Promyelinating drugs ameliorate oligodendrocyte pathologies in a mouse model of Krabbe disease.

Krabbe disease (KD) is a rare inherited demyelinating disorder caused by a deficiency in the lysosomal enzyme galactosylceramide (GalCer) ß-galactosidase. Most patients with KD exhibit fatal cerebral demyelination with apoptotic oligodendrocyte (OL) death and die before the age of 2-4 years. We have previously reported that primary OLs isolated from the brains of twitcher (twi) mice, an authentic mouse model of KD, have cell-autonomous developmental defects and undergo apoptotic death accompanied by abnormal accumulation of psychosine, an endogenous cytotoxic lyso-derivative of GalCer. In this study, we aimed to investigate the effects of the preclinical promyelinating drugs clemastine and Sob-AM2 on KD OL pathologies using primary OLs isolated from the brains of twi mice. Both agents specifically prevented the apoptotic death observed in twi OLs. However, while Sob-AM2 showed higher efficacy in restoring the impaired differentiation and maturation of twi OLs, clemastine more potently reduced the endogenous psychosine levels. These results present the first preclinical in vitro data, suggesting that clemastine and Sob-AM2 can act directly and distinctly on OLs in KD and ameliorate their cellular pathologies associated with myelin degeneration.

Reduction in miR-219 expression underlies cellular pathogenesis of oligodendrocytes in a mouse model of Krabbe disease.

Krabbe disease (KD), also known as globoid cell leukodystrophy, is an inherited demyelinating disease caused by the deficiency of lysosomal galactosylceramidase (GALC) activity. Most of the patients are characterized by early-onset cerebral demyelination with apoptotic oligodendrocyte (OL) death and die before 2 years of age. However, the mechanisms of molecular pathogenesis in the developing OLs before death and the exact causes of white matter degeneration remain largely unknown. We have recently reported that OLs of twitcher mouse, an authentic mouse model of KD, exhibit developmental defects and endogenous accumulation of psychosine (galactosylsphingosine), a cytotoxic lyso-derivative of galactosylceramide. Here, we show that attenuated expression of microRNA (miR)-219, a critical regulator of OL differentiation and myelination, mediates cellular pathogenesis of KD OLs. Expression and functional activity of miR-219 were repressed in developing twitcher mouse OLs. By using OL precursor cells (OPCs) isolated from the twitcher mouse brain, we show that exogenously supplemented miR-219 effectively rescued their cell-autonomous developmental defects and apoptotic death. miR-219 also reduced endogenous accumulation of psychosine in twitcher OLs. Collectively, these results highlight the role of the reduced miR-219 expression in KD pathogenesis and suggest that miR-219 has therapeutic potential for treating KD OL pathologies.

R3HDM1 haploinsufficiency is associated with mild intellectual disability.

R3HDM1 (R3H domain containing 1) is an uncharacterized RNA-binding protein that is highly expressed in the human cerebral cortex. We report the first case of a 12-year-old Japanese male with haploinsufficiency of R3HDM1. He presented with mild intellectual disability (ID) and developmental delay. He had a pericentric inversion of 46,XY,inv(2)(p16.1q21.3)dn with breakpoints in intron 19 of R3HDM1 (2q21.3) and the intergenic region (2p16.1). The R3HDM1 levels in his lymphoblastoid cells were reduced to approximately half that of the healthy controls. However, the expression of MIR128-1, in intron 18 of R3HDM1, was not affected via the pericentric inversion. Knockdown of R3HDM1 in mouse embryonic hippocampal neurons suppressed dendritic growth and branching. Notably, the Database of Genomic Variants reported the case of a healthy control with a 488-kb deletion that included both R3HDM1 and MIR128-1. miR-128 has been reported to inhibit dendritic growth and branching in mouse brain neurons, which directly opposes the novel functions of R3HDM1. These findings suggest that deleting both R3HDM1 and MIR128-1 alleviates the symptoms of the disease caused by loss-of-function mutations in R3HDM1 only. Thus, haploinsufficiency of R3HDM1 in the patient may be the cause of the mild ID due to the genetic imbalance between R3HDM1 and MIR128-1.

Developmental defects and aberrant accumulation of endogenous psychosine in oligodendrocytes in a murine model of Krabbe disease.

Krabbe disease (KD), or globoid cell leukodystrophy, is an inherited lysosomal storage disease with leukodystrophy caused by a mutation in the galactosylceramidase (GALC) gene. The majority of patients show the early onset form of KD dominated by cerebral demyelination with apoptotic oligodendrocyte (OL) death. However, the initial pathophysiological changes in developing OLs remain poorly understood. Here, we show that OLs of twitcher mice, an authentic mouse model of KD, exhibited developmental defects and impaired myelin formation in vivo and in vitro. In twitcher mouse brain, abnormal myelination and reduced expression of myelin genes during the period of most active OL differentiation and myelination preceded subsequent progressive OL death and demyelination. Importantly, twitcher mouse OL precursor cells proliferated normally, but their differentiation and survival were intrinsically defective. These defects were associated with aberrant accumulation of endogenous psychosine (galactosylsphingosine) and reduced activation of the Erk1/2 and Akt/mTOR pathways before apoptotic cell death. Collectively, our results demonstrate that GALC deficiency in developing KD OLs profoundly affects their differentiation and maturation, indicating the critical contribution of OL dysfunction to KD pathogenesis.

The effect of rapamycin, NVP-BEZ235, aspirin, and metformin on PI3K/AKT/mTOR signaling pathway of PIK3CA-related overgrowth spectrum (PROS).

The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway is critical for cellular growth and metabolism. Recently, mosaic or segmental overgrowth, a clinical condition caused by heterozygous somatic activating mutations in PIK3CA, was established as PIK3CA-related overgrowth spectrum (PROS). In this study, we report a Japanese female diagnosed with PROS, who presented with hyperplasia of the lower extremities, macrodactyly, multiple lipomatosis, and sparse hair. Sequencing and mutant allele frequency analysis of PIK3CA from affected tissues revealed that the patient had a heterozygous mosaic mutation (c.3140A>G [p.H1047R]) in PIK3CA and that there were higher mutant allele frequencies from samples with a larger amount of subcutaneous adipose tissue. We established two fibroblast cell lines from the patient, harboring high and low frequencies of the mosaic mutation, in which AKT and S6 showed higher level of phosphorylation compared with three control fibroblasts, indicating that PI3K/AKT/mTOR signaling is activated. We assessed the therapeutic effects of four compounds (rapamycin, NVP-BEZ235, aspirin, and metformin) on PI3K/AKT/mTOR signaling pathway and cell growth. All four compounds suppressed S6 phosphorylation and inhibited cell growth of the patient-derived fibroblast cell lines. However, only metformin mildly inhibited the growth of the control fibroblast cell lines. Since PROS is a congenital disorder, drugs for therapy should take into consideration the natural growth of children. Thus, metformin is a candidate drug for treating PROS in growing children.

Niemann-Pick disease type C1 predominantly involving the frontotemporal region, with cortical and brainstem Lewy bodies: an autopsy case.

Niemann-Pick disease type C (NPC) is an autosomal recessive neurovisceral lipid storage disorder. Two disease-causing genes (NPC1 and NPC2) have been identified. NPC is characterized by neuronal and glial lipid storage and NFTs. Here, we report a man with juvenile-onset progressive neurological deficits, including pyramidal signs, ataxia, bulbar palsy, vertical supranuclear ophthalmoplegia, and psychiatric symptoms; death occurred at age 37 before definitive clinical diagnosis. Post mortem gross examination revealed a unique distribution of brain atrophy, predominantly in the frontal and temporal lobes. Microscopically, lipid storage in neurons and widely distributed NFTs were observed. Lipid storage cells appeared in systemic organs and filipin staining indicated intracellular cholesterol accumulation in hepatic macrophages. Electron microscopy revealed accumulation of lipids and characteristic oligolamellar inclusions. These findings suggested an NPC diagnosis. Neuronal loss and gliosis were frequently accompanied by NFTs and occurred in the frontal and temporal cortices, hippocampus, amygdala, basal forebrain, basal ganglia, thalamus, substantia nigra and brain stem nuclei. Lewy bodies (LBs) were observed in most, but not all, regions where NFTs were evident. In contrast, neuronal lipid storage occurred in more widespread areas, including the parietal and occipital cortices where neurodegeneration with either NFTs or LBs was minimal. Molecular genetic analysis demonstrated that the patient had compound heterozygous mutations in the cysteine-rich loop (A1017T and Y1088C) of the NPC1 gene. To our knowledge there has been no previous report of the A1017T mutation. The pathological features of this patient support the notion that NPC has an aspect of α-synucleinopathy, and long-term survivors of NPC may develop a frontotemporal-predominant distribution of brain atrophy.

[New aspects of neuronal cell death: the molecular basis of neurodevelopmental and neurodegenerative disorders].

During the past two decades, the fundamental mechanism of neuronal cell death has been explained by building on the knowledge obtained from studies of other cell types, such as immune cells and cancer cells. However, recent advances in biotechnology allow us to show much more detailed molecular mechanisms which can reveal characteristics of neuronal cell death distinguished from other cell types, and pathogenesis of neurodevelopmental and neurodegenerative disorders, that may help to develop treatment for various neurological disorders. Here, I will review the recent advances in the research on neuronal cell death associated with neurodevelopmental and neurodegenerative disorders focusing on the defect of DNA repair and of neuron-astrocyte metabolic interaction.

Mutant huntingtin impairs Ku70-mediated DNA repair.

DNA repair defends against naturally occurring or disease-associated DNA damage during the long lifespan of neurons and is implicated in polyglutamine disease pathology. In this study, we report that mutant huntingtin (Htt) expression in neurons causes double-strand breaks (DSBs) of genomic DNA, and Htt further promotes DSBs by impairing DNA repair. We identify Ku70, a component of the DNA damage repair complex, as a mediator of the DNA repair dysfunction in mutant Htt-expressing neurons. Mutant Htt interacts with Ku70, impairs DNA-dependent protein kinase function in nonhomologous end joining, and consequently increases DSB accumulation. Expression of exogenous Ku70 rescues abnormal behavior and pathological phenotypes in the R6/2 mouse model of Huntington's disease (HD). These results collectively suggest that Ku70 is a critical regulator of DNA damage in HD pathology.

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 Huntington's 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.

[Disruption of amino acid metabolism in astrocyte and neurological disorders--possible implication of abnormal glia-neuron network in homocystineuria].

CBS is a vitamin B6-dependent transsulfuration enzyme needed to synthesize cysteine from methionine, catalyzing the condensation of serine with homocysteine to form cystathionine. A deficiency of CBS causes homocystinuria (MIM 236200), one of the most prevalent inborn errors, characterized by mental retardation, seizures, psychiatric disturbances, skeletal abnormalities and vascular disorders. Patients with CBS deficiency exhibit a major biochemical abnormality, hyperhomocysteinemia (HHcy), a condition associated with highly elevated plasma homocysteine levels. HHcy is recognized as a risk factor for several neurological diseases, such as cognitive impairment, dementia and Alzheimer's disease. Although the link between CBS deficiency and homocystinuria was first described over 40 years ago and mental retardation was the first clinical feature of the disease to be classified, very little is known about the role of CBS in the CNS. 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 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. 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. These results support the role of CBS in the development and maintenance of the CNS, and suggest that radial glia/astrocyte dysfunction might be involved in the complex neuropathological features associated with abnormal homocysteine 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 Huntington's 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 zeta) 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.

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.

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 Huntington's 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 (YAPDeltaCs) were sustained during TRIAD to suppress neuronal death in a dominant-negative fashion. YAPDeltaCs and activated p73 were colocalized in the striatal neurons of HD patients and mutant huntingtin (htt) transgenic mice. YAPDeltaCs 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.

Cystathionine beta-synthase is enriched in the brains of Down's patients.

Down's syndrome (DS) or trisomy 21 is the most common genetic cause of mental retardation, and adults with DS develop Alzheimer type of disease (AD). Cystathionine beta-synthase (CBS) is encoded on chromosome 21 and deficiency in its activity causes homocystinuria, the most common inborn error of sulfur amino acid metabolism and characterized by mental retardation and vascular disease. Here, we show that the levels of CBS in DS brains are approximately three times greater than those in the normal individuals. CBS is localized to astrocytes and those surrounding senile plaques in the brains of DS patients with AD. The over-expression of CBS may cause the developmental abnormality in cognition in DS children and that may lead to AD in DS adults.

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

Cystathionine beta-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-alpha, 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 beta-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 beta-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 beta-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.

MOCA induces membrane spreading by activating Rac1.

The modifier of cell adhesion protein (MOCA), or Dock3, initially identified as presenilin-binding protein (PBP), belongs to the Dock180 family of proteins and is localized specifically in neurons. Here we demonstrate that MOCA binds to Rac1 and enhances its activity, which leads to the activation of c-Jun NH(2)-terminal kinase (JNK) and causes changes in cell morphology. Farnesylated MOCA, which is localized in the plasma membrane, enhances the activation of Rac1 and JNK more markedly than wild-type MOCA, and cells expressing farnesylated MOCA show flattened morphology similar to those expressing a constitutive active mutant of Rac1, Rac1Q61L. On poly-d-lysine-coated dishes, endogenous MOCA is concentrated on the leading edge of broad membrane protrusions (lamellipodia) where actin filaments are co-localized. MOCA is also concentrated with actin on the growth cone in primary cultures of cortical neurons. These observations suggest that MOCA may induce cytoskeletal reorganization and changes in cell adhesion by regulating the activity of Rac1.

Regulation of Bax translocation through phosphorylation at Ser-70 of Bcl-2 by MAP kinase in NO-induced neuronal apoptosis.

The molecular mechanism of Bcl-2 phosphorylation and its relationship to Bax is largely unknown. Here we show that the phosphorylation of Bcl-2 is involved in the intracellular translocation of Bax from cytosol to mitochondria in NO-induced neuronal apoptosis. We examined how the phosphorylation of Bcl-2 is regulated during the apoptosis and found it to be mediated by the activation of p38 and ERK, members of the MAPK superfamily. Furthermore, we investigated whether Bcl-2 phosphorylation affected Bax translocation, using mutant Bcl-2 expression vectors. Cortical neuronal cells overexpressing the Bcl-2 mutant S70A (which cannot be phosphorylated) prevented the translocation of Bax. In contrast, transfection with Bcl-2 (S70D), a constitutively active Bcl-2 mutant, enhanced the translocation. Our results suggested that Bcl-2 phosphorylated at Ser-70 plays a critial role in the translocation of Bax from the cytosol to the mitochondria, and this may regulate NO-induced neuronal apoptosis.