Journal: Frontiers in Genetics

Article Title: Identification of a Functional Non-coding Variant in the GABA A Receptor α2 Subunit of the C57BL/6J Mouse Reference Genome: Major Implications for Neuroscience Research

doi: 10.3389/fgene.2019.00188

Figure Lengend Snippet: Strain variation in Gabra2 expression. (A) Low expression of Gabra2 in B6J brain relative to other inbred strains and substrains. B6J demonstrated the lowest expression of Gabra2 in hippocampus (data set GN110, probe set 1421738_at) compared to 15 inbred strains, including the D2 strain, and a closely related B6 substrain (C57BL/6ByJ). (B) Gabra2 levels measured by RNAseq are reduced in 10 mesocorticolimbic regions relative to D2, indicating a global reduction in B6J. BLA = basolateral amygdala, BST = bed nucleus stria terminalis, DLS = dorsolateral striatum, DMS = dorsomedial striatum, ILC = infralimbic cortex, NAc = nucleus accumbens core, NAs = nucleus accumbens shell, PrL = prelimbic cortex, VCX = visual cortex, VTA = ventral tegmented area. All P s < 0.001 for within region contrasts between B6J and D2. (C) Approximate time line seperating C57BL/6 substrains. (D) Average Gabra2 expression level (measured by qPCR) in the cortex, hippocampus, and striatum of D2, C57BL/6 substrains, and congenic strains relative to B6J shown at left. The B6N lineage (seperated in 1951 at generation F32 from B6J) includes C57BL/6JByJ (B6ByJ), C57BL/6NCrl (B6NCrl), and C57BL/6NJ (B6NJ) and all showed higher expression of Gabra2 relative to B6J. Even the C57BL/6EiJ (B6EiJ) substrain that diverged most recently from C57BL/6J in 1976 showed higher brain expression of Gabra2 . The only substrain with low Gabra2 expression similar to C57BL/6J is the B6(Cg)- Tyr c-2J /J (B6C2J) congenic strain. This albino mutation was detected in 1970 but the original strain harboring the spontaneous mutation was backcrossed to B6J creating a congenic line. All statistical contrasts performed relative to B6J. (E) Derivation of BXD strain cohorts. Each cohort was derived by separate crossing and inbreeding of female B6J (black) and male D2 (gray) parental inbred strains. The first cohort of BXD strains (1 through 32) was derived in the late 1970s. Another set of strains (33 through 42) was produced in the early 1990s. The last cohort of BXD strains (43 to 100) was created in the early 2000 s. (F) Hippocampal differences in Gabra2 expression between earlier and later derived BXD strains. There was little difference in the expression of Gabra2 between BXD strains with the B6 or D2 allele in the first cohort. However, inheritance of the B6 allele in the later two cohorts was associated with a dramatic reduction in Gabra2 expression. This suggests the appearance of a mutant allele in the B6J line that occurred after the late 1970’s and prior to 1990. Data from GeneNetwork data set GN110, probe set 1421738_at. (G) Protein levels of GABRA2 were reduced in B6J relative to D2 using an unbiased shotgun proteomics approach. Relative expression of GABRA2 protein shown as normalized relative intensity for GABRA2 in B6J and D2 hippocampus. Eight different peptide sequences that matched GABRA2 were used to generate normalized relative intensity counts for each sample. Each sample is 3 pooled animals (males only). (H) Hippocampal protein levels of GABRA2 are reduced in B6J ( n = 3) compared to the closely related B6N ( n = 3) substrain and D2 ( n = 2) strain (Western blot analysis). The experiment was repeated twice and both B6J sample 1 and B6N sample 1 were run each time. Tissue from Gabra2 KO mice and their wild type control strain (WT) were kindly provided by Y. Blednov at The University of Texas. These mice were originally maintained on a mixed B6J and 129/SvEv background but were backcrossed twice to B6J upon arrival at The University of Texas, thus the WT control strain has low GABRA2 protein expression similar to B6J. ANOVA for effect of strain is significant [ F (2,5) = 103.9, p < 0.001]. Post hoc tests show B6J is different from both B6N and D2 ( p < 0.001) but B6N and D2 are not significantly different from each other ( p = 0.15). Significance defined as: # p < 0.1, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: For chemiluminescence western blots, blots were incubated in primary antibody for GABRA2 (1:500 rabbit polyclonal, PhosphoSolutions, Cat #822-GA2CL) overnight at 4 ° C. Blots were washed three times (5 min each) with blocking buffer and incubated with horseradish-peroxidase conjugated antibody for 1 h at room temperature.

Techniques: Expressing, Mutagenesis, Derivative Assay, Produced, Western Blot, Control

Journal: Frontiers in Genetics

Article Title: Identification of a Functional Non-coding Variant in the GABA A Receptor α2 Subunit of the C57BL/6J Mouse Reference Genome: Major Implications for Neuroscience Research

doi: 10.3389/fgene.2019.00188

Figure Lengend Snippet: Identification of a B6J private variant in Gabra2 . (A) Position of variants near the Gabra2 gene locus. An intergenic SNP (yellow arrowhead) and a single nucleotide indel (red arrowhead) in the third intron were the only confirmed variants among B6J and other inbred mouse strains. Genotyping of strains with high (red) or low (blue) expression of Gabra2 revealed that the likely causal variant is the intronic indel (single base pair deletion in B6J). (B) Evidence for disruption of mRNA processing is shown as an accumulation of intronic reads in the affected intron three (designated with an asterisk) and downstream introns (four and five) in B6J (blue) compared to D2 (red). Normalized and binned read pileup of poly-A enriched striatal RNA-seq data from 10 to 11 B6 and D2 individuals, respectively, generated by Bottomly and colleagues and hosted for viewing and analysis at the GeneNetwork mirror of the UTHSC Genome Browser ( http://ucscbrowser.genenetwork.org/ ). Gabra2 gene model is shown above (5′ to 3′ orientation with dashed line representing introns and solid vertical bars representing exons). (C) Average hippocampal expression of Gabra2 exons and introns based on inheritance of B6J (B) and D2 (D) alleles in the BXD population. All coding exons and UTRs showed significantly higher expression associated with inheritance of the D allele. In contrast, introns 4, 5, and 6 showed significantly higher expression associated with inheritance of the B allele. The variant is located in intron 3. Introns 3, 6 (distal to the fifth coding exon), 7, and 8 are not significantly different based on expression of parental alleles. There was no evidence of a splicing defect resulting in loss of exons in B6J. GeneNetwork data set GN206, UMUTAffy Hippocampus Exon (Feb09) RMA was used for the analysis and includes 45 BXD strains from BXD33 and above. 15 strains inherited the B allele and 30 inherited the D allele at the Gabra2 locus. Significance was determined by a pair-wise comparison of B and D allele expression for each intron or exon using Student’s t -test. Significance defined as: # p < 0.1, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: For chemiluminescence western blots, blots were incubated in primary antibody for GABRA2 (1:500 rabbit polyclonal, PhosphoSolutions, Cat #822-GA2CL) overnight at 4 ° C. Blots were washed three times (5 min each) with blocking buffer and incubated with horseradish-peroxidase conjugated antibody for 1 h at room temperature.

Techniques: Variant Assay, Expressing, Disruption, RNA Sequencing, Generated, Comparison

Journal: Frontiers in Genetics

Article Title: Identification of a Functional Non-coding Variant in the GABA A Receptor α2 Subunit of the C57BL/6J Mouse Reference Genome: Major Implications for Neuroscience Research

doi: 10.3389/fgene.2019.00188

Figure Lengend Snippet: Repair of the private deletion in B6J is sufficient to restore GABRA2 protein and mRNA levels. (A) Site of repair of the private deletion in B6J (green), guide RNA (red) and PAM site (blue) are shown. The exon is highlighted in yellow. (B) Protein levels of GABRA2 are also restored in the hippocampus (top panel) and cortex (bottom panel) of the original Gabra2 KI/KI founder mouse ( n = 1) relative to B6J ( n = 3) and are similar to that of B6NJ ( n = 3). Western blot shown to the left and adjusted density bar graph shown to the right for each region. (C) Hippocampal and cortical Gabra2 mRNA levels were restored in the original Gabra2 KI/KI founder after insertion of a single nucleotide relative to B6J. Expression of Gabra2 in the founder mouse was similar to that of other B6 substrains. Expression was measured via qPCR and shown relative to B6J. Males and females were combined for the statistical analysis ( n = 5 to 6 per strain). Note that CRISPR founders can often be mosaic for the introduced mutation such that some cells may have the mutation, some may not, and other cells may have different mutations. We include molecular validation in the founder in order to demonstrate that our engineering approach worked, and that mosaicism is not evident in brain tissue of the Gabra2 KI/KI founder mouse. Tissue from Gabra2 knockout mice (KO, predominately B6J background mixed with 129/SvEv, see section Materials and Methods) was kindly provided by Y. Blednov from The University of Texas and used as a negative control. (D) Hippocampal, striatal, and cortical Gabra2 mRNA levels were also restored in heterozygous Gabra2 KI/- and homozygous knockin Gabra2 KI/KI mice after insertion of a single nucleotide relative to homozygous Gabra2 B6J/B6J mice. Expression measured via qPCR and data were analyzed at the ΔCt level (relative to the expression of the control gene Cyclin D). There is a significant main effect of genotype [ F (2,32) = 47.1, p = 2.9e-10] and brain region [ F (2,32) = 27.89, p = 9.7e-08]. The interaction was not significant. Pairwise significance between genotypes and within brain region were determined using Tukey’s HSD test. Significance defined as: # p < 0.1, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: For chemiluminescence western blots, blots were incubated in primary antibody for GABRA2 (1:500 rabbit polyclonal, PhosphoSolutions, Cat #822-GA2CL) overnight at 4 ° C. Blots were washed three times (5 min each) with blocking buffer and incubated with horseradish-peroxidase conjugated antibody for 1 h at room temperature.

Techniques: Western Blot, Expressing, CRISPR, Mutagenesis, Biomarker Discovery, Knock-Out, Negative Control, Knock-In, Control

Journal: Frontiers in Genetics

Article Title: Identification of a Functional Non-coding Variant in the GABA A Receptor α2 Subunit of the C57BL/6J Mouse Reference Genome: Major Implications for Neuroscience Research

doi: 10.3389/fgene.2019.00188

Figure Lengend Snippet: Expression of GABA-A receptor subunit mRNA. Expression generated using the Affymetrix Clariom D Assay (microarray platform). Only subunits with significant or suggestive ( p < 0.1) differential expression between B6J and KI Gabra2 genotypes are shown. Significance defined as: ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Fold change is indicated by color intensity with yellow representing increased expression in Gabra2 B6J/B6J (B6J allele) mice relative to Gabra2 KI/KI (KI allele) mice. In contrast, blue represents decreased expression in B6J allele mice relative to KI allele mice. Alterations in the mRNA levels of several alpha subunits, including the major Gabra1 subunit, are observed in the cortex and striatum of B6J allele mice which harbor a naturally occuring intronic deletion resulting in decreased Gabra2 expression. The only alpha subunit with significant and higher levels of mRNA expression (fold change >1.3) in B6J allele mice is Gabra4 (striatum). Beta and gamma subunit mRNAs are also altered at a suggestive level in cortex and hippocampus. In the hippocampus, γ1 transcript expression is significantly reduced in mice with the B6J allele. In the striatum, β1-2 and γ1-γ2 transcripts are also significantly reduced in mice with the B6J allele of Gabra2 . In contrast, striatal levels of delta subunit mRNA is increased in mice with the B6J allele relative to those with the KI allele. These results suggest brain region specific alterations in the abundance of certain classes of GABA-A receptors associated with inheritance of the Gabra2 B6J allele. For example, in the cortex, α1β2-containing receptors may be increased while α2γ1-containing subunits may be decreased. In the hippocampus, α2 and α3-containing receptors including β1 or γ1-γ2 subunits may be decreased. In the striatum there is evidence that there may be a general decrease in receptors containing α1, α2, α3 or α5; and β1 or 2; and γ1 or γ2. In contrast, there may be an increase in receptors containing α4 and δ subunits in striatum.

Article Snippet: For chemiluminescence western blots, blots were incubated in primary antibody for GABRA2 (1:500 rabbit polyclonal, PhosphoSolutions, Cat #822-GA2CL) overnight at 4 ° C. Blots were washed three times (5 min each) with blocking buffer and incubated with horseradish-peroxidase conjugated antibody for 1 h at room temperature.

Techniques: Expressing, Generated, Microarray, Quantitative Proteomics

Journal: Pharmacological research

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants.

doi: 10.1016/j.phrs.2024.107356

Figure Lengend Snippet: Fig. 2. Whole-cell patch-clamp recording of selected eight GABAA receptor α1 subunit DAVs. a HEK293T cells were transiently transfected with α1 (wild type or the indicated variants), β2, and γ2 cDNAs of GABAA receptors. Forty-eight hours post transfection, whole-cell currents were recorded using the IonFlux Mercury 16 ensemble plates at a holding voltage of −60 mV. Representative whole-cell voltage-clamp recording traces are shown. Application of GABA (100 μM, 3 s) is indicated by the horizontal bar above the current traces. Quantification of the peak currents (Imax) is shown on the right (n = 4–10). nA: nano Ampere. b Dose-response curves of GABA are shown for the calculation of EC50 values for wild type and the indicated α1 DAVs (n = 3–4). Each data point is reported as mean ± SEM. One-way ANOVA followed by post-hoc Tukey test was used for statistical analysis. Statistical significance was labelled for the comparison to the WT group. ***, p < 0.001.

Article Snippet: The pCMV6 plasmids containing human GABAA receptor α1 (Uniprot no. P14867–1) (catalog #: RC205390), β2 (isoform 2, Uniprot no. P47870–1) (catalog #: RC216424), γ2 (isoform 2, Uniprot no. P18507–2) (catalog #: RC209260), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene.

Techniques: Patch Clamp, Transfection, Comparison

Journal: Pharmacological research

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants.

doi: 10.1016/j.phrs.2024.107356

Figure Lengend Snippet: Fig. 4. Hispidulin and TP003 promote the functional surface expression of α1 DAVs. a Effect of Hispidulin (10 µM, 24 h) or TP003 (5 µM, 24 h) on the surface protein expression of the α1 variants in HEK293T cells expressing α1(A322D)β2γ2, α1(D219N)β2γ2, α1(P260L)β2γ2, α1(R214C)β2γ2, α1(S76R)β2γ2, α1(G251D)β2γ2, α1 (M263T)β2γ2, or α1(T289P)β2γ2 GABAA receptors according to surface biotinylation analysis. Na+/K+ ATPase serves as a plasma membrane protein loading control. Quantification of the surface α1 band intensities was shown on the bottom panels (n = 3). b Effect of Hispidulin (10 µM, 24 h) or TP003 (5 µM, 24 h) on GABA- induced peak current amplitudes in HEK293T cells expressing α1(A322D)β2γ2, α1(D219N)β2γ2, α1(P260L)β2γ2, α1(R214C)β2γ2, α1(S76R)β2γ2, α1(G251D)β2γ2, α1(M263T)β2γ2, or α1(T289P)β2γ2 GABAA receptors. Representative whole-cell voltage-clamp recording traces are shown. The holding voltage was set at −60 mV. Application of GABA (100 μM, 3 s) is indicated by the horizontal bar above the current traces. Quantification of the peak currents (Imax) are shown on the bottom panels (n = 4–11). pA: pico Ampere. c Comparison of the peak current amplitudes of compound (DMSO, Hispidulin, or TP003)-treated α1 variants with those in wild type (WT) receptors. Data was taken from Fig. 2a and Fig. 4b. Each data point is reported as mean ± SEM. One-way ANOVA followed by post-hoc Tukey test was used for statistical analysis. Statistical significance was labelled for the comparison to the DMSO group ((a, b) or WT group (a, b) or WT group (c). NS, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. Also see Supplementary Fig. S5.

Article Snippet: The pCMV6 plasmids containing human GABAA receptor α1 (Uniprot no. P14867–1) (catalog #: RC205390), β2 (isoform 2, Uniprot no. P47870–1) (catalog #: RC216424), γ2 (isoform 2, Uniprot no. P18507–2) (catalog #: RC209260), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene.

Techniques: Functional Assay, Expressing, Clinical Proteomics, Membrane, Control, Comparison

Journal: Pharmacological research

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants.

doi: 10.1016/j.phrs.2024.107356

Figure Lengend Snippet: Fig. 8. Proposed mechanism of action of Hispidulin and TP003 on rescuing α1 DAV trafficking. Hispidulin and TP003 act as pharmacological chaperones to bind GABAA receptor variants to stabilize them in the ER. Pharmacological chaperone treatment enhances the assembly of the variants and their forward trafficking to the Golgi and plasma membrane. The effect of Hispidulin and TP003 is also reflected from the enhanced folding and reduced degradation of the variants. Consistently, the interactions between the variants and pro-folding chaperones (BiP and calnexin) and a trafficking factor (LMAN1) are enhanced, whereas the interactions be tween the variants and ERAD factors (Grp94 and VCP) are reduced by pharmacological chaperone treatment.

Article Snippet: The pCMV6 plasmids containing human GABAA receptor α1 (Uniprot no. P14867–1) (catalog #: RC205390), β2 (isoform 2, Uniprot no. P47870–1) (catalog #: RC216424), γ2 (isoform 2, Uniprot no. P18507–2) (catalog #: RC209260), and pCMV6 Entry Vector plasmid (pCMV6-EV) (catalog #: PS100001) were obtained from Origene.

Techniques: Clinical Proteomics, Membrane

Journal: Frontiers in Pharmacology

Article Title: GABA Type A receptors expressed in triple negative breast cancer cells mediate chloride ion flux

doi: 10.3389/fphar.2024.1449256

Figure Lengend Snippet: Relative GABA A R-mediated Cl − flux in TNBC cells decreases after GABA A β 3 subunit knockdown. (A) Relative Cl − in HCC 1806 cells that have undergone GABA A β 3 subunit knockdown (scramble and KD constructs 2,3) exposed to 100 μM, 300 μM, and 1,000 μM GABA, n = 3. (B) Relative Cl − in HCC 1806 cells that have undergone GABA A β 3 subunit knockdown (scramble and KD constructs 2,3) exposed to 100 μM, 300 μM, and 1,000 μM GABA, n = 3. * represent significance compared to the scramble control within concentration of GABA, respectively. Data are presented as mean±SE, * p < 0.05, ** p < 0.01, **** p < 0.0001 (ANOVA).

Article Snippet: Cells were infected with transduction-ready scramble control shRNA lentiviral particles (#TR30021V, Origene, MD) or human GABA A R shRNA lentiviral particles (constructs # 2 and 3 targeting the GABA A β 3 gene, #TL304428V, Origene) for 24 h at multiplicity of infection (MOI) of 5.

Techniques: Knockdown, Construct, Control, Concentration Assay