TRPM2 and CaMKII Signaling Drives Excessive GABAergic Synaptic Inhibition Following Ischemia.

Excitotoxicity and the concurrent loss of inhibition are well-defined mechanisms driving acute elevation in excitatory/inhibitory (E/I) balance and neuronal cell death following an ischemic insult to the brain. Despite the high prevalence of long-term disability in survivors of global cerebral ischemia (GCI) as a consequence of cardiac arrest, it remains unclear whether E/I imbalance persists beyond the acute phase and negatively affects functional recovery. We previously demonstrated sustained impairment of long-term potentiation (LTP) in hippocampal CA1 neurons correlating with deficits in learning and memory tasks in a murine model of cardiac arrest/cardiopulmonary resuscitation (CA/CPR). Here, we use CA/CPR and an in vitro ischemia model to elucidate mechanisms by which E/I imbalance contributes to ongoing hippocampal dysfunction in male mice. We reveal increased postsynaptic GABAA receptor (GABAAR) clustering and function in the CA1 region of the hippocampus that reduces the E/I ratio. Importantly, reduced GABAAR clustering observed in the first 24 h rebounds to an elevation of GABAergic clustering by 3 d postischemia. This increase in GABAergic inhibition required activation of the Ca2+-permeable ion channel transient receptor potential melastatin-2 (TRPM2), previously implicated in persistent LTP and memory deficits following CA/CPR. Furthermore, we find Ca2+-signaling, likely downstream of TRPM2 activation, upregulates Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, thereby driving the elevation of postsynaptic inhibitory function. Thus, we propose a novel mechanism by which inhibitory synaptic strength is upregulated in the context of ischemia and identify TRPM2 and CaMKII as potential pharmacological targets to restore perturbed synaptic plasticity and ameliorate cognitive function.

Distinct mechanisms drive sequential internalization and degradation of GABAARs during global ischemia and reperfusion injury.

Synaptic inhibition is critical for controlling neuronal excitability and function. During global cerebral ischemia (GCI), inhibitory synapses are rapidly eliminated, causing hyper-excitability which contributes to cell-death and the pathophysiology of disease. Sequential disassembly of inhibitory synapses begins within minutes of ischemia onset: GABAARs are rapidly trafficked away from the synapse, the gephyrin scaffold is removed, followed by loss of the presynaptic terminal. GABAARs are endocytosed during GCI, but how this process accompanies synapse disassembly remains unclear. Here, we define the precise trafficking itinerary of GABAARs during the initial stages of GCI, placing them in the context of rapid synapse elimination. Ischemia-induced GABAAR internalization quickly follows their initial dispersal from the synapse, and is controlled by PP1α signaling. During reperfusion injury, GABAARs are then trafficked to lysosomes for degradation, leading to permanent removal of synaptic GABAARs and contributing to the profound reduction in synaptic inhibition observed hours following ischemia onset.

Stepwise disassembly of GABAergic synapses during pathogenic excitotoxicity.

GABAergic synaptic inhibition controls neuronal firing, excitability, and synaptic plasticity to regulate neuronal circuits. Following an acute excitotoxic insult, inhibitory synapses are eliminated, reducing synaptic inhibition, elevating circuit excitability, and contributing to the pathophysiology of brain injuries. However, mechanisms that drive inhibitory synapse disassembly and elimination are undefined. We find that inhibitory synapses are disassembled in a sequential manner following excitotoxicity: GABAARs undergo rapid nanoscale rearrangement and are dispersed from the synapse along with presynaptic active zone components, followed by the gradual removal of the gephyrin scaffold, prior to complete elimination of the presynaptic terminal. GABAAR nanoscale reorganization and synaptic declustering depends on calcineurin signaling, whereas disassembly of gephyrin relies on calpain activation, and blockade of both enzymes preserves inhibitory synapses after excitotoxic insult. Thus, inhibitory synapse disassembly occurs rapidly, with nanoscale precision, in a stepwise manner and most likely represents a critical step in the progression of hyperexcitability following excitotoxicity.

GluN2B S1303 phosphorylation by CaMKII or DAPK1: no indication for involvement in ischemia or LTP.

Binding of two different CaM kinases, CaMKII and DAPK1, to the NMDA-type glutamate receptor (NMDAR) subunit GluN2B near S1303 has been implicated in excitotoxic/ischemic neuronal cell death. The GluN2BΔCaMKII mutation (L1298A, R1300Q) is neuroprotective but abolishes only CaMKII but not DAPK1 binding. However, both kinases can additionally phosphorylate GluN2B S1303. Thus, we here tested S1303 phosphorylation for possible contribution to neuronal cell death. The GluN2BΔCaMKII mutation completely abolished phosphorylation by CaMKII and DAPK1, suggesting that the mutation could mediate neuroprotection by disrupting phosphorylation. However, S1303 phosphorylation was not increased by excitotoxic insults in hippocampal slices or by global cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation in vivo. In hippocampal cultures, S1303 phosphorylation was induced by chemical LTD but not LTP stimuli. These results indicate that the additional effect of the GluN2BΔCaMKII mutation on phosphorylation needs to be considered only in LTD but not in LTP or ischemia/excitotoxicity.

Reversal of Global Ischemia-Induced Cognitive Dysfunction by Delayed Inhibition of TRPM2 Ion Channels.

Hippocampal injury and cognitive impairments are common after cardiac arrest and stroke and do not have an effective intervention despite much effort. Therefore, we developed a new approach aimed at reversing synaptic dysfunction by targeting TRPM2 channels. Cardiac arrest/cardiopulmonary resuscitation (CA/CPR) in mice was used to investigate cognitive deficits and the role of the calcium-permeable ion channel transient receptor potential-M2 (TRPM2) in ischemia-induced synaptic dysfunction. Our data indicates that absence (TRPM2-/-) or acute inhibition of TRPM2 channels with tatM2NX reduced hippocampal cell death in males only, but prevented synaptic plasticity deficits in both sexes. Remarkably, administration of tatM2NX weeks after injury reversed hippocampal plasticity and memory deficits. Finally, TRPM2-dependent activation of calcineurin-GSK3ß pathway contributes to synaptic plasticity impairments. These data suggest persistent TRPM2 activity following ischemia contributes to impairments of the surviving hippocampal network and that inhibition of TRPM2 channels at chronic time points may represent a novel strategy to improve functional recovery following cerebral ischemia that is independent of neuroprotection.