Ischemic insult to cerebellar Purkinje cells causes diminished GABA_A receptor function and allopregnanolone neuroprotection is associated with GABA_A receptor stabilization

Abstract

Cerebellar Purkinje cells (PC) are particularly vulnerable to ischemic injury and excitotoxicity, although the molecular basis of this sensitivity remains unclear. We tested the hypothesis that ischemia causes rapid down‐regulation of GABAA receptors in cerebellar PC, thereby increasing susceptibility to excitotoxicity. Oxygen‐glucose deprivation (OGD) caused a decline in functional GABAA receptors, within the first hour of re‐oxygenation. Decreased amplitude of miniature inhibitory post‐synaptic potentials confirmed that OGD caused a significant decrease in functional synaptic GABAA receptors and quantitative Western blot analysis demonstrated the loss of GABAA receptor current was associated with a decline in total receptor protein. Interestingly, the potent neuroprotectant allopregnanolone (ALLO) prevented the decline in GABAA receptor current and protein. Consistent with our in vitro data, global ischemia in mice caused a significant decline in total cerebellar GABAA receptor protein and PC specific immunoreactivity. Moreover, ALLO provided strong protection of PC and prevented ischemia‐induced decline in GABAA receptor protein. Our findings indicate that ischemia causes a rapid and sustained loss of GABAA receptors in PC, whereas ALLO prevents the decline in GABAA receptors and protects against ischemia‐induced damage. Thus, interventions which prevent ischemia‐induced decline in GABAA receptors may represent a novel neuroprotective strategy.

Stabilization of GABAAReceptors at Endocytic Zones Is Mediated by an AP2 Binding Motif within the GABAAReceptor β3 Subunit

“…, respectively, indicating a high and similar level of cotransfection for these experiments (data not shown). Similar transfection effi- (Kelley et al, 2008; WT showed that the SEP ␥2 cluster fluorescence remained stable under control conditions (Fig. 5 A, B).…”

“…Several studies have reported a decrease in surface and synaptic GABA A R expression during ischemia, suggesting an increase in receptor endocytosis (Schwartz-Bloom and Sah, 2001;Mielke and Wang, 2005;Zhan et al, 2006;Kelley et al, 2008;Liu et al, 2010), but whether this is via an AP2-dependent pathway remains unclear.…”

Sex Differences in Neuroprotection Provided by Inhibition of TRPM2 Channels following Experimental Stroke

“…Knockdown of TRPM2 expression was confirmed by quantitative real-time polymerase chain reaction on ABI Prism 7000 using commercially available probe and primers (Applied Biosystems, Foster City, CA, USA) and standard Western blot analysis as previously described (Kelley et al, 2008), using anti-TRPM2 antibody from Novus Biologicals. Intrastriatal injection of lentivirus into the right striatum (0.5 mm anterior, 2.0 mm lateral, 3.5 mm deep) was performed under isoflurane anesthesia 2 TRPM2 in cerebral ischemia J Jia et al to 3 weeks before MCAO, as previously described (Cheng et al, 2011).…”

Examining the GABA_A receptor alpha-subunits in the neonatal pig brain: changes across development and the effect of seizures after hypoxic-ischaemic brain injury

“…In addition, the authors concluded that dephosphorylation of the GABAAR under ischemic conditions may regulate surface localisation and that internalisation may contribute to neuronal death (Mele et al 2014). In several studies by Kelley et al, they suggested that the ischaemia-induced reduction in α1 protein expression was attributable to protein degradation (Kelley et al 2008, Kelley et al 2013). In our HI-Sz animals at 24 h, the α1 protein was noticeably absent from cell processes.…”

“…cell populations respond to ischaemia may involve differential regulation of GABAAR internalisation and trafficking (Kelley et al 2008). In SE, internalisation of neurotransmitter receptors has been suggested as a mechanism for the self-sustaining nature of seizures and the development of pharmacoresistance to antiepileptic drugs (Brooks-Kayal et al 1998).…”