Abstract
Genetic epilepsy and neurodegenerative diseases are two common neurological disorders that are conventionally viewed as being unrelated. A subset of patients with severe genetic epilepsies who have impaired development and often go on to die of their disease respond poorly to anticonvulsant drug therapy, suggesting a need for new therapeutic targets. Previously, we reported that multiple GABAA receptor epilepsy mutations result in protein misfolding and abnormal receptor trafficking. We have now developed a model of a severe human genetic epileptic encephalopathy, the Gabrg2+/Q390X knock-in mouse. We found that, in addition to impairing inhibitory neurotransmission, mutant GABAA receptor γ2(Q390X) subunits accumulated and aggregated intracellularly, activated caspase 3 and caused widespread, age-dependent neurodegeneration. These findings suggest that the fundamental protein metabolism and cellular consequences of the epilepsy-associated mutant γ2(Q390X) ion channel subunit are not fundamentally different from those associated with neurodegeneration. Our results have far-reaching relevance for the identification of conserved pathological cascades and mechanism-based therapies that are shared between genetic epilepsies and neurodegenerative diseases.
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Acknowledgements
The authors would like to thank the Vanderbilt Translational Pathology core for tissue preparation and immunostaining, and K. Boyd for consultation on immunohistochemistry. We thank K.M. Verdier and A. Pimenta for animal husbandry, Z. Liu and J. Allison for assistance in neurobehavioral tests, and S. Zeng for computational assistance, as well as S. Joshi and J. Kapur (University of Virginia School of Medicine) for providing mouse monoclonal γ2 subunit antibody, L. Wang (Vanderbilt University) for providing consultation on statistics, and T. Abel (Vanderbilt University) for consultation on stereology and cell counting. This work was supported by grants from Citizen United for Research in Epilepsy, the Dravet Syndrome Foundation and Dravet.org (which was previously named IDEAleague), a Vanderbilt Clinical and Translation Science Award to J.-Q.K., grants from the National Institute of Neurological Disorders and Stroke (R01 NS082635 to J.-Q.K., R01 NS51590 to R.L.M. and R01 GM100701 to D.X.), and a grant from the National Institute of Child Health and Human Development P30HD15052) to Vanderbilt Kennedy Center.
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J.-Q.K. and R.L.M. conceived the project. J.-Q.K. designed and performed experiments, analyzed the data, coordinated the study, supervised the project, and wrote the manuscript. R.L.M. supervised the animal breeding to develop the congenic wild-type and mutant strains. W.S. collected the biochemical data and helped with colony maintenance, genotyping and tissue preparation. C.Z. collected and analyzed the electrophysiology data. D.X. performed the protein modeling and wrote the protein modeling part of the paper. R.L.M. supervised the project, critically reviewed the data and edited the manuscript. All of the authors discussed the results and commented on the manuscript.
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Integrated supplementary information
Supplementary Figure 1 A docking model suggests that truncated γ2(Q390X) subunits are prone to aggregate and form dimers with themselves or other partnering subunits that are potentially stable.
a-b Cartoons illustrate α1β2γ2 pentamers (a) and wild-type γ2 and mutant γ2(Q390X) subunits (b). c, Top 5 docking models for potential mutant γ2 subunit homodimers predicted by SymmDock are shown. In each panel, the two γ2 subunit chains are shown in red and green. d. Top 5 docking models of potential complexes between the mutant γ2 subunit (shown in green) and its wild-type partnering α1 subunit (shown in red) predicted by PatchDock are shown.
Supplementary Figure 2 Mutant γ2(Q390X) subunits accumulated in cortex and cerebellum in P0 Gabrg2+/Q390X KI mice.
Brains from P0 wild-type and KI mouse littermates (C57BL/6J strain) were paraffin embedded, sectioned at 5 µm and stained with rabbit anti-γ2 subunit antibody and visualized with DAB. The sections were counterstained with hematoxylin, and the equivalent areas in the cortices of both wt and heterozygous (het) KI mice were imaged under light microscopy. Large red boxes indicated the posterior lobe of the cerebellum (bottom).
Supplementary Figure 3 EEGs were obtained from wild-type and Gabrg2+/Q390X KI littermates in the C57BL/6J background.
Representative EEG recordings from a two month old wild-type mouse in the C57BL/6J background demonstrated no epileptiform activity (1) while its mutant littermate had ictal discharges (3). The boxed areas in 1 and 3 were expanded and illustrated in 2 and 4.
Supplementary Figure 4 Full-length gels.
Full-length gels for images presented in Figures 5a-d, 6d-e, 8e and supplementary Figure 10. The boxed region in each gel is the image presented in each corresponding figure. The colored image was shown for those gels with double colored protein molecular weight standards.
Supplementary Figure 5 Representative images showing that mutant γ2(Q390X) subunits formed caspase 3 positive aggregates in old, but not young, mice.
a, Images from cortices of 1 year old wild-type (wt) and heterozygous (het) KI littermates. The brains were paraffin embedded and stained with mouse anti-γ2 subunit antibody (red), rabbit cleaved caspase 3 (green) and cellular nucleus marker To-pro-3 (blue). b, The cortices of 1 year old wt and 2 month, 5 month and 1 year old het KI littermates were stained with rabbit cleaved caspase 3.
Supplementary Figure 6 Representative images showing that there was neuronal loss in cortical layers V and VI in the older Gabrg2+/Q390X KI mice.
a, b, Images from cortices of 18 month old wild-type (wt) and heterozygous (het) KI mouse littermates (C57BL/6J as well as in C57/BL/6J/S129/SvJ background). The brain sections were from paraffin embedded or short fixed tissues and stained with mouse anti-NeuN and rabbit anti-Er81 antibodies, the cortical layer V marker (Er81) (a) or rabbit anti Trb1, a layer VI marker, (b). To-pro-3 (blue) was used to stain cellular nuclei in both a and b. c, The average numbers of cells costained with NeuN and Er81 or with NeuN and Trb1 were reduced in the het mice. (Unpaired t test, p = 0.2071 for Er81+NeuN, p = 0.0318 for Tbr1+NeuN, n = 5 pair of mice from 14-18 months old) (* p < 0.05; vs wt).
Supplementary Figure 7 Representative images showing that there was neuronal loss in the somatosensory cortex in the older Gabrg2+/Q390X KI mice.
a, Images are presented showing a coronal brain section from a 16 month old heterozygous (het) KI mouse stained with anti-γ2 subunit (green), anti-NeuN (red) and To-pro-3 (blue) and the brain regions we surveyed for neuronal loss. (b). Enlarged image of cortex from a to demonstrate the cortical layers. c, d, The brain sections were from paraffin embedded tissues and stained with mouse anti-NeuN (c) or anti-NeuN and cellular nucleus marker To-pro-3 (blue) (d). e, The average number of cells stained with NeuN were reduced in the cortex in the het mice. (Unpaired t test, p = 0.03 for cortex I-II/III, p = 0.0012 for cortex IV-VI, p = 0.0226 for thal). f, The average number of cells stained with To-pro-3 or NeuN were unchanged in the dentate gyrus (DG), CA1 and CA3 regions in the het mice. To-pro-3 positive cells were quantified in DG because the cells were packed, and it was difficult to distinguish cell boundaries with the NeuN staining used in hippocampus (unpaired t test, p = 0.3590 for dg, p = 0.3924 for CA1, p = 0.7008 for CA3). g, for unbiased tissue imaging and cell counting, 6-8 consecutive brain sections from each mouse were prepared, the images were acquired automatically by computer and the core personnel who was blind to the mouse genotype. The images were scanned at original magnification of 20X, the images were viewed at 7X and the total cell numbers were measured per 5X105 um2 in the somatosensory cortex S1-2 region. For each mouse, the 6-8 consecutive sections were averaged and taken as n = 1 of 5 pairs. (Unpaired t test, p = 0.0074). (* p< 0.05; ** p< 0.01 vs wt, n = 5 pair of mice from 14-18 months old.).
Supplementary Figure 8 Representative images showing Purkinje neurons in the cerebellum in the older Gabrg2+/Q390X KI mice.
a, Image showing the sagittal section of cerebellum in the 16 month old heterozygous (het) KI mouse stained with anti-γ2 subunit (green), anti-NeuN (red) and To-pro-3 (blue). The white boxed region represented the area we surveyed for Purkinje cell loss, which was between cerebellar lobule VII-VIII. b, Enlarged image of the surveyed cerebellum region in c and d stained with anti-calbindin D-28K (green) and anti-NeuN (red) antibodies and with To-pro-3 (blue). We costained the cerebellar tissue with both anti-NeuN and anti-Calbindin D-28K antibody because we found that NeuN did not label Purkinje neurons well. c, Cerebellar sections from wild-type and het mice stained with mouse anti-NeuN (red) and anti-Calbindin D-28K. (d).The lobule VII in either wt or het cerebellar cortex was surveyed under 63X objective. The cell counts of the 6-8 consecutive sections were averaged and taken as one of 5 pairs of mice. Average numbers of cells stained with Calbindin D-28K were quantified (unpaired t test, p = 0.0515, n = 5 pair of mice from 14-18 months old.).
Supplementary Figure 9 Representative image showing that short fixed wild-type mouse brain tissues had good preservation of GABAA receptor γ2 subunit protein.
The image above shows the immunostaining of γ2 subunits (green) in the hippocampal CA1 region of a three month-old wild-type mouse with the short fix (30 min in 4% PFA) method. The blue staining in the image above is the nucleus marker, Topro-3. The γ2 subunit distribution pattern was similar to that of γ2YFP subunits in live cultured hippocampal neurons in the paper Figure 1f-g and Figure 6a-b.
Supplementary Figure 10 The old Gabrg2+/Q390X KI mice had increased caspase 3 expression.
The lysates from cytosolic fractions of forebrain tissues of wild-type (wt) and heterozygous (het) KI littermates were fractionated by SDS-PAGE and immunoblotted with rabbit polyclonal anti-active caspase 3. LC stands for NA+/K+ATPase internal loading control. Uncropped western blot for the image is shown in Supplementary Fig. 4.
Supplementary Figure 11 Representative images showing that no difference in caspase 3 expression in young wild-type and Gabrg2+/Q390X KI mice.
a, The brain sections from P0 old mice were stained with anti-caspase 3 (active form) antibody, and the nuclei were counterstained with hematoxylin. b, The caspase 3 positive cells in different brain regions were quantified. There was more positive caspase 3 staining in P0 old pups than in adult mice probably due to active neuronal pruning and apoptosis during early brain development (two way ANOVA and Bonferroni posttests, P>0.05 for all brain regions. Error bars are s.e.m. See Supplementary Mehods Checklist for full details of statistical tests.).
Supplementary Figure 12 Representative images showing that increased caspase 3 activation in the old mutant Gabrg2+/Q390X KI mice was likely due to mutant subunit accumulation.
a, Cortical brain sections from 1 year old mice were stained with anti-γ2 subunit (right panel) or anti-caspase 3 (left panel) antibodies, and nuclei were counterstained with hematoxylin. b, The caspase 3 positive cells in the cortex from mice at different ages were quantified. There were caspase 3 positive cells in the older KI mice but not in the young KI mice compared to the wild-type mice. There was more positive caspase 3 staining in P0 pups than in adult mice probably due to active neuronal pruning and apoptosis during early brain development (two way ANOVA and Bonferroni posttests, p>0.05 for p0, P>0.05 for 2-4 months, P>0.05 for 5-6 months, P<0.001 for ∼12 months. Error bars are s.e.m. See Supplementary Mehods Checklist for full details of statistical tests.).
Supplementary Figure 13 The GABRG2(Q390X) mutation produced more mutant subunit protein aggregates than the GABRG2(Q40X) mutation.
Total lysates from HEK 293T cells untransfected (con) or transfected with wild-type γ2 and two mutant γ2 subunits (Q40X and Q390X) alone associated with Dravet syndrome (DS) were analyzed by SDS-PAGE. The membrane was immunoblotted with rabbit polyclonal anti γ2 subunit antibody. LC stands for Na+/K+ ATPase loading control. There was much more mutant γ2(Q390X) subunit total protein than wild-type γ2 or mutant γ2(Q40X) subunit total protein in HEK 293T cells expressing the equal amounts of wild-type or mutant cDNAs.
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Supplementary Video 1
Gabrg2+/Q390X KI mice had generalized tonic clonic seizures and sudden death after seizures. A 19 day old KI mouse had spontaneous generalized seizures during handling (tailing). The mouse was found dead the next day, reminiscent of SUDEP in epilepsy patients. (MPG 16295 kb)
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Kang, JQ., Shen, W., Zhou, C. et al. The human epilepsy mutation GABRG2(Q390X) causes chronic subunit accumulation and neurodegeneration. Nat Neurosci 18, 988–996 (2015). https://doi.org/10.1038/nn.4024
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DOI: https://doi.org/10.1038/nn.4024
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