Yasushi Enokido

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.

Proteome analysis of soluble nuclear proteins reveals that HMGB1/2 suppress genotoxic stress in polyglutamine diseases

Nuclear dysfunction is a key feature of the pathology of polyglutamine (polyQ) diseases. It has been suggested that mutant polyQ proteins impair functions of nuclear factors by interacting with them directly in the nucleus. However, a systematic analysis of quantitative changes in soluble nuclear proteins in neurons expressing mutant polyQ proteins has not been performed. Here, we perform a proteome analysis of soluble nuclear proteins prepared from neurons expressing huntingtin (Htt) or ataxin-1 (AT1) protein, and show that mutant AT1 and Htt similarly reduce the concentration of soluble high mobility group B1/2 (HMGB1/2) proteins. Immunoprecipitation and pulldown assays indicate that HMGBs interact with mutant AT1 and Htt. Immunohistochemistry showed that these proteins were reduced in the nuclear region outside of inclusion bodies in affected neurons. Compensatory expression of HMGBs ameliorated polyQ-induced pathology in primary neurons and in Drosophila polyQ models. Furthermore, HMGBs repressed genotoxic stress signals induced by mutant Htt or transcriptional repression. Thus, HMGBs may be critical regulators of polyQ disease pathology and could be targets for therapy development.

The Induction Levels of Heat Shock Protein 70 Differentiate the Vulnerabilities to Mutant Huntingtin among Neuronal Subtypes

The reason why vulnerabilities to mutant polyglutamine (polyQ) proteins are different among neuronal subtypes is mostly unknown. In this study, we compared the gene expression profiles of three types of primary neurons expressing huntingtin (htt) or ataxin-1. We found that heat shock protein 70 (hsp70), a well known chaperone molecule protecting neurons in the polyQ pathology, was dramatically upregulated only by mutant htt and selectively in the granule cells of the cerebellum. Granule cells, which are insensitive to degeneration in the human Huntingtons disease (HD) pathology, lost their resistance by suppressing hsp70 with siRNA, whereas cortical neurons, affected in human HD, gained resistance by overexpressing hsp70. This indicates that induction levels of hsp70 are a critical factor for determining vulnerabilities to mutant htt among neuronal subtypes. CAT (chloramphenicol acetyltransferase) assays showed that CBF (CCAAT box binding factor, CCAAT/enhancer binding protein ζ) activated, but p53 repressed transcription of the hsp70 gene in granule cells. Basal and mutant htt-induced expression levels of p53 were remarkably lower in granule cells than in cortical neurons, suggesting that different magnitudes of p53 are linked to distinct induction levels of hsp70. Surprisingly, however, heat shock factor 1 was not activated in granule cells by mutant htt. Collectively, different levels of hsp70 among neuronal subtypes might be involved in selective neuronal death in the HD pathology.

Transcriptional repression induces a slowly progressive atypical neuronal death associated with changes of YAP isoforms and p73

Transcriptional disturbance is implicated in the pathology of polyglutamine diseases, including Huntingtons disease (HD). However, it is unknown whether transcriptional repression leads to neuronal death or what forms that death might take. We found transcriptional repression-induced atypical death (TRIAD) of neurons to be distinct from apoptosis, necrosis, or autophagy. The progression of TRIAD was extremely slow in comparison with other types of cell death. Gene expression profiling revealed the reduction of full-length yes-associated protein (YAP), a p73 cofactor to promote apoptosis, as specific to TRIAD. Furthermore, novel neuron-specific YAP isoforms (YAPΔCs) were sustained during TRIAD to suppress neuronal death in a dominant-negative fashion. YAPΔCs and activated p73 were colocalized in the striatal neurons of HD patients and mutant huntingtin (htt) transgenic mice. YAPΔCs also markedly attenuated Htt-induced neuronal death in primary neuron and Drosophila melanogaster models. Collectively, transcriptional repression induces a novel prototype of neuronal death associated with the changes of YAP isoforms and p73, which might be relevant to the HD pathology.

Mutant huntingtin impairs Ku70-mediated DNA repair

Mutant huntingtin prevents interaction of the DNA damage repair complex component Ku70 with damaged DNA, blocking repair of double-strand breaks.

Age-dependent change of HMGB1 and DNA double-strand break accumulation in mouse brain.

HMGB1 is an evolutionarily conserved non-histone chromatin-associated protein with key roles in maintenance of nuclear homeostasis; however, the function of HMGB1 in the brain remains largely unknown. Recently, we found that the reduction of nuclear HMGB1 protein level in the nucleus associates with DNA double-strand break (DDSB)-mediated neuronal damage in Huntingtons disease [M.L. Qi, K. Tagawa, Y. Enokido, N. Yoshimura, Y. Wada, K. Watase, S. Ishiura, I. Kanazawa, J. Botas, M. Saitoe, E.E. Wanker, H. Okazawa, Proteome analysis of soluble nuclear proteins reveals that HMGB1/2 suppress genotoxic stress in polyglutamine diseases, Nat. Cell Biol. 9 (2007) 402-414]. In this study, we analyze the region- and cell type-specific changes of HMGB1 and DDSB accumulation during the aging of mouse brain. HMGB1 is localized in the nuclei of neurons and astrocytes, and the protein level changes in various brain regions age-dependently. HMGB1 reduces in neurons, whereas it increases in astrocytes during aging. In contrast, DDSB remarkably accumulates in neurons, but it does not change significantly in astrocytes during aging. These results indicate that HMGB1 expression during aging is differentially regulated between neurons and astrocytes, and suggest that the reduction of nuclear HMGB1 might be causative for DDSB in neurons of the aged brain.

Omi / HtrA2 is relevant to the selective vulnerability of striatal neurons in Huntington's disease.

Selective vulnerability of neurons is a critical feature of neurodegenerative diseases, but the underlying molecular mechanisms remain largely unknown. We here report that Omi/HtrA2, a mitochondrial protein regulating survival and apoptosis of cells, decreases selectively in striatal neurons that are most vulnerable to the Huntington’s disease (HD) pathology. In microarray analysis, Omi/HtrA2 was decreased under the expression of mutant huntingtin (htt) in striatal neurons but not in cortical or cerebellar neurons. Mutant ataxin‐1 (Atx‐1) did not affect Omi/HtrA2 in any type of neuron. Western blot analysis of primary neurons expressing mutant htt also confirmed the selective reduction of the Omi/HtrA2 protein. Immunohistochemistry with a mutant htt‐transgenic mouse line and human HD brains confirmed reduction of Omi/HtrA2 in striatal neurons. Overexpression of Omi/HtrA2 by adenovirus vector reverted mutant htt‐induced cell death in primary neurons. These results collectively suggest that the homeostatic but not proapoptotic function of Omi/HtrA2 is linked to selective vulnerability of striatal neurons in HD pathology.

Loss of the xeroderma pigmentosum group A gene (XPA) enhances apoptosis of cultured cerebellar neurons induced by UV but not by low-K+ medium.

Abstract: To study the involvement of the xeroderma pigmentosum group A gene (XPA) in neuronal apoptosis, we cultured cerebellar neurons from mice lacking XPA gene (XPA−/−) and induced apoptosis by exposure to UV irradiation or medium containing a low concentration of potassium (low‐K+ medium). When cerebellar neurons from postnatal days 15–16 wild‐type mice were treated with UV irradiation, apoptotic neuronal death was observed after 24–48 h. About 60% of neurons survived 48 h after UV irradiation at a dose of 5 J/m2. On the other hand, neurons from XPA−/− mice showed a significantly increased vulnerability to UV irradiation, and >90% of neurons died 48 h after UV irradiation at a dose of 5 J/m2. In contrast, low‐K+ medium induced apoptosis of neurons from mice of each genotype with the same kinetics. These results suggest that the XPA gene is involved in neuronal DNA repair and that it thereby influences apoptosis induced by DNA damage in cultured cerebellar neurons.

Hepatoma-derived growth factor, a new trophic factor for motor neurons, is up-regulated in the spinal cord of PQBP-1 transgenic mice before onset of degeneration

Hepatoma‐derived growth factor (HDGF) is a nuclear protein homologous to the high‐mobility group B1 family of proteins. It is known to be released from cells and to act as a trophic factor for dividing cells. In this study HDGF was increased in spinal motor neurons of a mouse model of motor neuron degeneration, polyglutamine‐tract‐binding protein‐1 (PQBP‐1) transgenic mice, before onset of degeneration. HDGF promoted neurite extension and survival of spinal motor neurons in primary culture. HDGF repressed cell death of motor neurons after facial nerve section in newborn rats in vivo. We also found a significant increase in p53 in spinal motor neurons of the transgenic mice. p53 bound to a sequence in the upstream of the HDGF gene in a gel mobility shift assay, and promoted gene expression through the cis‐element in chloramphenicol acetyl transfer (CAT) assay. Finally, we found that HDGF was increased in CSF of PQBP‐1 transgenic mice. Collectively, our results show that HDGF is a novel trophic factor for motor neurons and suggest that it might play a protective role against motor neuron degeneration in PQBP‐1 transgenic mice.