Figure 8.
The levels of biomarkers of CNS pathology in the somatosensory cortex are normalized and brain storage of HS is reduced in HgsnatP304Lmice treated with glucosamine. (A) Reduction of granular autofluorescent ceroid material in cortical neurons. Panels show representative images of brain cortex (layers 4–5) of 4-mo-old WT and HgsnatP304L mice treated or not with glucosamine (GA) showing autofluorescent ceroid inclusions in the neurons (green). Scale bar equals 100 µm (upper panels) and 25 µm (lower panel). The graph shows quantification of autofluorescence with ImageJ software. (B) Reduction of GM2 ganglioside in cortical neurons. Panels show representative images of somatosensory cortex (layers 4–5) of 4-mo-old WT, and HgsnatP304L mice treated or not with glucosamine showing immunostaining for GM2 ganglioside and NeuN. DAPI was used as a nuclear counterstain. Scale bar equals 100 µm (upper panels) and 25 µm (lower panel). The bar graph shows quantification of GM2 staining with ImageJ software. (C) Reduction of misfolded SCMAS aggregates in cortical neurons. Panels show representative images of somatosensory cortex (layers 4–5), stained for SCMAS, of 4-mo-old WT and HgsnatP304L mice treated or not with glucosamine. DAPI (blue) was used as a nuclear counterstain. Scale bar equals 100 µm (upper panels) and 25 µm (lower panel). The bar graph shows quantification of SCMAS staining with ImageJ software. All graphs show individual data, means, and SD obtained for five mice per genotype per treatment (three areas/mouse). P values were calculated using nested one-way ANOVA test with Tukey post hoc test. (D) Levels of disaccharides produced by enzymatic digestion of HS (ΔDiHS-0S and ΔDiHS-NS), measured by MS/MS, are reduced in brain tissues of 4-mo-old HgsnatP304L mice treated with glucosamine. All graphs show individual data, means, and SD obtained for five to six mice per genotype per treatment. P values were calculated using two-way ANOVA test with Tukey post hoc test. (E) Reduction of HS+ and LAMP2+ puncta in cortical neurons. Panels show representative images of brain cortex (layers 4–5) and CA1 region of hippocampus, stained for LAMP2 (green) and HS (red), of 4-mo-old WT and HgsnatP304L mice, treated or not with glucosamine. Scale bar equals 25 µm. Graphs show quantification of HS and LAMP2 staining with ImageJ software. All graphs show individual results, means, and SD from experiments conducted with five mice (three panels per mouse) per genotype per treatment. 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.

The levels of biomarkers of CNS pathology in the somatosensory cortex are normalized and brain storage of HS is reduced in Hgsnat P304L mice treated with glucosamine. (A) Reduction of granular autofluorescent ceroid material in cortical neurons. Panels show representative images of brain cortex (layers 4–5) of 4-mo-old WT and HgsnatP304L mice treated or not with glucosamine (GA) showing autofluorescent ceroid inclusions in the neurons (green). Scale bar equals 100 µm (upper panels) and 25 µm (lower panel). The graph shows quantification of autofluorescence with ImageJ software. (B) Reduction of GM2 ganglioside in cortical neurons. Panels show representative images of somatosensory cortex (layers 4–5) of 4-mo-old WT, and HgsnatP304L mice treated or not with glucosamine showing immunostaining for GM2 ganglioside and NeuN. DAPI was used as a nuclear counterstain. Scale bar equals 100 µm (upper panels) and 25 µm (lower panel). The bar graph shows quantification of GM2 staining with ImageJ software. (C) Reduction of misfolded SCMAS aggregates in cortical neurons. Panels show representative images of somatosensory cortex (layers 4–5), stained for SCMAS, of 4-mo-old WT and HgsnatP304L mice treated or not with glucosamine. DAPI (blue) was used as a nuclear counterstain. Scale bar equals 100 µm (upper panels) and 25 µm (lower panel). The bar graph shows quantification of SCMAS staining with ImageJ software. All graphs show individual data, means, and SD obtained for five mice per genotype per treatment (three areas/mouse). P values were calculated using nested one-way ANOVA test with Tukey post hoc test. (D) Levels of disaccharides produced by enzymatic digestion of HS (ΔDiHS-0S and ΔDiHS-NS), measured by MS/MS, are reduced in brain tissues of 4-mo-old HgsnatP304L mice treated with glucosamine. All graphs show individual data, means, and SD obtained for five to six mice per genotype per treatment. P values were calculated using two-way ANOVA test with Tukey post hoc test. (E) Reduction of HS+ and LAMP2+ puncta in cortical neurons. Panels show representative images of brain cortex (layers 4–5) and CA1 region of hippocampus, stained for LAMP2 (green) and HS (red), of 4-mo-old WT and HgsnatP304L mice, treated or not with glucosamine. Scale bar equals 25 µm. Graphs show quantification of HS and LAMP2 staining with ImageJ software. All graphs show individual results, means, and SD from experiments conducted with five mice (three panels per mouse) per genotype per treatment. 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.

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