Fusako Usuki

In vivo protection of a water-soluble derivative of vitamin E, Trolox, against methylmercury-intoxication in the rat

Methylmercury (MeHg) is a well-known neurotoxicant. MeHg-intoxication causes a disturbance in mitochondrial energy metabolism in skeletal muscle and apoptosis in cerebellum. We report the first in vivo effectiveness of antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carhoxylic acid), a water soluble vitamin E analog, against the MeHg-induced cellular responses. Treatment with Trolox (6-hydroxy-2.5,7,8-tetramethylchroman-2-carboxylic acid) clearly protects MeHg-treated rat skeletal muscle against the decrease in mitochondrial electron transport system enzyme activities despite the retention of MeHg. Tdt-mediated dUTP nick-end-labeling method clarified that Trolox is effective for protecting cerebellum from MeHg-induced apoptosis. These data indicate that MeHg-mediated oxidative stress plays an important role in the in vivo pathological process of MeHg intoxication. Trolox may prevent some of clinical manifestations of MeHg-intoxication in humans.

Inhibition of nonsense‐mediated mRNA decay rescues the phenotype in Ullrich's disease

Nonsense‐mediated mRNA decay (NMD) is an mRNA surveillance system that eliminates aberrant mRNAs containing premature translation termination codons (PTCs). We evaluated the role of NMD in of Ullrichs disease. The patient has a frameshift mutation with a PTC in the collagen VI α2 gene causing the loss of collagen VI and functional defects in extracellular matrix (ECM). The pharmacological block of NMD caused upregulation of the mutant collagen VI α2 subunit, resulting in collagen VI assembly and partially functional ECM formation. Our results suggest that NMD inhibitors can be used as a therapeutic tool to rescue some human genetic diseases exacerbated by NMD.

Post-transcriptional Defects of Antioxidant Selenoenzymes Cause Oxidative Stress under Methylmercury Exposure

Methylmercury (MeHg) toxicity is a continuous environmental problem to human health. The critical role of oxidative stress in the pathogenesis of MeHg cytotoxicity has been clarified, but the molecular mechanisms underlying MeHg-mediated oxidative stress remain to be elucidated. Here we demonstrate a post-transcriptional effect of MeHg on antioxidant selenoenzymes by using a MeHg-susceptible cell line. MeHg-induced selenium deficiency leads to failure of the recoding of a UGA codon for selenocysteine and results in degradation of the major antioxidant selenoenzyme glutathione peroxidase 1 (GPx1) mRNA by nonsense-mediated mRNA decay (NMD), a cellular mechanism that detects the premature termination codon (PTC) located 5′-upstream of the last exon-exon junction and degrades PTC-containing mRNAs. In contrast, thioredoxin reductase 1 (TrxR1), another antioxidant selenoenzyme of the thioredoxin system, was likely skipped by NMD because of a UGA codon in the last exon. However, TrxR1 activity was decreased despite mRNA up-regulation, which was probably due to the synthesis of aberrant TrxR1 protein without selenocysteine. Changes in selenoenzyme GPx1 and TrxR1 mRNAs were observed earlier than was the incidence of oxidative stress and up-regulation of other antioxidant enzyme mRNAs. Results indicated that the MeHg-induced relative selenium-deficient condition affects the major antioxidant selenoenzymes GPx1 and TrxR1 through a post-transcriptional effect, resulting in the disturbance of cellular redox systems and the incidence of oxidative stress. Treatment with ebselen, a seleno-organic compound, effectively suppressed oxidative stress and protected cells against MeHg-induced relative selenium deficiency and cytotoxicity.

Methylmercury induces neuropathological changes with tau hyperphosphorylation mainly through the activation of the c-jun-N-terminal kinase pathway in the cerebral cortex, but not in the hippocampus of the mouse brain.

Methylmercury (MeHg) is a well-known neurotoxicant inducing neuronal degeneration in the central nervous system. This in vivo study investigated the involvement of tau hyperphosphorylation in MeHg-induced neuropathological changes in the mouse brain, because abnormal tau hyperphosphorylation causes significant pathological changes associated with some neurodegenerative diseases. Mice that were administrated to 30 ppm MeHg in drinking water for 8 weeks exhibited neuropathological changes, e.g. a decrease in the number of neuron; an increase in the number of migratory astrocytes and microglia/macrophages; necrosis and apoptosis in the cerebral cortex, particularly the deep layer of primary motor cortex and prelimbic cortex. Western blotting revealed that MeHg exposure increased tau phosphorylation at Thr-205, Ser-396 and Ser-422 in the cerebral cortex, consistent with the phosphorylation patterns noted in Alzheimers disease and frontotemporal dementia. Immunohistochemical analyses revealed that the distribution of tau-phosphorylated (Thr-205) neurons corresponded with the areas showing considerable neuropathological changes. Among the kinases and phosphatases related to tau hyperphosphorylation, the activation of mitogen-activated protein kinase kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK) was recognized. Neither neuropathological changes nor tau hyperphosphorylation was detected in the hippocampus in this study although the mercury concentration here was twice that in the cerebral cortex. These findings suggest that MeHg exposure induces tau hyperphosphorylation at specific sites of tau mainly through the activation of JNK pathways, leading to neuropathological changes in the cerebral cortex selectively, but not in the hippocampus of mouse brain.

Inhibition of the Rho/ROCK pathway prevents neuronal degeneration in vitro and in vivo following methylmercury exposure.

Methylmercury (MeHg) is an environmental neurotoxicant which induces neuropathological changes in both the central nervous and peripheral sensory nervous systems. Our recent study demonstrated that down-regulation of Ras-related C3 botulinum toxin substrate 1 (Rac1), which is known to promote neuritic extension, preceded MeHg-induced damage in cultured cortical neurons, suggesting that MeHg-mediated axonal degeneration is due to the disturbance of neuritic extension. Therefore we hypothesized that MeHg-induced axonal degeneration might be caused by neuritic extension/retraction incoordination. This idea brought our attention to the Ras homolog gene (Rho)/Rho-associated coiled coil-forming protein kinase (ROCK) pathway because it has been known to be associated with the development of axon and apoptotic neuronal cell death. Here we show that inhibition of the Rho/ROCK pathway prevents MeHg-intoxication both in vitro and in vivo. A Rho inhibitor, C3 toxin, and 2 ROCK inhibitors, Fasudil and Y-27632, significantly protected against MeHg-induced axonal degeneration and apoptotic neuronal cell death in cultured cortical neuronal cells exposed to 100 nM MeHg for 3 days. Furthermore, Fasudil partially prevented the loss of large pale neurons in dorsal root ganglia, axonal degeneration in dorsal spinal root nerves, and vacuolar degeneration in the dorsal columns of the spinal cord in MeHg-intoxicated model rats (20 ppm MeHg in drinking water for 28 days). Hind limb crossing sign, a characteristic MeHg-intoxicated sign, was significantly suppressed in this model. The results suggest that inhibition of the Rho/ROCK pathway rescues MeHg-mediated neuritic extension/retraction incoordination and is effective for the prevention of MeHg-induced axonal degeneration and apoptotic neuronal cell death.

Methylmercury exposure downregulates the expression of Racl and leads to neuritic degeneration and ultimately apoptosis in cerebrocortical neurons.

Methylmercury (MeHg) has been recognized as a neurotoxicant targeted on the central nervous system including cerebellum and cerebral cortex. Some molecular targets of MeHg have been identified using cerebellar neuronal cells, but little is known in the cerebrocortical neuronal cells. In this study, the molecular mechanism underlying MeHg-induced cell death in cerebrocortical neurons was investigated using a primary culture of embryonic rat cortical neuronal cells. The cultured cells exhibited apoptosis 3 days after exposure to 100 nM MeHg, suggesting the involvement of caspase-dependent apoptotic pathways. We demonstrated for the first time that neuritic degeneration precedes MeHg-induced apoptotic death in neurons exposed to 100 nM MeHg. Immunocytochemical and ELISA analyses for neurite-specific proteins namely, tau and MAP2, showed that injury to tau-positive axons was first induced followed by damage to the dendrites and cellular bodies. To further investigate the factors responsible for neuronal death, we investigated the expression levels of Rho-family proteins (Rac1, Cdc42, and RhoA), which regulate neuritic functions and apoptosis in neurons. Western blot analysis demonstrated that MeHg downregulated the expression levels of Rac1 and Cdc42 but did not affect RhoA. The exposure concentration and time course studies confirmed that Rac1 is targeted during an early stage of MeHg-induced cytotoxicity. The results indicate that neuritic degeneration, in particular axonal degeneration triggered by the downregulation of Rac1 expression, contributes to MeHg-induced apoptotic cell death in cultured cerebrocortical neurons.

Expanded CTG repeats in myotonin protein kinase increase susceptibility to oxidative stress

THE effect of oxidative stress on myogenic cells with expanded CTG repeats in the myotonin protein kinase (MtPK) gene was investigated using MtPK cDNA-transformants in order to investigate the disease process underlying myotonic dystrophy. We employed methylmercury as a model for reagents that produce reactive oxygen species (ROS). Mutant MtPK cDNA transformants containing 46 CTG repeats treated with 1 μM methylmercury for 24 h underwent cell death showing the characteristics of apoptosis. In contrast, methylmercury-induced cytotoxicity was weaker in wild type MtPK cDNA transformants. Antioxidants such as N-acetyl-L-cysteine and trolox suppressed methylmercury-induced apoptosis, indicating that the intracellular generation of ROS plays an important role. These studies suggest that expanded CTG repeats in MtPK increase the susceptibility of cells to oxidative stress.

Beneficial effects of mild lifelong dietary restriction on skeletal muscle: prevention of age-related mitochondrial damage, morphological changes, and vulnerability to a chemical toxin

The effect of mild lifelong dietary restriction (DR) on age-related changes was investigated in rats. Histopathological findings were compared between 25-month-old male rats fed ad libitum and 25-month-old male rats that were calorie restricted (80% of ad libitum calories; protein 15%) from 9 weeks of age. DR-fed rats retained motor activity even in old age compared with ad libitum-fed rats. Histopathological studies on soleus muscles clarified myopathic changes in the ad libitum-fed rats, including variations in fiber size and an increase in the number of central nuclei. Increased non-grouping atrophic angulated fibers were also observed. The specimens revealed a confused arrangement of the mitochondria and decreased mitochondrial electron transduction enzyme activities, indicating mitochondrial insults in the ad libitum-fed rats. In contrast, no myopathic changes, little mitochondrial insult, and fewer angulated fibers were recognized in the DR-fed rats. The accumulations of heme oxygenase-1, αβ crystallin, 8-hydroxydeoxyguanosine, and heat shock protein 27 were recognized in ad libitum-fed rats, indicating the attack of oxidative stress. In contrast, the expressions of these proteins were suppressed in DR-fed rats. The results suggest that even mild calorie restriction is enough to attenuate oxidative stress and age-related morphological changes in skeletal muscle. Additionally, DR was effective in protecting against methylmercury-induced pathological changes. Small fiber size and suppression of mitochondrial electron transduction enzyme activities in skeletal muscle and degenerative changes in peripheral nerves were milder in methylmercury-exposed DR-fed rats. The results indicate that mild lifelong DR also protects skeletal muscle and peripheral nerves against a chemically-induced form of oxidative stress.

Differing effects of toxicants (methylmercury, inorganic mercury, lead, amyloid β, and rotenone) on cultured rat cerebrocortical neurons: differential expression of rho proteins associated with neurotoxicity.

Methylmercury (MeHg), inorganic mercury (IHg), lead (Pb), amyloid-β peptide (Aβ), and rotenone (RTN) are well-known toxicants. Here, we demonstrate that these five toxicants exhibit differing effects on cerebrocortical neurons. The concentration responsible for 30% loss of viability (EC30) values 3 days after exposure was approximately 100nM for MeHg, IHg, and RTN and 10μM for Aβ. Neuritic degeneration and subsequent apoptotic cell death were observed in these toxicant-treated cells. In contrast, the EC30 value 3 days after exposure to Pb was > 10μM. We clarified the differential expression of Ras homolog gene (Rho) family proteins (Ras-related C3 botulinum toxin substrate 1 [Rac1], cell division cycle 42, and Ras homolog gene family, member A [RhoA]) upon exposure to these five toxicants. Exposure to 100nM MeHg, IHg, or RTN downregulated the expression of Rac1, related to neuritic extension, but did not affect RhoA, related to retraction. At a higher concentration (1μM), IHg and RTN also acted through the suppression of Rac1, whereas increased MeHg toxicity was not associated with the expression of Rho family proteins. On the other hand, Pb and Aβ showed no effects on the expression of Rho proteins. Modification of the balance of neuritic extension and retraction by the suppression of Rho A rescued the neurotoxicity of 100nM MeHg, IHg, and RTN. The results indicate that the imbalance of neuritic extension and retraction by the suppression of Rac1 by 100nM MeHg, IHg, and RTN causes cerebrocortical neuron axonal degeneration and cell death. By contrast, the neurotoxicities of Pb, Aβ, and MeHg (at higher concentrations) are conferred by other toxic mechanisms.

Cardiomyopathy, mental retardation, and autophagic vacuolar myopathy. Abnormal MRI findings in the head.

A 21-year-old man with childhood-onset mental retardation, non-obstructive hypertrophic cardiomyopathy, and vacuolar myopathy is presented. A histopathological study of biopsied skeletal muscle showed lysosomal glycogen storage mimicking acid maltase deficiency, but biochemical analysis showed normal acid alpha-glucosidase activity. Glycogenosomes were also recognized in endothelial cells on electronmicroscopic examination of biopsied skeletal muscle. Magnetic resonance imaging (MRI) findings in the head revealed the involvement of the central nervous system. This is a new type of lysosomal glycogen storage disease with multisystemic involvement. The specific biochemical defect in this disorder remains to be elucidated.