2 in this process.

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