Yoshie Yamamoto

Chemical chaperone therapy for brain pathology in GM1-gangliosidosis

We synthesized a galactose derivative, N-octyl-4-epi-β-valienamine (NOEV), for a molecular therapy (chemical chaperone therapy) of a human neurogenetic disease, β-galactosidosis (GM1-gangliosidosis and Morquio B disease). It is a potent inhibitor of lysosomal β-galactosidase in vitro. Addition of NOEV in the culture medium restored mutant enzyme activity in cultured human or murine fibroblasts at low intracellular concentrations, resulting in a marked decrease of intracellular substrate storage. Short-term oral administration of NOEV to a model mouse of juvenile GM1-gangliosidosis, expressing a mutant enzyme protein R201C, resulted in significant enhancement of the enzyme activity in the brain and other tissues. Immunohistochemical stain revealed a decrease in the amount of GM1 and GA1 in neuronal cells in the fronto-temporal cerebral cortex and brainstem. However, mass biochemical analysis did not show the substrate reduction observed histochemically in these limited areas in the brain probably because of the brief duration of this investigation. Chemical chaperone therapy may be useful for certain patients with β-galactosidosis and potentially other lysosomal storage diseases with central nervous system involvement.

Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type I

Adult T-cell leukemia-lymphoma (ATLL) is a group of T-cell malignancies caused by infection with human T-lymphotropic virus type I (HTLV-I). Although the pathogenesis of ATLL remains incompletely understood, the viral regulatory protein Tax is centrally involved in cellular transformation. Here we describe the generation of HTLV-I Tax transgenic mice using the Lck proximal promoter to restrict transgene expression to developing thymocytes. After prolonged latency periods, transgenic mice developed diffuse large-cell lymphomas and leukemia with clinical, pathological and immunological features characteristic of acute ATLL. Transgenic mice were functionally immunocompromised and they developed opportunistic infections. Fulminant disease also developed rapidly in SCID mice after engraftment of lymphomatous cells from transgenic mice. Flow cytometry showed that the cells were CD4− and CD8−, but CD44+, CD25+ and cytoplasmic CD3+. This phenotype is indicative of a thymus-derived pre–T-cell phenotype, and disease development was associated with the constitutive activation of NF-κB. Our model accurately reproduces human disease and will provide a tool for analysis of the molecular events in transformation and for the development of new therapeutics.

Early death of mice cloned from somatic cells.

Here we report that the lifespan of mice cloned from somatic cells is significantly shorter than that of genotype- and sex-matched controls, most likely due to severe pneumonia and hepatic failure. This finding demonstrates the possibility of long-term deleterious effects of somatic-cell cloning, even after normal birth.

Effects of Donor Cell Type and Genotype on the Efficiency of Mouse Somatic Cell Cloning

Abstract Although it is widely assumed that the cell type and genotype of the donor cell affect the efficiency of somatic cell cloning, little systematic analysis has been done to verify this assumption. The present study was undertaken to examine whether donor cell type, donor genotype, or a combination thereof increased the efficiency of mouse cloning. Initially we assessed the developmental ability of embryos that were cloned from cumulus or immature Sertoli cells with six different genotypes (i.e., 2 × 6 factorial). Significantly better cleavage rates were obtained with cumulus cells than with Sertoli cells (P < 0.005, two-way ANOVA), which probably was due to the superior cell-cycle synchrony of cumulus cells at G0/G1. After embryo transfer, there was a significant effect of cell type on the birth rate, with Sertoli cells giving the better result (P < 0.005). Furthermore, there was a significant interaction (P < 0.05) between the cell type and genotype, which indicates that cloning efficiency is determined by a combination of these two factors. The highest mean birth rate (10.8 ± 2.1%) was obtained with (B6 × 129)F1 Sertoli cells. In the second series of experiments, we examined whether the developmental ability of clones with the wild-type genotype (JF1) was improved when combined with the 129 genotype. Normal pups were cloned from cumulus and immature Sertoli cells of the (129 × JF1)F1 and (JF1 × 129)F1 genotypes, whereas no pups were born from cells with the (B6 × JF1)F1 genotype. The present study clearly demonstrates that the efficiency of somatic cell cloning, and in particular fetal survival after embryo transfer, may be improved significantly by choosing the appropriate combinations of cell type and genotype.

BETA -GALACTOSIDASE-DEFICIENT MOUSE AS AN ANIMAL MODEL FOR GM1-GANGLIOSIDOSIS

GM1-gangliosidosis is a progressive neurological disease in humans caused by deficiency of lysosomal acid β-galactosidase, which hydrolyses the terminal β-galactosidic residue from ganglioside GM1 and other glycoconjugates. In this study, we generated a mouse model for GM1-gangliosidosis by gene targeting in embryonic stem cells. The mouse homozygous for the disrupted β-galactosidase gene showed β-galactosidase deficiency, presented with progressive spastic diplegia, and died of emaciation at 7–10 months of age. Pathologically, PAS-positive intracytoplasmic storage was observed in neuronal cells of various areas in the brain. Biochemical analysis revealed a marked accumulation of ganglioside GM1 and asialo GM1 in brain tissue. This animal model will be useful for pathogenetic analysis and therapeutic trial of human GM1-gangliosidosis.

Decreased Matrix Metalloproteinase Activity in the Kidneys of Hereditary Nephrotic Mice (ICGN Strain)

Abnormalities of extracellular matrix (ECM) metabolism, i.e., overproduction and/or inhibition of ECM breakdown, may contribute to progression of fibrotic degeneration in the kidney. Earlier studies revealed that major ECM components, type I, III, and IV collagens, etc., were accumulated in glomeruli and tubulointerstitium in kidneys of Institute of Cancer Research (ICR) derived glomerulonephritis (ICGN) mice which are a novel inbred strain of mice with a hereditary nephrotic syndrome of unknown etiology and are considered to be a good model of human idiopathic nephrotic syndrome. In the present study, we compared the activities of matrix metalloproteinases (MMPs), a family of enzymes that degrade ECM components, in the kidneys of aged ICGN mice and age-matched ICR mice as normal controls. We biochemically measured interstitial collagenase (MMP-1), gelatinase (MMP-2 and MMP-9), and stromelysin (MMP-3) activities in the kidney tissues. Lower activities of MMP-1 and MMP-2 and MMP-9 were demonstrated in the kidneys of ICGN mice as compared with those of ICR mice, but there were no significant differences in the MMP-3 activities between these strains. These results show that decreased MMP activities cause abnormal accumulation of ECM in ICGN mouse kidneys.

Hereditary nephrotic syndrome with progression to renal failure in a mouse model (ICGN strain): clinical study.

The clinical course of murine hereditary nephrotic syndrome (ICGN strain) was determined by examining 201 animals under different conditions. In the early stage, significant hypoproteinemia and hypoalbuminemia developed (p < 0.001) in parallel with a progressive rise in urinary protein concentration (p < 0.001). In the middle stage, the concentrations of total cholesterol, triglyceride, and beta-lipoprotein markedly increased (p < 0.01, p < 0.001, and p < 0.001, respectively), suggesting that type IIb hyperlipoproteinemia developed as in human nephrotic patients. Systemic edema appeared in 8 of 24 animals. In the terminal stage, both BUN and creatinine values greatly increased (p < 0.001), indicating rapid deterioration of renal function. The present study suggests that ICGN mice could be a useful model to study the pathophysiology of human nephrotic syndrome and its progression to renal failure.

Fertilization of Oocytes and Birth of Normal Pups Following Intracytoplasmic Injection with Spermatids in Mastomys (Praomys coucha)

Abstract The mastomys is a small laboratory rodent that is native to Africa. Although it has been used for research concerning reproductive biology, in vitro fertilization (IVF) and intracytoplasmic sperm injection are very difficult in mastomys because of technical problems, such as inadequate sperm capacitation and large sperm heads. The present study was undertaken to examine whether mastomys spermatids could be used to fertilize oocytes in vitro using a microinsemination technique, because spermatids are more easily injected than mature spermatozoa into oocytes. Most mastomys oocytes (80%–90%) survived intracytoplasmic injection with either round or elongated spermatids. Round spermatids had little oocyte-activating capacity, similar to those of mice and rats, and exogenous stimuli were needed for normal fertilization. Treatment with an electric pulse in the presence of 50 μM Ca2+ followed by culture in 10 mM SrCl2 led to successful oocyte activation. After injection of round spermatids into preactivated oocytes, 93% of oocytes were normally fertilized (male and female pronuclei formed), and 100% of cultured oocytes developed to the 2-cell stage. However, none reached term after transfer into recipient females. Elongated spermatids, which correspond to steps 9–11 in rats, activated oocytes on injection without additional activation treatment. After embryo transfer, five offspring (6% per transfer) developed to term. These results indicate that microinsemination with spermatids is a feasible alternative in animal species that are refractory to IVF and sperm injection and that using later-stage spermatids may lead to increased production of viable embryos that can develop into normal offspring.

Transforming growth factor-β1 mediated up-regulation of lysyl oxidase in the kidneys of hereditary nephrotic mouse with chronic renal fibrosis

Lysyl oxidase (LOX), an extracellular emzyme, plays a key role in the post-translational modification of collagens and elastin, catalyzing inter- and intra-crosslinking reactions. Because the crosslinked extracellular matrices (ECMs) are highly resistant to degradative enzymes, it is considered that the over-expression of LOX may cause severe fibrotic degeneration. In the present study, we addressed the role of LOX-mediated crosslinking in chronic renal tubulointerstitial fibrosis using an animal model of hereditary nephrotic syndrome, the Institute of Cancer Research (ICR)-derived glomerulonephritis (ICGN) mouse. Ribonuclease protection assay (RPA) revealed that LOX mRNA expression was up-regulated in the kidneys of ICGN mice as compared with control ICR mice. High-level expression of LOX and transforming growth factor (TGF)-β1 (an up-regulator of LOX) mRNA was detected in tubular epithelial cells of ICGN mouse kidneys by in situ hybridization. Type-I and -III collagens, major substrates for LOX, were accumulated in tubulointerstitium of ICGN mouse kidneys. The present findings imply that TGF-β1 up-regulates the production of LOX in tubular epithelial cells of ICGN mouse kidneys, and the excessive LOX acts on interstitial collagens and catalyzes crosslinking reactions. As a result, the highly crosslinked collagens induce an irreversible progression of chronic renal tubulointerstitial fibrosis in the kidneys of ICGN mice.

Augmented cytoplasmic Smad4 induces acceleration of TGF-β1 signaling in renal tubulointerstitial cells of hereditary nephrotic ICGN mice with chronic renal fibrosis; possible role for myofibroblastic differentiation

The Institute of Cancer Research (ICR)-derived glomerulonephritis (ICGN) mouse is a hereditary model animal for nephrotic syndrome with chronic renal tubulointerstitial fibrosis. In most fibrotic diseases, myofibroblastic differentiation is considered to play crucial roles in pathogenesis of fibrosis and is dominantly regulated by the transforming growth factor (TGF)-β1 signaling system. To reveal the pathogenic mechanism of chronic renal fibrosis in ICGN mice, we examined the expression and localization of TGF-β1 signal transducer proteins (TGF-β receptor-I and -II, Smad2/3 and Smad4) in kidney sections and in primarily cultured tubulointerstitial fibroblasts (TIFs). In kidneys of ICGN mice, many tubulointerstitial cells were differentiated to myofibroblastic cells and were α-smooth muscle actin (αSMA)-positive. The numbers of αSMA-positive TIFs prepared from kidneys of ICGN mice (ICGN-TIFs), but not those of ICR control mice (ICR-TIFs), increased during cell culture. No significant differences in production or activation of TGF-β1 between ICGN-TIFs and ICR-TIFs were seen by enzyme-linked immunosorbent assay. In vitro transcriptional reporter assay for TGF-β1 and Western immunoblotting for TGF-β1 signal transducers showed no notable differences in the expression levels of TGF-β receptor-I or -II or Smad2/3 between these TIFs. However, augumented cytoplasmic Smad4 protein in ICGN-TIFs, but not ICR-TIFs, seemed to cause hypersensitivity against TGF-β1, and the eventual nuclear localization of Smad2/3-Smad4 complex was increased in ICGN-TIFs. Thus, the abnormal cytoplasmic augmentation of Smad4 induces acceleration of TGF-β1 signaling in the renal tubulointerstitial cells of ICGN mice.