Dystroglycan synaptic localization and glycosylation in mouse models of dystroglycanopathy.

(A) Schematic depiction of Dystroglycan in different mouse models. The IIH6 antibody recognizes the matriglycan repeats on extracellular αDag1. Hippocampal CA1 of P30 mice immunostained for Dystroglycan glycosylation (IIH6, green) and nuclear marker Hoechst (blue) show puncta of glycosylated Dystroglycan localized to the perisomatic region of pyramidal cells and to blood vessels (magenta arrowheads); scale bar = 50μm. Lower panels show cell bodies in SP; scale bar = 25μm. CA1 layers: SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. (B) WGA-enriched lysates from P0 forebrain were immunoblotted for IIH6, β-Dag1, and β-tubulin. (C-D) Quantification of immunoblot in (B). Error bars show mean + SEM.

Dystroglycan is required for cortical neuron migration in a glycosylation-dependent manner and independent of intracellular interactions.

Immunostaining for cortical layer markers (A) Cux1 (layers 2-4) and (B) Tbr1 (layers 3 and 6) in P30 somatosensory cortex (scale bar = 200μm). Layer markers are shown in green. Nuclear marker Hoechst is shown in magenta. Quantification of fluorescence intensity of layer markers shown for (C) Cux1 and (D) Tbr1. Shaded regions of intensity profile illustrate ± SEM. See Supplementary Table 1 for Ns. Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, NS = p ≥ 0.05. Abbreviations: CC, corpus callosum; A.U., arbitrary units.

Dag1 is required for CCK+/CB1R+ basket IN perisomatic axon targeting in stratum pyramidale of hippocampal CA1-3.

(A) Nuclear marker Hoechst (upper panels) shows hippocampal morphology. Granule cell migration is disrupted in dentate gyrus of Emx1Cre;Dag1cKOs and Emx1Cre;POMT2cKOs(yellow arrows). CA1-3 gross morphology is normal in all models. CB1R immunostaining (lower panels) shows abnormal CCK+/CB1R+ basket interneuron targeting in CA1-3 to varying degrees across models (scale bar = 400μm). (B) Higher magnification view of CB1R immunostaining in CA1 (scale bar = 50μm). (C) Quantification of CA1 CB1R fluorescence intensity profile. Shaded regions of intensity profile illustrate ± SEM. Gray region highlights SP. See Supplementary Table 1 for Ns. Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, NS = p ≥ 0.05. Abbreviations: A.U., arbitrary units; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

Dag1 is required for CCK+/CB1R+ IN axon targeting during early postnatal development.

(A) Immunostaining for CB1R+ axon terminals (green) in the hippocampus of Emx1Cre;Dag1 controls (left) and cKOs (right) at ages P5-P30. Nuclear marker Hoechst is shown in magenta. White arrowheads indicate migration errors in dentate granule cells Emx1Cre;Dag1cKOmice. Scale bar = 500μm. (B) Higher magnification images of CB1R+ axon terminals in CA1 of Emx1Cre;Dag1 controls (left) and cKOs (right) at ages P5-P30. Scale bar = 100μm. (C) Quantification of CB1R fluorescence intensity profile in CA1. Shaded regions of intensity profile illustrate ± SEM. Gray region highlights SP. See Supplementary Table 1 for Ns. Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, NS = p ≥ 0.05. Abbreviations: A.U., arbitrary units; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

Dag1 and Pomt2 cKOs exhibit impaired CB1R+ basket synapse formation in stratum pyramidale of hippocampal CA1.

P30 coronal sections immunostained for (A) CB1R and (B) VGAT in hippocampal CA1; merged image in (C) shows CB1R in magenta and VGAT in green. (A-C) Scale bar = 50μm. (Higher magnification view of SP in (C’); scale bar = 10μm.) (D) Quantification of VGAT puncta density in SP expressed as a percent of control. (E) Quantification of CB1R puncta density in SP expressed as a percent of control. (F) Quantification of co-localization between VGAT and CB1R in SP to estimate putative CB1R+ basket cell synapse formation. Error bars show mean + SEM. (For quantification of puncta densities and co-localization in SO and SR see Fig. 5 Supp. 1.) See Supplementary Table 1 for Ns. Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, NS = p ≥ 0.05. Abbreviations: SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

Dag1 is required for CCK+/CB1R+ IN synapse function in hippocampal CA1 in a manner dependent on both glycosylation and intracellular interactions.

(A) Representative traces showing 15 seconds of sIPSC recordings at baseline (top) and after the addition of carbachol (bottom). (B) Quantification of average sIPSC frequency at baseline and after the addition of carbachol. (C) Quantification of the change in sIPSC frequency with the addition of carbachol. Error bars show mean + SEM. See Supplementary Table 1 for Ns. Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, NS = p ≥ 0.05. Abbreviations: sIPSC, spontaneous inhibitory postsynaptic current; CCh, carbachol.

Reduced seizure induction threshold in models of dystroglycanopathy.

(A)Quantification of latency (in seconds) to generalized tonic clonic seizure upon exposure to 10% flurothyl delivered at a constant rate. Open points denote statistical outliers. See Supplementary Table 1 for Ns. Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, NS = p ≥ 0.05.

Mouse strains

Breeding schemes

Primary antibodies used for immunohistochemistry

Emx1Credrives recombination in forebrain excitatory neurons and astrocytes, but not interneurons or microglia.

(A) Left; endogenous red fluorescence in the forebrain of P60 Emx1Cre;R26LSL-H2B-mCherryreporter mice (scale bar = 1mm). Right; coronal section of the forebrain from Emx1Cre;R26LSL-H2B-mCherry mice showing robust nuclear mCherry signal (magenta) in the cortex and hippocampus (scale bar = 500μm). (B-E) mCherry+ nuclei shown with markers of multiple cell types in the brain including astrocytes (GFAP, green) (B), microglia (Iba1, green) (C), and neurons (NeuN, green) (D) (scale bar = 100μm). Insets show enlarged images of mCherry+ nuclei with cell type markers (scale bar = 20μm). (E) mCherry+ nuclei with markers for different interneuron subtype markers (yellow arrowheads) (scale bar = 100μm) (VIP = Vasoactive intestinal peptide, NECAB = Neuronal calcium-binding protein 1, SOM = Somatostatin). CA1 layers: SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

Dystroglycan glycosylation is required for synaptic localization.

(A) Immunostaining for matriglycan (IIH6, green) and β-Dag1 (magenta) in CA1 pyramidal cells of WT controls, Emx1Cre;Dag1cKOs, Emx1Cre;Pomt2cKOs, and Dag1cyto/- mutants. Scale bars = 25μm. (B) WGA-enriched lysates from P21-P30 hippocampus were immunoblotted for IIH6 and β-Dag1. (C-D) Quantification of immunoblot in (B). Error bars show mean + SEM.

Cortical migration is disrupted at midline of B4gat1M155T/M155T but not FkrpP448L/P448L mutants.

(A) Immunostaining for cortical layer marker Cux1 (layers 2-4) in P30 somatosensory cortex (scale bar = 500μm). Cux1 is shown in green. Nuclear marker Hoechst is shown in magenta. Cortical migration is normal in both B4gat1 and Fkrp point mutants except at midline, where the B4gat1 mutants show a migration phenotype not seen in the Fkrp point mutants. This phenotype was observed with 100% penetrance in the B4gat1 mutants and 0% in the Fkrp mutants.

Laminin immunoreactivity in adult neocortex appears discontinuous in Dag1 mutants. (A-C)

Immunostaining for ECM protein Laminin (green). Nuclear marker Hoechst is shown in blue. Scale bar = 1000μm.

CCK+/CB1R+ IN axon targeting phenotypes in hippocampal CA1 of various Dag1cKOs.

CB1R immunostaining in hippocampal CA1 of control mice (left panels) and Dag1cKOs(right panels) generated using Emx1Cre (forebrain progenitors, E10.5) (A), NestinCre(neural stem cells in the central and peripheral nervous systems, E11.5) (B), and NEXCre (forebrain postmitotic excitatory neurons, E12.5) (C). Scale bar = 50μm. Abbreviations: SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

CCK+/CB1R+ IN cell numbers are unchanged in Emx1Cre;Dag1cKOs.

(A) Immunostaining in hippocampal CA1 for NECAB1 (left) and PV (right) in Emx1Cre;Dag1Ctrland Emx1Cre;Dag1cKO mice. NECAB1 labeling is evident in PV+ cells (example shown in blue inset) and in an additional PV- population that contains the CCK+/CB1R+ population (example shown in magenta inset). The average number of NECAB1+;PV- cells per 100μm of SP is quantified in (B). Immunostaining for NECAB2 (C) and PV (D) with cell number quantification in (E) and (F). Scale bar = 100μm. Error bars show mean + SEM. See Supplementary Table 1 for Ns. Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, NS = p ≥ 0.05. Abbreviations: SP, stratum pyramidale; PV, parvalbumin.

Parvalbumin+ basket INs do not require Dag1 for proper axon targeting in hippocampal CA1.

(A) Parvalbumin (PV) immunostaining in P30 hippocampal CA1 shows that PV+ basket interneurons exhibit normal distribution in CA1 for all genetic models except for an increase in PV intensity within stratum pyramidale of Emx1Cre;Dag1cKOs (scale bar = 50μm). (B) Quantification of CA1 PV fluorescence intensity profile. Shaded regions of intensity profile illustrate ± SEM. Gray region highlights SP. See Supplementary Table 1 for Ns. Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, NS = p ≥ 0.05. Abbreviations: A.U., arbitrary units; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

Altered CB1R expression in cortex and basolateral amygdala of Dag1 and POMT2 mutants.

(A) CB1R immunostaining in P30 coronal sections showing somatosensory cortex (Ctx) and CA1 of hippocampus (Hipp) (scale bar = 400μm). (B) CB1R immunostaining (green) shown with nuclear marker Hoechst (magenta) in basolateral amygdala (BLA) (scale bar = 250μm).

Extended quantification of images in Figure 5A-C.

Quantification within hippocampal CA1 SO and SR regions and all regions pooled together. (A) Quantification of VGAT puncta density expressed as a percent of control. (B) Quantification of CB1R puncta density expressed as a percent of control. (C) Quantification of co-localization between VGAT and CB1R to estimate putative CB1R+ basket cell synapse formation. Error bars show mean + SEM. (To see quantification of puncta densities and co-localization in SP see Fig. 5D-F.) See Supplementary Table 1 for Ns. Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, NS = p ≥ 0.05. Abbreviations: SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

Dag1 and Pomt2 cKOs exhibit increased PV+ basket synapse formation in stratum pyramidale of hippocampal CA1.

P30 coronal sections immunostained for (A) PV and (B) VGAT in hippocampal CA1; merged image in (C) shows PV in magenta and VGAT in green. Yellow arrows indicate PV+ interneuron cell bodies. (A-C) Scale bar = 50μm. (Higher magnification view of SP in (C’); scale bar = 10μm.) (D) Quantification of PV puncta density in SP expressed as a percent of control. (E) Quantification of co-localization between VGAT and PV in SP to estimate putative PV+ basket synapse formation. Error bars show mean + SEM. (To see quantification of puncta densities and co-localization in SO and SR see Fig. 5 Supp. 2.) See Supplementary Table 1 for Ns. Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, NS = p ≥ 0.05. Abbreviations: PV, parvalbumin; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

Extended quantification of images in Fig. 5 Supp. 2A-C.

Quantification within hippocampal CA1 SO and SR regions and all regions pooled together. (A) Quantification of PV puncta density expressed as a percent of control. (B) Quantification of co-localization between VGAT and PV to estimate putative PV+ basket cell synapse formation. Error bars show mean + SEM. (To see quantification of puncta densities and co-localization in SP see Fig. 5 Supp. 2D-E.) See Supplementary Table 1 for Ns. Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, NS = p ≥ 0.05. Abbreviations: PV, parvalbumin; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

Additional quantification of sIPSC recordings.

(A) Quantification of average sIPSC amplitude at baseline and after the addition of carbachol. Error bars show mean + SEM. (B) Quantification of the proportion of carbachol-sensitive cells within each genotype. Non-responsive cells are shown in lighter color, responsive cells are shown in darker color. See Supplementary Table 1 for Ns. Significance: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, NS = p ≥ 0.05. Abbreviations: sIPSC, spontaneous inhibitory postsynaptic current; CCh, carbachol.

Extended seizure induction threshold data.

(A) Proportion of mice that died as a direct result of a flurothyl-induced seizure. Survivors are depicted in darker colors, fatalities in lighter colors. (B) To minimize the number of animals needed for this experiment, certain genotypes were pooled together. Here we show that pooled genotypes are not different. Statistical tests were run where Ns were sufficient to provide meaningful comparison. (C) Seizure latencies split by sex. Note: only the Emx1Cre;Dag1 group is powered sufficiently to statistically compare sexes. Open points denote statistical outliers. Error bars show mean + SEM.