Aggravated pathological alterations in the gene expression and increased levels of protein markers of UPR and the ER stress in the brains of Hgsnat ^P304L mice. (A–C) Hippocampal mRNA profiling in 4-mo-old MPS IIIC mice reveals increased expression of genes involved in lysosomal, lipid synthesis, and proinflammatory processes and reduced expression of genes involved in synaptic transmission, vesicular transport, and neurogenesis. Dot plots (left) show significantly enriched GO terms (biological processes, molecular functions, and cellular components) and heatmaps (right) of the genes significantly upregulated and downregulated in the hippocampi of Hgsnat^P304L mice compared with WT mice (A), Hgsnat-Geo mice compared with WT mice (B), and Hgsnat^P304L mice compared with Hgsnat-Geo mice (C). GO terms are plotted in the order of gene ratios, and each pathway is shown as a circle with the color representing the P values (−log₁₀) and the size representing the number of differentially expressed genes. The heatmap colors and their intensity show changes in gene expression levels. Data were obtained by sequencing mRNA samples extracted from three mice per genotype. (D) Brain cortex of Hgsnat^P304L 6-mo-old mice shows increased levels of O-GlcNAc–modified proteins compared with Hgsnat-Geo and WT mice. Panels show representative images of brain cortex (layers 4–5) immunostained for O-GlcNAc (green). DAPI (blue) was used as a nuclear counterstain. Scale bar equals 25 µm. Graphs show results of quantification performed using ImageJ software. Individual data, means, and SD obtained for five mice per genotype (three areas/mouse) are shown. P values were calculated using nested one-way ANOVA test with Tukey post hoc test. (E) Increased levels of ubiquitinated protein aggregates are detected in the brain homogenates of Hgsnat^P304L mice by immunoblotting. Graphs show combined intensities (individual values, means, and SD) of protein ubiquitin⁺ bands, quantified with ImageJ software and normalized by either intensity of tubulin bands or bands of ubiquitin monomers. Three mice per genotype were analyzed. P values were calculated using ANOVA with Tukey post hoc test. (F) Somatosensory cortex (layers 4–5) of Hgsnat^P304L mice shows increased levels of pyramidal neurons containing cytoplasmic ubiquitin⁺ materials. Panels show representative confocal microscopy images of brain tissues, stained for ubiquitin, of 6-mo-old Hgsnat-Geo, Hgsnat^P304L, and WT mice. Scale bar equals 25 µm. Graph shows results of quantification performed using ImageJ software. Individual data, means, and SD obtained for five mice per genotypes (three areas/mouse) are shown. P values were calculated using nested one-way ANOVA test with Tukey post hoc test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Source data are available for this figure: SourceData F5.