Gephyrin Cleavage in In Vitro Brain Ischemia Decreases GABAA Receptor Clustering and Contributes to Neuronal Death

Abstract

GABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the central nervous system, and changes in GABAergic neurotransmission modulate the activity of neuronal networks. Gephyrin is a scaffold protein responsible for the traffic and synaptic anchoring of GABAA receptors (GABAAR); therefore, changes in gephyrin expression and oligomerization may affect the activity of GABAergic synapses. In this work, we investigated the changes in gephyrin protein levels during brain ischemia and in excitotoxic conditions, which may affect synaptic clustering of GABAAR. We found that gephyrin is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with glutamate, as well as after intrahippocampal injection of kainate, giving rise to a stable cleavage product. Gephyrin cleavage was also observed in cultured hippocampal neurons subjected to transient oxygen-glucose deprivation (OGD), an in vitro model of brain ischemia, and after transient middle cerebral artery occlusion (MCAO) in mice, a model of focal brain ischemia. Furthermore, a truncated form of gephyrin decreased the synaptic clustering of the protein, reduced the synaptic pool of GABAAR containing γ2 subunits and upregulated OGD-induced cell death in hippocampal cultures. Our results show that excitotoxicity and brain ischemia downregulate full-length gephyrin with a concomitant generation of truncated products, which affect synaptic clustering of GABAAR and cell death.

Calpains and neuronal damage in the ischemic brain: The swiss knife in synaptic injury

“…Most studies that have been published so far reported no alteration in synaptophysin expression level under ischemic and excitotoxic conditions. This was the case in cultured hippocampal neurons subjected to transient OGD and in the ischemic region induced by transient MCAO (Costa et al, 2015;Curcio et al, 2015;Lobo et al, 2011;Mele et al, 2014). Similar results were obtained in cultured hippocampal neurons subjected to excitotoxic stimulation with glutamate Gomes et al, 2011) and following intrahippocampal injection of kainate .…”

“…A calpain-mediated downregulation of GABAAR subunits (α1, β3, α2 and γ2) and gephyrin was observed in cultured hippocampal neurons subjected transiently to OGD (Figure 6) (Mele et al, 2014). Moreover, it was shown that calpains cleave gephyrin under excitotoxic conditions, both in cultured hippocampal neurons and in vivo, giving rise to a stable cleavage product with ~47 kDa (Costa et al, 2015). Indeed, chemical inhibition of calpain abrogated the cleavage of gephyrin observed in hippocampal neurons subjected to OGD or to excitotoxic injury.…”

“…The extrasynaptic pool of NMDAR is also selectively coupled to calpain-mediated cleavage of gephyrin, a protein involved in the anchoring of GABAAR at the synapse. The disruption of the gephyrin lattice decreases GABAAR clustering at the synapse, and the resulting decrease in GABAergic synaptic transmission was found to contribute to cell death in hippocampal neurons subjected to OGD (Costa et al, 2015) (see section 5.2).…”

“…In particular the regulation of GABAAR stability at the synapse, which largely depends on the interaction of the receptors with scaffold protein gephyrin (Tyagarajan and Fritschy, 2014), is a critical process for the control of GABAergic synaptic strength. The downregulation of GABAergic synapses observed in in vivo and in vitro models of brain ischemia (Costa et al, 2015;Mele et al, 2016;Mele et al, 2014;Schwartz-Bloom and Sah, 2001) has been associated to different mechanisms, including the calpain-mediated cleavage of proteins belonging to the inhibitory postsynaptic density (Costa et al, 2015;Mele et al, 2016;Mele et al, 2014).…”

Cocaine Withdrawal Reduces Gamma-Aminobutyric Acid-Ergic Transmission and Gephyrin Expression at Medial Prefrontal Cortex in Cocaine-Conditioned Place-Preference Rats, Which Shows Increased Cocaine Seeking

“…Downregulation of gephyrin expression by gene targeting or gene silencing leads to the rapid disappearance of postsynaptic GABA A R clustering, and loss of inhibitory postsynaptic currents (IPSCs) [22][23][24] . Thus, gephyrin promotes the stabilization of GABA A Rs at postsynaptic sites and serves as an important regulator of GABAergic neurotransmission [25][26][27][28] . However, the role of gephyrin in cocaine addiction has not been studied.…”