Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) SLC35A2 encodes a transporter for UDP-galactose, which is required for the synthesis of O-GalNAc glycans. B) The lectin VVA binds terminal alpha-GalNAc (Tn-antigen), while PNA binds terminal Gal of Core 1 O-GalNAc glycans (T-antigen), which can be removed by O-glycosidase (OGA). C) Breeding strategy of Emx1-Cre mice with floxed- Slc35a2 mice, with blue regions highlighting the forebrain expression Emx1 . D) VVA binding is minimal in protein blots of wild-type mouse cortex; female (+/+) and male (+/y) with floxed- Slc35a2 . In contrast, mice lacking Slc35a2 in cells expressing the forebrain-specific promoter Emx1 ( Emx1-Cre ) display robust VVA binding in both females (+/-) and males (-/y), consistent with inhibition of O-GalNAc extension. Binding of PNA after Neuraminidase A (NeuA) treatment was significantly reduced in both +/- and -/y lines, and specificity of PNA binding was confirmed via OGA treatment of a +/+ sample. ConA, which binds all N-glycans, was unaffected in all mouse lines, and specificity was confirmed using peptide N-glycosidase F (PNGase F) treatment of a +/+ sample. Each lane contains 15 µg cortical protein lysate from an individual mouse of the corresponding genotype. Experiment was replicated three times with similar results. E) Quantification of lectin binding from D normalized to total protein (TP) staining within the same lane of each blot. Data presented as the mean binding intensity +/- SEM, with data points for each individual mouse included, N = 3/genotype. One-way ANOVA confirmed significant group differences ( p < 0.05) for VVA and PNA but not ConA, followed by post hoc Student’s t-tests to confirm genotype effects in each sex: ** p -value = 0.01, **** p -value = 0.0001. F) Schematic of coronal mouse brain section highlighting the cortex (CTX), corpus callosum (CC), and basal ganglia (BG). G) VVA binding is minimal across all regions in wildtype mouse cortex (+/y), while PNA binding localizes to neuronal tracts of the CC and axon bundles in the BG. In contrast, mice lacking forebrain expression of Slc35a2 (-/y) display a build-up of truncated O-GalNAc glycans in cortex that fail to properly localize. Mature O-GalNAc glycans are entirely absent from the CTX and CC, but preserved in the BG, which does not express Emx1 -Cre. H) Relative quantification of lectin binding across brain regions (CTX, CC, BG) from G. Data presented as the mean binding intensity +/- SEM, with individual points representing the average of 6 measures across independent mice. Colored bars correspond with the specific brain region indicated in (F): CTX, purple; CC, orange; BG, blue. N = 2 for +/y VVA; N = 4 for +/- PNA, -/y VVA, and -/y PNA. Fluorescence was quantified as arbitrary intensity (a.i.) after subtracting background signal from each sample.

Article Snippet: In brief, floxed homozygous Slc35a2 female mice were crossed with Emx1 -Cre and Olig2-Cre male mice obtained from Jackson Laboratory (#005628, #025567).

Techniques: Expressing, Binding Assay, Inhibition, Staining, Quantitative Proteomics, Fluorescence

Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) Fluorescent images of +/y and -/y primary neuron cultures at 11 days in vitro (DIV11) show robust VVA binding in Slc35a2 -deleted cultures only. Scale bar = 75 μm. B) Quantification of raw integrated density values of VVA fluorescence (VVA Int.). ROIs were selected via automated module. N = 5 independent cultures/genotype. C) High magnification of the ROI from A (white box), highlighting that while binding is high in many MAP2+ neurons, some appear VVA negative. C) Representative raster plots from 2 minutes of high-density multi-electrode array (HD-MEA) recordings of DIV11 +/y and -/y primary cultures, with active units representing cells firing ≥0.1 spikes/second. Each point represents an individual detected action potential, and colored vertical lines indicate a time when > 20% of active units were recruited during a network bursting event. E) Quantification of spontaneous network activity measured by HD-MEA shows that while +/y cultures show clear periods of uniform network activity that recruit many active units, -/y cultures are hyperactive and demonstrate aberrant network activity. N = 4 and 5 independent cultures for +/y and -/y cultures, respectively. F) Colocalization of the axon initial segment (AIS) marker Ankyrin G (AnkG) with PNA shows a reduction of properly localized O-GalNAc glycans to the AIS (white arrow, AnkG+/MAP2-). Scale bar = 10μm. G) Quantification of the mean PNA value along the AIS from F. N = 3 independent cultures for +/y and -/y cultures, respectively, where 31-32 AISs were measured per condition and average PNA profiles at the AIS were log transformed to fit the normality assumption of the statistical test. Data are shown as mean ± SEM. p value = *<0.05, **<0.01, ***<0.001, ****<0.0001 via unpaired two-tailed t-test.

Article Snippet: In brief, floxed homozygous Slc35a2 female mice were crossed with Emx1 -Cre and Olig2-Cre male mice obtained from Jackson Laboratory (#005628, #025567).

Techniques: In Vitro, Binding Assay, Fluorescence, Activity Assay, Marker, Transformation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) Schematic depicting affinity purification and elution of VVA-bound glycoproteins in mouse cortex, including controls for elution (+ GlcNAc) and binding (no lectin), analyzed by LC-MS/MS. B) The total number of peptides with spectral count > 0 detected in each sample. Biological triplicates for +/y and -/y were included, as well as a single +/- sample, all eluted with 200 mM GalNAc. Controls for lectin specificity and non-specific binding of a -/y sample were performed in parallel, specifically elution with 200 mM GlcNAc and sample with no VVA lectin. C) Among the detected 443 distinct glycopeptides detected, HexNAc(1) modified peptides (+ 203.079 Da) were almost exclusively detected in Slc35a2- deleted samples. D) HexNAc modified glycopeptides mapped to 60 genes, of which lecticans including Vcan, Bcan, and Ncan were most abundant. E) Volcano plot of significantly upregulated (blue) and downregulated (red) proteins in +/y vs -/y cortex, mutants compared to WT. Dotted line indicates significance threshold of p = 0.05. F) Ingenuity Pathway Analysis (IPA) highlighting the top 10 canonical pathways increased by Slc35a2 deletion.

Article Snippet: In brief, floxed homozygous Slc35a2 female mice were crossed with Emx1 -Cre and Olig2-Cre male mice obtained from Jackson Laboratory (#005628, #025567).

Techniques: Affinity Purification, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Modification

Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) Sagittal adult mouse brain sections labeled with WFA for CSPGs and PNA for O-GalNAc glycans display almost no colocalization despite both being bound to lecticans in the brain. B) High magnification images of a deep cerebellar nuclei (ROI from A; white box) where both WFA and PNA are present show non-overlapping signals, with WFA densely surrounding cells where PNA is excluded. Scale bar = 20 μm. Nuclei are stained with Hoechst-33324. C) ConA, GNL, AAL, and PNA + Neuraminidases A (NeuA) binding (top) from brain cortex samples of wild-type male mice (+/y) and human brain cortex that does not harbor SLC35A2 mutations. Treatment with PNGase F (#) and O-Glycosidase (#*) of a human sample in the last lane confirms the lectin specificity. D) Quantification of lectin binding from C normalized to TP staining within the same lane of each blot. Compared to mouse cortex, human cortex had significantly reduced ConA binding of 27%. GNL staining was decreased even further in humans relative to mice by 55%, while AAL showed no significant difference. In contrast, PNA binding after NeuA treatment was significantly increased in human cortex relative to mice by 44%. Data presented as the mean binding intensity +/- SEM, with data points for each individual mouse and human samples included. N = 4 independent samples per group, with 15 µg cortical protein lysate loaded per lane. Experiment replicated twice with similar results. Student’s t-tests: *p-value = 0.05, **p-value = 0.01, ***p-value = 0.001.

Article Snippet: In brief, floxed homozygous Slc35a2 female mice were crossed with Emx1 -Cre and Olig2-Cre male mice obtained from Jackson Laboratory (#005628, #025567).

Techniques: Labeling, Staining, Binding Assay

Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) Surgically resected tissue from human SLC35A2 -associated focal epilepsy was analyzed for VVA positivity using fluorescence microscopy, lectin blots, and confirmatory DNA sequencing. B) VVA binding across the full tissue block of a high mutation case (VAF = 62.6%) shows variable intensity. Nuclei are stained with Hoechst-33324, and the green signal represents auto-fluorescent red blood cells in the 488ƛ channel. Scale bar = 1 mm. C) ROI from B shows scattered binding, with areas of both isolated and diffuse signal. Autofluorescent red blood cells can be seen in the lumen of vessels at the 488ƛ channel. Scale bar = 100 µm. D) High magnification images of VVA staining from the same sample, which shows areas of diffuse mesh-like binding around multiple cells and some individual cells. Scale bar = 10 µm. Complementary images with individual channels are available in the Supplementary Material. E) Random 500 x 500 µm fluorescent intensity measures of VVA and H-33324 signal from B, plotted as arbitrary intensity (a.i.) normalized to average background signal adjacent to tissue block. Bar indicates median, N = 82 and 80.

Article Snippet: In brief, floxed homozygous Slc35a2 female mice were crossed with Emx1 -Cre and Olig2-Cre male mice obtained from Jackson Laboratory (#005628, #025567).

Techniques: Fluorescence, Microscopy, DNA Sequencing, Binding Assay, Blocking Assay, Mutagenesis, Staining, Isolation

Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) Surgically resected tissue from a SLC35A2 -associated focal epilepsy patient (VAF = 3.4-5.2%) were analyzed for VVA positivity via lectin-based histochemistry. Using laser capture microdissection (LCM), VVA+ areas were isolated from VVA-areas and both were re-sequenced for the patient specific variant. B) Variant allele frequency (VAF) from areas positive and negative for VVA binding was measured using amplicon sequencing of DNA isolated via laser capture microdissection (LCM). Areas of diffuse VVA+ show enrichment for the patient-specific variant in SLC35A2 , which were absent in both VVA-areas and reference. C) Image of the SLC35A2- associated epilepsy tissue sample stained with VVA, visualized using DAB histochemistry and counterstained with hematoxylin/eosin. Scale bar = 2000 μm. D) High magnification images from (C) of areas of both low density VVA+ with individual cells (i), diffuse high density VVA+ (ii), and hypercellularity in white matter (iii). Scale bar = 100 μm.

Article Snippet: In brief, floxed homozygous Slc35a2 female mice were crossed with Emx1 -Cre and Olig2-Cre male mice obtained from Jackson Laboratory (#005628, #025567).

Techniques: Laser Capture Microdissection, Isolation, Variant Assay, Binding Assay, Amplification, Sequencing, Staining

Journal: bioRxiv

Article Title: Disrupted O-GalNAc glycosylation as a mechanism and biomarker of SLC35A2 -associated epilepsy

doi: 10.64898/2026.03.02.708854

Figure Lengend Snippet: A) VVA lectin blot (both high and low exposure) from human cortex lysate from surgical biopsies of eight cases of SLC35A2 -associated epilepsy, as well as multiple unrelated controls. The percentage of SLC35A2 variant allele frequency (VAF) measured on the same same is indicated, as well as controls (-). 15 µg of brain protein lysate was loaded per lane, with total protein (TP) stain indicated in the right panel. B) Simple linear regression plot of normalized VVA intensity vs VAF % shows a strong positive linear correlation in SLC35A2 -associated focal epilepsy. C) Linear regression after removal of the highest VAF sample (62.6%) as a potential outlier in B maintains a strong correlation.

Article Snippet: In brief, floxed homozygous Slc35a2 female mice were crossed with Emx1 -Cre and Olig2-Cre male mice obtained from Jackson Laboratory (#005628, #025567).

Techniques: Variant Assay, Staining

Journal: Epilepsia

Article Title: Mouse models of Slc35a2 brain mosaicism reveal mechanisms of mild malformations of cortical development with oligodendroglial hyperplasia in epilepsy

doi: 10.1111/epi.18166

Figure Lengend Snippet: Emx1‐Cre‐mediated Slc35a2 conditional knockout (cKO) mice have developmental delays and impaired growth and survival. (A) Emx1‐ Cre mice were crossed with Slc35a2 floxed mice to produce forebrain‐specific loss of SLC35A2 from excitatory neurons and glia (blue). Cx, cortex; Hp, hippocampus; OB, olfactory bulb. (B) Slc35a2 gene expression measured by real‐time quantitative polymerase chain reaction was significantly reduced in the cortices of cKO mice compared to floxed controls ( n = 4 per group; one‐way analysis of variance [ANOVA], F 3,12 = 21.16, p < .0001, Bonferroni post hoc comparisons shown). (C) SLC35A2 protein expression measured by Western blot was significantly reduced in the cortices of cKO male mice compared to floxed male controls ( n = 3–4 per group; one‐way ANOVA, F 3,11 = 4.228, p < .05, Bonferroni post hoc comparisons shown). (D, E) Male cKO mice were smaller (D) and had significantly reduced body weight (E) beginning at postnatal day 9 ( n = 7–24 mice per group; mixed‐effect model [genotype × time] with Tukey post hoc comparisons, p < .05). (F) Both male and female cKO mice had reduced survival compared to floxed controls ( p < .0001), and hemizygous males had reduced survival compared to heterozygous females ( p < .0001; n = 32–88 per group; Kaplan–Meier survival differences evaluated by the log‐rank test). (G–J) Neonatal cKO mice exhibited developmental delays including delayed eye opening (G), delayed ear twitch flattening reflex (H), delayed auditory startle reflex (I), and abnormal hind limb clasping reflex (J; n = 6–23 per group; two‐tailed t ‐test). * p < .05, ** p < .01, **** p < .0001. Data are shown as mean ± SEM.

Article Snippet: Briefly, single guide (sg) RNA sequences targeting upstream and downstream of Slc35a2 exon 3 were designed using the Benchling design tool ( www.benchling.com ).

Techniques: Knock-Out, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Two Tailed Test

Journal: Epilepsia

Article Title: Mouse models of Slc35a2 brain mosaicism reveal mechanisms of mild malformations of cortical development with oligodendroglial hyperplasia in epilepsy

doi: 10.1111/epi.18166

Figure Lengend Snippet: Cortical architecture and oligodendroglial cell density in Emx1‐Cre‐mediated Slc35a2 conditional knockout (cKO) mice. (A) Representative images of coronal sections through the postnatal day 8 brain stained with 4,6‐diamidino‐2‐phenylindole (DAPI). Lines indicate measurements of cortex (ctx) thickness and corpus callosum (cc) thickness. (B) Both female and male cKO mice had reduced cortex thickness (one‐way analysis of variance [ANOVA] with Bonferroni post hoc comparisons, F 3,16 = 31.51, p < .0001; post hoc p < .001 in females and p < .0001 in males). (C) No differences in corpus callosum thickness were observed. (D–F) Representative images of coronal sections through the cortex stained with layer markers BRN2 (layers II/III), SATB2 (layers II–IV), and CTIP2 (layers V/VI). Cortex was divided into six equal bins from the ventricle to cortical surface to analyze distribution of positive immunofluorescent area. (G) BRN2 staining was more widely distributed in female and male cKO mice compared to floxed controls, with significantly decreased BRN2 positivity in bin 1 of both sexes and increased BRN2 staining found in bin 2 ( n = 3–4 per group, 2–3 sections per brain; two‐way ANOVA with Bonferroni post hoc comparisons; interaction of genotype × cortical layer, F 15,96 = 12.55, p < .0001; post hoc comparisons shown). (H) SATB2 staining was reduced in male cKO mice compared to floxed controls in bin 2 and bin 3 ( n = 3–4 per group, 2–3 sections per brain; two‐way ANOVA with Bonferroni post hoc comparisons; effect of genotype, F 3,90 = 3.618, p < .05; post hoc comparisons shown). (I) No significant differences were observed in CTIP2 staining across cortical bins. (J) OLIG2 immunoreactivity was quantified in an region of interest containing the cortex–white matter junction (blue box). (K) OLIG2‐positive cell density was significantly increased in the cortex–white matter junction of male and female cKO mice compared to floxed controls ( n = 3–4 per group, 2–3 sections per brain; two‐tailed t ‐tests, p < .05). Data are shown as mean ± SEM. * p < .05, *** p < .001, **** p < .0001.

Article Snippet: Briefly, single guide (sg) RNA sequences targeting upstream and downstream of Slc35a2 exon 3 were designed using the Benchling design tool ( www.benchling.com ).

Techniques: Knock-Out, Staining, Two Tailed Test

Journal: Epilepsia

Article Title: Mouse models of Slc35a2 brain mosaicism reveal mechanisms of mild malformations of cortical development with oligodendroglial hyperplasia in epilepsy

doi: 10.1111/epi.18166

Figure Lengend Snippet: Neuronal migration is delayed in Emx1‐Cre‐mediated Slc35a2 conditional knockout (cKO) mice. (A) To determine whether a defect in neuronal migration was responsible for the changes in cortical layering observed within the cortex, newborn neurons destined for layer II/III were labeled by EdU injection at embryonic day 15.5 (E15.5) and brains were subsequently examined at postnatal day 1 (P1) or P8. (B, C) EdU signal was more prevalent in the white matter (WM) and ventricular zone (VZ) compared to cortex in cKO mice at P1, whereas most signal was found in the outer cortex in floxed control mice ( n = 3 per group, 2–3 sections per brain; two‐way analysis of variance [ANOVA] with Bonferroni post hoc tests; interaction of genotype × cortical layer, F 6,24 = 64.99, p < .0001; post hoc comparisons shown). (D, E) By P8, EdU signal was concentrated in the outer cortex in both floxed control and cKO mice, indicating a delay in neuronal migration ( n = 3 mice per group, 2–4 sections per brain; two‐way ANOVA with Bonferroni post hoc tests, no significance). Data are shown as mean ± SEM. * p < .05, *** p < .001, **** p < .0001. DAPI, 4,6‐diamidino‐2‐phenylindole.

Article Snippet: Briefly, single guide (sg) RNA sequences targeting upstream and downstream of Slc35a2 exon 3 were designed using the Benchling design tool ( www.benchling.com ).

Techniques: Migration, Knock-Out, Labeling, Injection, Control

Journal: Epilepsia

Article Title: Mouse models of Slc35a2 brain mosaicism reveal mechanisms of mild malformations of cortical development with oligodendroglial hyperplasia in epilepsy

doi: 10.1111/epi.18166

Figure Lengend Snippet: Emx1‐Cre‐mediated Slc35a2 conditional knockout (cKO) mice exhibit spontaneous seizure activity. (A) Representative electroencephalographic (EEG) traces over 5 min of recording from floxed control and Emx1‐Cre‐mediated Slc35a2 cKO mice between 15 and 25 weeks of age. cKO mice exhibited spontaneous bursts of electrographic activity correlating with behavioral abnormalities (highlighted by black bars) and abnormal interictal spike discharges. (B) Highlighted regions of EEG activity are shown in more detail. Emx1‐Cre‐mediated Slc35a2 cKO mice demonstrated rhythmic ictal spiking with clear onset and offset. (C) An average of 26 electrographically confirmed seizures were observed in cKO mice during the 72‐h video‐EEG recordings compared to none in the floxed controls (Mann–Whitney test, * p < .05). (D) These events coincided with behavioral signs of seizure, including leg/head jerking (score = 2–3) and clonic activity (e.g., falling to the side; score = 4–5; Mann–Whitney test, ** p < .01). Male and female mice are pooled. Data shown are as mean ± SEM.

Article Snippet: Briefly, single guide (sg) RNA sequences targeting upstream and downstream of Slc35a2 exon 3 were designed using the Benchling design tool ( www.benchling.com ).

Techniques: Knock-Out, Activity Assay, Control, MANN-WHITNEY

Journal: Epilepsia

Article Title: Mouse models of Slc35a2 brain mosaicism reveal mechanisms of mild malformations of cortical development with oligodendroglial hyperplasia in epilepsy

doi: 10.1111/epi.18166

Figure Lengend Snippet: Olig2‐Cre‐mediated Slc35a2 conditional knockout (cKO) mice reveal contributions of oligodendrocytes to abnormal electroencephalographic (EEG) activity. (A) Olig2‐Cre mice were crossed with Slc35a2 floxed mice to delete SLC35A2 from oligodendrocytes. (B) Olig2‐Cre‐mediated Slc35a2 cKO mice had reduced body mass compared to floxed controls ( n = 7–15 mice per group; one‐way analysis of variance with Tukey multiple comparisons; effect of genotype, F 3,814 = 170.2, p < .0001, post hoc p < .05 in +/y vs. −/y males day 7–21, +/+ vs. −/+ females day 11–21, −/+ females vs. −/y males day 13–15 and day 19–21). (C) Olig2‐Cre‐mediated Slc35a2 cKO males, but not females, had significantly reduced survival. Kaplan–Meier survival differences were evaluated by the log‐rank test ( p < .01). (D) OLIG2 immunoreactivity was quantified in the cortex–white matter junction (blue box). (E) OLIG2‐positive cell density was significantly increased in the cortex–white matter junction of male cKO mice ( n = 3–5 per group, 2–4 sections per brain; two‐tailed t ‐tests, p < .05). (F) Representative EEG traces over 5 min of recording from floxed control and Olig2‐Cre‐mediated Slc35a2 cKO mice between 15 and 25 weeks of age. The same floxed control mice were used for both Figure and Figure comparisons, replotted here for direct comparison. cKO mice exhibit spontaneous bursts of >10 s. Ictal spiking activity correlating with behavioral abnormalities (highlighted by black bars) and abnormal spike discharges are shown. (G) Highlighted regions of EEG activity are shown in more detail. Olig2‐Cre‐mediated Slc35a2 cKOs exhibited bursts of irregular spikes. (H) An average of 13 ictal discharges were observed over the 72‐h recording period in Olig2‐Cre‐mediated Slc35a2 cKO mice (Mann–Whitney test, p < .05). (I) Behavioral correlates of these events corresponded to mild twitching or jerking (score = 2–3; Mann–Whitney test, p < .05). Male and female mice are pooled. Data are shown as mean ± SEM. * p < .05.

Article Snippet: Briefly, single guide (sg) RNA sequences targeting upstream and downstream of Slc35a2 exon 3 were designed using the Benchling design tool ( www.benchling.com ).

Techniques: Knock-Out, Activity Assay, Two Tailed Test, Control, Comparison, MANN-WHITNEY