Huntingtin mediates anxiety/depression-related behaviors and hippocampal neurogenesis

Abstract

Huntington disease (HD) is associated with early psychiatric symptoms including anxiety and depression. Here, we demonstrate that wild-type huntingtin, the protein mutated in HD, modulates anxiety/depression-related behaviors according to its phosphorylation at serines 1181 and 1201. Genetic phospho-ablation at serines 1181 and 1201 in mouse reduces basal levels of anxiety/depression-like behaviors. We observe that the reduction in anxiety/depression-like phenotypes is associated with increased adult hippocampal neurogenesis. By improving the attachment of molecular motors to microtubules, huntingtin dephosphorylation increases axonal transport of BDNF, a crucial factor for hippocampal adult neurogenesis. Consequently, the huntingtin-mediated increased BDNF dynamics lead to an increased delivery and signaling of hippocampal BDNF. These results support the notion that huntingtin participates in anxiety and depression-like behavior and is thus relevant to the etiology of mood disorders and anxiety/depression in HD.

Figure 1.

Generation of Hdh^{S1181A/S1201A} and Hdh^{S1181D/S1201D} Mice. A, Schematic representation of the Hdh locus. B, Mouse HTT sequence for wild-type, Hdh^{S1181A/S1201A} and Hdh^{S1181D/S1201D} around the serines of interest. Data correspond to the noncoding strand. C, Representative PCR genotyping. Top, photo of a gel obtained after the migration of PCR products. hetero, heterozygous; homo, homozygous. Bottom, primers location and size of PCR products obtained for wild-type, Hdh^{S1181A/S1201A} on the left and Hdh^{S1181D/S1201D} on the right. D, HTT (4C8) immunoprecipitates (IP) of extracts from wild-type, Hdh^{S1181A/S1201A} and Hdh^{S1181D/S1201D} cortices are immunoblotted using anti-HTT (4C8), anti-P-HTT-S1181, and anti-P-HTT-S1201 antibodies.

Figure 2.

Hdh^{S1181A/S1201A} mice show reduced anxiety/depression-like behavior compared with Hdh^{S1181D/S1201D}and wild-type mice. A, Open Field test. Anxiety is measured as the mean of the total time spent in the center (ANOVA F_(2,146) = 12.505; ***p < 0.0001) and the distance in the center over total ambulatory distance (ANOVA F_(2,146) = 10.609; ***p < 0.0001). Representative trajectories followed by a mouse of each genotype during one session are shown in the three squares. B, Novelty suppressed feeding paradigm. The anxious/depressed-like phenotype of each group of mice is measured as the latency to feed (ANOVA F_(2,144) = 7.448; ***p < 0.001). The food consumption during a 5 min session is shown for each group of mice (ANOVA F_(2,144) = 0.104; p = 0.901). C, Splash test. The depression-related phenotype is measured by the grooming duration after 10% sucrose solution is sprayed on the mouse coat (ANOVA F_(2,144) = 4.491; *p < 0.05). All graphs: n > 40 mice per group; Values plotted are mean ±SEM. Significant ANOVAs were followed up with Fisher's PLSD test: NS, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3.

The behavioral effects observed in mice expressing unphosphorylated HTT are mediated by a neurogenesis-dependent mechanism. A, Quantification of the total number of TUNEL-positive cells in the dentate gyrus per mouse (n = 3) from each genotype (ANOVA F_(2,6) = 0.833; p = 0.4793). B, Protocol used to measure neuronal survival and representative immunostaining for BrdU-positive cells in the dentate gyrus of wild-type, Hdh^{S1181A/S1201A} and Hdh^{S1181D/S1201D} mice. Nuclei are counterstained with hematoxylin. Scale bar, 50 μm. The graph represents the quantitative assessment of the mean total number of BrdU-positive cells in the dentate gyrus per mouse (n ≥ 6) from each genotype (ANOVA F_(2,17) = 7.700; **p < 0.01). C, Quantification of the mean total number of DCX-positive neurons in the dentate gyrus per mouse (n = 4) from each genotype (ANOVA F_(2,9) = 0.297; p = 0.7498). D, Illustration of a DCX-positive neuron with quinary dendrites. White arrowheads point to dendritic nodes. Scale bar, 10 μm. Histograms show the quantification of the mean total number of neurons with quinary dendrite per mouse (n ≥ 3) from each genotype (ANOVA F_(2,8) = 8.848; **p < 0.01), and the maturation index (ANOVA F_(2,8) = 6.211; *p < 0.05). The maturation index is a normalization of the number of neurons with quinary dendrites over the total number of DCX-positive neurons. E, Sholl analysis of DCX-positive neurons. The graphs indicate the number of intersections (left graph) and the dendritic length (right graph) according to the distance from the soma for each condition (n = 3 per group). All graphs: Values plotted are mean ±SEM. Significant ANOVAs were followed up with Fisher's PLSD test; *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4.

BDNF expression and signaling is increased in Hdh^{S1181A/S1201A} hippocampi. A, BDNF total protein levels in the hippocampus of wild-type, Hdh^{S1181A/S1201A} and Hdh^{S1181D/S1201D} mice are measured by BDNF immunoenzyme assays (n ≥ 13 hippocampi per group; ANOVA F_(2,39) = 3.242; *p < 0.05). B, Hippocampal extracts from mice of the different genotypes were analyzed by immunoblotting for the presence of BDNF. The graph represents the quantitative assessment of the ratio of mature-BDNF/pro-BDNF (n = 3 hippocampi per group; ANOVA F_(2,6) = 12.500; **p < 0.01). C, Hippocampal extracts from mice (n = 3 hippocampi per group) of the different genotypes are analyzed by immunoblotting for the presence of phosphorylated-Erk (phospho-Ser133-Erk, pErk), Erk, phosphorylated-CREB (phospho-Thr202/Tyr204-CREB, pCREB), and CREB. The graphs represent the quantitative assessments of the ratio of activated Erk/CREB over total Erk/CREB (pErk/Erk ratio: ANOVA F_(2,6) = 7.410; *p < 0.05, and pCREB/CREB ratio: ANOVA F_(2,5) = 126.737; ***p < 0.001). D, bdnf exon IIA, IIB, IIC, and IV transcripts levels as determined by quantitative PCR analysis (n ≥ 6 hippocampi per group; exon IIA: ANOVA F_(2,15) = 0.137, p = 0.8730; exon IIB: ANOVA F_(2,15) = 0.47, p = 0.6336; exon IIC: ANOVA F_(2,15) = 0.257, p = 0.7769; exon IV: ANOVA F_(2,17) = 4.365, p = 0.0295). All graphs, Values plotted are mean ±SEM. Significant ANOVAs were followed up with Fisher's PLSD test; *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 5.

S1181A/S1201A-HTT promotes microtubule-based BDNF transport and release. A, Anterograde and retrograde transports of BDNF-mCherry-containing vesicles are analyzed in primary cultures of rat hippocampal neurons coelectroporated as indicated with constructs expressing the first N-terminal 1301 aa of wild-type HTT with HTT-1301 (HTT), HTT-1301-S1181A/S1201A (HTT-AA), or HTT-1301-S1181D/S1201D (HTT-DD), siRNA targeting HTT (siHTT) and BDNF-mCherry (vesicles number: n ≥ 42 for each condition; anterograde velocity: ANOVA F_(2,132) = 9.376; ***p < 0.001; retrograde velocity: ANOVA F_(2,132) = 7.613; ***p < 0.001 and pausing time: ANOVA F_(2,132) = 6.443; **p < 0.01). Data were obtained from three independent experiments. B, Transport-dependent BDNF release is evaluated after two KCl-induced depolarizations in hippocampal neurons coelectroporated as indicated with siRNA targeting HTT (siHTT) and HTT-1301 (HTT), or HTT-1301-S1181A/S1201A (HTT-AA) or HTT-1301-S1181D/S1201D (HTT-DD) and BDNF. Release is expressed as a K2/L (L, lysate) ratio (ANOVA F_(2,6) = 11,390; **p < 0.01). Data were obtained from three independent experiments. C, Anterograde and retrograde transports of BDNF-mCherry-containing vesicles are analyzed in primary cultures of cortical neurons from wild-type, Hdh^{S1181A/S1201A} and Hdh^{S1181D/S1201D} mice electroporated with BDNF-mCherry (vesicles number: wild-type n = 46; Hdh^{S1181A/S1201A} n = 59; Hdh^{S1181D/S1201D} n = 32; anterograde velocity: ANOVA F_(2,134) = 8.906; ***p < 0.001; retrograde velocity: ANOVA F_(2,134) = 4.735; *p < 0.05 and pausing time: ANOVA F_(2,134) = 4.872; **p < 0.01). Data were obtained from three independent experiments. D, Representative kymographs of BDNF-mCherry-containing vesicles dynamics. E, Transport-dependent BDNF release is evaluated after two KCl-induced depolarizations of cultured cortical neurons from wild-type, Hdh^{S1181A/S1201A}, and Hdh^{S1181D/S1201D} mice electroporated with BDNF. Release is expressed as a K2/L ratio (ANOVA F_(2,6) = 10.311; *p < 0.05). Data were obtained from three independent experiments. All graphs, Values plotted are mean ±SEM. Significant ANOVAs were followed up with Fisher's PLSD test; *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 6.

The S1181/S1201 phosphorylation status of HTT regulates motors and BDNF vesicles attachment to microtubules. A, MT fractionation of HeLa cells expressing HTT-1301 (HTT), HTT-1301-S1181A/S1201A (HTT-AA), or HTT-1301-S1181D/S1201D (HTT-DD). Samples are analyzed by immunoblotting for the presence of HTT, p150^Glued, and dynein (DIC, dynein intermediate chain). The graphs represent the ratios of HTT (ANOVA F_(2,12) = 4.335; *p < 0.05), p150^Glued (ANOVA F_(2,12) = 4.372; *p < 0.05), and DIC to tubulin (ANOVA F_(2,12) = 4.028; *p < 0.05). Values refer to band densities. S, supernatant; P, pellet corresponding to the MT-enriched fraction. Data are from five independent experiments. B, Cells express HTT-1301 (HTT), HTT-1301-S1181A/S1201A (HTT-AA), or HTT-1301-S1181D/S1201D (HTT-DD) and BDNF-mCherry. Cells are immunostained for the presence of BDNF (red) and α-tubulin (green). Scale bar, 5 μm and for the magnification: 1 μm. The graph represents the quantifications of BDNF-mCherry vesicles localized on MTs for each conditions (ANOVA F_(2,106) = 3234; *p < 0.05). Data are from three independent experiments. All graphs, Values plotted are mean ±SEM. Significant ANOVAs were followed up with Fisher's PLSD test; *p < 0.05, **p < 0.01, and ***p < 0.001.