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Name of the department: The First Department of Biochemistry, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi
The members of department: Shuichiro Maeda (Head of the department), Tadashi Mabuchi, Goro Kato, Keiko Takemoto
Name of the research projects:
1)Use of genetically altered mice to define factors implicated in amyloidogenesis (S. Maeda):
To facilitate defining factors that affect amyloid deposition and aid in developing effective treat￢ments, we used several genetically altered mice as follows.
(1) Using the serum amyloid P component (Sap, encoded by Apcs)-deficient mice, we showed that Sap significantly promotes the amyloid deposition. Thus, Sap may play an important role in the pathogenesis of human amyloidoses. However, no enhancement in the rate of regression of splenic AA amyloid was observed in the Sap-deficient mice relative to wild-type mice. We suggest that dissociation of bound Sap from amyloid deposits would not significantly accelerate regression of the deposits in vivo.
(2) We asked if administration of transthyretin (TTR)-amyloid fibrils (ATTR) extracted from the heart of a familial amyloidotic polyneuropathy (FAP) type I patient would accelerate ATTR deposition in transgenic mice expressing the human mutant TTR gene responsible for FAP type I and indeed the administration did accelerate deposition of apolipoprotein A-II- amyloid fibrils (AApoAII), and not ATTR. We suggest that intake of ATTR may not explain differences in the mean age at onset and the genetic anticipation which occurs in FAP.
(3) To elucidate the role of Ttr and Sap in the A? amyloid deposition, we generated mouse lines carrying a null mutation either at the endogenous Ttr or Apcs locus and human mutant amyloid precursor protein gene responsible for familial Alzheimer's disease.
(4) A Val 30 Met point mutation in TTR is associated with FAP. To establish
a better animal model of FAP, we generated a mouse line carrying the Val
30 Met point mutation in the endogenous Ttr gene through a novel gene targeting
procedure. The expression pattern of the mutated gene is equivalent to
that of the normal gene. The novel mouse model will add to the armamentarium
for evaluating the efficacy of gene therapy such as targeted gene correction
by chimeric RNA/DNA oligonucleotides..
Recent Publications:
Terazaki, H. et al., Immunization in familial amyloidotic polyneuropathy: counteracting deposition by immunization with a Y78F TTR mutant. Lab Invest 2006, 86: 23-31.
Tamaoki, T. et al., Avoiding the effect of linked genes is crucial to elucidate the role of Apcs in autoimmunity. Nature Medicine 2005, 11: 11-12.
Nakamura, M. et al., Targeted conversion of the transthyretin gene in vitro
and in vivo. Gene Ther 2004, 11: 838-846..
2) Functional and morphological analyses of mitochondria (T. Mabuchi):
(1) Elucidation of regulatory mechanisms of mitochondrial ATP synthase.
Mitochondria are essential organelle for eukaryotes, in which most of energy is produced by ATP synthase. While the enzyme activity is controlled in response to the energy demands of the cell, little is known about the regulation of the enzyme complex. Our main research focuses on the study of mechanisms that regulate the ATP synthase activity through the alpha subunit of the enzyme using yeast Saccharomyces cerevisiae as a model organism. We have already characterized several mutants and isolated extragenic suppressors of the mutations in the ATP1 gene (encoded for alpha subunit). We have revealed that one of the suppressors for the point mutant, atp1-2, was RAS2, a well known regulator of cell proliferation and signal transduction, indicating the presence of a relationship between a growth regulatory pathway involving RAS and mitochondrial energy transduction (Mabuchi et a., J. Biol. Chem., 275, 10492-10497, 2000). We are now investigating how RAS2 regulates ATP synthase activity, through the elucidation of suppression mechanisms of RAS2 on respiratory deficiency caused by the atp1-2 mutation.
Takeda M. et al., Current Genetics, 47, 265-272, 2005
Ohnishi K. et al., Yeast, 20, 943-954, 2003
Takeda M. et al.,Yeast, 15, 873-878, 1999
(2) Study on the morphological and functional changes of mitochondria
during spermatogenesis, fertilization and the early developments.
Mitochondria are also essential organelle for conservation of species in eukaryotes. We study on the morphological and functional changes of mitochondria during spermatogenesis, fertilization and the early developments using mouse, rat and human (Haraguchi S. et al., Biol. Reprod., 69, 885-895, 2003; Haraguchi H. et al., J. Histochem. Cytochem., 53, 455-465, 2005; Nagai S. et al., Tohoku J. Exp. Med., 210,137-44, 2006). This project is carried out by collaboration with Biology Laboratory and Department of Obstetrics and Gynecology.
Haraguchi M. et al., J. Histochem. Cytochem., 55, 2007
Nagai S. et al., Hum Cell, 17, 195-201,2004
Haraguchi M. et al, J. Histochem. Cytochem., 52, 1393-1403, 2004
Shoda, T. et al., In Proceedings of 9th International Symposium on Spermatology. ed. by Horst, G. V-. D., Franken, D., Bornman, R., Bornman, T., Dyer, S., pp.173-176, Monduzzi Editore, Bologna, Italy, 2002
Hirata S. et al., Reproductive Medicine and Biology, 1, 41-47, 2002
Mabuchi, T., Nishikawa, S. Electrophoresis, 21, 865-873,2000,erratum Electrophoresis, 21, 1631, 2000
Other Publications:
Mabuchi T. et al., J. Forensic Sci., 52, 355-363, 2007
Susukida R. et al., Electrophoresis, 28, 309-316, 2007
Kaneko N. et al., Neuropsychopharmacology, 31, 2619-2626, 2006
Watanabe A. et al., Journal of Neuro-Oncology, 77, 25-32, 2006
Satoh E. et al., J. Neurosurgery, 104, 264-271, 2006
Zhang L. et al., Neurol. Med. Chir. (Tokyo), 44, 637-645, 2004
Haraguchi M. et al., Acta Histochem. Cytochem., 36, 465-469, 2003
Haraguchi, M. et al., J. Histochem. Cytochem., 51, 215-216, 20
3) Functions of Cdk5-mediated phosphorylation of Src (G. Kato):.
(1)Src protein： Src is a membrane-associated 60-kDa tyrosine kinase,
which is involved in growth regulation and post-mitotic character. Src
is expressed ubiquitously in tissues and is highly expressed in neural
tissues such as brain and retina. Src activity is implicated in neurodegeneration
and tumorigenesis. However, a Src knockout mouse have not showed detectable
abnormalities in neural tissues and not provided a full understanding of
the physiological role of Src. This may reflect functional compensation
by other tyrosine kinases related to src. To overcome this limitation,
we introduced a point mutation into the src allele at a site connected
with regulation of specific function of Src as described below.
(2)Phosporylation of Src at Ser75 ： We have found that one of the mitotic phosphorylation sites in the unique domain of human SRC, Ser75, is phosphorylated in a mitosis-independent manner in human retinoblastoma cells. This phosphorylation occurs in some neuronal and non-neuronal cultured cells. We have previously showed that Cdk5 kinase and its activator, p35, is responsible for the novel phosphorylation in the retinoblastoma cells [1]. Cdk5/p35 kinase is expressed primarily in neurons, and in some non-neural tissues such as cornea, lens and muscles. We are now studying the biochemical functions of the Ser75 phosphorylation using cultured cells expressing mutant Src.
(3)Mutant mice： To clarify the physiological role of Src, we have
established two types of mutant mice: one with a Ser75 to Asp (SD) mutation,
mimicking the phosphorylated form and the other with a Ser75 to Ala (SA)
mutation, and lacking the phosphorylation site. These knock-in mice were
produced by our previously reported procedure for introducing a point mutation
into one allele [2]. These mice are expected to comparatively show detectable
abnormalities because of introducing a point mutation into the unique domain,
the sequences of which share no homology with those of other src family
kinases. We are now analyzing retinal neurodegeneration and corneal abnormality
of these mice.
1. Kato, G. and Maeda, S. (1999) J. Biochem. 126(5):957-961.
2. Kato, G. and Maeda, S. (2003) J. Biochem. 133(5): 563-569.
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