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Muscle disuse induces atrophy through increased reactive oxygen species (ROS) released from damaged mitochondria. Mitophagy, the autophagic degradation of mitochondria, is associated with increased ROS production. However, the mitophagy activity status during disuse-induced muscle atrophy has been a subject of debate. Here, we developed a new mitophagy reporter mouse line to examine how disuse affected mitophagy activity in skeletal muscles. Mice expressing tandem mCherry-EGFP proteins on mitochondria were then used to monitor the dynamics of mitophagy activity. The reporter mice demonstrated enhanced mitophagy activity and increased ROS production in atrophic soleus muscles following a 14-day hindlimb immobilization. Results also showed an increased expression of multiple mitophagy genes, including Bnip3, Bnip3l, and Park2. Our findings thus conclude that disuse enhances mitophagy activity and ROS production in atrophic skeletal muscles and suggests that mitophagy is a potential therapeutic target for disuse-induced muscle atrophy.

Parkinson's disease is characterized by loss of nigral dopaminergic neurons and presence of Lewy bodies, whose major component is alpha-synuclein. In the present study, we generated transgenic mice termed Syn130m that express truncated human alpha-synuclein (amino acid residue number: 1-130) in dopaminergic neurons. Notably, dopaminergic neurons were selectively diminished in the substantia nigra pars compacta of Syn130m, while transgenic mice that expressed comparable amount of full-length human alpha-synuclein did not develop such pathology. Therefore, the truncation of human alpha-synuclein seems to be primarily responsible for the loss of nigral dopaminergic neurons. The nigral pathology resulted in impairment of axon terminals in the striatum and concomitant decrease in striatal dopamine content. Behaviorally, spontaneous locomotor activities of Syn130m were reduced, but the abnormality was ameliorated by treatment with L-DOPA. The loss of nigral dopaminergic neurons was not progressive and seemed to occur during embryogenesis along with the onset of expression of the transgene. Our results indicate that truncated human alpha-synuclein is deleterious to the development and/or survival of nigral dopaminergic neurons.

SummaryCytokines may be crucially involved in the pathogenesis of inflammatory bowel diseases (IBD), but it remains controversial whether interferon (IFN)-γ, a typical proinflammatory cytokine, is an essential mediator to cause the disorders. In the present study, IFN-γ–/– and wild-type (WT) C57BL/6 mice were fed 2·5% dextran sodium sulphate (DSS) in drinking water for 7 days, in order to investigate DSS-induced intestinal inflammation. The DSS-treated WT mice exhibited a robust production of IFN-γ in the gut, a remarkable loss of body weight, as well as high rate of mortality (60%). In striking contrast, IFN-γ deficient mice did not develop DSS-induced colitis, as indicated by the maintenance of body weight and survival rate of 100%. Severe intestinal inflammation was demonstrated exclusively in WT animals in terms of the shortening of the bowel as well as the elevation of the disease activity index, myeloperoxidase (MPO) activity and serum haptoglobin level. Histological study of DSS-treated WT intestine revealed disruption of mucosal epithelium and massive infiltration of inflammatory cells, while the organ from IFN-γ–/– mice remained virtually normal in appearance. Enzyme-linked immunosorbent assay (ELISA) analyses indicated abundant production of three chemokines, i.e. monokine induced by interferon-γ (MIG), interferon-inducible protein 10 (IP-10) and monocyte chemoattractant protein-1 (MCP-1), in the DSS-irritated intestine of WT but not of IFN-γ–/– mice. The present results demonstrate clearly that IFN-γ plays indispensable roles in the initiation of DSS colitis, and some chemokines are produced in an IFN-γ-dependent fashion.

Meltrin alpha (ADAM12) is a metalloprotease-disintegrin whose specific expression patterns during development suggest that it is involved in myogenesis and the development of other organs. To determine the roles Meltrin alpha plays in vivo, we generated Meltrin alpha-deficient mice by gene targeting. Although the number of homozygous embryos are close to the expected Mendelian ratio at embryonic days 17 to 18, ca. 30% of the null pups born die before weaning, mostly within 1 week of birth. The viable homozygous mutants appear normal and are fertile. Most of the muscles in the homozygous mutants appear normal, and regeneration in experimentally damaged skeletal muscle is unimpeded. In some Meltrin alpha-deficient pups, the interscapular brown adipose tissue is reduced, although the penetrance of this phenotype is low. Impaired formation of the neck and interscapular muscles is also seen in some homozygotes. These observations suggest Meltrin alpha may be involved in regulating adipogenesis and myogenesis through a linked developmental pathway. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a candidate substrate of Meltrin alpha, and we found that TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ectodomain shedding of HB-EGF is markedly reduced in embryonic fibroblasts prepared from Meltrin alpha-deficient mice. We also report here the chromosomal locations of Meltrin alpha in the mouse and rat.

Caspase-8 plays the role of initiator in the caspase cascade and is a key molecule in death receptor-induced apoptotic pathways. To investigate the physiological roles of caspase-8 in vivo, we have generated caspase-8-deficient mice by gene targeting. The first signs of abnormality in homozygous mutant embryos were observed in extraembryonic tissue, the yolk sac. By embryonic day (E) 10.5, the yolk sac vasculature had begun to form inappropriately, and subsequently the mutant embryos displayed a variety of defects in the developing heart and neural tube. As a result, all mutant embryos died at E11.5. Importantly, homozygous mutant neural and heart defects were rescued by ex vivo whole-embryo culture during E10.5-E11.5, suggesting that these defects are most likely secondary to a lack of physiological caspase-8 activity. Taken together, these results suggest that caspase-8 is indispensable for embryonic development.

Parkinson's disease is neuropathologically characterized by the presence of Lewy bodies, whose major component is alpha-synuclein. We had previously generated transgenic mice that expressed human alpha-synuclein carrying an Ala53Thr point mutation (halpha-syn140m) under the control of the rat tyrosine hydroxylase promoter and found that halpha-syn140m was localized not only in the cytoplasm but also in the nuclei of mesencephalic dopaminergic neurons. In the present study, we carried out immunohistochemical analysis of the brain of Tg mice using anti-PSer129, an antibody that specifically recognizes alpha-synuclein phosphorylated at Ser129. The antibody detected only phosphorylated halpha-syn140m, whereas phosphorylation of endogenous alpha-synuclein, if any, was below the detection limit of the method employed. The analysis showed that approximately one-third of the halpha-syn140m-positive neurons in the midbrain of heterozygous Tg mice were concomitantly reactive to anti-PSer129. The ratio almost doubled in homozygotes, indicating that the phosphorylation level depends directly on the amount of substrate. In addition, the ratio did not change at least up to 48 weeks of age. These data strongly suggest that halpha-syn140m underwent constitutive phosphorylation and that the phosphorylation level was maintained to a certain level until the aged stages. Remarkably, halpha-syn140m localized in the nuclei seemed to be preferentially phosphorylated compared with that in the cytoplasm. Among kinases that have been reported to be involved in the phosphorylation of alpha-synuclein, the beta subunit of casein kinase-2 was detected in the nuclei by immunohistochemistry. These data imply that at least casein kinase-2 is involved in the phosphorylation of halpha-syn140m in the Tg mice.

The HNK-1 carbohydrate epitope, a sulfated glucuronic acid at the non-reducing terminus of glycans, is expressed characteristically on a series of cell adhesion molecules and is synthesized through a key enzyme, glucuronyltransferase (GlcAT-P). We generated mice with a targeted deletion of the GlcAT-P gene. The GlcAT-P -/- mice exhibited normal development of gross anatomical features, but the adult mutant mice exhibited reduced long term potentiation at the Schaffer collateral-CA1 synapses and a defect in spatial memory formation. This is the first evidence that the loss of a single non-reducing terminal carbohydrate residue attenuates brain higher functions.

Six family genes are homologues of Drosophila melanogaster sine oculis, one of the homeobox genes essential for compound eye formation (5). They are characterized by the presence of the Six domain and Six-type homeodomain in the encoded proteins, which confer specific DNA binding activity and function as transcription factors (12, 28). In mammals, six members of the family have so far been identified (2–4, 7, 8, 10, 12, 13, 31, 32, 34, 41; for a review, see reference 14). Each member of the family is expressed in a spatiotemporally regulated manner during embryogenesis. In mice, Six3 and Six6 are exclusively expressed in the developing forebrain and eyes (10, 22, 31, 40). In contrast, Six1, Six2, and Six5 show relatively broader expression patterns (15, 32). Six1 is expressed in the cranial and dorsal root ganglia, somites, otic and nasal placodes, branchial arches, limbs, Rathke's pouch, and nephrogenic cords (32). Six2 is expressed in head mesenchyme, branchial arches, limbs, and some mesenchymal regions surrounding the gastrointestinal tract (32). Six5 shows a broad expression in branchial arches, limb buds, telencephalon, eye, sclerotomes, and cartilages (15). Such distribution suggests that these genes play specific roles in embryogenesis. Overexpression and misexpression experiments showed that Six3 and Six6 genes are involved in forebrain and eye organogenesis (18, 21, 30, 48). Consistently, SIX3 mutations cause holoprosencephaly, a severe malformation of the brain in humans (42). SIX5 is located immediately downstream of the CTG trinucleotide repeats whose expansion causes myotonic dystrophy (3). Downregulation of the gene was observed in myotonic dystrophy patients and is thought to be responsible for some of the symptoms of the disease such as cataracts (16, 44). In agreement with this, Six5-heterozygous and -homozygous mutant mice develop cataracts (15, 35). Six4 was originally identified as a binding factor to the positive regulatory element of the Na+,K+-ATPase α1 subunit gene (Atp1a1) (12, 38). Immunostaining showed the presence of Six4 protein in some populations of neuronal cells in developing mouse embryos and developing retina (27, 29). In adult mice, Six4 protein is localized in skeletal muscles, as demonstrated by gel retardation assay (37). These observations suggest that this gene is involved in neurogenesis, myogenesis, and probably the development of other organs. In myogenesis, myogenin plays an important role along with other myogenic genes such as MyoD, Myf5, and MRF4 (33). Promoter analysis with transgenic mice demonstrated that the MEF3 site in the promoter region is essential for myogenin expression (37). Mouse Six1 and Six4 proteins are present in the developing somites in which myogenin expression is activated and bind to the MEF3 site in the myogenin promoter, as shown by gel retardation assay (37). Furthermore, Six4 can activate the myogenin gene promoter alone or in synergy with the specific cofactor, Eya, through direct binding to the MEF3 site in cultured cells (28). These results support the notion that Six4 is one of the genes that control myogenesis through activation of myogenin. In addition, Eya1, one of the specific cofactors of Six4, is implicated in branchio-oto-renal syndrome, a dominantly inherited disorder characterized by hearing loss and branchial arch and renal anomalies in humans (1). Eya1-deficient mice lack ears and kidneys, and heterozygous mutant mice show hearing loss and renal anomalies, as seen in human branchio-oto-renal syndrome (46). Because immunostaining showed the presence of Six4 protein in acoustic ganglia and otic vesicles (29), it is possible that Six4 is involved in the development of the ear in association with Eya1. Nevertheless, the biological function of Six4 in development is not clear, due to the lack of natural mutants, knockout models, and overexpression experiments. To access the biological function of Six4, we generated Six4-deficient mice and analyzed phenotypic changes both in adults and in embryos. We found no apparent changes in morphology and expression patterns of some marker genes in Six4-deficient mice. Functionally, hearing ability was normal. The reason for the lack of phenotype in Six4-deficient mice and the possible compensation among Six family genes is discussed.

To study the mutually exclusive expression of odorant receptor (OR) genes, we generated transgenic mice that carried the murine OR gene MOR28. Expression of the transgene and the endogenous MOR28 was distinguished by using two different markers, beta-galactosidase and green fluorescent protein (GFP), respectively. Double staining of the olfactory epithelium revealed that the two genes were rarely expressed simultaneously in individual olfactory neurons. A similar exclusion was also observed between differently tagged but identical transgenes integrated into the same locus of one particular chromosome. Although allelic inactivation has been reported for the choice between the maternal and paternal alleles, this is the first demonstration of mutually exclusive activation among non-allelic OR gene members with identical coding and regulatory sequences. Such an unusual mode of gene expression, monoallelic and mutually exclusive, has previously been shown only for the antigen-receptor genes of the immune system.

The cell-surface molecule Cd9, a member of the transmembrane-4 superfamily, interacts with the integrin family and other membrane proteins. and is postulated to participate in cell migration and adhesion. Expression of Cd9 enhances membrane fusion between muscle cells and promotes viral infection in some cells. Fertilization also involves membrane fusion, between gametes. In mammals, the sperm binds to microvilli on the egg surface, and sperm-egg membrane fusion first occurs around the equatorial region of the sperm head12. The fused membrane is then disrupted, and the sperm nucleus as well as the cytoplasm is incorporated into the egg. Cd9 is expressed on the plasma membrane of the mouse egg, and an anti-Cd9 monoclonal antibody inhibits sperm-egg surface interactions. We generated Cd9 mice and found that homozygous mutant females were infertile. Sperm-egg binding was normal, but sperm-egg fusion was almost entirely inhibited in eggs from Cd9 females. Intracellular Ca2 oscillations, which signal fertilization, were absent in almost all mutant eggs; in rare cases, a response occurred after a long time period. In normal animals, Cd9 molecules were expressed on the egg microvilli and became densely concentrated at the sperm attachment site. Thus, our results show that Cd9 is important in the gamete fusion process at fertilization.

Leukocyte cell-derived chemotaxin 2 (LECT2) was originally identified for its possible chemotactic activity against human neutrophils in vitro. It is a 16-kDa protein that is preferentially expressed in the liver. Its homologues have been widely identified in many vertebrates. Current evidence suggests that LECT2 may be a multifunctional protein like cytokines. However, the function of LECT2 in vivo remains unclear. To elucidate the role of this protein in vivo, we have generated LECT2-deficient (LECT2−/−) mice. We found that the proportion of NKT cells in the liver increased significantly in LECT2−/− mice, although those of conventional T cells, NK cells, and other cell types were comparable with those in wild-type mice. Consistent with increased hepatic NKT cell number, the production of IL-4 and IFN-γ was augmented in LECT2−/− mice upon stimulation with α-galactosylceramide, which specifically activates Vα14 NKT cells. In addition, NKT cell-mediated cytotoxic activity against syngeneic thymocytes increased in hepatic mononuclear cells obtained from LECT2−/− mice in vitro. Interestingly, the hepatic injury was exacerbated in LECT2−/− mice upon treatment with Con A, possibly because of the significantly higher expression of IL-4 and Fas ligand. These results suggest that LECT2 might regulate the homeostasis of NKT cells in the liver and might be involved in the pathogenesis of hepatitis.