SAHA (Vorinostat) Corrects Inhibitory Synaptic Deficits Caused by Missense Epilepsy Mutations to the GABAA Receptor γ2 Subunit

Abstract

The GABAA receptor (GABAAR) α1 subunit A295D epilepsy mutation reduces the surface expression of α1A295Dβ2γ2 GABAARs via ER-associated protein degradation. Suberanilohydroxamic acid (SAHA, also known as Vorinostat) was recently shown to correct the misfolding of α1A295D subunits and thereby enhance the functional surface expression of α1A295Dβ2γ2 GABAARs. Here we investigated whether SAHA can also restore the surface expression of γ2 GABAAR subunits that incorporate epilepsy mutations (N40S, R43Q, P44S, R138G) known to reduce surface expression via ER-associated protein degradation. As a control, we also investigated the γ2K289M epilepsy mutation that impairs gating without reducing surface expression. Effects of mutations were evaluated on inhibitory postsynaptic currents (IPSCs) mediated by the major synaptic α1β2γ2 GABAAR isoform. Recordings were performed in neuron-HEK293 cell artificial synapses to minimise contamination by GABAARs of undefined subunit composition. Transfection with α1β2γ2N40S, α1β2γ2R43Q, α1β2γ2P44S and α1β2γ2R138G subunits produced IPSCs with decay times slower than those of unmutated α1β2γ2 GABAARs due to the low expression of mutant γ2 subunits and the correspondingly high expression of slow-decaying α1β2 GABAARs. SAHA pre-treatment significantly accelerated the decay time constants of IPSCs consistent with the upregulation of mutant γ2 subunit expression. This increase in surface expression was confirmed by immunohistochemistry. SAHA had no effect on either the IPSC kinetics or surface expression levels of α1β2γ2K289M GABAARs, confirming its specificity for ER-retained mutant γ2 subunits. We also found that α1β2γ2K289M GABAARs and SAHA-treated α1β2γ2R43Q, α1β2γ2P44S and α1β2γ2R138G GABAARs all mediated IPSCs that decayed at significantly faster rates than wild type receptors as temperature was increased from 22 to 40°C. This may help explain why these mutations cause febrile seizures (FS). Given that SAHA is approved by therapeutic regulatory agencies for human use, we propose that it may be worth investigating as a treatment for epilepsies caused by the N40S, R43Q, P44S and R138G mutations. Although SAHA has already been proposed as a therapeutic for patients harbouring the α1A295D epilepsy mutation, the present study extends its potential utility to a new subunit and four new mutations.

Effects of GABAA Receptor α3 Subunit Epilepsy Mutations on Inhibitory Synaptic Signaling

“…This is corroborated by previous work in oocytes which showed reduced whole-cell currents for these mutants (Niturad et al, 2017). Decreased cell surface expression reduces overall inhibition, resulting in the manifestation of epilepsy phenotypes (Fisher, 2004;Durisic et al, 2018;Shi et al, 2019). However, it may also result in the preferential expression of receptors lacking the α3 subunit which could in turn alter IPSC kinetics.…”

“…This apparent gain-of-function does not explain the associated pathology. However, further studies have shown that γ2 R43Q is retained in the endoplasmic reticulum (Durisic et al, 2018), resulting in reduced surface expression and synaptic targeting of the assembled receptor complexes (Frugier et al, 2007), which may explain the resulting loss of inhibitory control underlying the epileptic phenotypes.…”

“…However, a detailed study examining the subcellular localization of these mutants and quantification of cell-surface expression levels using biotinylation or differential labeling is still warranted. Defects in receptor trafficking could be corrected using drugs such as SAHA, which can rescue misfolded proteins from the ER and restore expression levels of low-expressing GABA A R mutants (Chen et al, 2017a;Durisic et al, 2018). Patients carrying mutations like Q242L, which causes low expression levels without affecting channel kinetics, could potentially benefit from these types of therapies.…”

The GABRG2 F343L allele causes spontaneous seizures in a novel transgenic zebrafish model that can be treated with suberanilohydroxamic acid (SAHA)

“…As a broad HDACs inhibitor, SAHA has been shown to protect against seizure-induced brain damage through histone acetylation regulation (44). Intriguingly, SAHA was reported to enhance epilepsyassociated mutant GABA A receptor proteostasis in vitro (12)(13)(14). It has been demonstrated that SAHA increased the BiP protein level and the interaction between the mutant α1 subunit and calnexin, and thus partially corrected the transcription, folding and trafficking of mutant GABA A receptors (12).…”

“…Suberanilohydroxamic acid (SAHA, also known as Vorinostat) is an orally available histone deacetylases (HDACs) inhibitor that was approved by the US Food and Drug Administration for management of cutaneous T cell lymphoma (11). In vitro SAHA treatment has previously been shown to effectively enhance proteostasis, and thereby, partially correct the impaired function of GABA A receptors produced by epilepsy mutations associated with endoplasmic reticulum (ER) -associated protein degradation (12)(13)(14). Since SAHA crosses the blood brain barrier, we investigated its anticonvulsant efficacy and that of several established anticonvulsant drugs (valproic acid, levetiracetam, clonazepam, and carbamazepine) in vivo using the Tg(hGABRG2 F343L ) and Tg(hGABRG2 wt ) zebrafish models.…”