Proteostasis maintenance of ␥-aminobutyric acid type A (GABA A ) receptors dictates their function in controlling neuronal inhibition in mammalian central nervous systems. However, as a multisubunit, multispan, integral membrane protein, even wild type subunits of GABA A receptors fold and assemble inefficiently in the endoplasmic reticulum (ER). Unassembled and misfolded subunits undergo ER-associated degradation (ERAD), but this degradation process remains poorly understood for GABA A receptors. Here, using the ␣1 subunits of GABA A receptors as a model substrate, we demonstrated that Grp94, a metazoan-specific Hsp90 in the ER lumen, uses its middle domain to interact with the ␣1 subunits and positively regulates their ERAD. OS-9, an ER-resident lectin, acts downstream of Grp94 to further recognize misfolded ␣1 subunits in a glycan-dependent manner. This delivers misfolded ␣1 subunits to the Hrd1-mediated ubiquitination and the valosin-containing protein-mediated extraction pathway. Repressing the initial ERAD recognition step by inhibiting Grp94 enhances the functional surface expression of misfolding-prone ␣1(A322D) subunits, which causes autosomal dominant juvenile myoclonic epilepsy. This study clarifies a Grp94-mediated ERAD pathway for GABA A receptors, which provides a novel way to finely tune their function in physiological and pathophysiological conditions.

Background:The ␣1 subunit harboring the A322D mutation is subject to excessive ERAD. Results: VCP inhibition using Eeyarestatin I reduces the ERAD of ␣1(A322D) subunits, and coapplication of SAHA additively enhances their proteostasis. Conclusion: Combining ERAD inhibition and folding enhancement yields significant functional rescue. Significance: This combination represents a new, promising strategy to treat epilepsy resulting from GABA A receptor misfolding.

Background Microglial activation contributes to neuroinflammation and neuronal damage in neurodegenerative disorders including Alzheimer's and Parkinson's diseases. It has been suggested that neurodegenerative disorders may be improved if neuroinflammation can be controlled. trans-cinnamaldehyde (TCA) isolated from the stem bark of Cinnamomum cassia possesses potent anti-inflammatory capability; we thus tested whether TCA presents neuroprotective effects on improving neuronal survival by inhibiting neuroinflammatory responses in BV2 microglial cells. Results To determine the molecular mechanism behind TCA-mediated neuroprotective effects, we assessed the effects of TCA on lipopolysaccharide- (LPS-) induced proinflammatory responses in BV2 microglial cells. While LPS potently induced the production and expression upregulation of proinflammatory mediators, including NO, iNOS, COX-2, IL-1β, and TNF-α, TCA pretreatment significantly inhibited LPS-induced production of NO and expression of iNOS, COX-2, and IL-1β and recovered the morphological changes in BV2 cells. TCA markedly attenuated microglial activation and neuroinflammation by blocking nuclear factor kappa B (NF-κB) signaling pathway. With the aid of microglia and neuron coculture system, we showed that TCA greatly reduced LPS-elicited neuronal death and exerted neuroprotective effects. Conclusions Our results suggest that TCA, a natural product, has the potential of being used as a therapeutic agent against neuroinflammation for ameliorating neurodegenerative disorders.

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