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. 2024 Apr 23;25(9):4601.
doi: 10.3390/ijms25094601.

Modulating Endoplasmic Reticulum Chaperones and Mutant Protein Degradation in GABRG2(Q390X) Associated with Genetic Epilepsy with Febrile Seizures Plus and Dravet Syndrome

Sarah Poliquin  1   2 , Gerald Nwosu  2   3   4 , Karishma Randhave  4 , Wangzhen Shen  4 , Carson Flamm  4 , Jing-Qiong Kang  2   4   5   6
Affiliations

Modulating Endoplasmic Reticulum Chaperones and Mutant Protein Degradation in GABRG2(Q390X) Associated with Genetic Epilepsy with Febrile Seizures Plus and Dravet Syndrome

Sarah Poliquin et al. Int J Mol Sci. .

Abstract

A significant number of patients with genetic epilepsy do not obtain seizure freedom, despite developments in new antiseizure drugs, suggesting a need for novel therapeutic approaches. Many genetic epilepsies are associated with misfolded mutant proteins, including GABRG2(Q390X)-associated Dravet syndrome, which we have previously shown to result in intracellular accumulation of mutant GABAA receptor γ2(Q390X) subunit protein. Thus, a potentially promising therapeutic approach is modulation of proteostasis, such as increasing endoplasmic reticulum (ER)-associated degradation (ERAD). To that end, we have here identified an ERAD-associated E3 ubiquitin ligase, HRD1, among other ubiquitin ligases, as a strong modulator of wildtype and mutant γ2 subunit expression. Overexpressing HRD1 or knockdown of HRD1 dose-dependently reduced the γ2(Q390X) subunit. Additionally, we show that zonisamide (ZNS)-an antiseizure drug reported to upregulate HRD1-reduces seizures in the Gabrg2+/Q390X mouse. We propose that a possible mechanism for this effect is a partial rescue of surface trafficking of GABAA receptors, which are otherwise sequestered in the ER due to the dominant-negative effect of the γ2(Q390X) subunit. Furthermore, this partial rescue was not due to changes in ER chaperones BiP and calnexin, as total expression of these chaperones was unchanged in γ2(Q390X) models. Our results here suggest that leveraging the endogenous ERAD pathway may present a potential method to degrade neurotoxic mutant proteins like the γ2(Q390X) subunit. We also demonstrate a pharmacological means of regulating proteostasis, as ZNS alters protein trafficking, providing further support for the use of proteostasis regulators for the treatment of genetic epilepsies.

Keywords: Dravet syndrome; E3 ubiquitin ligase; GABAA receptor; endoplasmic-reticulum-associated protein degradation (ERAD); epilepsy; intracellular trafficking; proteostasis.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
The GABRG2(Q390X) mutation resulted in the γ2 subunit dimers and reduced expression of α1 and β2 subunits. A-C. HEK293T cells were transfected with wildtype γ2 or γ2 truncation mutations and wildtype α1 and β2 (total cDNA: 3 µg per 60 mm dish). Con is untransfected control. 48 h after transfection, cells were harvested and lysed. Lysates were subjected to SDS-PAGE. (A) Immunoblot for γ2 (1:1000). Monomers of γ2 can be seen on the bottom half of the membrane, and dimers and larger multimers can be seen on the top half. γ2 runs slightly below the predicted size of 55 kDa, consistently running at ~45 kDa. The band near 60 kDa is nonspecific. (B) Immunoblot for α1 (1:500) and graph showing integrated density values (IDVs) normalized to the loading control and then to the wildtype α1β2γ2 condition. (C) Immunoblot for β2 (1:1000) and graph of normalized IDVs. (D) Cartoon demonstrating that the γ2(Q390X) mutant subunit forms oligomers and retains partnering wildtype α1, β2, and γ2 subunits intracellularly, preventing proper trafficking of GABAARs to the cell surface. N = 5–6 separate transfections. Unpaired t-tests were used to evaluate statistical significance. * p < 0.05, ** p < 0.01. Values are expressed as the mean ± S.E.M.
Figure 2
Figure 2
Overexpression of the E3 ubiquitin ligase HRD1-enhanced γ2(Q390X) subunit degradation. (A–E) HEK293T cells were transfected with γ2 or γ2(Q390X) (3 μg) and pcDNA, HA-RNF34, HRD1-myc, HA-NEDD4L, or UBE3A-HA (0.5 μg). (A) Here, 48 h post-transfection, cells were collected, lysed, and subjected to SDS-PAGE. The membrane was immunoblotted with an anti-γ2 antibody (1:1000). (B) A graph of the IDVs of the wildtype γ2 band ~45 kDa, normalized first to the loading control ATPase (1:1000) and then to the γ2 + pcDNA condition. (C) A graph of the IDVs of the γ2(Q390X) monomers (lower band, 39 kDa). (D) A graph of the IDVs of the γ2(Q390X) dimers (upper band, ~80 kDa). E. A graph of the total γ2(Q390X) signal, the sum of the upper and lower bands. (C–E) IDVs were normalized to ATPase (1:1000) and then to the γ2(Q390X) + pcDNA control condition. (F–H) HEK293T cells were transfected with γ2 and γ2(Q390X) (1.5 μg each). They were cotransfected with HRD1-myc (labeled HRD) or HA-SEL1L (labeled SEL) or both HRD1-myc and HA-SEL1L (labeled H+S), using 0.5 μg of these plasmids. Total cDNA was normalized to 4 μg with empty vector pcDNA. pc is γ2 and γ2(Q390X) + pcDNA. UT are untransfected controls. (F) An SDS-PAGE membrane was immunoblotted with γ2 (1:1000). Wildtype γ2 runs at ~45 kDa, and γ2(Q390X) runs at ~39 kDa and can thus be seen separately. (G) Graph showing γ2 IDVs normalized first to the loading control, ATPase (1:1000), and then to the control condition, γ2 + γ2(Q390X) + pcDNA. The sum of both wildtype γ2 and mutant γ2(Q390X) is presented here. (H) Normalized IDVs of the wildtype γ2 and mutant γ2(Q390X) bands in (F) are shown individually, with wildtype as the solid bars and mutant as the outlined bars. N = 8 separate transfections for (A–E); 7 separate transfections for (F–G). One-way ANOVA and Šídák’s (A–E) or Tukey’s (F–G) test for post-hoc analysis, corrected for multiple comparisons, were used to evaluate statistical significance. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Values are expressed as the mean ± S.E.M.
Figure 3
Figure 3
ZNS facilitated the trafficking of GABAAR subunits to the cell surface. (A–C) HEK293T cells were transfected with α1, β2, and γ2 subunit cDNAs and empty vector pcDNA (3 μg each) (labeled as WT). UT are untransfected controls. For all other conditions, the cells were transfected with α1, β2, γ2, and γ2(Q390X) (3:3:1.5:1.5 μg). They were cotransfected with HRD1-myc (labeled HRD) or HA-SEL1L (labeled SEL) or both HRD1-myc and HA-SEL1L (labeled H+S), using 1.5 μg of these plasmids. Total cDNA was normalized to 12 μg with empty vector pcDNA. pc is α1, β2, γ2, γ2(Q390X), and pcDNA (3:3:1.5:1.5 μg). ZNS is α1, β2, γ2, γ2(Q390X), and pcDNA (3:3:1.5:1.5 μg) treated with 3 μM ZNS 24 h before harvesting. Living cells were treated with EZ-Link Sul-fo-NHS-SS-biotin to biotinylate surface proteins, which were then purified and run on polyacrylamide gels. A. Membranes were immunoblotted for γ2 (1:1000) (A), α1 (1:500) (B), or β2 (1:1000) (C) or ATPase (1:1000) as loading control. Protein IDVs were normalized first to the loading control and then to the WT condition (D–F). N = 5–6 separate transfections in different batches of cells. One-way ANOVA and Tukey’s test for post-hoc analysis, corrected for multiple comparisons, were used to evaluate statistical significance. * p < 0.05, **** p < 0.0001. Values are expressed as the mean ± S.E.M.
Figure 4
Figure 4
ZNS altered the total expression of the γ2 subunit, but not the α1 or β2 subunit in the mutant α1β2γ2/γ2(Q390X) receptors. (A–H). HEK293T cells were transfected with α1, β2, and γ2 (1 μg each per 60 mm dish) (WT); α1, β2, and mixed γ2 and γ2(Q390X) (1:1:0.5:0.5); or α1, β2, and γ2(Q390X) (1:1:1) (mutant). Untransfected cells were used as controls (con). Here, 24 h before harvesting, 3 μM ZNS or vehicle was applied. IDVs were normalized to ATPase (1:1000) and then to vehicle-treated WT. The membranes were immunoblotted for α1 (1:500) (A) or β2 (1:1000) (C) or γ2 (1:1000) subunit (E) antibodies. In (B,D,F,G,H), protein IDVs were normalized first to the loading control and then to the WT condition. (A) Immunoblot for α1 (1:500) and graph of normalized IDVs. (B) Immunoblot for β2 (1:1000) and graph of normalized IDVs. (C) Immunoblot for γ2 (1:1000) and graph of normalized IDVs. Quantification is for all γ2 signals: wildtype γ2 at 45 kDa, mutant γ2 at 39 kDa, and dimers between 80 and 100 kDa. (D) Graph of normalized IDVs of only monomeric wildtype γ2 at 45 kDa. (B,D,F,G,H) Normalized α1 (B), β2 (D), the total γ2 (F), the total wildtype γ2 (G), or the total γ2(Q390X) subunit protein (H) IDVs were plotted. In (F,G,H), the purple arrow-pointed band was not included as it is likely nonspecific. For (H), N = 7–8 separate transfections. Two-way ANOVA and Šídák’s multiple comparisons, examining simple effects within drug treatments, were used to evaluate statistical significance. * p < 0.05. Values are expressed as the mean ± S.E.M.
Figure 5
Figure 5
HRD1 dose-dependently enhanced degradation of the mutant γ2(Q390X) subunits. (A,B) Untransfected HEK293T cells were treated with 0.3, 1, 3, 10, or 30 μM ZNS or vehicle 48 h after passaging and 24 h before harvesting. (A) The SDS-PAGE membrane was immunoblotted for HRD1 (1:1000). (B) HRD1 IDVs were normalized to the average of all vehicle-treated dishes (ZNS 0 µM). This average was taken as 1. (C–E) HEK293T cells were transfected with γ2(Q390X) (0.5, 1, and 1.5 µg) with different HRD1 cDNA amounts. The total amount of cDNA in each condition was normalized with the vector pcDNA. The membranes were immunoblotted with an anti-γ2 subunit or HRD1 antibody. ATPase was used as a loading control. (D) The γ2(Q390X) subunit protein (D) or HRD1 (E) in cells cotransfecting HRD1 0.5 µg was normalized to that of HRD1 0.25 µg. For (A,B), N = 5. For (C–E), N = 6 separate transfections. Two-way ANOVA and Šídák’s multiple comparisons were used to evaluate statistical significance. In (B), * p < 0.05; ** p < 0.01 vs. ZNS 0 (untreated). In (D,E), * p < 0.05; ** p < 0.01; ***p < 0.001 vs. HRD1 0.25 µg. Values are expressed as the mean ± S.E.M.
Figure 6
Figure 6
ZNS reduced seizures in Gabrg2+/Q390X mice. (A) Schematic showing EEG headmount affixation and recordings. (B) Representative traces from a Gabrg2+/Q390X mouse experiencing a 5–7 Hz spike-and-wave discharge (SWD) at baseline and after 7 days of ZNS treatment (20 mg/kg/day, administered intraperitoneally). A 60 s trace is zoomed in on a 10 s window. (C) Total number of 5–7 Hz SWDs in 24 h, during baseline and after ZNS treatment. (D) Average duration of 5–7 SWD events, during baseline and after ZNS treatment. (E) Total time spent seizing in 5–7 Hz SWDs in 24 h, during baseline and after ZNS treatment. N = 3 female heterozygous Gabrg2+/Q390X mice. One-tailed paired t-tests were used to determine significance. * p < 0.05 vs. baseline.
Figure 7
Figure 7
ZNS selectively altered γ2 subunit expression in Gabrg2+/Q390X mice. (A) Schematic depicting the experimental protocol for ZNS administration. (B–E) Gabrg2+/Q390X mice and wildtype littermates of 1–1.5 months old were treated with 20 mg/kg ZNS or an equal volume of DMSO/saline vehicle, with daily intraperitoneal injections for 7 days. Brains were dissected, and lysates of the somatosensory cortex (cor), cerebellum (cb), thalamus (thal), and hippocampus (hip) were used for SDS-PAGE. The membranes after SDS-PAGE were immunoblotted for γ2 (1:1000) (B) or α1 (1:500) (D) subunit antibodies. Only the band of the wildtype γ2 subunit was quantified, as the γ2(Q390X) subunit is not always visible. In (C, E), specific protein IDVs were normalized to the loading control, ATPase (1:1000), and then to a paired vehicle-treated wildtype animal. N = 6–8 animals. Two-way ANOVA and Šídák’s multiple comparisons, examining simple effects within drug treatments, were used to evaluate statistical significance. ** p < 0.01. Values are expressed as the mean ± S.E.M.
Figure 8
Figure 8
BiP was upregulated in the mutant Gabrg2+/Q390X mice and had a differential response to ZNS compared to the wildtype mice. (A–D) The wildtype and Gabrg2+/Q390X mouse littermates at post-natal day 30–45 were treated with 20 mg/kg ZNS or an equal volume of DMSO/saline vehicle, with daily intraperitoneal injections for 7 days. Brains were dissected, and lysates of the somatosensory cortex (cor), cerebellum (cb), thalamus (thal), and hippocampus (hip) were used for SDS-PAGE. The membranes after SDS_PAGE were immunoblotted for BiP (1:500) (A,C) or Calnexin (1:500) (B,D). In (C,D), specific protein IDVs were normalized to the loading control, ATPase (1:1000), and then to a paired vehicle-treated wildtype animal. (C,D) N = 6–8 animals. Two-way ANOVA and Šídák’s multiple comparisons. * p < 0.05 vs. wt ZNS of the same brain region; δ p <0.05; δδδ p < 0.001 vs. wt vehicle of the same brain region. Values are expressed as the mean ± S.E.M.
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