Yu Tezuka

Phenotype of cardiomyopathy in cardiac-specific heat shock protein B8 K141N transgenic mouse.

Background: A lys141Asn (K141N) missense mutation in heat shock protein (HSP) B8 causes distal hereditary motor neuropathy (HMN). Results: HSPB8 K141N transgenic mice exhibited mild hypertrophy and apical fibrosis as well as slightly reduced cardiac function. Conclusion: A single point mutation of HSPB8, such as K141N, can cause cardiac disease. Significance: The cardiomyopathy phenotype was observed in cardiac-specific HSPB8 K141N transgenic mice. A K141N missense mutation in heat shock protein (HSP) B8, which belongs to the small HSP family, causes distal hereditary motor neuropathy, which is characterized by the formation of inclusion bodies in cells. Although the HSPB8 gene causes hereditary motor neuropathy, obvious expression of HSPB8 is also observed in other tissues, such as the heart. The effects of a single mutation in HSPB8 upon the heart were analyzed using rat neonatal cardiomyocytes. Expression of HSPB8 K141N by adenoviral infection resulted in increased HSPB8-positive aggregates around nuclei, whereas no aggregates were observed in myocytes expressing wild-type HSPB8. HSPB8-positive aggresomes contained amyloid oligomer intermediates that were detected by a specific anti-oligomer antibody (A11). Expression of HSPB8 K141N induced slight cellular toxicity. Recombinant HSPB8 K141N protein showed reactivity against the anti-oligomer antibody, and reactivity of the mutant HSPB8 protein was much higher than that of wild-type HSPB8 protein. To extend our in vitro study, cardiac-specific HSPB8 K141N transgenic (TG) mice were generated. Echocardiography revealed that the HSPB8 K141N TG mice exhibited mild hypertrophy and apical fibrosis as well as slightly reduced cardiac function, although no phenotype was detected in wild-type HSPB8 TG mice. A single point mutation of HSPB8, such as K141N, can cause cardiac disease.

Regulation of Neurite Growth by Inorganic Pyrophosphatase 1 via JNK Dephosphorylation

Neural cell differentiation during development is controlled by multiple signaling pathways, in which protein phosphorylation and dephosphorylation play an important role. In this study, we examined the role of pyrophosphatase1 (PPA1) in neuronal differentiation using the loss and gain of function analysis. Neuronal differentiation induced by external factors was studied using a mouse neuroblastoma cell line (N1E115). The neuronal like differentiation in N1E115 cells was determined by morphological analysis based on neurite growth length. In order to analyze the loss of the PPA1 function in N1E115, si-RNA specifically targeting PPA1 was generated. To study the effect of PPA1 overexpression, an adenoviral gene vector containing the PPA1 gene was utilized to infect N1E115 cells. To address the need for pyrophosphatase activity in PPA1, D117A PPA1, which has inactive pyrophosphatase, was overexpressed in N1E115 cells. We used valproic acid (VPA) as a neuronal differentiator to examine the effect of PPA1 in actively differentiated N1E115 cells. Si-PPA1 treatment reduced the PPA1 protein level and led to enhanced neurite growth in N1E115 cells. In contrast, PPA1 overexpression suppressed neurite growth in N1E115 cells treated with VPA, whereas this effect was abolished in D117A PPA1. PPA1 knockdown enhanced the JNK phosphorylation level, and PPA1 overexpression suppressed it in N1E115 cells. It seems that recombinant PPA1 can dephosphorylate JNK while no alteration of JNK phosphorylation level was seen after treatment with recombinant PPA1 D117A. Enhanced neurite growth by PPA1 knockdown was also observed in rat cortical neurons. Thus, PPA1 may play a role in neuronal differentiation via JNK dephosphorylation.

Effect of antithyroid drug on chick embryos during the last week of development: delayed hatching and decreased cerebellar acetylcholinesterase activity.

Aim:  Hypothyroid state during embryogenesis disturbs normal growth and brain development, influencing later life. To evaluate the harmful consequences of the state during embryogenesis using an animal model, we inhibited thyroid hormone biosynthesis in chick embryos by using methimazole (MMI).

Effect of Astaxanthin on Cataract Formation Induced by Glucocorticoids in the Chick Embryo

Abstract Purpose: To examine whether astaxanthin (AST) prevent the cataract formation induced by glucocorticoid in chick embryo. Materials and methods: Hydrocortisone hemisuccinate sodium (HC) (0.5 μmol/egg) was administered directly into the air chamber in the egg shell of chick embryo day 15. The eggs were then kept in an incubator at same conditions and administered 100 μL of 50 (HC + AST50 group), 80 (HC + AST80 group), 100 (HC + AST100 group) mg/mL of AST solutions dissolved in dimethyl sulfoxide (DMSO) 3 h after administration of HC. In addition, non-HC treated group (treated with physiological saline without HC and 100 μL of DMSO), HC-alone group (treated with 0.5 μmol of HC and 100 μL of DMSO), and AST100 group (treated with physiological saline without HC and 100 μL of DMSO) were also incorporated. After 48 h of treatment, lenses were removed from embryo and classified into five stages according to developed opacity. The amounts of reduced glutathione in the lenses and the blood glucose levels were measured. Results: The average scores of lens opacitiy were 2.63 ± 1.02 nmol/lens (HC-alone), 2.78 ± 0.97 nmol/lens (HC + AST50), 2.22 ± 1.20 nmol/lens (HC + AST80) and 1.84 ± 0.83 nmol/lens (HC + AST100; p < 0.05), respectively. Administration of AST decreased the lens opacity dose-dependently. The amounts of reduced glutathione in lenses were 11.6 ± 2.8 nmol/lens (HC-alone), 11.3 ± 2.7 nmol/lens (HC + AST50), 13.4 ± 2.4 nmol/lens (HC + AST80) and 13.7 ± 3.1 nmol/lens (HC + AST100; p < 0.05), respectively. Higher levels of AST prevented loss of reduced glutathione from the lens. Conclusion: These findings support that AST protects glucocorticoid-induced cataract in chick embryo.

Lovastatin Alters TGF-β-Induced Epithelial-Mesenchymal Transition in Porcine Lens Epithelial Cells

Purpose: To determine whether lovastatin affects the epithelial-mesenchymal transition (EMT) in porcine lens epithelial cells (LECs) induced by transforming growth factor-β (TGF-β). Materials and Methods: Porcine LECs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) for 24 h. The cultured cells were then exposed or not exposed to lovastatin (10 µM) for 18 h and then stimulated with or not stimulated with TGF-β2 (5 ng/ml) for 24 h. The expression of α-smooth muscle actin (α-SMA), a marker of myofibroblasts, was determined by real-time PCR, and the expression of α-SMA protein was determined by Western blot. The effect of lovastatin on the expression of the mRNA of collagen type 1 (COL1) was determined by real-time PCR. To assess cell contractility, LECs were cultured in collagen gel with or without pretreatment of lovastatin and exposure of TGF-β2. The longest and shortest diameters of the gels were measured and the area was determined. Results: Exposure of LECs to TGF-β2 increased the expression of the mRNA and protein of α-SMA and the mRNA of COL1A1. TGF-β2 increased the degree of contraction of collagen gel. These findings indicated that TGF-β2 promoted EMT, and the pretreatment of the LECs with lovastatin blocked these changes induced by TGF-β2. Conclusion: Lovastatin inhibits the TGF-β-induced EMT of cultured porcine LECs. This suggests that lovastatin should be considered as a new agent to prevent postoperative complications associated with EMT of LECs.

Effects of glucocorticoid on brain acetylcholinesterase of developing chick embryos

Aim:  Fetal exposure to excessive or deficient glucocorticoids may alter the programming in differentiation and maturation of various tissues including the brain and nervous system, leading to dysfunctions later in life. For further exploration of this possibility, we established an animal model using developing chick embryos.

Upregulation of inorganic pyrophosphatase 1 as a JNK phosphatase in hypothyroid embryonic chick cerebellum

AIM Thyroid hormones play important roles in vertebrate neuronal development and differentiation. In our previous study, we showed that fetal thyroid dysfunction led to impaired social behaviors of hatchlings on post-hatch day 3, as well as to impaired learning and memory determined by the imprinting preference. However, little is known about the mechanisms underlying the direct adverse effects of fetal thyroid dysfunction on neuronal development. MATERIALS AND METHODS We used a chick embryo as a fetal model to investigate the effects of prenatal exposure to antithyroid drugs on neuronal development in the chick cerebellum. Methimazole (MMI) at a dose of 20μmol/egg was administered to eggs on day 14, while the control was given only a vehicle. In order to address the underlying mechanisms of the impaired behavior, proteomic approaches were employed in the chick cerebellum two days after MMI treatment. KEY FINDINGS In this experiment, we found that inorganic pyrophosphatase 1 (PPA1) was upregulated in the chick cerebellum treated with MMI, and we confirmed this upregulation of PPA1 by Western blot analysis as well as by RT-PCR analysis. Concomitant with the upregulation of PPA1, a marked reduction in JNK activity, as well as of phospho-JNK level, was detected in the MMI-treated chick cerebellum. SIGNIFICANCE Since PPA1 can dephosphorylate JNK, these results suggest that the upregulation of PPA1 during neuronal development in the hypothyroid chick cerebellum may lead to impaired social behaviors as well as to impaired learning and memory via JNK dephosphorylation and inactivation in the chick cerebellum.

Effect of Geranylgeranylacetone on Ultraviolet Radiation Type B-Induced Cataract in Heat-Shock Transcription Factor 1 Heterozygous Mouse

ABSTRACT Purpose: We investigated whether heat-shock transcription factor 1 (HSF1) was involved in ultraviolet radiation type B (UVR-B)-induced lens opacity (cataract) using HSF1 heterozygous mice. We also examined the effects of geranylgeranylacetone (GGA), an inducer of heat-shock proteins via activation of HSF, on the UVR-B-induced cataract. Material and Methods: Male HSF1+/– and WT mice were unilaterally exposed to UVR-B (total: 1200mJ) at 16 weeks of age. At 48 h after the last UVR-B irradiation, the lens was isolated and the induction of the cataract was quantified as the cataract area ratio (opacity area/anterior capsule). GGA was orally administered at a dosage of 500 mg/kg once a day for two days before the first UVR-B exposure until the end of the experiment (21days in total). Results: The HSF1 expression was more greatly decreased in the lens from HSF1+/– mice than in that from WT mice (p < 0.01). UVR-B exposure could mainly induce cataracts in the anterior capsule in both HSF1+/– and WT mice, while the opacity of the lens was markedly enhanced in HSF1+/– mice compared to that in WT mice(p (0.01). GGA treatment could prevent the induction of lens opacity by UVR-B exposure in both WT and HSF1+/– mice as compared with the non-administration group (p < 0.01). No obvious alteration by the UVR-B radiation was seen in lens protein levels of αA-crystallin, αB-crystallin, or γ-crystallin with or without GGA administration among all groups of mice. In contrast to the crystallins, the lens protein level of HSP25 was decreased by UVR-B exposure in both HSF1+/– and WT mice, and was significantly recovered in WT mice by the GGA treatment (p < 0.01). The induction of HSP25 was suppressed in HSF1+/– mice compared with that in WT mice. Conclusions: These data suggest that HSF1 plays an important role in the occurrence of UVR-B-induced cataracts, possibly via regulation of HSPs such as HSP25.