VGLUT3 Deletion Rescues Motor Deficits and Neuronal Loss in the zQ175 Mouse Model of Huntington's Disease

Abstract

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disease characterized by progressive motor and cognitive impairments, with no disease-modifying therapies yet available. HD pathophysiology involves evident impairment in glutamatergic neurotransmission leading to severe striatal neurodegeneration. The vesicular glutamate transporter-3 (VGLUT3) regulates the striatal network that is centrally affected by HD. Nevertheless, current evidence on the role of VGLUT3 in HD pathophysiology is lacking. Here, we crossed mice lacking Slc17a8 gene (VGLUT3 ^-/-) with heterozygous zQ175 knock-in mouse model of HD (zQ175:VGLUT3 ^-/-). Longitudinal assessment of motor and cognitive functions from 6 to 15 months of age reveals that VGLUT3 deletion rescues motor coordination and short-term memory deficits in both male and female zQ175 mice. VGLUT3 deletion also rescues neuronal loss likely via the activation of Akt and ERK1/2 in the striatum of zQ175 mice of both sexes. Interestingly, the rescue in neuronal survival in zQ175:VGLUT3 ^-/- mice is accompanied by a reduction in the number of nuclear mutant huntingtin (mHTT) aggregates with no change in the total aggregate levels or microgliosis. Collectively, these findings provide novel evidence that VGLUT3, despite its limited expression, can be a vital contributor to HD pathophysiology and a viable target for HD therapeutics.SIGNIFICANCE STATEMENT Dysregulation of the striatal network centrally contributes to the pathophysiology of Huntington's disease (HD). The atypical vesicular glutamate transporter-3 (VGLUT3) has been shown to regulate several major striatal pathologies, such as addiction, eating disorders, or L-DOPA-induced dyskinesia. Yet, our understanding of VGLUT3's role in HD remains unclear. We report here that deletion of the Slc17a8 (Vglut3) gene rescues the deficits in both motor and cognitive functions in HD mice of both sexes. We also find that VGLUT3 deletion activates neuronal survival signaling and reduces nuclear aggregation of abnormal huntingtin proteins and striatal neuron loss in HD mice. Our novel findings highlight the vital contribution of VGLUT3 in HD pathophysiology that can be exploited for HD therapeutic management.

Keywords: Huntington's disease; glutamate; motor dysfunction; neurodegeneration, microglia.

Figure 1.

VGLUT3 loss improves novelty memory impairments in zQ175 Huntington's disease mice. Data are mean ± SEM of the recognition index for exploring a novel object versus a familiar object in novel object recognition test at (A) 6, (B) 9, (C) 12, and (D) 15 months of age in male and female WT and heterozygous zQ175 HD mice in the presence or absence of VGLUT3 expression (n = 10-13 mice per group). *p < 0.05 (two-way ANOVA followed by Tukey's post hoc test for multiple comparisons).

Figure 2.

Loss of VGLUT3 rescues motor coordination deficits in zQ175 mice. (A) Latency to fall from accelerating rotarod, (B) percentage of errors made on the horizontal ladder, and (C) forepaw grip strength (gram-force [gf]) of male and female WT and heterozygous zQ175 HD mice in the presence or absence of VGLUT3 tested at 6, 9, 12, and 15 months of age (n = 10-13 mice per group). *p < 0.05, significant difference compared with WT mice (two-way ANOVA followed by Dunnett's test for multiple comparisons).

Figure 3.

VGLUT3 loss in mice impairs anxiety-mediated locomotor performance in the open field test. (A) Percent time mobile and (B) frequency of entries to the center of open field box in male and female WT and heterozygous zQ175 HD mice in the presence or absence of VGLUT3 tested at 6, 9, 12, and 15 months of age (n = 10-13 mice per group). *p < 0.05, significant difference compared with zQ175 or VGLUT3^–/– mice (two-way ANOVA followed by Dunnett's test for multiple comparisons).

Figure 4.

Loss of VGLUT3 mitigates striatal neuronal loss in zQ175 mice. Representative images and quantification of neuronal nuclei (NeuN) immunostaining in the striatal regions of (A) male and (B) female WT and heterozygous zQ175 HD mice in the presence or absence of VGLUT3. Quantification of NeuN-labeled cells is presented as mean ± SEM and was done in 2 striatal (300 μm × 300 μm) regions from six brain slices per mouse (n = 5 for each group). Scale bar, 50 μm. *p < 0.05, significantly different from WT VGLUT3^+/+ values (two-way ANOVA followed by Tukey's post hoc test). ^#p < 0.05, significantly different from zQ175:VGLUT3^+/+ values (two-way ANOVA followed by Tukey's post hoc test).

Figure 5.

Loss of VGLUT3 activates ERK1/2 and Akt signaling in the zQ175 mouse striatum. Representative Western blots and quantification of ERK1/2-pT202/Y204 and Akt-pS473 levels with the corresponding total protein in the striatum of (A) male and (B) female WT and heterozygous zQ175 HD mice in the presence or absence of VGLUT3. ERK1/2-pT202/Y204 was normalized to total ERK1/2, and Akt-pS473 was normalized to total Akt (n = 5 for each group). Values are expressed as a fraction of mean VGLUT3^+/+ WT values. *p < 0.05, significantly different from WT VGLUT3^+/+ values (two-way ANOVA followed by Tukey's post hoc test). ^#p < 0.05, significantly different from zQ175:VGLUT3^+/+ values (two-way ANOVA followed by Tukey's post hoc test).

Figure 6.

Autophagy initiation is suppressed in zQ175 and zQ175:VGLUT3^–/– mouse striatum. Representative Western blots and quantification of ULK1-pS757 phosphorylation levels with the corresponding loading controls in the striatum of (A) male and (B) female WT and heterozygous zQ175 HD mice in the presence or absence of VGLUT3. ULK1-pS757 was normalized vinculin (n = 5 for each group). Values are expressed as a fraction of mean VGLUT3^+/+ WT values. *p < 0.05, significantly different from WT VGLUT3^+/+ values (two-way ANOVA followed by Tukey's post hoc test). ^#p < 0.05, significantly different from zQ175:VGLUT3^+/+ values (two-way ANOVA followed by Tukey's post hoc test).

Figure 7.

VGLUT3 deletion reduces mHTT nuclear accumulation with no impact on microgliosis in zQ175 mice. Representative images of mHTT staining using EM48 antibody in coronal whole-brain slice and magnified area of the striatum of (A) male and (B) female zQ175 and zQ175:VGLUT3^–/– mice. Quantification as mean ± SEM of the total number of EM48-positive aggregates and percentage of EM48-positive nuclei was done in 2 striatal regions (300 μm × 300 μm) from six brain slices per mouse (n = 5 for each group). Scale bars: whole slice, 500 μm; magnified area of the striatum, 20 μm. Statistical significance was assessed by unpaired Student's t test. Representative images of microglial Iba1 immunostaining in striatal regions of (C) male and (D) female WT and heterozygous zQ175 HD mice in the presence or absence of VGLUT3. Iba1-positive cell quantification is presented as mean ± SEM and was done in two striatal regions (300 μm × 300 μm) from six brain slices per mouse (n = 5 for each group). Scale bar, 50 μm. *p < 0.05, significantly different from WT VGLUT3^+/+ values (two-way ANOVA followed by Tukey's post hoc test).