Kazuhiko Namekata

Murine cystathionine γ-lyase: complete cDNA and genomic sequences, promoter activity, tissue distribution and developmental expression

Cystathionine gamma-lyase (CSE) is the last key enzyme in the trans-sulphuration pathway for biosynthesis of cysteine from methionine. Cysteine could be provided through diet; however, CSE has been shown to be important for the adequate supply of cysteine to synthesize glutathione, a major intracellular antioxidant. With a view to determining physiological roles of CSE in mice, we report the sequence of a complete mouse CSE cDNA along with its associated genomic structure, generation of specific polyclonal antibodies, and the tissue distribution and developmental expression patterns of CSE in mice. A 1.8 kb full-length cDNA containing an open reading frame of 1197 bp, which encodes a 43.6 kDa protein, was isolated from adult mouse kidney. A 35 kb mouse genomic fragment was obtained by lambda genomic library screening. It contained promoter regions, 12 exons, ranging in size from 53 to 579 bp, spanning over 30 kb, and exon/intron boundaries that were conserved with rat and human CSE. The GC-rich core promoter contained canonical TATA and CAAT motifs, and several transcription factor-binding consensus sequences. The CSE transcript, protein and enzymic activity were detected in liver, kidney, and, at much lower levels, in small intestine and stomach of both rats and mice. In developing mouse liver and kidney, the expression levels of CSE protein and activity gradually increased with age until reaching their peak value at 3 weeks of age, following which the expression levels in liver remained constant, whereas those in kidney decreased significantly. Immunohistochemical analyses revealed predominant CSE expression in hepatocytes and kidney cortical tubuli. These results suggest important physiological roles for CSE in mice.

The potential role of glutamate transporters in the pathogenesis of normal tension glaucoma

Glaucoma, a progressive optic neuropathy due to retinal ganglion cell (RGC) degeneration, is one of the leading causes of irreversible blindness. Although glaucoma is often associated with elevated intraocular pressure (IOP), IOP elevation is not detected in a significant subset of glaucomas, such as normal tension glaucoma (NTG). Moreover, in some glaucoma patients, significant IOP reduction does not prevent progression of the disease. Thus, understanding IOP-independent mechanisms of RGC loss is important. Here, we show that mice deficient in the glutamate transporters GLAST or EAAC1 demonstrate spontaneous RGC and optic nerve degeneration without elevated IOP. In GLAST-deficient mice, the glutathione level in Müller glia was decreased; administration of glutamate receptor blocker prevented RGC loss. In EAAC1-deficient mice, RGCs were more vulnerable to oxidative stress. These findings suggest that glutamate transporters are necessary both to prevent excitotoxic retinal damage and to synthesize glutathione, a major cellular antioxidant and tripeptide of glutamate, cysteine, and glycine. We believe these mice are the first animal models of NTG that offer a powerful system for investigating mechanisms of neurodegeneration in NTG and developing therapies directed at IOP-independent mechanisms of RGC loss.

Cystathionine β-synthase, a key enzyme for homocysteine metabolism, is preferentially expressed in the radial glia/astrocyte lineage of developing mouse CNS

Cystathionine β‐synthase (CBS; EC 4.2.1.22) is a key enzyme in the generation of cysteine from methionine. A deficiency of CBS leads to homocystinuria, an inherited human disease characterized by mental retardation, seizures, psychiatric disturbances, skeletal abnormalities, and vascular disorders; however, the underlying mechanisms remain largely unknown. Here, we show the regional and cellular distribution of CBS in the adult and developing mouse brain. In the adult mouse brain, CBS was expressed ubiquitously, but it is expressed most intensely in the cerebellar molecular layer and hippocampal dentate gyrus. Immunohistochemical analysis revealed that CBS is preferentially expressed in cerebellar Bergmann glia and in astrocytes throughout the brain. At early developmental stages, CBS was expressed in neuroepithelial cells in the ventricular zone, but its expression changed to radial glial cells and then to astrocytes during the late embryonic and neonatal periods. CBS was most highly expressed in juvenile brain, and a striking induction was observed in cultured astrocytes in response to EGF, TGF‐α, cAMP, and dexamethasone. Moreover, CBS was significantly accumulated in reactive astrocytes in the hippocampus after kainic acid‐induced seizures, and cerebellar morphological abnormalities were observed in CBS‐deficient mice. Taken together, these results suggest that CBS plays a crucial role in the development and maintenance of the CNS and that radial glia/astrocyte dysfunction might be involved in the complex neuropathological features associated with abnormal homocysteine metabolism.

Abnormal Lipid Metabolism in Cystathionine β-Synthase-deficient Mice, an Animal Model for Hyperhomocysteinemia

Hyperhomocysteinemia (HHCY) is a consequence of impaired methionine/cysteine metabolism and is caused by deficiency of vitamins and/or enzymes such as cystathionine β-synthase (CBS). Although HHCY is an important and independent risk factor for cardiovascular diseases that are commonly associated with hepatic steatosis, the mechanism by which homocysteine promotes the development of fatty liver is poorly understood. CBS-deficient (CBS–/–) mice were previously generated by targeted deletion of the Cbs gene and exhibit pathological features similar to HHCY patients, including endothelial dysfunction and hepatic steatosis. Here we show abnormal lipid metabolism in CBS–/– mice. Triglyceride and nonesterified fatty acid levels were markedly elevated in CBS–/– mouse liver and serum. The activity of thiolase, a key enzyme in β-oxidation of fatty acids, was significantly impaired in CBS–/– mouse liver. Hepatic apolipoprotein B100 levels were decreased, whereas serum apolipoprotein B100 and very low density lipoprotein levels were elevated in CBS–/– mice. Serum levels of cholesterol/phospholipid in high density lipoprotein fractions but not of total cholesterol/phospholipid were decreased, and the activity of lecithin-cholesterol acyltransferase was severely impaired in CBS–/– mice. Abnormal high density lipoprotein particles with higher mobility in polyacrylamide gel electrophoresis were observed in serum obtained from CBS–/– mice. Moreover, serum cholesterol/triglyceride distribution in lipoprotein fractions was altered in CBS–/– mice. These results suggest that hepatic steatosis in CBS–/– mice is caused by or associated with abnormal lipid metabolism.

Regulation of the severity of neuroinflammation and demyelination by TLR-ASK1-p38 pathway

Apoptosis signal‐regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen‐activated protein kinase (MAPK) kinase kinase which plays important roles in stress and immune responses. Here, we show that ASK1 deficiency attenuates neuroinflammation in experimental autoimmune encephalomyelitis (EAE), without affecting the proliferation capability of T cells. Moreover, we found that EAE upregulates expression of Toll‐like receptors (TLRs) in activated astrocytes and microglia, and that TLRs can synergize with ASK1‐p38 MAPK signalling in the release of key chemokines from astrocytes. Consequently, oral treatment with a specific small molecular weight inhibitor of ASK1 suppressed EAE‐induced autoimmune inflammation in both spinal cords and optic nerves. These results suggest that the TLR‐ASK1‐p38 pathway in glial cells may serve as a valid therapeutic target for autoimmune demyelinating disorders including multiple sclerosis.

ASK1 deficiency attenuates neural cell death in GLAST-deficient mice, a model of normal tension glaucoma

Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase and has an important role in stress-induced retinal ganglion cell (RGC) apoptosis. In the mammalian retina, glutamate/aspartate transporter (GLAST) is a major glutamate transporter, and the loss of GLAST leads to optic nerve degeneration similar to normal tension glaucoma (NTG). In GLAST−/− mice, the glutathione level in the retina is decreased, suggesting the involvement of oxidative stress in NTG pathogenesis. To test this hypothesis, we examined the histology and visual function of GLAST+/−:ASK1−/− and GLAST−/−:ASK1−/− mice by multifocal electroretinograms. ASK1 deficiency protected RGCs and decreased the number of degenerating axons in the optic nerve. Consistent with this finding, visual function was significantly improved in GLAST+/−:ASK1−/− and GLAST−/−:ASK1−/− mice compared with GLAST+/− and GLAST−/− mice, respectively. The loss of ASK1 had no effects on the production of glutathione or malondialdehyde in the retina or on the intraocular pressure. Tumor necrosis factor (TNF)-induced activation of p38 MAPK and the production of inducible nitric oxide synthase were suppressed in ASK1-deficient Müller glial cells. In addition, TNF-induced cell death was suppressed in ASK1-deficient RGCs. These results suggest that ASK1 activation is involved in NTG-like pathology in both neural and glial cells and that interrupting ASK1-dependent pathways could be beneficial in the treatment of glaucoma, including NTG.

Glia- and neuron-specific functions of TrkB signalling during retinal degeneration and regeneration

Glia, the support cells of the central nervous system, have recently attracted considerable attention both as mediators of neural cell survival and as sources of neural regeneration. To further elucidate the role of glial and neural cells in neurodegeneration, we generated TrkBGFAP and TrkBc-kit knockout mice in which TrkB, a receptor for brain-derived neurotrophic factor (BDNF), is deleted in retinal glia or inner retinal neurons, respectively. Here, we show that the extent of glutamate-induced retinal degeneration was similar in these two mutant mice. Furthermore in TrkBGFAP knockout mice, BDNF did not prevent photoreceptor degeneration and failed to stimulate Müller glial cell proliferation and expression of neural markers in the degenerating retina. These results demonstrate that BDNF signalling in glia has important roles in neural protection and regeneration, particularly in conversion of Müller glia to photoreceptors. In addition, our genetic models provide a system in which glia- and neuron-specific gene functions can be tested in central nervous system tissues in vivo.

Dock3 Stimulates Axonal Outgrowth via GSK-3β-Mediated Microtubule Assembly

Dock3, a new member of the guanine nucleotide exchange factors, causes cellular morphological changes by activating the small GTPase Rac1. Overexpression of Dock3 in neural cells promotes axonal outgrowth downstream of brain-derived neurotrophic factor (BDNF) signaling. We previously showed that Dock3 forms a complex with Fyn and WASP (Wiskott–Aldrich syndrome protein) family verprolin-homologous (WAVE) proteins at the plasma membrane, and subsequent Rac1 activation promotes actin polymerization. Here we show that Dock3 binds to and inactivates glycogen synthase kinase-3β (GSK-3β) at the plasma membrane, thereby increasing the nonphosphorylated active form of collapsin response mediator protein-2 (CRMP-2), which promotes axon branching and microtubule assembly. Exogenously applied BDNF induced the phosphorylation of GSK-3β and dephosphorylation of CRMP-2 in hippocampal neurons. Moreover, increased phosphorylation of GSK-3β was detected in the regenerating axons of transgenic mice overexpressing Dock3 after optic nerve injury. These results suggest that Dock3 plays important roles downstream of BDNF signaling in the CNS, where it regulates cell polarity and promotes axonal outgrowth by stimulating dual pathways: actin polymerization and microtubule assembly.

Inhibition of ASK1-p38 pathway prevents neural cell death following optic nerve injury

Optic nerve injury (ONI) induces retinal ganglion cell (RGC) death and optic nerve atrophy that lead to visual loss. Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase and has an important role in stress-induced RGC apoptosis. In this study, we found that ONI-induced p38 activation and RGC loss were suppressed in ASK1-deficient mice. Sequential in vivo retinal imaging revealed that post-ONI treatment with a p38 inhibitor into the eyeball was effective for RGC protection. ONI-induced monocyte chemotactic protein-1 production in RGCs and microglial accumulation around RGCs were suppressed in ASK1-deficient mice. In addition, the productions of tumor necrosis factor and inducible nitric oxide synthase in microglia were decreased when the ASK1-p38 pathway was blocked. These results suggest that ASK1 activation in both neural and glial cells is involved in neural cell death, and that pharmacological interruption of ASK1-p38 pathways could be beneficial in the treatment of ONI.

Dock3 attenuates neural cell death due to NMDA neurotoxicity and oxidative stress in a mouse model of normal tension glaucoma

Dedicator of cytokinesis 3 (Dock3), a new member of the guanine nucleotide exchange factors for the small GTPase Rac1, promotes axon regeneration following optic nerve injury. In the present study, we found that Dock3 directly binds to the intracellular C-terminus domain of NR2B, an N-methyl-D-aspartate (NMDA) receptor subunit. In transgenic mice overexpressing Dock3 (Dock3 Tg), NR2B expression in the retina was significantly decreased and NMDA-induced retinal degeneration was ameliorated. In addition, overexpression of Dock3 protected retinal ganglion cells (RGCs) from oxidative stress. We previously reported that glutamate/aspartate transporter (GLAST) is a major glutamate transporter in the retina, and RGC degeneration due to glutamate neurotoxicity and oxidative stress is observed in GLAST-deficient (KO) mice. In GLAST KO mice, the NR2B phosphorylation rate in the retina was significantly higher compared with Dock3 Tg:GLAST KO mice. Consistently, glaucomatous retinal degeneration was significantly improved in GLAST KO:Dock3 Tg mice compared with GLAST KO mice. These results suggest that Dock3 overexpression prevents glaucomatous retinal degeneration by suppressing both NR2B-mediated glutamate neurotoxicity and oxidative stress, and identifies Dock3 signaling as a potential therapeutic target for both neuroprotection and axonal regeneration.