Loss of Deacetylation Activity of Hdac6 Affects Emotional Behavior in Mice

Acetylation is mediated by acetyltransferases and deacetylases, and occurs not only on histones but also on diverse proteins. Although histone acetylation in chromatin structure and transcription has been well studied, the biological roles of non-histone acetylation remain elusive. Histone deacetylase 6 (Hdac6), a member of the histone deacetylase (HDAC) family, is a unique deacetylase that localizes to cytoplasm and functions in many cellular events by deacetylating non-histone proteins including α-tubulin, Hsp90, and cortactin. Since robust expression of Hdac6 is observed in brain, it would be expected that Hdac6-mediated reversible acetylation plays essential roles in CNS. Here we demonstrate the crucial roles of Hdac6 deacetylase activity in the expression of emotional behavior in mice. We found that Hdac6-deficient mice exhibit hyperactivity, less anxiety, and antidepressant-like behavior in behavioral tests. Moreover, administration of Hdac6-specific inhibitor replicated antidepressant-like behavior in mice. In good agreement with behavioral phenotypes of Hdac6-deficient mice, Hdac6 dominantly localizes to the dorsal and median raphe nuclei, which are involved in emotional behaviors. These findings suggest that HDAC6-mediated reversible acetylation might contribute to maintain proper neuronal activity in serotonergic neurons, and also provide a new therapeutic target for depression

Kbus/Idr, a mutant mouse strain with skeletal abnormalities and hypophosphatemia: Identification as an allele of 'Hyp'

Abstract Background The endopeptidase encoded by Phex (phosphate-regulating gene with homologies to endopeptidases linked to the X chromosome) is critical for regulation of bone matrix mineralization and phosphate homeostasis. PHEX has been identified from analyses of human X-linked hypophosphatemic rickets and Hyp mutant mouse models. We here demonstrated a newly established dwarfism-like Kbus/Idr mouse line to be a novel Hyp model. Methods Histopathological and X-ray examination with cross experiments were performed to characterize Kbus/Idr. RT-PCR-based and exon-directed PCR screening performed to identify the presence of genetic alteration. Biochemical assays were also performed to evaluate activity of alkaline phosphatase. Results Kbus/Idr, characterized by bone mineralization defects, was found to be inherited in an X chromosome-linked dominant manner. RT-PCR experiments showed that a novel mutation spanning exon 16 and 18 causing hypophosphatemic rickets. Alkaline phosphatase activity, as an osteoblast marker, demonstrated raised levels in the bone marrow of Kbus/Idr independent of the age. Conclusions Kbus mice should serve as a useful research tool exploring molecular mechanisms underlying aberrant Phex-associated pathophysiological phenomena.</p

Abnormal emotional behaviors in <i>Hdac6</i> KO mice.

<p>(<b>A</b>) Hyperactivity of <i>Hdac6</i> KO mice in the open field test. Total distance traveled (left), an index of activity, and the number of entries into the center zone (right), an index of anxiety, are indicated (<i>n</i> = 24 and 23 for WT and <i>Hdac6</i> KO mice, respectively). (<b>B</b>) Anxiolytic-like behavior of <i>Hdac6</i> KO mice in the elevated plus-maze test. <i>Hdac6</i> KO mice showed increased number of entries into the open arms (OAs, left) and spent more time in OAs (middle) compared with WT mice, but total distance traveled, an index of activity, was not significantly different between genotypes (right; <i>n</i> = 24 and 23 for WT and KO, respectively). (<b>C</b>) Antidepressant-like behavior of <i>Hdac6</i> KO mice in the tail suspension test. <i>Hdac6</i> KO mice showed decreased immobility compared with WT mice (<i>n</i> = 12 for WT and KO mice). (<b>D</b>) Normal home cage activity of <i>Hdac6</i> KO mice. Home cage activity of KO mice in both light and dark periods was not distinguishable from that of WT mice (<i>n</i> = 5 and 6 for WT and KO, respectively). (<b>E</b>) Normal stress response of <i>Hdac6</i> KO mice. No genotype differences were found in serum corticosterone levels in both basal and stressful conditions (<i>n</i> = 9, 10, 10, and 10 for control WT, control KO, +stress WT, and +stress KO, respectively). (<b>F</b>) Effect of fluoxetine on the immobility of WT and <i>Hdac6</i> KO mice in the tail suspension test (<i>n</i> = 18, 18, 12, and 13 for saline WT, saline KO, fluoxetine WT, and fluoxetine KO, respectively).</p

Strong expression of Hdac6 in raphe nuclei.

<p>(<b>A–C</b>) Hdac6 distribution in mouse brain. Hdac6 protein is visualized by an antibody to Hdac6 (green: each right panel) in the coronal sections of adult mouse brain spanning raphe nuclei (<b>A</b>), hippocampus (<b>B</b>), and motor cortex (<b>C</b>). Nucleus is visualized by DAPI staining (each left panel). Arrowheads indicate Hdac6-positive cells magnified in insets. (<b>D</b>) Coexpression of Hdac6 with Tph2 in dorsal raphe neuron. The coronal brain sections of the dorsal raphe from WT (upper panels) and <i>Hdac6</i> KO mice (lower panels) were double immunostained for Hdac6 (red) and Tph2 (green), a marker of mature serotonin neuron. No signal was detected with anti-Hdac6 in <i>Hdac6</i> KO mice. Merged images were shown in right panels (blue: DAPI staining). (<b>E</b>) HDAC6 expression in human brainstem. HDAC6 is visualized by antibody to HDAC6 in the horizontal sections of the postmortem human brainstem spanning raphe nuclei, locus ceruleus, and substantia nigra. DR, dorsal raphe nucleus; MnR, median raphe nucleus; Aq, aqueduct. Scale bars, 100 µm.</p

Inhibition of Hdac6 deacetylase activity causes antidepressant-like effect.

<p>(<b>A</b>) A NCT-14b (14b) treatment of the HeLa cells increased the amount of acetylated-α-tubulin (Ac-α-Tub) but not that of histone H3. HDAC6 and actin were shown as loading controls. (<b>B</b>) Effect of NCT-14b on the immobility of WT and <i>Hdac6</i> KO mice in the tail suspension test (<i>n</i> = 14, 21, 16, and 16 for saline WT, saline KO, NCT-14b WT, and NCT-14b KO, respectively). (<b>C</b>) Effect of NCT-14b on α-tubulin acetylation in mouse brain. Amount of acetylated α-tubulin in the brain extract after the tail suspension test in (<b>B</b>) was determined by Western blotting. Lower panel shows quantification of Ac-α-Tub normalized by actin.</p