Hgsnat ^P304L mice treated with glucosamine show significant increase of HGSNAT activity in brain and liver tissues, reveal delay in development of deficits in memory and learning, and partial rescue of synaptic protein markers in the CA1 area of the hippocampus. (A) HGSNAT activity, measured using the fluorogenic substrate, muf-β-D-glucosaminide, was increased in cultured MEF cells of homozygous Hgsnat^P304L mice with glucosamine (+GA) for 5 d compared with untreated cells. Graph shows individual results, means, and SD of experiments conducted with four to six different cell cultures, each established from pooled tissues of three mice. (B–D) HGSNAT activity is also increased in the brain (B and C) and liver (D) tissue homogenates of 4-mo-old Hgsnat^P304L mice treated with glucosamine (+GA) for 1 wk (B) or 13 wk (C and D) compared with untreated Hgsnat^P304L mice of the same age. (E and F) No decrease in total β-hexosaminidase activity in both organs was detected for Hgsnat^P304L mice treated with glucosamine for 13 wk. Individual results, means and SD from experiments performed with 6–10 mice per genotype, per treatment are shown. P values were calculated using an unpaired t test. (G–I)Hgsnat^P304L mice, treated with glucosamine, show rescue or a trend for improvement of deficits in spatial/short-term memory in the YM test (G) and short-term memory in the NOR test at the age of 4 mo (H and I) compared with untreated Hgsnat^P304L mice. Individual results, means and SD from experiments performed with 24 mice per genotype, per treatment are shown. P values were calculated using one-way ANOVA with Tukey post hoc test. (J) Deficient levels of protein markers of glutamatergic synaptic neurotransmission, VGUT1 and PSD-95, are rescued in the CA1 hippocampal area of Hgsnat^P304L mice, treated with glucosamine at the age of 4 mo. Panels show representative images of brain cortex (layers 4–5) and CA1 area of the hippocampus, stained for PSD-95 (red) and VGLUT1 (green), of 4-mo-old WT and Hgsnat^P304L mice treated or not with glucosamine. Scale bar equals 15 µm. The graph shows quantification of fluorescence with ImageJ software. Individual results, means, and SD from experiments performed with five mice per genotype (three areas/mouse), per treatment are shown. P values were calculated using nested one-way ANOVA test with Tukey post hoc test. (K) Deficient level of synaptic vesicular protein Syn-1 is rescued in the somatosensory cortex area of Hgsnat^P304L mice, treated with glucosamine at the age of 4 mo. Panels show representative images of brain cortex (layers 4–5) and CA1 area of the hippocampus, stained for PSD-95 (red) and Syn1 (green), of 4-mo-old WT, and Hgsnat^P304L mice treated or not with glucosamine. Scale bar equals 15 µm. The graph shows quantification of fluorescence with ImageJ software. Individual results, means, and SD from experiments performed with five mice per genotype (three areas/mouse), per treatment 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.