Efficient signal transmission in the central nervous system is essential for higher brain functions. Inhibitory signaling in the brain primarily takes place at GABAergic (γ-aminobutyric acid) synapses and balances the activity of excitatory synapses. GABA type A receptors (GABAARs) are clustered at the synapse by a scaffold with the peripheral membrane protein gephyrin as major postsynaptic protein. One key feature of synapses is their ability to adapt in response to neuronal network activities, though underlying mechanisms how these changes are orchestrated under normal and pathological conditions are poorly understood.
The first part of the study focuses on gephyrin’s membrane association in neurons with the aim to elucidate the molecular mechanisms and significance of membrane tethering. Gephyrin from Sf9 insect cells, but not E. coli, bound to liposomes and specifically to phosphatidylinositol 4-phosphate in protein-lipid overlay assays. Furthermore, gephyrin was identified to be mainly associated with cholesterol-rich membrane microdomains in mouse brains. Posttranslational lipid modifications of synaptic proteins regulate their trafficking and membrane localization and contribute to synaptic plasticity. Using different experimental approaches, gephyrin was identified to be palmitoylated in vivo. Palmitoylation of gephyrin was crucial for its localization at synapses and influenced the size of gephyrin clusters and the architecture of the inhibitory synapse. Membrane release of gephyrin upon inhibition of palmitoylation led to reduced surface quantities of synaptic GABAAR subunits. Additionally, the membrane detachment made gephyrin more susceptible to cleavage by the protease calpain I resulting in an accelerated turnover of the protein. Gephyrin palmitoylation was identified to be regulated by GABAAR activity leading to rapid changes in gephyrin palmitoylation levels. Palmitoylation screens in hippocampal neurons identified the neurite-localized palmitoyl transferase DHHC12 and Golgi-resident DHHC16 as gephyrin palmitoylating enzymes, which increase gephyrin cluster size and its amount in synapses. Gephyrin was also identified to be physiologically S-nitrosylated by NO, which is produced by neuronal nitric oxide synthase (nNOS). Pharmacological nNOS activity modulation in HEK-293 cells and hippocampal neurons reciprocally regulated gephyrin palmitoylation. Together, these findings identify differential modification of gephyrin by palmitoylation and nitrosylation and suggest that palmitoylation dynamics of gephyrin contribute to the regulation of GABAergic activity-dependent plasticity.
Inhibitory signaling is crucial to counterbalance excitatory transmission. Anomalous inhibitory circuits and particularly irregular gephyrin expression have been linked to epileptic disorders. Patients with idiopathic generalized epilepsy have been screened for mutations in the GPHN gene. In the second part, a patient was identified with a hemizygous mutation in GPHN resulting in the expression of a truncated gephyrin variant that failed to oligomerize at inhibitory synapses. This pathogenic variant acted dominant-negatively on regular gephyrin and disrupted the normal gephyrin scaffold and synaptic GABAAR clustering in hippocampal neurons. The results suggest that mutations in genes coding for proteins of the inhibitory synapse is an important mechanism in the pathophysiology of monogenetic epilepsy forms
