Proteostasis Regulators Restore Function of Epilepsy-Associated GABAA Receptors.
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ABSTRACT:
SUBMITTER: Di XJ
PROVIDER: S-EPMC7855620 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Recent advances in genetic diagnosis identified variants in genes encoding GABAA receptors as causative for genetic epilepsy. Here, we selected eight disease-associated variants in the α1 subunit of GABAA receptors causing mild to severe clinical phenotypes and showed that they are loss of function, mainly by reducing the folding and surface trafficking of the α1 protein. Furthermore, we sought client protein-specific pharmacological chaperones to restore the function of pathogenic receptors. Applications of positive allosteric modulators, including Hispidulin and TP003, increase the functional surface expression of the α1 variants. Mechanism of action study demonstrated that they enhance the folding and assembly and reduce the degradation of GABAA variants without activating the unfolded protein response in HEK293T cells and human iPSC-derived neurons. Since these compounds cross the blood-brain barrier, such a pharmacological chaperoning strategy holds great promise to treat genetic epilepsy in a GABAA receptor-specific manner.
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Project description:GABA(A) receptors are the primary inhibitory ion channels in the mammalian central nervous system. The A322D mutation in the ?1 subunit of GABA(A) receptors is known to result in its degradation and reduce its cell surface expression, leading to loss of GABAA receptor function in autosomal dominant juvenile myoclonic epilepsy. Here, we show that SAHA, a FDA-approved drug, increases the transcription of the ?1(A322D) subunit, enhances its folding and trafficking posttranslationally, increases its cell surface level, and restores the GABA-induced maximal current in HEK293 cells expressing ?1(A322D)?2?2 receptors to 10% of that for wild-type receptors. To enhance the trafficking efficiency of the ?1(A322D) subunit, SAHA increases the BiP protein level and the interaction between the ?1(A322D) subunit and calnexin. SAHA is a drug that enhances epilepsy-associated GABAA receptor proteostasis.
| S-EPMC3872227 | biostudies-literature
Project description:The endoplasmic reticulum membrane complex (EMC) plays a critical role in the biogenesis of tail-anchored proteins and a subset of multi-pass membrane proteins in the endoplasmic reticulum (ER). However, because of nearly exclusive expression of neurotransmitter-gated ion channels in the central nervous system (CNS), the role of the EMC in their biogenesis is not well understood. In this study, we demonstrated that the EMC positively regulates the surface trafficking and thus function of endogenous γ-aminobutyric acid type A (GABAA) receptors, the primary inhibitory ion channels in the mammalian brain. Moreover, among ten EMC subunits, EMC3 and EMC6 have the most prominent effect, and overexpression of EMC3 or EMC6 is sufficient to restore the function of epilepsy-associated GABAA receptor variants. In addition, EMC3 and EMC6 demonstrate endogenous interactions with major neuroreceptors, which depends on their transmembrane domains, suggesting a general role of the EMC in the biogenesis of neuroreceptors.
| S-EPMC9352529 | biostudies-literature
Project description:BackgroundGenetic variants in the subunits of the gamma-aminobutyric acid type A (GABAA) receptors are implicated in the onset of multiple pathologic conditions including genetic epilepsy. Previous work showed that pathogenic GABAA subunits promote misfolding and inefficient assembly of the GABAA receptors, limiting receptor expression and activity at the plasma membrane. However, GABAA receptors containing variant subunits can retain activity, indicating that enhancing the folding, assembly, and trafficking of these variant receptors offers a potential opportunity to mitigate pathology associated with genetic epilepsy.ResultsHere, we demonstrate that pharmacologically enhancing endoplasmic reticulum (ER) proteostasis using small molecule activators of the ATF6 (Activating Transcription Factor 6) signaling arm of the unfolded protein response (UPR) increases the assembly, trafficking, and surface expression of variant GABAA receptors. These improvements are attributed to ATF6-dependent remodeling of the ER proteostasis environment, which increases protein levels of pro-folding ER proteostasis factors including the ER chaperone BiP (Immunoglobulin Binding Protein) and trafficking receptors, such as LMAN1 (Lectin Mannose-Binding 1) and enhances their interactions with GABAA receptors. Importantly, we further show that pharmacologic ATF6 activators increase the activity of GABAA receptors at the cell surface, revealing the potential for this strategy to restore receptor activity to levels that could mitigate disease pathogenesis.ConclusionsThese results indicate that pharmacologic ATF6 activators offer an opportunity to restore GABAA receptor activity in diseases including genetic epilepsy and point to the potential for similar pharmacologic enhancement of ER proteostasis to improve trafficking of other disease-associated variant ion channels implicated in etiologically-diverse diseases.
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Project description:GABAA receptors containing the α5 subunit mediate tonic inhibition and are widely expressed in the limbic system. In animals, activation of α5-containing receptors impairs hippocampus-dependent memory. Temporal lobe epilepsy is associated with memory impairments related to neuron loss and other changes. The less selective PET ligand [11C]flumazenil has revealed reductions in GABAA receptors. The hypothesis that α5 subunit receptor alterations are present in temporal lobe epilepsy and could contribute to impaired memory is untested. We compared α5 subunit availability between individuals with temporal lobe epilepsy and normal structural MRI ('MRI-negative') and healthy controls, and interrogated the relationship between α5 subunit availability and episodic memory performance, in a cross-sectional study. Twenty-three healthy male controls (median ± interquartile age 49 ± 13 years) and 11 individuals with MRI-negative temporal lobe epilepsy (seven males; 40 ± 8) had a 90-min PET scan after bolus injection of [11C]Ro15-4513, with arterial blood sampling and metabolite correction. All those with epilepsy and six controls completed the Adult Memory and Information Processing Battery on the scanning day. 'Bandpass' exponential spectral analyses were used to calculate volumes of distribution separately for the fast component [V F; dominated by signal from α1 (α2, α3)-containing receptors] and the slow component (V S; dominated by signal from α5-containing receptors). We made voxel-by-voxel comparisons between: the epilepsy and control groups; each individual case versus the controls. We obtained parametric maps of V F and V S measures from a single bolus injection of [11C]Ro15-4513. The epilepsy group had higher V S in anterior medial and lateral aspects of the temporal lobes, the anterior cingulate gyri, the presumed area tempestas (piriform cortex) and the insulae, in addition to increases of ∼24% and ∼26% in the ipsilateral and contralateral hippocampal areas (P < 0.004). This was associated with reduced V F:V S ratios within the same areas (P < 0.009). Comparisons of V S for each individual with epilepsy versus controls did not consistently lateralize the epileptogenic lobe. Memory scores were significantly lower in the epilepsy group than in controls (mean ± standard deviation -0.4 ± 1.0 versus 0.7 ± 0.3; P = 0.02). In individuals with epilepsy, hippocampal V S did not correlate with memory performance on the Adult Memory and Information Processing Battery. They had reduced V F in the hippocampal area, which was significant ipsilaterally (P = 0.03), as expected from [11C]flumazenil studies. We found increased tonic inhibitory neurotransmission in our cohort of MRI-negative temporal lobe epilepsy who also had co-morbid memory impairments. Our findings are consistent with a subunit shift from α1/2/3 to α5 in MRI-negative temporal lobe epilepsy.
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Project description:A study was made of the "rundown" of GABA(A) receptors, microtransplanted to Xenopus oocytes from surgically resected brain tissues of patients afflicted with drug-resistant human mesial temporal lobe epilepsy (mTLE). Cell membranes, isolated from mTLE neocortex specimens, were injected into frog oocytes that rapidly incorporated functional GABA(A) receptors. Upon repetitive activation with GABA (1 mM), "epileptic" GABA(A) receptors exhibited a GABA(A)-current (I(GABA)) rundown that was significantly enhanced by Zn(2+) (</=250 microM), and practically abolished by the high-affinity GABA(A) receptor inverse agonist SR95531 (gabazine; 2.5-25 microM). Conversely, I(GABA) generated by "control" GABA(A) receptors microtransplanted from nonepileptic temporal lobe, lesional TLE, or authoptic disease-free tissues remained stable during repetitive stimulation, even in oocytes treated with Zn(2+). We conclude that rundown of mTLE epileptic receptors depends on the presence of "phasic GABA(A) receptors" that have low sensitivity to antagonism by Zn(2+). Additionally, we found that GABA(A) receptors, microtransplanted from the cerebral cortex of adult rats exhibiting recurrent seizures, caused by pilocarpine-induced status epilepticus, showed greater rundown than control tissue, an event also occurring in patch-clamped rat pyramidal neurons. Rundown of epileptic rat receptors resembled that of human mTLE receptors, being enhanced by Zn(2+) (40 microM) and sensitive to the antiepileptic agent levetiracetam, the neurotrophin brain-derived neurotrophic factor, and the phosphatase blocker okadaic acid. Our findings point to the rundown of GABA(A) receptors as a hallmark of TLE and suggest that modulating tonic and phasic mTLE GABA(A) receptor activity may represent a useful therapeutic approach to the disease.
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Project description:A number of epilepsy-causing mutations have recently been identified in the genes of the α1, β3, and γ2 subunits comprising the γ-aminobutyric acid type A (GABAA) receptor. These mutations are typically dominant, and in certain cases, such as the α1 and β3 subunits, they may lead to a mix of receptors at the cell surface that contain no mutant subunits, a single mutated subunit, or two mutated subunits. To determine the effects of mutations in a single subunit or in two subunits on receptor activation, we created a concatenated protein assembly that links all five subunits of the α1β3γ2 receptor and expresses them in the correct orientation. We created nine separate receptor variants with a single-mutant subunit and four receptors containing two subunits of the γ2R323Q, β3D120N, β3T157M, β3Y302C, and β3S254F epilepsy-causing mutations. We found that the singly mutated γ2R323Q subunit impairs GABA activation of the receptor by reducing GABA potency. A single β3D120N, β3T157M, or β3Y302C mutation also substantially impaired receptor activation, and two copies of these mutants within a receptor were catastrophic. Of note, an effect of the β3S254F mutation on GABA potency depended on the location of this mutant subunit within the receptor, possibly because of the membrane environment surrounding the transmembrane region of the receptor. Our results highlight that precise functional genomic analyses of GABAA receptor mutations using concatenated constructs can identify receptors with an intermediate phenotype that contribute to epileptic phenotypes and that are potential drug targets for precision medicine approaches.
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Project description:Normal organismal physiology depends on the maintenance of proteostasis in each cellular compartment to achieve a delicate balance between protein synthesis, folding, trafficking, and degradation while minimizing misfolding and aggregation. Defective proteostasis leads to numerous protein misfolding diseases. Pharmacological chaperones are cell-permeant small molecules that promote the proper folding and trafficking of a protein via direct binding to that protein. They stabilize their target protein in a protein-pharmacological chaperone state, increasing the natively folded protein population that can effectively engage trafficking machinery for transport to the final destination for function. Here, as regards the application of pharmacological chaperones, we focus on their capability to promote the folding and trafficking of lysosomal enzymes, G protein coupled receptors (GPCRs), and ion channels, each of which is presently an important drug target. Pharmacological chaperones hold great promise as potential therapeutics to ameliorate a variety of protein misfolding diseases.
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Project description:Numerous studies recently showed that the inhibitory neurotransmitter, γ-aminobutyric acid (GABA), can stimulate cerebral angiogenesis and promote neurovascular coupling by activating the ionotropic GABAA receptors on cerebrovascular endothelial cells, whereas the endothelial role of the metabotropic GABAB receptors is still unknown. Preliminary evidence showed that GABAA receptor stimulation can induce an increase in endothelial Ca2+ levels, but the underlying signaling pathway remains to be fully unraveled. In the present investigation, we found that GABA evoked a biphasic elevation in [Ca2+]i that was initiated by inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-dependent Ca2+ release from neutral and acidic Ca2+ stores, respectively, and sustained by store-operated Ca2+ entry. GABAA and GABAB receptors were both required to trigger the endothelial Ca2+ response. Unexpectedly, we found that the GABAA receptors signal in a flux-independent manner via the metabotropic GABAB receptors. Likewise, the full Ca2+ response to GABAB receptors requires functional GABAA receptors. This study, therefore, sheds novel light on the molecular mechanisms by which GABA controls endothelial signaling at the neurovascular unit.
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Project description:Epilepsy animal models indicate pronounced changes in the expression and rearrangement of GABAA receptor subunits in the hippocampus and in para-hippocampal areas, including widespread downregulation of the subunits α5 and δ, and upregulation of α4, subunits that mediate tonic inhibition of GABA. In this case-control study, we investigated changes in the expression of subunits α4, α5 and δ in hippocampal specimens of drug resistant temporal lobe epilepsy patients who underwent epilepsy surgery. Using in situ hybridization, immunohistochemistry and α5-specific receptor autoradiography, we characterized expression of the receptor subunits in specimens from patients with and without Ammon's horn sclerosis compared to post-mortem controls. Expression of the α5-subunit was abundant throughout all subfields of the hippocampus, including the dentate gyrus, sectors CA1 and CA3, the subiculum and pre- and parasubiculum. Significant but weaker expression was detected for subunits α4 and δ notably in the granule cell/molecular layer of control specimens, but was faint in the other parts of the hippocampus. Expression of all three subunits was similarly altered in sclerotic and non-sclerotic specimens. Respective mRNA levels were increased by about 50-80% in the granule cell layer compared with post-mortem controls. Subunit α5 mRNA levels and immunoreactivities were also increased in the sector CA3 and in the subiculum. Autoradiography for α5-containing receptors using [3H]L-655,708 as ligand showed significantly increased binding in the molecular layer of the dentate gyrus in non-sclerotic specimens. Increased expression of the α5 and δ subunits is in contrast to the previously observed downregulation of these subunits in different epilepsy models, whereas increased expression of α4 in temporal lobe epilepsy patients is consistent with that in the rodent models. Our findings indicate increased tonic inhibition likely representing an endogenous anticonvulsive mechanism in temporal lobe epilepsy.
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