Atsuko Hanai

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.

Possible involvement of myosin-X in intercellular adhesion: Importance of serial pleckstrin homology regions for intracellular localization

Subcellular fractionation experiments with mouse hepatocytes, combined with sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (PAGE)–immunoblot analysis using antibodies against two different tail regions of mouse myosin‐X demonstrated a 240 kDa molecular mass to be associated with the plasma membrane‐rich P2 fraction. The basolateral plasma membrane fraction, but not the brush border fraction, isolated from renal cortices also contained the 240 kDa form of myosin‐X. In an attempt to assess relative contributions of possible functional domains in the tail of myosin‐X to localization and function, cDNA corresponding to all three pleckstrin homology (PH) domains and different regions (PH1, 2 and 3, and the two subdomains of PH1: PHS1 and PHS2), as well as the myosin tail homology 4 domain (MyTH4) and the band4.1/ezrin/radixin/moesin‐like domain (FERM) were separately inserted into the pEGFP vector and expressed in cultured COS‐1 cells. As a result, two distinct regions responsible for localization were identified with regard to PH: one covers all three forms that tends to localize to regions of dynamic actin, such as membrane ruffles, lamellipodia and thick cortical actin bundles at the sites of cell–cell adhesion in a Rac‐ and Cdc42‐dependent manner. The other covers PHS1 and PH2 that localizes to filopodia, filopodial puncta and the sites of intercellular adhesion in a Cdc42‐dependent manner. Expression of green fluorescent protein (GFP)‐MyTH4 fusion protein resulted in formation of phalloidin‐positive granules, while GFP‐FERM affected the actin cytoskeletal system in a distinctly different way. Taken altogether, the results lend support to the view that myosin‐X is involved in cell–cell adhesion‐associated signaling‐linked membrane and/or cytoskeleton reorganization.

Chromosomal localization of a gene responsible for vestibulocochlear defects of BUS/Idr mice: identification as an allele of waltzer.

Mice of the bustling mutant strain BUS/Idr have vestibulocochlear defects. bus/bus homozygotes, but not heterozygotes, are hyperactive and display an abnormal behavior such as circling, head bobbing and head tilting. To characterize BUS mice further, the auditory brain-stem response of the mutant was examined. In +/bus heterozygotes as well as control animals, the auditory brain-stem response was developmentally first recorded as early as 11 days of age and heterozygous and normal adults showed typical auditory brain-stem responses with five peaks in a threshold of 40-45 dB SPL. In contrast, bus/bus homozygotes showed no auditory brain-stem response at any age in response to stimuli up to 130 dB SPL, indicating that they are deaf throughout life. Linkage analysis revealed that the responsible gene, originally designated as bus, maps on chromosome 10, 1.09+/-0.9 cM distal to D10Mit127 and D10Mit59, and 0.72+/-0.51 cM proximal to three markers, D10Mit48, D10Mit112 and D10Mit258, at a site indistinguishable from that of the Albany waltzer, v(A/b). The results of allelism tests between BUS and Albany waltzer indicated that bus is allelic with v(Alb). From these data, we propose here that the bus mutation could represent another allele of waltzer, now designated v(bus).

BUS/ldr, a Mutant Mouse Strain Exhibiting Abnormal Behaviors: Behavioral Similarities of BUS Mice and Chemically Labyrinthectomized Mice

The behavioral abnormalities of homozygotes (bus/bus) of mutant bustling mice, BUS/ldr, were characterized in comparison with those of heterozygotes (+/bus) and of chemically labyrinthectomized mice. Homozygotes were 4‐fold more active than heterozygotes, as evaluated in an open field (11 × 17 cm) for 24 hr by means of Animex. Homozygotes revealed the lack of righting reflex and of head nystagmus on a rotating board, the frequent backward‐moving and the inability to swim, in addition to characteristic behaviors such as circling, head bobbing and head tossing. Bilaterally labyrinthectomized mice behaviorally mimicked BUS homozygotes: The behavioral patterns of operated animals were essentially the same as those of BUS homozygotes with respect to all reflex and locomotor indices employed, suggesting that some peripheral vestibular dysfunction, though unidentified yet, is attributed to abnormal behaviors of BUS mice. Unilaterally labyrinthectomized mice were not hyperactive, nor exhibited circling behavior.

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

BackgroundThe 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.MethodsHistopathological 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.ResultsKbus/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.ConclusionsKbus mice should serve as a useful research tool exploring molecular mechanisms underlying aberrant Phex-associated pathophysiological phenomena.

A new allele at the lpr gene on mouse Chromosome 19 expresses properties different from the original recessive mutation

aDepartment of Biochemistry, Institute for Developmental Research, Aichi Prefecture Colony, Kamiyacho, Kasugai, Aichi 480-03, Japan 2Laboratory of Experimental Animals, Yagi Memorial Park, Mitake, Gifu 505-01, Japan 3Research Laboratories, Nippon Shinyaku Co., Sakanotsujicbo, Oyake, Yamashinaku, Kyoto 607, Japan 4Laboratory Animal Research Center, Institute of Medical Science, University of Tokyo, Tokyo 108, Japan

Defective Myosin Genes in Mutant Mice and Human Diseases

Myosins are highly divergent actin‐based molecular motors. In five of eight classes expressed in mammals, defects in genes have been identified in mutant mice and/or human diseases. A mutated myosin II‐7 gene is one of the causes of human familial hypertrophic cardiomyopathy (FHC). The defective myosin Va gene is responsible for Griscelli disease, which is characterized by partial albinism and immunodeficiency, while in its mouse homologue coat color dilution is seen with or without neurological defects. There are three classes of myosins, VI, VII and XV, that are essential in the inner ear function. In humans, mutations in the VIIa gene are associated with three deafness‐related diseases, Usher 1B/DFNB2/DFNA11, providing the first example of exhibition of recessive‐ and dominant‐inherited disorders by different mutations in a single myosin gene.