[Background and Recent Research Activities]

Our group is involved in development of therapy against intractable diseases including neuromuscular diseases such as muscular dystrophy and related disorders, psychiatric disorders, diabetes and cancers. Development of rational molecular therapy against muscular dystrophy and related disorders is one of the main projects. We are also interested in understanding the molecular mechanism of differentiation of adipocytes from mesenchymal progenitors. We have a number of up-to-date technologies to study intractable diseases, such as fluorescence activated cell sorting (FACS) and differentiation culture, proteomics analyses and nanomedicine. These technologies are indispensable for interdisciplinary cooperation among medical and clinical sciences, pharmaceutical science and material science. Our major research activities were summarized bellow.

I. Development of therapy for muscular dystrophy and related diseases

Muscular dystrophy is a prototype of intractable neuromuscular diseases. In spite of progress of the characterization of responsible genes such as dystrophin, calpain-3, and caveolin3, no therapy has not been realized yet. In addition to exon skipping of dystrophin genes, increasing skeletal mass is a promising therapeutic strategy. Myostatin, a member of the TGF-β superfamily is regarded as a novel drug target for muscular disorders. We have developed a number of effective myostatin inhibitors, and made transgenic mice expressing these myostatin inhibitors. We have shown that myostatin inhibitors are effective to increase skeletal muscle mass both by hypertrophy and hyperplasia. Transgenic expression of developed myostatin inhibitor prevents muscle degeneration and atrophy of mdx of Duchenne muscular dystrophy model mice and several other muscular dystrophy models (Faseb J 2008; Acta Myologica 2008; Cell Communication and Signaling 2009). We are currently characterizing the mechanism how myostatin inhibition leads to skeletal myogenesis by miRNA analysis and proteome analysis. Furthermore, we found that both visceral and subcutaneous adipose tissue masses decrease in myostatin inhibitor transgenic mice. Studies by electron microscopes indicate that adipocytes both in WAT and BAT are smaller in size in transgenic mice compared with control mice. Interestingly, we also found that numbers of mitochondria in adipocytes are increased by myostatin inhibition. Expression levels of acetyl-CoA carboxylase and stearoyl-CoA desaturase 1, essential enzymes for fatty acid synthesis were reduced in adipose tissues of transgenic mice. Transgenic mice with myostatin inhibition showed resistance to high fat diet-induced adiposity and fatty liver. Our data indicates that inhibition of myostatin leads to reduction of essential enzymes for lipid metabolism, which explains one mechanism of reduced adiposity by myostatin inhibition.

II. Characterization of mesenchymal progenitors involved in adipogenesis in skeletal muscle

To understand the mechanism how adipocytes are generated from mesenchymal progenitors is important for therapy for diabetes and obesity.

Using sophisticated cell sorting and culture system, we prospectively identified PDGFRα+(Pα+) mesenchymal progenitors, distinct from muscle satellite cells, in the muscle interstitium. We show that, of the muscle-derived cell populations, only Pα+cells exhibit efficient adipogenic differentiation both in vitro and in vivo. Reciprocal transplantations between regenerating and degenerating muscles showed that in glycerol-induced fatty degeneration condition, transplanted Pα+cells became adipocyte in vivo. Pα+cells are likely the major contributor to ectopic fat formation in skeletal muscle. Co-culture experiments reveal that adipogenesis of Pα+cells is strongly inhibited by the presence of satellite cell-derived myofibers. Pα+cells proliferate and may induce myogenesis in a microenvironment-dependent manner, and determine the balance between myogenesis and adipogenesis(Nature Cell Biology 2010).

III. Development of novel model animals for memory formation and neuropsychotic diseases

Activin A, a member of the TGF-β superfamily, is increased in activated neuronal circuits and regulates dendritic spine morphology and protects neuron from ischemic damage. To clarify the role of activin in the synaptic plasticity of adult brain, we examined the effect of inhibiting or enhancing activin function on hippocampal LTP. We found that follistatin, an inhibitor of activin, blocked the maintenance of Late-LTP (L-LTP) in the hippocampus. In contrast, administration of activin facilitated the maintenance of early LTP. We generated forebrain-specific activin- or follistatin-transgenic mice in which transgene expression is under the control of Tet-OFF system. Maintenance of hippocampal L-LTP was blocked in the follistatin-transgenic mice. In the contextual fear conditioning test, we found that follistatin blocked the formation of long-term memory without affecting short-term memory. Furthermore, consolidated memory was selectively weakened by the expression of follistatin during retrieval, but not during the maintenance phase. On the other hand, the maintenance of memory was also influenced by activin overexpression during the retrieval phase. Thus, the level of activin in the brain during the retrieval phase plays a key role in the maintenance of long-term memory. We also established forebrain-specific activin or follistatin expressing me.

Behavioral analyses revealed that follistatin-overexpressing mice showed enhanced anxiety, whereas activin-overexpressing mice exhibited reduced anxiety. In follistatin overexpressors, survival of newly-formed neurons was significantly reduced. The established mouse models would be useful to study novel molecular mechanism to explore memory formation and anxiety disorders (PLos One 2008; Learning & Memory 2010).

IV. Nanomedicine and cancer therapy

Nanotechnology is one of the hopeful technologies for development of drug delivery systems. We are working on nanoparticles as drug carriers and development of novel drug delivery systems for anticancer therapies in vivo. Water-dispersed carbon nanohorns and cell penetrating lipoproteins were prepared. They were intratumorally administered to cancer cell bearing mice. When intratumorally injected, developed nanoparticles with anticancer drugs caused significant growth retardation of tumor growth associated with drug retention in the tumor. We found that carbon nanoparticles migrate to lymph node. Lymph node metastasis could be monitored by using nanoparticles (PNAS 2008; nanomedicine 2010).

V. Cooperative studies with joint research laboratories

Our studies are conducted with help of joint research laboratories in Fujita Health University.

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