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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 13, 2005 as doi:10.1096/fj.05-3724fje. |
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,1
* Department of Molecular Genetics,
Department of Neurochemistry, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan;
Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan; and
Department of Neuropathology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
1 Correspondence: Department of Neuropathy, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. E-mail: enokido.npat{at}mri.tmd.ac.jp
SPECIFIC AIMS
A deficiency of cystathionine ß-synthase (CBS; EC 4.2.1.22) causes homocystinuria (MIM 236200), one of the most prevalent inborn errors, characterized by mental retardation, seizures, psychiatric disturbances, skeletal abnormalities, and vascular disorders; however, the underlying mechanisms remain largely unknown. To elucidate the role of CBS in the nervous system, we examined the regional and cellular expression of CBS in the developing and adult mouse CNS. We also examined the effect of CBS deficiency on the normal development of brain, using Cbs-deficient mice.
PRINCIPAL FINDINGS
1. CBS is preferentially expressed in the radial glia/astrocyte lineage in the adult and developing mouse brain
To examine the regional and cellular distribution of CBS in the adult and developing mouse brain, we performed a histochemical analysis. In the adult mouse brain, CBS was expressed ubiquitously, but most intensely in the cerebellar molecular layer and hippocampal dentate gyrus. Immunohistochemical analysis revealed that CBS is preferentially expressed in cerebellar Bergman glia and in astrocytes throughout the brain. Early in development, CBS was expressed in neuroepithelial cells constituting the ventricular zone. Thereafter, its expression was transmitted to radial glia and then to astrocytes; however, CBS was hardly detectable in neuronal populations (Fig. 1
). Immunoblot analysis revealed that the CBS protein level markedly increases from the late embryonic period, reaches a maximum in the early postnatal period, and then decreases during maturation. Notably, in the cerebellum where the amount of CBS protein is 
2 to 3-fold that in other regions of the brain; the level was very low in the prenatal period, but drastically increased during the first 3 wk after birth when cerebellar histogenesis extensively occurs. These results suggest that CBS is associated with the generation and/or differentiation of the radial glia/astrocyte lineage cells in the developing CNS.
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2. CBS is up-regulated in reactive astrocytes after kainic acid-induced seizures
To assess the involvement of CBS in neuropathological conditions, we examined CBS expression after kainic acid (KA)-induced seizures which represent an animal model for human temporal lobe epilepsy. Immunoblot analysis demonstrated that the CBS protein level was significantly increased in the hippocampus after KA treatment. The CBS expression began to increase from 1 day, and the increased levels were sustained for at least 5 days after KA treatment. In parallel, we also observed a striking induction of heat shock protein (HSP) 25 (also known as HSP27), a marker for reactive astrocytes. In contrast, no significant change of CBS and HSP25 expression was observed in the hippocampus of saline-treated control animals at any point examined. Immunohistochemical analysis revealed that immunoreactivity for CBS was heavily increased in cells with a large body and numerous stellate processes in whole hippocampus treated with KA. Double immunofluorescent staining showed that the CBS-positive cells were also positive for GFAP, a marker for astrocytes, and for HSP25, indicating that CBS was predominantly expressed in reactive astrocytes. In saline-treated control hippocampus, CBS immunoreactivity was unchanged in comparison with that in untreated animals, and there was no HSP25 immunoreactivity except for faint background staining.
3. CBS induction in astrocytes is mediated by EGFR ligands, cAMP, and glucocorticoid
To address the regulatory mechanism of CBS expression, we examined the effect of various cell growth/differentiation factors on the expression in cultured astrocytes. The treatments with EGF and TGF-
, ligands for the EGFR, CPT-cAMP, an analog for cAMP, and dexamethasone, a synthetic glucocorticoid, specifically increased the CBS protein level in cultured astrocytes, whereas there was no effect of KA or glutamate on CBS. Moreover, we tested the effect of several cell-stressing stimuli, such as serum deprivation, oxidative stress (L-buthionine-(S,R)-sulfoximine and H2O2), ER-Golgi stress (tunicamycin), DNA damage (etoposide), and homocysteine on CBS protein expression in cultured astrocytes; however, CBS was not affected by any of the stimuli tested. These results suggest that the expression of CBS in astrocytes is not directly mediated by glutamate or cell stressing stimuli, but some exogenous factors produced by neighboring cells such as neurons or microglia, or by astrocytes themselves may mediate the induction.
4. CBS deficiency affects normal cerebellar development of mice in the early postnatal period
To examine the role of CBS in the developing CNS, we focused on the postnatal development of the mouse cerebellum, one of the best characterized regions with respect to brain development, in which CBS is most prominently expressed, by using Cbs-deficient (CBS–/–) mice. Although a slight reduction in size was observed, the gross appearance of the brain of CBS–/– mice (n=14) seemed normal (e.g., the cerebrum and olfactory bulbs). However, the cerebellum was remarkably smaller than that of wild-type (CBS+/+) mice (n=15) at P21. These abnormalities were not observed at birth but became apparent around P8, suggesting that cell depletion takes place during early postnatal development. An analysis of thionine-stained sagittal sections confirmed the histological defects in CBS–/– cerebellum; a gross reduction in size and impaired foliation and sulcus formation (Fig. 2
A, B). Immunostaining for calbindin-D revealed that the structural organization of the cerebellar layers was ordered, but a striking reduction in the thickness of the ML and IGL and stunted PC dendrites were observed, indicating impaired PC differentiation (n=6 for CBS+/+ and CBS–/– mice) (Fig. 2C
).
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Because cerebellar morphogenesis largely depends on the proliferation of granular neuron precursors in the external granular layer (EGL), which is completed by the end of the third postnatal week in the mouse, we further examined the cell proliferation in the EGL based on the incorporation of BrdU in dividing cells followed by immunohistochemistry. At P8, when the neurogenesis in cerebellar granule cells in the EGL reaches a maximum, the structural organization of cerebellar layers seemed normal; however, the thickness of the EGL was markedly reduced in CBS–/– mice in comparison with the CBS+/+ EGL (n=5 for each genotype) (Fig. 2D-F
). Moreover, a significant reduction in the number of BrdU-positive cells in the EGL was observed in CBS–/– mice. In contrast, we could detect few BrdU-positive cells in the EGL of CBS+/+ and CBS–/– mice at P21 (n=6 for each genotype) (Fig. 2G, H
). These results suggest that the deficiency of CBS does not delay the disappearance of the EGL, but rather impairs cell proliferation in the EGL, which may be associated with the reduced size of the CBS–/– cerebellum.
CONCLUSIONS AND SIGNIFICANCE
CBS is a vitamin B6-dependent transsulfuration enzyme needed to synthesize cysteine from methionine, catalyzing the condensation of serine with homcysteine to form cystathionine. The link between a deficiency of CBS and homocystinuria was first described over 40 years ago, and mental retardation was the first clinical feature of the disease to be classified; however, the localization and function of CBS in the nervous system have long been controversial. Moreover, accumulating evidence suggests that abnormal homocysteine metabolism is associated with various neurological disorders, such as depression, schizophrenia, and Alzheimer's disease. In the present study, we have demonstrated that CBS is preferentially expressed in the radial glia/astrocyte lineage (Fig. 3
). In addition to the morphological abnormalities, PC dendritic abnormality and impaired cell proliferation in the EGL were observed in early postnatal CBS–/– mouse cerebellum.
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Our findings may support the idea that a deficiency of CBS causes various neurodevelopmental defects which result in complex neuropathological features associated with abnormal homocysteine metabolism, and also suggest that radial glia/astrocyte lineage cells might be a new therapeutic target for preventing and treating them.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3724fje;
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