OGTC921Y mice show postnatal growth development delay and increased spontaneous activity.

Significance is shown as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p<0.0001. (a) Body weight of male OGTWT (n=15) and OGTC921Y (n=16) mice from 3 weeks to 20 weeks old. Two-way ANOVA (Alpha, 0.05) followed by Tukey’s column comparisons. (b) Line chart representing the cumulative locomotion index (x-axis, days) for 75 days. Two-way ANOVA (Alpha, 0.05) followed by Tukey’s column comparisons (OGTWT, n=10 housed in 5 cages; OGTC921Y, n=10 housed in 5 cages). (c) Line chart representing the cumulative locomotion index with light (6am-6pm) and dark (6pm-6am) periods in DVC cages over a longitudinal period up to 75 days (x-axis showing time in hours, 24-h format). The arrows indicate increased nocturnal activity by OGTC921Y mice compared to WT littermates. (d) Heat maps representation of the cumulative locomotion index with light and dark periods in DVC cages for 75 days (x-axis showing time in hours over 24-hour period each day, y-axis showing days with Day 31 as the starting point on top). The arrow indicates increased nocturnal activity by OGTC921Y mice compared to WT littermates. (e) Representative tracking plot over three consecutive days of a male OGTWT and male OGTC921Y mice in the open field arena. (f) Distance travelled over three consecutive days of male OGTWT (n = 15) and male OGTC921Y (n = 16) mice in the open field arena. Two-way ANOVA (Alpha, 0.05) followed by Tukey’s column comparisons. (g) Maximal speed displayed by male OGTWT (n = 15) and male OGTC921Y (n = 16) mice in the open field arena. Two-way ANOVA (Alpha, 0.05) followed by Tukey’s column comparisons.

Motor, digging and burying activity.

Significance is shown as * p < 0.05, ** p < 0.01 and *** p < 0.001. Student t test was used for statistics. (a) Representative image of experimental set-up for the static rods test. (b) Time to t-turn of male OGTWT (n = 15) and OGTC921Y (n = 16) during the static rods test. (c) Delta time defined as (Total time – time to turn) of male OGTWT (n = 15) and OGTC921Y (n = 16) during the static rods test. (d) Representative images of the marble test at 0 min (T0) and 30 min (T30). (e) Number of buried marbles by male OGTWT (n = 15) and OGTC921Y (n = 16) during the marble test. (f) Representative image of the litter burrowing test. (g) Percentage of litter removed by male OGTWT (n = 15) and OGTC921Y (n = 16) during the litter burrowing test. (h) Number of digging events by male OGTWT (n = 15) and OGTC921Y (n = 16) during 3 min observation. (i) Time spent digging by male OGTWT (n = 15) and OGTC921Y (n = 16) during 3 min observation. (j) Number of rearing events by male OGTWT (n = 15) and OGTC921Y (n = 16) during 3 min observation. (k) Time spent rearing by male OGTWT (n = 15) and OGTC921Y (n = 16) during 3 min observation.

OGTC921Y show impulsivity, enhanced long term memory and delayed aversive associated learning.

Significance is shown as *p < 0.05, **p < 0.01 and ***p < 0.001. (a) Schematic of the T-maze paradigm. Mice are free to explore both arms for 7 additional trials. Each choice and latency were recorded. (b) Percentage of correct alternation (L-R/R-L sequences) of OGTWT (n = 15) and OGTC921Y (n = 16) during the T-maze test. Student t test was used for statistics. (c) Latency of arm entry of OGTWT (n = 15) and OGTC921Y (n = 16) during the T-maze test. Student t test was used for statistics. (d) Representative image of the Novel Object Recognition (NOR) test. During the familiarization phase, mice are free to explore an arena with two identical objects. During the test phase, mice were free to explore the same arena where one of the familiar objects has been replaced by a novel object to assess short (90 min) and long (24 h) memory. (e) Discrimination index of OGTWT (n = 15) and OGTC921Y (n = 16) during the short term (90 min) NOR test. Student t test was used for statistics. (f) Discrimination index of OGTWT (n = 15) and OGTC921Y (n = 16) during the long term (24h) NOR test. Student t test was used for statistics. (g) Diagram showing the behavioural paradigm (top), tone, conditioned stimulus (CS), trace period, and unconditioned stimulus, foot shock (US) lengths (bottom). (h) Freezing behaviour of OGTWT and OGTC921Y (n = 9 per group) during the acquisition session of the aversive conditioning. ITI, Intertrial interval. Two-way ANOVA time x genotype interaction p < 0.0001, F(11,176) = 5.572. (i) Locomotor activity of OGTWT and OGTC921Y (n = 9 per group) evoked by the US. The data represent the 3 s bin following the foot shock. Two-way ANOVA time x genotype interaction p = 0.3323, F(3,48) = 1.166 (j) Freezing behaviour of OGTWT and OGTC921Y (n = 9 per group) in the recall session where the CS were presented without US. The trace period refers to the 25 s following the CS presentation. Two-way ANOVA time x genotype interaction p = 0.5337, F(11,176) = 0.9082.

Micro-computed tomography of OGT-ID mouse skulls indicates a reduced endocast volume and shorter skull length.

Significance is shown as * p < 0.05, ** p < 0.01 and *** p < 0.001. (a) Heatmaps obtained of the average OGTC921Y skull (n = 16) to the average OGTWT skull (n = 14). Red and blue regions indicate that the average OGTC921Y skull is smaller or larger respectively than the average OGTWT in those areas. (b) PCA biplot of PC1 and PC2 of the surface landmarks of 20 weeks old male OGTWT and OGTC921Y skulls (top). Percentages in the axis indicate the explained variance. Deformations in PC1 showing shape differences across this axis are shown (bottom). (c) Volume of the skulls of 8 weeks and 20 weeks old male OGTWT and OGTC921Y mice. Student t test was used for statistics. (d) Volume of the extracted endocranial cavity of 8 weeks and 20 weeks old male OGTWT and OGTC921Y mice. Student t test was used for statistics.

OGTC921Y mice show changes in regional brain volume and reduced cortical thickness.

Volumetry and cortical thickness from high resolution T1-weighted Magnetic Resonance (MR) images. (a) Total brain volume of 20 weeks old male OGTWT and OGTC921Y mice. Groupwise total brain volume where each dot represents one subject. The horizontal lines correspond to group extrema and median. Asterisks (*) mark significance (p<0.05) based on permutation tests (1M permutations) of either the mean (green asterisk) or median (red asterisk) of the two groups. Heatmap Cortical thickness. Statistical maps of group differences in cortical thickness between wild type and OGTC921Y. Maps are effect sizes (top row), p values of significant differences both uncorrected for multiple comparisons (middle row) and corrected with family-wise error (FWE). (b) Heatmaps of group differences in cortical thickness between male OGTWT and OGTC921Y mice. Maps are effect sizes (top row), p-values of significant differences both uncorrected for multiple comparisons (middle row) and corrected with family-wise error (FWE). n=15 & 15 one mutant was not perfused correctly (c) Regional brain volumes represented as percentage of whole brain volume of male OGTWT and OGTC921Y mice. Asterisks (*) indicate uncorrected significance (p<0.05), based on permutation tests (100k permutations for each region). Pound symbols (#) indicate significance below alpha (0.05) divided by total number regions tested (40) for mean (green) and median (red), respectively. Section signs (§) indicate significance (p<0.05) with p-values adjusted for false discovery rate (Benjamini-Hochsberg, BH). Y-axes are scaled to individual ROIs to highlight group variation and difference.

Pseudosulcus formation, cortical dysplasia and ectopic white matter in the cingulate cortex of OGTC921Y mice.

(a) Representative images of low magnification (10X) sagittal views showing cortical malformations in the cingulate cortex of two OGTC921Y mice by H&E staining. The black filled arrows point to instances of pseudosulcus formation and the yellow arrows point to instances of cortical dysplasia observed in the layers II-IV (scale bar = 500 µm). (b) Representative images of high magnification (40X) views extracted from the insets in A (OGTC921Y mice) and a WT control (scale bar = 100 µm). (c) Representative images of low magnification (10X) sagittal view showing ectopic white matter (red arrows) in the cingulate cortex of OGTC921Y mice by luxol fast staining. Notice the correspondence (indicated by thin yellow projection arrows) between nodular malformations (H&E image in A) and ectopic white matter in a section cut ∼100 µm lateral to the midline (interaural distance, ∼1.3). Also notice the normal flattening of the cortical surface (green arrow) in a corresponding region. (d) Representative images of low magnification (20X) sagittal views showing normal luxol fast staining in cortex of WT mice and distinct patterns of ectopic white matter (red arrows) in the cingulate cortex of OGTC921Y mice in relation to a cortical malformation in layers II-IV (scale bar = 100 µm). (e) Regional cell density assessed in 10X views (H&E staining) of two-serial sections from WT and OGTC921Y mice. Error bars indicate Mean ± SEM (n = 6/group; Mann-Whitney test, none significant). Regions examined include: (coronal sections) primary motor cortex M1/M2, primary somatosensory cortex S1/S2, auditory cortex (A); (sagittal sections) visual cortex V1 and cingulate cortex (C). Also see Fig. S10 depicting coordinates and region demarcations (dashed rectangles) used in the cell density analyses.

Quantitative proteomics analyses revealed distinctly perturbed molecular pathways in prefrontal cortex of the OGTC921Y mice.

(a) List of Top 50 proteins up-regulated (cut off criteria p value 0.05, log 2 change 1.45). (b) List of Top 50 proteins down-regulated (cut off criteria p value 0.05, log 2 change-1.33). (c) Up-regulated biological pathways enrichment in the STRING database. (d) Down-regulated biological pathways enrichment in the STRING database.

Possible mechanisms underlying OGT-ID.

(a) Graphical representation of possible mechanisms underlying OGT-ID using Biorender. Mechanisms include loss of O-GlcNAcylation on OGT substrates important for brain function and development, OGT aggregation due to reduced OGT stability, impaired OGT interactome, misprocessing of HCF1, or loss of OGA. These hypotheses are not mutually exclusive and can individually or in combination lead to cognitive dysfunction due to impaired brain development, defects in synaptogenesis/synaptic pruning and/or neural transmission impairment. This panel was created using BioRender.com. (b) Graphical summary of the neurodevelopmental, structural and behaviour defects identified in the OGTC921Y mice. This panel was created using BioRender.com.

OGTC921Y mice show similar anxiety-like behaviour and increased exploration

Significance is shown as *p < 0.05, **p < 0.01 and ***p < 0.001. (a) Distance travelled in periphery by male OGTWT (n = 15) and OGTC921Y (n = 16) mice in the open field arena. Two-way ANOVA (Alpha, 0.05) followed by Tukey’s column comparisons. (b) Time spent in periphery by male OGTWT (n = 15) and OGTC921Y (n = 16) mice in the open field arena. Two-way ANOVA (Alpha, 0.05) followed by Tukey’s column comparisons. (c) Distance travelled in centre by male OGTWT (n = 15) and OGTC921Y (n = 16) mice in the open field arena. Two-way ANOVA (Alpha, 0.05) followed by Tukey’s column comparisons. (d) Time spent in centre by male OGTWT (n = 15) and OGTC921Y (n = 16) mice in the open field arena. Two-way ANOVA (Alpha, 0.05) followed by Tukey’s column comparisons. (e) Representative tracking plot of male OGTWT and OGTC921Y mice in the elevated plus maze (EPM). (f) Total distance travelled of male OGTWT (n = 15) and OGTC921Y (n = 16) mice in the EPM. Student t test was used for statistics. (g) Number of centre entries of male OGTWT (n = 15) and OGTC921Y (n = 16) mice in the EPM. Student t test was used for statistics. (h) Ratio between time spent in open and close arms by male OGTWT (n = 15) and OGTC921Y (n = 16) mice in the EPM. Student t test was used for statistics. (i) Representative tracking plot of male OGTWT and OGTC921Y mice in the light compartment during the dark-light paradigm (D/L). (j) Total distance travelled of male OGTWT (n = 15) and OGTC921Y (n = 16) mice in the light compartment during the dark-light paradigm (D/L). Two-way ANOVA (Alpha, 0.05) followed by Tukey’s column comparisons. (k) Number of centre entries of male OGTWT (n = 15) and OGTC921Y (n = 16) mice in the light compartment during the dark-light paradigm (D/L). Two-way ANOVA (Alpha, 0.05) followed by Tukey’s column comparisons. (l) Time spent of male OGTWT (n = 15) and OGTC921Y (n = 16) mice in the light compartment during the dark-light paradigm (D/L). Two-way ANOVA (Alpha, 0.05) followed by Tukey’s column comparisons.

OGTC921Y mice show similar motor skills

Significance is shown as *p < 0.05, **p < 0.01 and ***p < 0.001. (a) Mean score reached by male OGTWT (n = 15) and OGTC921Y (n = 16) mice on the rotarod. (b) Mean time spent by male OGTWT (n = 15) and OGTC921Y (n = 16) mice on the rotarod. (c) Score reached by male OGTWT (n = 15) and OGTC921Y (n = 16) mice during the pole test. (d) Number of self-grooming events male OGTWT (n = 15) and OGTC921Y (n = 16) mice during 3 min observation. (e) Time spent self-grooming by male OGTWT (n = 15) and OGTC921Y (n = 16) mice during 3 min observation. (f) Percentage of material removed from a cotton pad by male OGTWT (n = 15) and OGTC921Y (n = 16) mice during 3 min during the nesting test.

OGTC921Y mice show reduced distances in the cranial base.

(a) Lateral and superior views of a representative microCT 3D reconstruction of a mouse skull presenting the 45 landmarks used for Euclidean distance matrix analysis (EDMA) (left) and surface markups used for Principal Components Analysis and average shapes generation (right). (b) Lateral and inferior views of representative 3D reconstructions of mouse skulls presenting the linear distances that are at least 5% shorter in 2 and 5 months old OGTC921Y skulls (p < 0.01, two-tailed unpaired t-test used)

Volumetry from segmentation of T1-weighted structural images (FLASH) from Magnetic Resonance Imaging (MRI) illustrated as absolute volumes.

Asterisks (*) indicate uncorrected significance (p < 0.05), based on permutation tests (100k permutations for each region). Pound symbols (#) indicate significance below alpha (0.05) divided by total number regions tested (40) for mean (green) and median (red), respectively. Section signs (§) indicate significance (p < 0.05) with p values adjusted for false discovery rate (Benjamini-Hochsberg, BH). Y-axes are scaled to individual ROIs to highlight group variation and difference.

Relative volumetry from T1 segmentation.

Volumetry from segmentation of T1-weighted structural images (FLASH) from Magnetic Resonance Imaging (MRI) illustrated as regional relative volumes (divided by individual total brain volume). Asterisks (*) indicate uncorrected significance (p < 0.05), based on permutation tests (100k permutations for each region). Pound symbols (#) indicate significance below alpha (0.05) divided by total number regions tested (40) for mean (green) and median (red), respectively. Section signs (§) indicate significance (p < 0.05) with p values adjusted for false discovery rate (Benjamini-Hochsberg, BH). Y-axes are scaled to individual ROIs to highlight group variation and difference.

OGTC921Y mice show conserved DKI metrics for Neocortex

(a) Middle coronal section of subject:207 illustrating the different calculated DKI metrics: Mean Diffusivity (MD), Fractional Anisotropy (FA) and Mean Kurtosis (MK). (b) Extracted DKI metrics (MD, FA and MK) for Neocortex of all subjects in each group for neocortex of all mice.

Subject-wise median of extracted Fractional Anisotropy (FA) values in 20 selected regions of interest (ROI).

Subject-wise median of extracted Mean Diffusivity (MD) values in 20 selected regions of interest (ROI).

Subject-wise median of extracted Mean Kurtosis (MK) values in 20 selected regions of interest (ROI).

Panoramic views of the H&E stained sagittal and coronal brain sections depicting neuroanatomical landmarks used for measurements of cortical thickness and cell density.

(a) Representative images showing coronal panoramic views of the brain sections used for the measurements of cortical cell density. The sections were used to sample regions in the cortex at the level of Begma 0.14 and Bregma-2.06. Regional cell counts were obtained from primary motor cortex M1/M2 and primary somatosensory cortex S1/S2, and from the auditory cortex. (b) Representative images showing sagittal panoramic views of the brain sections used for the measurements of cortical cell density. The sections were used to sample medial (interaural, 0.36, in C) and lateral (interaural, 2.28, in D) regions of the cortex. Regional cell counts were obtained from cingulate cortex and visual cortex V. Abbreviations: AuD/AuV, auditory cortex (in B); CPu, caudate putamen; superior colliculus. Neuroanatomical annotations are based on the Paxinos and Franklin’s Mouse Brain in Stereotaxic Coordinates, Elsevier Publishing, 4th Edition.

Low magnification (10X) views of the H&E stained sagittal and coronal brain sections used for assessing cortical organization.

Representative images showing low magnification (10X) views of select cortical regions from OGTWT (in A) and OGTC921Y (in B) mice. Areas shown include primary motor cortex M1/M2, primary somatosensory cortex S1/S2, auditory cortex and visual cortex V1. Notice a mild degree of cortical dysplasia in layers II-III in the auditory cortex of OGTC921Y mice (yellow arrow). The images were obtained from coronal sections (Bregma, 0.14, for M1/M2 and S1/S2; Bregma,-2.06, auditory cortex) and sagittal sections (Interaural 2.28, visual cortex V1). Scale bar = 200 µm. Also see Fig. S10. Neuroanatomical annotations are based on the Paxinos and Franklin’s Mouse Brain in Stereotaxic Coordinates, Elsevier Publishing, 4th Edition.

OGTC921Y mice show altered OGT/OGA ratio and global reduction in O-GlcNAc levels in prefrontal cortex

Significance is shown as * p < 0.05, ** p < 0.01 and *** p < 0.001 (n = 3 per group) (a) Western blot of OGA and OGT level in male prefrontal cortex of OGTWT and OGTC921Y mice. Actin antibodies were used as loading control. (b) Quantification of OGA proteins levels from the Western blot in panel a). Student t test was used for statistics. (c) Quantification of OGT proteins levels from the Western blot in panel a). Student t test was used for statistics. (d) OGT/OGA protein ratio from the Western blot in panel b). Student t test was used for statistics. (e) Western blot of O-GlcNAc levels in prefrontal cortex of male OGTWT and OGTC921Y mice. Actin antibodies were used as loading control. (f) Quantification of O-GlcNAc levels from the Western blot in panel e). Student t test was used for statistics.

Perturbed proteomic signatures in the prefrontal cortex corresponded to distinct Monarch enrichment profiles in the OGTC921Y mice

(a) Up-regulated human phenotypes enrichment in the Monarch database. (b) Down-regulated human phenotypes enrichment in the Monarch database.