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doi: 10.1093/hmg/ddw168 Advance Access Publication Date: 23 June 2016 Original Article

ORIGINAL ARTICLE

Differential molecular and behavioural alterations in mouse models of GABRG2 haploinsufficiency versus dominant negative mutations associated with human epilepsy Timothy A. Warner1, Wangzhen Shen1, Xuan Huang4, Zhong Liu1, Robert L. Macdonald1,2,3,5 and Jing-Qiong Kang1,5,* 1

Departments of Neurology, 2Molecular Physiology and Biophysics, 3Pharmacology, 4The Graduate Program of Neuroscience and 5The Vanderbilt Brain Institute, Vanderbilt University Medical Center Nashville, TN 37212, USA *To whom correspondence should be addressed at: Vanderbilt University Medical Center, 6140 Medical Research Building III 465 21st Ave, South, Nashville, TN 37232-8552, USA. Tel: þ615 936 8399; Fax: þ615 322 5517; Email: [email protected]

Abstract Genetic epilepsy is a common disorder with phenotypic variation, but the basis for the variation is unknown. Comparing the molecular pathophysiology of mutations in the same epilepsy gene may provide mechanistic insights into the phenotypic heterogeneity. GABRG2 is an established epilepsy gene, and mutations in it produce epilepsy syndromes with varying severities. The disease phenotype in some cases may be caused by simple loss of subunit function (functional haploinsufficiency), while others may be caused by loss-of-function plus dominant negative suppression and other cellular toxicity. Detailed molecular defects and the corresponding seizures and related comorbidities resulting from haploinsufficiency and dominant negative mutations, however, have not been compared. Here we compared two mouse models of GABRG2 loss-of-function mutations associated with epilepsy with different severities, Gabrg2þ/Q390X knockin (KI) and Gabrg2þ/- knockout (KO) mice. Heterozygous Gabrg2þ/Q390X KI mice are associated with a severe epileptic encephalopathy due to a dominant negative effect of the mutation, while heterozygous Gabrg2þ/- KO mice are associated with mild absence epilepsy due to simple haploinsufficiency. Unchanged at the transcriptional level, KI mice with severe epilepsy had neuronal accumulation of mutant c2 subunits, reduced remaining functional wild-type subunits in dendrites and synapses, while KO mice with mild epilepsy had no intracellular accumulation of the mutant subunits and unaffected biogenesis of the remaining wild-type subunits. Consequently, KI mice with dominant negative mutations had much less wild-type receptor expression, more severe seizures and behavioural comorbidities than KO mice. This work provides insights into the pathophysiology of epilepsy syndrome heterogeneity and designing mechanism-based therapies.

Received: March 22, 2016. Revised: May 19, 2016. Accepted: May 23, 2016 C The Author 2016. Published by Oxford University Press. V

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Introduction Genetic epilepsy is a common neurological disorder with substantial phenotypic heterogeneity, a feature shared with many other inherited human diseases. It has been a challenge to understand the basis for epilepsy phenotypic heterogeneity given that multiple ion channel or non-ion channel genes are associated with genetic epilepsies (1,2), and those genes have different functions and developmental and regional expressions. It is also intriguing that mutations in the same gene produce different phenotypes. This phenomenon can occur even in families with the same mutation where the phenotype among relatives is different. For example, mutations in SCN1A and GABRG2 are associated with both severe epilepsy (e.g. Dravet syndrome) and less severe epilepsy (e.g. generalized seizure with febrile seizures plus, GEFSþ (3,4). This thus hinders the development of effective treatments for genetic epilepsies. Familial and sporadic mutations in GABAA receptor subunit genes (GABRs) have been frequently associated with epilepsy (5–7) and as such have been recognized as human epilepsy genes (5). Additionally, multiple loss-of-function mutations in GABRG2 have been associated with different epilepsy syndromes with various severities (4,8,9). Loss-of-function mutations often refer to nonsense mutations that result in premature translation-termination codons (PTCs) and comprise about one third of mutations associated with human diseases (10,11). Besides nonsense mutations, frame shift mutations produced by insertion or deletions, splice site mutations, or missense mutations can result in a loss or impaired function of the gene. Many epileptic encephalopathies, the most severe type of genetic epilepsy, are associated with loss-of-function mutations in diverse genes (12–15). It is unknown why this subset of epilepsies is more severe and has a poor prognosis, whereas other epilepsies are relatively mild and have better outcomes. The pathophysiology of loss-of-function mutations associated with epilepsy syndromes with different severities has never been compared directly. In our previous study in vitro, we demonstrated that different GABRG2 mutations may result in dissimilar molecular defects due to differences in mutant protein metabolism (16). In humans, there are multiple mutations in GABRG2 that may cause similar pathology and present as a mild epilepsy syndrome probably due to simple functional haploinsufficiency. These loss-of-function mutations in GABRG2 include, but are not limited to, R136X (8) and W429X (9,17) mutations that are associated with relatively mild epilepsy phenotypes. Other mutations like Q40X and Q390X are associated with more severe epilepsy, Dravet syndrome (14) probably due to functional haploinsufficiency with other toxic cellular effects, referred to as dominant negative mutations. In the present study, we hypothesize that the simple loss-offunction mutations will cause mild epilepsy, while the dominant negative mutations will cause severe epilepsy syndromes in vivo. Gabrg2þ/- KO mice represent a model of a mild epilepsy, absence epilepsy (20) while Gabrg2þ/Q390X KI mice represent a model of a severe epilepsy, epileptic encephalopathy (4,19). The GABRG2(Q390X) mutation has also been designated GABRG2(Q351X) when the 39 amino acid signal peptide is not included (4,19). We compared directly two Gabrg2 loss-offunction mouse models at both molecular and behavioural levels. We have determined the expression of GABRG2 transcripts, wild-type and mutant GABAA receptor subunit protein in total and in cortical synaptosomes, dendrites and synapses in the two mouse models. We also characterized anxiety, locomotor

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activity, social ability and cognition in these two mouse models. The study represents a conceptual advance in understanding phenotypic heterogeneity by providing novel mechanistic insights for epilepsy phenotypic variation and for many other inherited human diseases.

Results Mutant Gabrg2(Q390X) subunit mRNA was not subject to nonsense mediated mRNA decay (NMD) PTCs resulting from nonsense mutations often activate NMD, and thus eliminate the mutant transcripts at the mRNA level if in early exons or at least 50 to 55 nucleotides 5’ to an exon-exon junction (20). However, the PTC in GABRG2(Q390X) is located in the last exon and should not be subject to NMD. Gabrg2 has 9 exons before the 3’-untranslated region (UTR; Fig. 1A upper panel). A DraI restriction endonuclease site TTTAAA was generated in Gabrg2 exon 9 in the mutant allele of the Gabrg2þ/Q390X KI mice, which could be used to distinguish the mutant allele from the wild-type allele in KI mice (Fig. 1A, middle panel). The C to T mutation in Gabrg2 exon 9 was confirmed by sequencing the genotyping PCR product in Gabrg2þ/Q390X KI mice (Fig. 1B). With primers flanking the 5th exon and the junction region of the 9th exon and 3’-UTR, a band at 841 bp was detected in both wild-type and mutant KI mice. Equal amounts of cDNA from wild-type and mutant KI mice were digested with DraI restriction endonuclease. In the heterozygous KI mice, two extra bands of lower molecular mass (594 bp and 247 bp) were produced, which were the two DraI digestion products (Fig. 1C). In the Gabrg2þ/- KO mice, exon 8 was replaced by the phosphoglycerate kinase (PGK)-neo cassette to generate the heterozygous KO mouse (21) (Fig. 1A lower panel). With the same primers used above, the PCR products from Gabrg2þ/- KO mice displayed two bands, one at 841 bp (wild type allele) and one at 650 bp (mutant allele). The wild-type mouse only displayed one band at 841 bp, as expected (Fig. 1D). This is consistent with the presence of one full length, functional allele and one shortened, nonfunctional allele that was reduced in size due to the replacement of exon 8 by the PGK-neo cassette. With qPCR, the total Gabrg2 mRNA abundance in Gabrg2þ/ Q390X KI mice and Gabrg2þ/- KO mice was the same as their wildtype littermates (1.03 6 0.08 vs 1.18 6 0.08, n ¼ 3 for KI; 1.17 6 0.16 vs 1.13 6 0.11, n ¼ 3 for KO; Fig. 1E), suggesting that NMD was not activated in either KI or KO mice. Similarly, there was no difference for Gabra1 mRNA abundance in Gabrg2þ/Q390X KI mice (1.09 6 0.06 vs 0.98 6 0.14, n ¼ 4) or Gabrg2þ/- KO mice (1.05 6 0.07 vs 1.03 6 0.19, n ¼ 4).

Mutant c2(Q390X) subunits were produced and impaired biogenesis of wild-type partnering subunits in Gabrg2þ/ Q390X KI mice, while no mutant c2 subunits were detected in Gabrg2þ/- KO mice Because the c2(Q390X) subunit mutation did not activate NMD and the mutant subunits had a slow degradation rate (22), we determined mutant c2 subunit expression levels. The total amount of c2 subunit protein was similar between Gabrg2þ/Q390X KI and wild-type mouse cortices (0.93 6 0.14 vs 1 ¼ wt, n ¼ 4), and the mutant subunit was detected (Fig. 2A,F). In contrast, in the Gabrg2þ/- KO mice, no mutant c2 subunit was detected, and the total c2 subunit level was reduced (0.76 6 0.09 vs 1 ¼ wt, n ¼ 5; Fig. 2B,F).

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Figure 1. The GABRG2(Q390X) mutation did not activate NMD, and Gabrg2 mRNA abundance was unchanged in both Gabrg2þ/Q390X KI and Gabrg2þ/- KO mice. (A) Schematic illustrations of the wild-type Gabrg2 allele, the mutant Gabrg2(390X) allele in KI mice and the mutant allele in Gabrg2þ/- in KO mice are presented. Gabrg2 has 9 exons before the untranslated 3’ region (UTR; top panel). A DraI site was generated in the mutant Gabrg2(Q390X) allele (middle panel). Exon 8 was replaced by a PGK-neo cassette in the mutant allele in the KO mice (low panel). (B) The C to T mutation was confirmed in KI mice by Sanger sequencing, and an equal ratio of C/T was present. A DraI endonuclease site was generated due to the C to T mutation. (C, D) The total RNAs from the forebrain of one month old wild-type mice and KI (C) or KO (D) mice were extracted and transcribed to cDNAs. The transcripts with primers flanking exons 5 and 9 and the adjacent UTR were amplified for both KI and KO mice. The PCR products from KI mice were digested with DraI endonuclease for 5 hrs in C. In panel C, U stands for undigested while DraI stands for DraI digested. In panel D, two bands were detected in PCR products in KO mice, but only one band was detected in the wild-type mice. (E, F) The relative abundance of Gabrg2 (E) and Gabra1 (F) mRNA was determined by quantitative real-time PCR with probes amplifying Gabrg2 and Gabra1, and the relative abundance of Gabrg2 and Gabra1 mRNA in each condition was normalized to actin, an endogenous control.

Since we previously identified more impairment of GABAergic mIPSCs in layer VI cortical pyramidal neurons in Gabrg2þ/Q390X KI mice than in Gabrg2þ/- KO mice (23), we determined the wild-type c2 subunit levels in cortical synapses from both KO and KI mice. We used subcellular fractionation to purify synaptosomes from somatosensory cortex and detected the c2 subunits with an antibody that recognized both wild-type and mutant subunits or an antibody that only recognized wild-type subunits. While the total c2 subunits were reduced in synaptosomes from both KI (0.58 6 0.06 vs 1 ¼ wt, n ¼ 6) and KO (0.78 6 0.04 vs 1 ¼ wt, n ¼ 6) mice, the reduction was greater in KI mice (Fig. 2C,G). Similar to c2 subunits, a1 subunits were more reduced in synaptosomes from KI mice (0.63 6 0.09 vs 1 ¼ wt, n ¼ 5) than from KO mice (0.89 6 0.21 vs 1 ¼ wt n ¼ 5; Fig. 2D,G). In contrast, b2 subunits were increased in synaptosomes from KO (1.19 60.16 vs 1 ¼ wt, n ¼ 5), and decreased in synaptosomes from KI (0.89 60.11 vs 1 ¼ wt, n ¼ 5) mice (Fig. 2E,G). Because the mutant c2(Q390X) subunits could not traffick beyond the endoplasmic reticulum (ER) and were trapped intracellularly, we thus determined if the c2 subunits in cortical synaptosomes were the wild-type c2 subunits. Using an antibody that only recognized

the wild-type c2 subunit (Fig. 2H), we found a similar amount of c2 subunits in synaptosomes of KI (0.53 6 0.08 vs 1 ¼ wt n ¼ 7) and KO mice (0.79 6 0.11 vs 1 ¼ wt n ¼ 6) as we detected with the antibody that recognizes both wild-type and mutant c2 subunits (Fig. 2G,I). To further confirm that the GABAA receptor subunits from the synaptosomal fraction were truly synaptic, we compared the abundance of the inhibitory synaptic marker gephyrin in different cytosolic fractions including S1, S2, S3 and synaptosomes. We found both gephyrin and c2 subunits were highly abundant in synaptosomes (Fig. 2J). In summary, this suggests that the cortical synaptosomes had minimal contamination with other cellular compartments, and that the mutant c2 subunits were not present in synaptosomes, likely due to ER retention of them.

Mutant c2(Q390X) subunits reduced levels of wild-type partnering subunits, likely by altering posttranslational modifications We hypothesized that altered synaptic levels of partnering a1 and b2 subunits were due to posttranslational rather than

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Figure 2. Total levels of mutant and wild-type GABAA receptor a1, b2 and c2 subunits in synaptosomes in Gabrg2þ/Q390X KI and Gabrg2þ/- KO mice were different. (A, B) c2 subunits from mouse cortices were immunopurified with guinea pig polyclonal anti-c2 subunit antibody (A) or total lysates directly homogenized from mouse cortices were analysed by SDS-PAGE and immunoblotted with the rabbit polyclonal anti-c2 subunit antibody that recognized both wild-type and mutant c2 subunits. (C-E) Synaptosomes from mouse forebrains were isolated by subcellular fractionation. The samples were then fractionated by SDS-PAGE and immunoblotted by rabbit polyclonal anti-c2 subunit antibody, (C), mouse monoclonal anti-a1 subunit (D) and rabbit polyclonal anti-b2 subunit (E). (F, G) The total protein IDVs of each subunit in cortex (F) or in synaptosomes (G) were normalized to total protein IDVs from wild-type mice, which were arbitrarily taken as 1. In panel A, the arrows designate the wt c2 or mut c2(Q390X) subunit. In panels A-E, LC stands for loading control. (H, I), Synaptosomes from mouse forebrains were isolated by subcellular fractionation. The samples were then fractionated by SDS-PAGE and immunoblotted by rabbit polyclonal anti-c2 subunit antibody, which recognizes only the wild-type c2 subunit (H). (I) The total protein IDVs of the wild-type c2 subunit were normalized to total protein IDVs from wild-type mice, which were arbitrarily taken as 1. In panel J, the mouse forebrains were subcellular fractionated with sucrose gradient and centrifugation. Total, also called S1, represented the fraction without membranes and nuclei, S2 represented cytosol and light membrane, S3 represents cytosol with crude synaptic vesicles. SPM represents synaptosome. Proteins from different layers were analysed for the presence of c2 subunits and the inhibitory postsynaptic marker gephyrin. In panels F,G and I, * P < 0.05, **P < 0.01, *** P < 0.001 vs wt, §P < 0.05, vs het KO.

genomic mechanisms; we therefore expressed wild-type or mutant c2 subunits alone or with a1 and b2 subunits in HEK 293T cells. We compared a transfection with a half wild-type c2 subunit cDNA concentration to mimic the simple loss-of-function hemizygous condition (hemi; heterozygous KO) to a transfection with equal amounts of wild-type and mutant c2(Q390X)

subunit cDNAs to mimic the heterozygous condition (het) (Fig. 3A,C). With expression alone (Fig. 3A,C) or with a1 and b2 subunits (Fig. 3A,D), wild-type c2 subunits were reduced in both hemi (0.6 6 0.09 vs 1 ¼ wt, n ¼ 4) and het (0.59 6 0.05 vs 1 ¼ wt, n ¼ 4) conditions. However, mutant c2(Q390X) subunits accumulated and were abundantly present in both het (2 fold of wt)

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Figure 3. The changes of wild-type GABAA receptor a1, b2 and c2 subunits in Gabrg2þ/Q390X KI mice were likely due to posttranslational protein modifications in the presence of the c2(Q390X) mutant protein. (A, B) HEK 293T cells were transfected with wild-type (wt) c2 or mutant c2(Q390X) subunit alone or in combination with a1 and b2 subunits (receptor condition). For wt receptors, an a1:b2:c2 1:1:1 cDNA ratio was used. For “hemizygous” (hemi) receptors, a 1:1:0.5 cDNA ratio was used. For mutant homozygous (hom) receptors, an a1:b2:c2(mut) 1:1:1 cDNA ratio was used. Total cell lysates were analysed by SDS-PAGE and immunoblotted with anti-c2 (A) and anti-a1 or anti-b2 (B) subunit antibodies. In panel A, arrows denote the wild-type (wt) or mutant (mut) c2 subunit bands. (C, D) Wild-type and mutant c2 subunit (C) or a1 and b2 subunits (D) IDVs were quantified, and the data were normalized to internal control ATPase IDVs (LC) first and then normalized to the wt condition, which was arbitrarily taken as 1 (*P < 0.05, **P < 0.01, ***P < 0.001; §§ P < 0.01 vs wt in het). (E, F) Total cortical lysates were undigested or digested with Endo-H or PNGase (F) for 3 hrs and then subjected to SDS-PAGE and immunoblotted with anti-a1 subunit antibody (E). The protein IDVs were quantifiedand the data were normalized to internal control ATPase IDVs (LC) and then to the undigested wild-type band that was arbitrarily taken as 1. (G, H) Mouse forebrains were subcellular fractionated with sucrose gradient and centrifugation. Membrane represents the fraction without cytosol, S1 represents the fraction without membrane and nuclei, S2 represents cytosol and light membrane, and S3 represents cytosol with crude synaptic vesicles (In panels F and H, * P < 0.05 vs wt; ** P < 0.01 vs wt).

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and homozygous (hom) ( 3 fold of wt) conditions as monomers and protein aggregates. Both a1 and b2 subunits were also reduced in het (0.78 6 0.09 for a1 and 0.79 6 0.06 for b2, n ¼ 4) and hom (0.42 6 0.04 for a1 and 0.53 6 0.12 for b2, n ¼ 4) conditions, but not in the hemi condition (Fig. 3B,D). Because we used recombinant receptors that were expressed acutely in cells with equivalent genomic background and no difference in the mutant protein synthesis was found with pulse labeling (22), these findings suggest that the reduction of the wild-type partnering subunits was likely due to posttranslational protein modifications. Consistent with the finding that wild-type a1 subunit expression was reduced in vitro, the data in the Gabrg2þ/Q390X mice indicated that a1 subunits were not reduced at the mRNA level (Fig. 1F) but were reduced at the protein level for total undigested (U), endo-H treated (H) and PNGase F (F) treated lysates (1 vs 0.586 0.06 for U, 0.89 6 0.05 vs 0.54 6 0.11 for H, 0.84 6 0.12 vs 0.50 6 0.05 for F, n ¼ 4; Fig. 3E,F). We fractionated the whole forebrain and identified that a1 subunits were reduced in the membrane (1 vs 0.69 6 0.04, n ¼ 4) but increased in the cytosolic fractions (0.12 6 0.04 vs 0.21 6 0.03 for S1, 0.04 6 0.012 vs 0.09 6 0.05, n ¼ 4 for S2) (Fig. 3G,H). This is consistent with the previous in vitro finding that wild-type a1 subunits were subject to endoplasmic reticulum retention in the mutant condition (19), and supports the conclusion that reduction of the wild-type partnering subunits was mediated by a posttranslational mechanism.

In layers IV-VI of somatosensory cortex, Gabrg2þ/Q390X KI mice had more reduced c2 puncta in neuronal dendrites than Gabrg2þ/- KO mice To further validate the reduction of c2 subunits in synapses, we co-labelled the c2 subunits with microtubule-associated protein 2 (Map2), a cytoskeleton member that mainly labels dendrites (24). We surveyed the whole brain but specifically investigated layers IV-VI in the somatosensory cortex (as shown in Fig. 4A). We co-labelled the mouse brain tissues with the mouse monoclonal anti-c2 subunit antibody in combination with the rabbit polyclonal Map2 antibody (Fig. 4B,C). The c2 subunit signal was normalized to Map2 (Fig. 4D). The het KI (1.12 6 0.16, n ¼ 6 vs 1.58 6 0.07, n ¼ 6 for wt), had more reduced c2 subunits relative to Map2 than the KO the KO (1.38 6 0.10 vs 1.62 6 0.20, n ¼ 6). Similarly, compared to the wild-type, the total puncta of c2 subunits were more reduced in the het KI (5.50 6 1.2 vs 17.1 6 2.6, n ¼ 6) than in the het KO (11.6 6 1.10 vs 19.3 6 3.2, n ¼ 10) mice (Fig. 4E). This suggests that the c2 subunit was more reduced in synapses in het KI than in het KO mice.

c2 Subunits had reduced colocalization with gephyrin in KI, but not KO, mice, suggesting the reduced presence of c2 subunits in inhibitory synapses Since c2 subunits are present postsynaptically in inhibitory synapses, we determined the localization of c2 subunits (Fig. 5A, second column) and the inhibitory postsynaptic marker gephyrin (Fig. 5A third, column) in cortical neuronal synapses by colabeling them (Fig. 5A, first column). The neuronal nuclei were marked by TO-PRO–3 (Fig. 5A, fourth column). The c2 subunit fluorescence colocalizing with gephyrin fluorescence relative to the total c2 subunit fluorescence was determined by measuring the c2 subunit signal overlapping gephyrin (Fig. 5A, first column and 5B). The percent of c2 subunit signal colocalizing with gephyrin was reduced in KI (46.0 6 3.90 for het, n ¼ 6 vs

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71.8 6 3.84, n ¼ 5 for wt), but not in KO (80.17 6 2.01 for het vs 72.5 6 4.24 for wt, n ¼ 6), mice (Fig. 5A,B). The staining of c2 subunits and gephyrin in the wild-type controls for the KO mice was almost identical to the wild-type controls for the KI mice, and thus the images of wild-type KO mice were not presented. There was no difference in the overlap of c2 subunits with gephyrin between wild-type and het KO mice, suggesting that the distribution of the c2 subunits in inhibitory synapses in the het KO mice was not changed except for the reduction of the amount of c2 subunits.

In layers IV-VI of somatosensory cortex, c2 subunits were increased in the neuronal somata of Gabrg2þ/Q390X KI mice, reduced in the somata of Gabrg2þ/- KO mice, and reduced in the neuropil of both mice The lack of mutant c2(Q390X) subunits in synaptosomes was consistent with our in vitro study that demonstrated that the mutant subunit was trafficking deficient and retained in the ER. To further confirm this in the KI mice, we determined the somatic and neuropil presence of total c2 subunits in neurons in somatosensory cortical layers IV-VI in both KI and KO mice (Fig. 5C,D). There was an increase in c2 subunits in the neuronal somata of KI mice (wt 56.40 6 7.9 vs KI 78.40 6 11.30, n ¼ 30) (Fig. 5C), but a reduction in c2 subunits in the neuronal somata of KO mice (wt 52.46 6 6.58 vs KO 27.89 6 8.3, n ¼ 30) (Fig. 5D). The c2 subunits in the cortical neuropil region were reduced in both KO (wt 48.4 6 3.4 vs KO 38.1 6 3.6, n ¼ 30) and KI (wt 47.3 6 8.4 vs KI 24.70 6 6.50, n ¼ 30; Fig. 5D) mice. These findings were consistent with endoplasm reticulum retention of the mutant c2(Q390X) subunits and the reduction of the wild-type c2 subunits in synaptosomes in both KI and KO mice. Interestingly, we noticed there were some cell morphological changes in the deep layers (IV-VI) of the cortex in the KI mice but not in the wild-type or KO mice. The cellularity change has also been reported in human epilepsy patients (25). This suggests the existence of a genetic mutation like GABRG2(Q390X) may result in previously uncharacterized cellular changes. However, the nature of the cellularity changes needs to be further elucidated.

Gabrg2þ/Q390X KI mice had a more severe epilepsy phenotype than Gabrg2þ/- KO mice The KI mice had spontaneous generalized tonic clonic seizures, myoclonic jerks and increased mortality throughout life (23), representing a mouse model of a GABRG2 dominant negative mutation, while the KO mouse was reported to have absence seizures in a seizure-prone mouse background (18) or anxiety (26), representing a mouse model of GABRG2 haploinsufficiency. We have observed multiple forms of seizures including generalized tonic clonic and myoclonic seizures in KI mice (Supplementary video, Fig. 6A). However, we have only observed absence-like activity in the KO mice. It is worth noting that we observed SWDs in KO mice in both C57BL/6J N8-10 and DBA N8 backgrounds, but SWDs were more pronounced (i.e., higher voltage and increased incidence) in DBA mice. This is consistent with a previous report that mouse genetic background affects SWD and epilepsy severity., Epilepsy was more severe in mice with the C3H background compared with the C57BL/6J background (27). In addition, no SWDs were recorded from KO mice in with the C57BL/6J >N20 background in a previous study (18). This suggests that even in the same mouse background,

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Figure 4. The c2 subunits in Gabrg2þ/Q390X KI mice were more reduced in dendrites compared with Gabrg2þ/- KO mice. (A) A sample image of mouse cortex and the region of interest in the somatosensory cortex as boxed. The sections were then stained with rabbit anti-microtubule-associated protein 2 (Map 2) (green) and counterstained with TO-PRO-3 (blue). (B) Mouse brains from 3-4 month old KI, KO, and their respective wild-type littermates were short-fixed (30 min exposure to 4% paraformaldehyde) and sectioned on a cryostat at 15 to 30 lm. The sections were then stained with rabbit anti-microtubule-associated protein 2 (Map 2) (green) and mouse monoclonal anti-c2 subunits (red) antibodies. The nuclei were stained with TO-PRO-3. Only the wild-type and het KI images were presented because the images of the het KO were similar to those of the wild-type. (C) The box regions in B were enlarged. (D) The images were analysed by MetaMorph software. The c2 subunit and Map2 fluorescence signals were measured, and the ratios of c2 subunit fluorescent signals colocalizing with Map2 were quantified. (E) The total number of c2 subunit puncta per 50 mm2 was quantified (* P < 0.05, **P < 0.01; vs wt, §P < 0.05 vs het KO).

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Figure 5. The c2 subunit in Gabrg2þ/Q390X KI mice was less colocalized with gephyrin and was more accumulated in the somatic regions than in wild-type or Gabrg2þ/KO mice. (A) Mouse brains from 3-4 month old KI, KO, and their respective wild-type littermates were short-fixed (30 min exposure to 4% paraformaldehyde) and sectioned on a cryostat at 15 to 30 lm. The sections were then stained with rabbit anti-c2 subunit (green) and mouse monoclonal anti-gephyrin (red) antibodies. The nuclei were stained with TO-PRO-3. Only the wild-type KI images were presented because the images of the wild-type KO were almost identical to those of the wild-type KI. (B) The images were analysed by MetaMorph software. The c2 subunit and gephyrin fluorescence signals were measured, and the percentages of c2 subunit fluorescent signals colocalizing with gephyrin were quantified. (C, D) Fluorescence intensities of the c2 subunits in the neuronal somatic regions (C) or neuropil regions (D) in cortical brain sections were quantified by ImageJ. The c2 subunit signal from the nuclei was used as background and was subtracted for both somata and neuropil c2 subunit signals. (* P < 0.05; **P < 0.01; ***P < 0.001 vs wt, §P < 0.05, §§P < 0.01, vs het KO).

different founder generations could affect seizure phenotype. Since we focused on the effect of mutations instead of the mouse background, we only reported the SWDs in C57BL/6J mice (Fig. 6B). Since spontaneous seizures were rarely observed, and thus hard to quantify, we compared the seizure activity with pentylenetetrazol (PTZ) induction. Since severe epilepsies like Dravet syndrome are often refractory to treatment. We also determined the response of seizures in KI mice to diazepam (DZP) treatment. DZP was administered via intraperitoneal injection at 0.3 mg/kg 30 min before PTZ (50 mg/kg) administration, and EEGs were recorded for 2 hrs before and after DZP treatment.

We chose to administer 50 mg/kg PTZ in the KI mice because a higher dose would result in 100% mortality within a few minutes. We used a modified Racine scale to evaluate the seizure stages as in our previous study (28). We also assessed seizure activity following DZP administration because the GABRG2(Q390X) mutation is associated with Dravet syndrome in humans, and seizures of Dravet syndrome are often refractory to treatment. All the KI mice reached stage 3-4 and stage 56 in the saline-treated group. While all of the mice reached stage 3-4 in the DZP-treated group (Fig. 6C) and fewer mice died afterwards than saline treated group (data not shown), only 37.5% of mice (6 out of 16 mice) reached stage 5-6. In contrast,

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Figure 6. Gabrg2þ/Q390X KI mice had multiple seizure semiologies and more severe seizures after pentylenetetrazol treatment than Gabrg2þ/- KO mice. (A) Representative EEG recordings show that the Gabrg2þ/Q390X KI mouse had spontaneous generalized tonic clonic seizures and myoclonic jerks. (B) Representative EEG recordings show that the Gabrg2þ/- KO mice had slow spike-wave discharges (SWDs). The boxed areas in B were expanded for a more detailed view (lower panel). (C, D) Percentage of Gabrg2þ/Q390X KI (C) and Gabrg2þ/- KO (D) mice that reached different stages of the modified Racine scale after intraperitoneal pentylenetetrazol (PTZ) administration (50 mg/kg) 30 min following normal saline or diazepam (DZP, 0.3 mg/kg) treatment was plotted.

all of the KO mice reached stage 3-4 but only 45.5% (5 out of 11) reached stage 5-6 in the saline-treated group. In in the DZPtreated group, only 55.5% (6 out of 11) reached stage 3-4 and none reached stage 5-6. The data indicate the KO mice were more susceptible to PTZ-induced seizures than the wild-type mice, but the seizures in KO mice were less severe than those in KI mice and more responsive to diazepam treatment.

Gabrg2þ/Q390X KI, but not Gabrg2þ/- KO, mice displayed hyperactivity and social deficits Patients with epileptic encephalopathies often have behavioural comorbid conditions including attention deficit hyperactivity disorder (ADHD), anxiety, autism and cognitive impairment. Both the KO and KI mice had heightened anxiety that was not reported in this study because this aspect has been addressed in previous reports for both KO and KI mice. To evaluate behavioural comorbidities in the KI and KO mice, we first assessed their locomotor activity using activity chambers. KI (wt:1607 6 109 cm, n ¼ 35; het: 2209 6 276 cm, n ¼ 27), but not KO (wt:1508 6 346 cm, n ¼ 14; het:1887 6 473 cm, n ¼ 15), mice travelled a significantly greater distance, suggesting hyperactivity of KI mice (Fig. 7A,B). In addition to hyperactivity, impaired social interaction is common among patients with epileptic encephalopathies like Dravet syndrome (29). We thus assessed social interactions for the KI and KO mice. For sociability, there was no difference between the wild-type (133.61 6 12.79 s, n ¼ 13) and KI (129.56 6 14.71 s, n ¼ 9) or wild-type (159.81 6 13.93 s, n ¼ 9) and KO (177.19 6 17.23 s, n ¼ 10) mice interacting with the novel object. However, only KI mice had reduced time (219.78 6 20.15 s,

n ¼ 9) interacting with the novel mouse compared to their wild-type littermates (272.39 6 12.39 s, n ¼ 13) as there was no difference between KO mice (268.73 6 17.63 s, n ¼ 10) and their wild-type littermates (252.78 6 17.25 s, n ¼ 9) (Fig. 8B). For social novelty, there was no difference in the time spent with the familiar mice for the KO mice (148.99 6 16.45 s, n ¼ 10) and their wild-type littermates (154.43 6 22.35 s, n ¼ 9), nor for the KI mice (131.19 6 6.63 s, n ¼ 9) and their wild-type littermates (122.67 6 10.27 s, n ¼ 13). Similar to sociability, only KI mice (172.99 6 17.69, n ¼ 9) had reduced interaction with the novel mouse compared to their wild-type littermates (234.15 6 12.75, n ¼ 13; Fig. 7C), while KO mice (236.56 6 9.58 s, n ¼ 10) and their wild-type littermates (226.43 6 17.32 s, n ¼ 9; Fig. 7C) spent a comparable amount of time with the novel mouse. The findings indicated that KI mice expressed hyperactivity and engaged in less social interactions with novel mice, consistent with patients with epileptic encephalopathy.

Gabrg2þ/Q390X KI and Gabrg2þ/- KO mice had slowed learning but only KI mice had impaired memory as the cognitive demands escalated Since impaired cognition is another common comorbidity associated with epileptic encephalopathies, we tested the learning and memory of the wild-type, KI, and KO mice with the Y-maze (Fig. 8A) and Barnes maze (Fig. 8C,D) tests, which are commonly used for studying spatial learning and memory. In the Y-maze test, both KI (86.81 6 10.43 s, n ¼ 9) and KO (85.35 6 12.74 s, n ¼ 10) mice spent less time in the novel arm compared with their respective (126.77 6 10.50 s, n ¼ 13) or KO (132.58 6 14.64 s, n ¼ 9)

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deficiency was more pronounced for the KI mice as the cognitive demands escalated.

Discussion

Figure 7. Gabrg2þ/Q390X KI, but not Gabrg2þ/- KO, mice displayed hyperactivity and reduced social interaction. (A) The total distance travelled (cm) in an activity chamber over a 10-min session was recorded for KI (A) and KO mice to assess locomotor activity. (B) Three-chamber social interaction test was performed with wt and het KI and KO mice to test for sociability. Each mouse was presented the option of exploring an empty pencil cup or one that contained a stranger mouse. All exploratory behaviours within a 1 cm radius of the noted stimuli were recorded. (C) Three-chamber social interaction test was performed with wt and het KI and KO mice to test for social novelty. Each mouse had the option to investigate a familiar mouse (stranger mouse during sociability testing) or a novel mouse in their respective pencil cups. All exploratory behaviours within a 1 cm radius of the noted stimuli for the three-chamber test were recorded (*P < 0.05 vs wt).

wild-type littermates (Fig. 8B). Since reduced time in the novel arm in the Y maze is indicative of impairment in spatial working memory (30), these results indicated spatial memory deficits for both KI and KO mice. To further assess cognition in these two mouse models, we also utilized the Barnes maze test in which the animals learned to escape and locate the target hole (Fig. 8C,D). In both the KI and KO mice, the escape latency was prolonged in the first two days (day 1 ¼ 40.85 6 3.53 s, day 2 ¼ 23.83 6 2.39 s, n ¼ 9 for KI het; day 1 ¼ 39.49 6 3.75 s, day 2 ¼ 25.82 6 3.34 s, n ¼ 10 for KO het) compared to the respective wild-type littermates (day 1 ¼ 27.90 6 2.74 s, day 2 ¼ 16.74 6 2.09 s, n ¼ 13 for KI; day 1 ¼ 27.31 6 3.19 s, day 2 ¼ 14.45 6 1.55 s for KO), but this disparity between groups was no longer present after the second day (Fig. 8E). This indicated a slower acquisition in learning for both the KI and KO mice. However, during the probe trial, only the KI mice (26.58 6 2.58 s, n ¼ 9) had reduced time spent in the target hole area compared with their wild-type littermates (39.23 6 2.86 s, n ¼ 13), while the KO mice spent a similar amount of time in the target hole area as the wild-type littermates (Fig. 8F). This suggested that both the Gabrg2þ/Q390X KI and Gabrg2þ/- KO mice had impaired cognition, but the cognitive

Accumulating evidence from genetic studies indicates substantial phenotypic heterogeneity for genetic epilepsies as well as many other genetic diseases. Understanding those inter- or intra-familial phenotypic variations presents a great challenge. This study directly compared the molecular basis and behavioural phenotypes of mouse models of GABRG2 simple haploinsufficiency versus dominant negative mutations, the Gabrg2þ/- KO mouse and the Gabrg2þ/Q390X KI mouse. Based on our studies in vitro, there are some epilepsy mutations in GABRG2 that produce a molecular phenotype similar to haploinsufficiency while others that produce a much more impaired phenotype that includes haploinsufficiency and dominant negative suppression plus cellular toxicity. In this study, the KI mouse is associated with a severe epileptic encephalopathy and the production of stable, mutant c2 subunits, representing a dominant negative mutation. In contrast, the KO mouse is associated with mild epilepsy in a seizure-prone mouse background (18) or hyperanxiety (26) and no production of mutant c2 subunits, representing a haploinsufficiency mutation. Since mutations in a single gene are often associated with a wide spectrum of disease phenotypes (16), the comparisons of the molecular changes and seizure/behavioural phenotypes of these two mouse models would provide insight into the molecular basis of epilepsy phenotype heterogeneity. KO mice have been used widely to study human diseases associated with loss-of-function mutations including those associated with epileptic encephalopathies (20,31) and have yielded critical insights into disease mechanisms. This is probably true for many mutations in which the functional and cellular consequences of the mutation are indeed similar to the gene KO condition. We previously demonstrated in vitro that different truncated c2 subunits with loss-of-function were associated with different severities of epilepsy syndromes, had a different protein metabolism and reduced wild-type partnering subunits to different extents (17). In the present study, we further demonstrate in vivo the distinctive phenotypes caused by different mutations in GABRG2, because of differential remaining GABAA receptor expression in different subcellular compartments that led to differential seizure/EEG abnormalities and neuropsychiatric comorbidities.

The GABRG2(Q390X) mutation did not activate NMD to eliminate mutant subunit mRNA NMD of mutant mRNA is a potential mechanism to attenuate disease phenotypes caused by nonsense mutations in Marfan’s syndrome (32). A similar disease phenotype attenuation by NMD has also been observed in neurological diseases such as neurocristopathy including peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome and Hirschsprung disease (PCWH), Waardenburg-Shah syndrome associated with sex determining region Y-box 10 (SOX10) mutations, or myelinopathies including Charcot-MarieTooth 1B associated with myelin protein zero (MPZ) mutations (33). We previously compared two GABAA receptor mutations, GABRA1(975delC, S326fs328X) associated with childhood absence epilepsy and GABRA1(A322D) associated with juvenile myoclonic epilepsy, and proposed a similar phenotype

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Figure 8. Gabrg2þ/Q390X KI, but not Gabrg2þ/- KO, mice had impaired memory, although both had slowed learning. (A) The Y-maze apparatus was set up differently for training and testing trials. One arm was blocked during training trial but opened during testing trial after a 5 min interval. (B) Time spent in the novel arm (blocked arm in training trial) was assessed for mice in each genotype. (C) A flow chart depicts an overview of the Barnes maze. (D) The Barnes maze had one target hole and eleven non-target holes. Mice were trained to find the target hole to escape during training sessions, which was hidden during the probe trial. (E) Time spent to locate the target hole was recorded and quantified for each day in each mouse genotype. (F) An hour after the last training trial, each mouse was allotted a 300 s session to find the target hole. The total time spent at each of the 12 holes was assessed (*P < 0.05 vs wt). All data were recorded by an over-head camera and generated by ANY-Maze software.

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modifying mechanisms in epilepsy (34). Our findings in the present study, however, confirmed that the GABRG2(Q390X) mutation did not activate NMD in Gabrg2þ/Q390X KI mice. The mutant Gabrg2(Q390X) subunit mRNA was not degraded and was translated into mutant subunits, which were stable, had slowed turn-over, produced dominant negative suppression of wildtype subunits and accumulated with aging (23). In contrast, although they were not eliminated at the mRNA level in Gabrg2þ/KO mice, the mutant c2 subunits with replacement of exon 8 by the PGK-neo cassette were degraded rapidly. This resulted in no dominant negative suppression of biogenesis of wild-type subunits or other cellular toxicity.

GABAergic synaptic transmission was more impaired in KI than in KO mice due to the production of mutant c2(Q390X) subunits and impairment of wild-type subunits The presence of the nonfunctional mutant c2(Q390X) subunits in the KI mice and the reduction of wild-type c2 subunits in the KO mice may form the bases for the different levels of wild-type partnering GABAA receptor subunit expression in synaptosomes and subsequent reduction of GABAergic neurotransmission. The mutant c2(Q390X) subunit was detected in KI mice, but no mutant c2 subunits were detected in KO mice. The expression levels of wild-type c2 subunits, as well as of a1 and b2 subunits, were different in the two mice, suggesting differentially impaired GABAergic synaptic transmission. The upregulation of b2 subunits in KO mice is consistent with the formation of a1b2 receptors at the expense of a1b2c2 receptors with b2 subunits replacing the c2 subunits when c2 subunit levels were reduced.

Only KI mice had intracellular retention of mutant c2 subunits Mutant c2(Q390X) subunits were present mainly in cortical neuronal somata and were not present in synaptosomes. Because c2(Q390X) subunits could not traffick to the cell surface (19), the increased c2 subunit signal in the neuronal somata was likely due to ER retention of the mutant subunits. Chronic accumulation of the mutant subunits and sustained ER stress could lead to neurodegeneration. We demonstrated that the c2(Q390X) subunits formed intracellular aggregates in neurons in old, but not in young, Gabrg2þ/Q390X mice (23). The c2(Q390X) subunits were co-localized with the active form of caspase 3, suggesting the initiation of cellular apoptosis by the c2(Q390X) subunits (23). The greater reduction of wild-type c2 subunit in synaptosomes in KI than in KO mice may explain also why GABAergic mIPSCs were more impaired in KI than in KO mice (23). While there was an increased presence of c2(Q390X) subunits in neuronal somata in KI, but not in KO, mice, we cannot exclude the possibility that some wild-type c2 subunits were also trapped intracellularly due to oligomerization with the c2(Q390X) subunit because the c2 subunit antibody used for immunohistochemistry recognized the N-terminal motif of both wild-type and mutant c2 subunits. Nevertheless, the differences in somatic presence of c2 subunits were evident among wild-type, KI and KO neurons. Similar to mutations associated with cystic fibrosis (35), protein misfolding, glycosylation arrest and trafficking deficiency are common defects for GABAA receptor subunit mutations (36). Based on our previous in vitro study of multiple GABRG2 nonsense mutations in the last exon, we demonstrated that ER retention and loss of surface expression were common

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phenomena for the trafficking deficient c2 subunits (17). However, the levels of ER stress and subsequent cellular injury caused by the mutant c2 subunits may differ depending on the mutant protein stability (17). Some mutant c2 subunits such as c2(Q390X) subunits were very stable and could cause chronic subunit accumulation, sustained ER stress and cell death, while other c2 subunits like the mutant c2 subunits in the Gabrg2þ/knockout condition were very unstable and disposed of easily by the cellular protein degradation machinery. The mutant c2(Q390X) subunits had glycosylation arrest and were retained in the ER in multiple cell types (19) and were not present in synaptosomes in KI mice. In contrast, mutant c2 subunits were not stable in KO mice. The primary defect in KO mice was the reduction of wild-type c2 subunits. These two mouse models are very representative, and thus serve as good examples to understand the pathophysiology caused by the two groups of mutation.

KI mice had a more severe epilepsy phenotype than the KO mice The KI mice had spontaneous generalized tonic clonic seizures and spontaneous death, likely related to seizures, while the KO mice have been reported to be seizure free (26) or to display only absence seizures in a seizure prone mouse background (18). We noticed that the frequency of SWDs in KO mice was 4-7 Hz, which was slower than the standard range of frequency (7-9 Hz) seen in rodent models of typical absence epilepsy (37) and SWDs identified in another GABAA receptor subunit absence, the mouse model Gabra1þ/- KO mouse (38). It has been reported that the slow absence-like EEG discharges indicate involvement of the hippocampus and is associated with cognitive deficits (39). This is not unexpected given the abundant presence of c2 subunits in the hippocampus throughout development and the cognitive impairment seen in patients harbouring the c2(Q390X) subunit mutation (4).

KI mice had more severe neurobehavioural comorbidities than KO mice In addition to severe and intractable seizures, epileptic encephalopathies, like Dravet syndrome, often have multiple neurobehavioural comorbidities such as autistic traits, anxiety, hyperactivity, impaired social interactions as well as cognitive impairment (40). The GABRG2(Q390X) mutation is associated with Dravet syndrome (4) while other loss-of-function mutations in the same gene resulted in less mutant subunit accumulation and are associated with relatively milder epilepsy phenotypes (8,9). It is not surprising that the KI mice, but not the KO mice, displayed hyperactivity and impaired social interactions. Nor is it surprising that as spatial learning and memory demands escalated, KI mice revealed greater cognitive deficits than the KO mice,

Conclusions In conclusion, this study has characterized the molecular and behavioural phenotypes of two mouse models with different levels of epilepsy severity associated with mutations in a common human epilepsy gene GABRG2. The Gabrg2þ/Q390X KI mouse is associated with epileptic encephalopathy representing dominant negative mutations and the Gabrg2þ/- KO mouse is associated with mild absence epilepsy representing functional haploinsufficiency. Although the mutant alleles were

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unchanged at the transcription level and were nonfunctional in both KI and KO mice, the mutant c2 subunits were produced only and accumulated intracellularly in KI, but not in KO, mice. Consequently, the KI mice had less remaining wild-type GABAA receptors in dendrites and synapses than KO mice, leading to a more severe epilepsy phenotype. This suggests that therapeutic strategies to reduce or eliminate the mutant protein may attenuate the disease phenotype.

more background with the rabbit polyclonal anti-c2 antibody from Synaptic Systems (224 003) in HEK 293T cells. Thus, we used the anti-c2 subunit antibodies from Synaptic Systems for mouse tissue and the rabbit polyclonal anti-c2 subunit antibody from Alomone for HEK 293T cells. We used mouse monoclonal anti-c2 subunit antibody from Synaptic Systems (224 011) only for immunohistochemistry.

Quantitative Real-time Polymerase Chain Reaction (qPCR)

Materials and Methods Mice The Gabrg2þ/Q390X KI mouse line was recently developed (41) and the Gabrg2þ/- KO mouse line was reported before (21). The mice used in the study were bred into C57Bl/6J mice for 8 to 10 generations. The mice used for gene expression were 1 month old. All mice used for immunohistochemistry, biochemistry, EEG recordings and behavioural tests were between 2-4 months old.

GABAA receptor subunit plasmids, expression, Western blot analysis and antibodies The cDNAs encoding human GABAA receptor a1, b2 and c2 subunits were as described previously (42,43). The short form of the GABAA receptor c2 subunit was used in this study. Transfection of human embryonic kidney (HEK) 293T cells and Western blots were also as previously described (22). The antibodies were obtained from the following sources, and the clone or catalogue number and concentrations used for the Western blots are listed below in Table 1. The experimental procedures were carried out with our standard laboratory protocols, and the reagents were from LI-COR Biosciences. The blots were imaged on an infrared fluorescent imaging system (LI-COR Biosciences).

Validation of c2 antibodies We have validated the specificity of all the three anti-c2 subunit antibodies used in this study with Gabrg2-/- KO mice for biochemistry and collaborated with the Vanderbilt University Medical Center Translational Pathology Shared Resource for immunohistochemistry. There is a nonspecific band around 50 KDa in the gel if detected with the rabbit polyclonal anti-c2 subunit antibody from Alomone for mouse tissue, while there is

The relative mRNA abundance of GABRG2 was determined with a protocol modified from previous studies (34,44). Freshly extracted mouse brains, using sagittal halves, were dissected, and total RNA was isolated using a commercial silica membrane column (Purelink). Using 200 ng of RNA, we generated correspondR III firsting cDNAs with reverse transcriptase using SuperScriptV strand synthesis system (Invitrogen). We performed quantitative real-time PCR using an Applied Biosystems 7900 with the TaqMan Universal Master Mix and with 6-carboxyfluorescein (FAM)-labelled probes (Ambion) for the GABRG2 (Mm 00433487) and a VIC-labelled probe b-actin as an endogenous control (4352341E). For real-time PCR, we incubated at 95  C for 10 min, ran 40 cycles that consisted of denaturation at 95  C for 15 s, and annealing and extension at 60  C for 60 s. The GABRG2 mRNA expression was normalized to the endogenous control actin, and the values were calculated with the DCt cycle threshold method. GABRG2 primers encoding 5’ of exon 5 and 3’ of exon 9 bordering untranslated region (UTR) were used to amplify the fragment of exon 5 to exon 9 of mouse GABRG2 mRNA. PCR products amplified from the Gabrg2Q390X/þ KI mouse and its littermate were further digested by the endonuclease Dra1 for 5 hrs.

Brain slice immunohistochemistry and related quantifications The brain sections were from freshly prepared brain tissues. Briefly, mice were not perfused, and the brains were directly blocked and briefly fixed in 4% paraformaldehyde for 30 min and then maintained in a 30% sucrose solution before sectioning on a cryostat. The brains were sectioned at 15 to 30 mm. The sectioned tissues were stored at -20 before staining. The brain slices were permeabilized with 0.4% triton for 10 min and blocked with 0.2%/0.2% BSA/Triton for 1 hr followed by immunoreaction overnight with specifically targeted antibodies. The slices were then gently washed with 0.1% BSA/PBA three times

Table 1. Primary and secondary antibodies, and nucleic acid stain Target Protein

Species

Source, clone/catalog number

Application(s)

Dilution

GABAAR a1 GABAAR a1 GABAAR b2 GABAAR c2 GABAAR c2 GABAAR c2 Gephyrin Gephyrin Microtuble-associated protein 2 (MAP2) GAPDH Alexa Fluor 488 Rhodamine Red-X TO-PRO-3 Iodide

Mouse Mouse Rabbit Rabbit Mouse Rabbit Mouse Rabbit Rabbit

NeuroMab, N95/35 Millipore, BD24 Millipore, AB5561 Synaptic Systems, 224 003 Synaptic Systems, 224 011 Alomone, AGA-005 Synpatic Systems, 147 011 Synpatic Systems, 147 103 Cell Signaling Technology, 4542 Millipore CB1001 Molecular Probes, A11034 Jackson ImmunoResearch, 115295003 Molecular Probes, T3605

WB, WB WB WB, IHC IHC WB IHC WB IHC

1:250 1:300 1:100 1:100 1:100 1:100 1:200 1:300 1:200

WB IHC IHC IHC

1:2000 1:400 1:60 1:500

Mouse Goat Goat

WB ¼ Western blot, IHC ¼ Immunohistochemistry.

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before incubating with secondary antibodies. The cell nuclei were labelled with TO-PRO-3 (Molecular Probes) with a 1:500 dilution for 1 hr. After washing, the slices were directly sealed with ProLong Gold antifade mounting medium (Molecular Probes). The samplings and quantifications of immunohistochemistry were based on the previous study. As for the c2 puncta quantification, we chose the region of interest along Map2 positive staining for which the length of the dendrites was more than 10 mm. The total c2 puncta in an area of 50 mm2 was counted. Three to five areas were quantified in each image and the average of the counts from each image was taken as n ¼ 1.

Subcellular fractionation and isolation of synaptosomes and postsynaptic density region The procedures of subcellular fractionation were modified from a previous study for synaptosome preparation (45). The synaptosome layer (spm) was at the 1.0/1.2 M sucrose interface. To prepare postsynaptic densities, the spm fraction was diluted to 0.32 M sucrose by adding 2.5xvol of 4 mM Hepes (pH 7.4) and balanced with Hepes buffered sucrose (HBS). The diluted spm preparation was then centrifuged at 150,000 g for 30 min (TH 641:29,600 rpm). After centrifugation, the pellet was collected and suspended by adding 4 ml 0.5% triton-100 solution containing 50 mM Hepes, 2 mM ethylenediaminetetraacetic acid (EDTA) and protease inhibitors rotated for 15 min.

EEG surgery, recording, analysis and related drug administration The surgery to implant the EEG headmount and the video-monitoring synchronized EEG recordings were conducted as previously described (23,38). We administered pentylenetetrazol (PTZ; 50 mg/kg, ip), heterozygous KI mice to test the seizure threshold in the KI and KO mice. We administered diazepam (0.3 mg/kg, ip) 30 min before PTZ injection to determine the effect of diazepam on the seizure activity in KI and KO mice. Normal saline was applied as control vehicle. The reason we chose a lower dose of PTZ was due to the high mortality rate of Gabrg2þ/Q390X mice at higher doses of PTZ (70 mg/kg or 85 mg/kg).

Locomotor activity The protocol for locomotor activity was as previously described (23,46,47).

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Modified Y-maze The protocol used was described previously (50). Briefly, the test consisted of a two-trial spatial memory task where mice were placed in an apparatus with three equally-sized arms. One arm was randomly blocked for the first trial (training trial), while all three arms were open for the second trial (testing trial). Mice were given 5 min to freely explore the maze during each trial. Time spent in the novel arm, which was the blocked arm in trial 1, was used to assess spatial memory.

Barnes Maze The test was based on the protocol described in the previous studies (51,52). The procedure includes three components: pretraining, training, and a memory probe, and each is briefly outlined. Pretraining: the mouse was placed in a black start box for 30 s and then guided to the target hole where the mouse was able to descend into the escape box. After 30 s, the mouse was removed from the escape box and placed back in the start box for an additional three trials. The pretraining session only occurred on the first day of Barnes maze testing. Training: similar to pretraining, the mouse was placed in the start box for 30 s. After 30 s in the start box, the mouse was allowed to freely explore the maze in search of the target hole using extra-maze cues for a maximum of 300 s. On each training trial, 11 of the 12 holes were blocked. Mice were exposed to 4 training trials per day for 5 consecutive days, and training begin immediately after pretraining on day 1. Probe: A 300 s probe test was conducted 1 hr after the final training trial on the fifth day using the same parameters described during the training session, except that all 12 holes were now blocked.

Statistical analysis Protein integrated density values (IDVs) were quantified by using the Quantity One or Odyssey fluorescence imaging system (Li-Cor). The fluorescence intensity values were quantified by using ImageJ. Data were expressed as mean 6 S.E.M values and analysed with GraphPad Prism 5.0 software. Analysis of variance (ANOVA), including one-way and two-way ANOVA, and unpaired student t tests were used for imaging, gene expression, biochemistry and EEG analysis. All behavioural data were analysed with SPSS 22.0 software. Independent-sample t tests were used in the analyses of locomotor activity, modified Y-maze, and the probe trials of the Barnes maze. A two-way ANOVA was used for the threechamber test of sociability and social novelty. A repeated measures ANOVA was used to analyse the training trials of the Barnes maze. Post hoc and a priori Bonferroni comparisons were conducted to evaluate individual mean comparisons where appropriate. All analyses used an alpha level of 0.05 to determine statistical significance.

Three-chamber social interaction The protocol was based on a previous study (48) and is briefly outlined here. Habituation: A subject mouse freely explored entire apparatus for 10 min. Sociability: A subject mouse freely explored a stranger mouse (novel mouse) and empty pencil cup (novel object) for 10 min. Social Novelty: A subject mouse freely explored a novel mouse and familiar mouse for 10 min. Mice, excluding the subject mouse, were contained in inverted pencil cups in the apparatus (48,49). Time that the subject mouse spent within 1 cm of each stimulus was recorded by an opensource laboratory timer.

Supplementary Material Supplementary Material is available at HMG online.

Acknowledgements The authors would like to thank Drs. John D. Allison, Fiona A. Harrison, Jeremy Veenstra-VanderWeele and Gregg D. Stanwood for their suggestions for the behavioural procedures, and Dr. John P. Christianson for sharing his open-source

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laboratory timer. The authors would like to thank Dr. Martin J. Gallagher for sharing reagents and making critical comments for the EEG analysis. The authors would also like to thank Kelienne A. Verdier for mouse colony maintenance and genotyping. All the behavioural tests and EEG recordings were done in Vanderbilt University Medical Center (VUMC) Murine Neurobehaviour Core. Imaging data were performed in part through the use of the VUMC Cell Imaging Shared Resource. Conflict of Interest statement. None declared.

Funding This work was supported by research grants from Citizen United for Research in Epilepsy (CURE), Dravet syndrome foundation (DSF), IDEAleague (Dravet organization) and Vanderbilt Clinical and Translation Science Award (CTSA) NINDS R01 NS082635 to KJQ and NINDS NIH grant R01 NS51590 to RLM.

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