CRY Drives Cyclic CK2-Mediated BMAL1 Phosphorylation to Control the Mammalian Circadian Clock.

Intracellular circadian clocks, composed of clock genes that act in transcription-translation feedback loops, drive global rhythmic expression of the mammalian transcriptome and allow an organism to anticipate to the momentum of the day. Using a novel clock-perturbing peptide, we established a pivotal role for casein kinase (CK)-2-mediated circadian BMAL1-Ser90 phosphorylation (BMAL1-P) in regulating central and peripheral core clocks. Subsequent analysis of the underlying mechanism showed a novel role of CRY as a repressor for protein kinase. Co-immunoprecipitation experiments and real-time monitoring of protein-protein interactions revealed that CRY-mediated periodic binding of CK2ß to BMAL1 inhibits BMAL1-Ser90 phosphorylation by CK2α. The FAD binding domain of CRY1, two C-terminal BMAL1 domains, and particularly BMAL1-Lys537 acetylation/deacetylation by CLOCK/SIRT1, were shown to be critical for CRY-mediated BMAL1-CK2ß binding. Reciprocally, BMAL1-Ser90 phosphorylation is prerequisite for BMAL1-Lys537 acetylation. We propose a dual negative-feedback model in which a CRY-dependent CK2-driven posttranslational BMAL1-P-BMAL1 loop is an integral part of the core clock oscillator.

Role of hippocalcin in mediating Aß toxicity.

Alzheimer's disease (AD) is the most common cause of dementia, and amyloid-ß (Aß) plaques and tau-containing tangles are its histopathological hallmark lesions. These do not occur at random; rather, the neurodegenerative process is stereotyped in that it is initiated in the entorhinal cortex and hippocampal formation. Interestingly, it is the latter brain area where the calcium-sensing enzyme hippocalcin is highly expressed. Because calcium deregulation is a well-established pathomechanism in AD, we aimed to address the putative role of hippocalcin in human AD brain and transgenic mouse models. We found that hippocalcin levels are increased in human AD brain and in Aß plaque-forming APP23 transgenic mice compared to controls. To determine the role of hippocalcin in Aß toxicity, we treated primary cultures derived from hippocalcin knockout (HC KO) mice with Aß and found them to be more susceptible to Aß toxicity than controls. Likewise, treatment with either thapsigargin or ionomycin, both known to deregulate intracellular calcium levels, caused an increased toxicity in hippocampal neurons from HC KO mice compared to wild-type. We found further that mitochondrial complex I activity increased from 3 to 6months in hippocampal mitochondria from wild-type and HC KO mice, but that the latter exhibited a significantly stronger aging phenotype than wild-type. Aß treatment induced significant toxicity on hippocampal mitochondria from HC KO mice already at 3months of age, while wild-type mitochondria were spared. Our data suggest that hippocalcin has a neuroprotective role in AD, presenting it as a putative biomarker.

Down-regulation of MutS homolog 3 by hypoxia in human colorectal cancer.

Down-regulation of hMSH3 is associated with elevated microsatellite alterations at selected tetranucleotide repeats and low levels of microsatellite instability in colorectal cancer (CRC). However, the mechanism that down-regulates hMSH3 in CRC is not known. In this study, a significant association between over-expression of glucose transporter 1, a marker for hypoxia, and down-regulation of hMSH3 in CRC tissues was observed. Therefore, we examined the effect of hypoxia on the expression of hMSH3 in human cell lines. When cells with wild type p53 (wt-p53) were exposed to hypoxia, rapid down-regulation of both hMSH2 and hMSH3 occurred. In contrast, when null or mutated p53 (null/mut-p53) cells were exposed to hypoxia, only hMSH3 was down-regulated, and at slower rate than wt-p53 cells. Using a reporter assay, we found that disruption of the two putative hypoxia response elements (HREs) located within the promoter region of the hMSH3 abrogated the suppressive effect of hypoxia on reporter activity regardless of p53 status. In an EMSA, two different forms of HIF-1α complexes that specifically bind to these HREs were detected. A larger complex containing HIF-1α predominantly bound to the HREs in hypoxic null/mut-p53 cells whereas a smaller complex predominated in wt-p53 cells. Finally, HIF-1α knockdown by siRNA significantly inhibited down-regulation of hMSH3 by hypoxia in both wt-p53 and mut-p53 cells. Taken together, our results suggest that the binding of HIF-1α complexes to HRE sites is necessary for down-regulation of hMSH3 in both wt-p53 and mut-p53 cells.

CLOCK-mediated acetylation of BMAL1 controls circadian function.

Regulation of circadian physiology relies on the interplay of interconnected transcriptional-translational feedback loops. The CLOCK-BMAL1 complex activates clock-controlled genes, including cryptochromes (Crys), the products of which act as repressors by interacting directly with CLOCK-BMAL1. We have demonstrated that CLOCK possesses intrinsic histone acetyltransferase activity and that this enzymatic function contributes to chromatin-remodelling events implicated in circadian control of gene expression. Here we show that CLOCK also acetylates a non-histone substrate: its own partner, BMAL1, is specifically acetylated on a unique, highly conserved Lys 537 residue. BMAL1 undergoes rhythmic acetylation in mouse liver, with a timing that parallels the downregulation of circadian transcription of clock-controlled genes. BMAL1 acetylation facilitates recruitment of CRY1 to CLOCK-BMAL1, thereby promoting transcriptional repression. Importantly, ectopic expression of a K537R-mutated BMAL1 is not able to rescue circadian rhythmicity in a cellular model of peripheral clock. These findings reveal that the enzymatic interplay between two clock core components is crucial for the circadian machinery.

Dicalcin suppresses invasion and metastasis of mammalian ovarian cancer cells by regulating the ganglioside-Erk1/2 axis.

Metastasis, a multistep process including cancer cell migration and invasion, is the major cause of mortality in patients with cancer. Here, we investigated the effect of dicalcin, a Ca2+-binding protein, on the invasion and metastasis of ovarian cancer (OC) cells. Extracellularly administered dicalcin bound to the membrane of OV2944 cells, mouse OC cells, and suppressed their migration in vitro; however, cell viability or proliferation were unaffected. Repeated intraperitoneal injection of a partial peptide of dicalcin (P6) prolonged the survival, and reduced the number of microcolonies in the livers of cancer-bearing mice. P6 bound to the ganglioside GM1b in a solid-phase assay; treatment with P6 inhibited the constitutive activation of Erk1/2 in OC cells, whereas excess administration of GM1b augmented Erk activity and cancer cell migration in vitro. Thus, dicalcin, a novel suppressor of invasion and metastasis of OC cells, acts via the GM1b-Erk1/2 axis to regulate their migration.

Hippocalcin and KCNQ channels contribute to the kinetics of the slow afterhyperpolarization.

The calcium-activated slow afterhyperpolarization (sAHP) is a potassium conductance implicated in many physiological functions of the brain including memory, aging, and epilepsy. In large part, the sAHP's importance stems from its exceedingly long-lasting time-course, which integrates action potential-induced calcium signals and allows the sAHP to control neuronal excitability and prevent runaway firing. Despite its role in neuronal physiology, the molecular mechanisms that give rise to its unique kinetics are, to our knowledge, still unknown. Recently, we identified KCNQ channels as a candidate potassium channel family that can contribute to the sAHP. Here, we test whether KCNQ channels shape the sAHP rise and decay kinetics in wild-type mice and mice lacking Hippocalcin, the putative sAHP calcium sensor. Application of retigabine to speed KCNQ channel activation accelerated the rise of the CA3 pyramidal neuron sAHP current in both wild-type and Hippocalcin knockout mice, indicating that the gating of KCNQ channels limits the sAHP activation. Interestingly, we found that the decay of the sAHP was prolonged in Hippocalcin knockout mice, and that the decay was sensitive to retigabine modulation, unlike in wild-type mice. Together, our results demonstrate that sAHP activation in CA3 pyramidal neurons is critically dependent on KCNQ channel kinetics whereas the identity of the sAHP calcium sensor determines whether KCNQ channel kinetics also limit the sAHP decay.

Hippocalcin mediates calcium-dependent translocation of brain-type creatine kinase (BB-CK) in hippocampal neurons.

Hippocalcin (Hpca) is a Ca(2+)-binding protein that is expressed in neurons and contributes to neuronal plasticity. We purified a 48 kDa Hpca-associated protein from rat brain and identified it to be the creatine kinase B (CKB) subunit, which constitutes brain-type creatine kinase (BB-CK). Hpca specifically bound to CKB in a Ca(2+)-dependent manner, but not to the muscle-type creatine kinase M subunit. The N-terminal region of Hpca was required for binding to CKB. Hpca mediated Ca(2+)-dependent partial translocation of CKB (approximately 10-15% of total creatine kinase activity) to membranes. N-myristoylation of Hpca was critical for membrane translocation, but not for binding to CKB. In cultured hippocampal neurons, ionomycin treatment led to colocalization of Hpca and CKB adjacent to the plasma membrane. These results indicate that Hpca associates with BB-CK and that together they translocate to membrane compartments in a Ca(2+)-dependent manner.

Immunohistochemical Characterization of S100A6 in the Murine Ovary.

S100 proteins comprise a large family of Ca(2+)-binding proteins and exhibit a variety of intra- and extracellular functions. Despite our growing knowledge about the biology of S100 proteins in some tissues such as brain and smooth muscle, little is known about S100 proteins in the normal mammalian reproductive tissue. In the present study, we investigated the distribution pattern of S100A6 (alternatively named calcyclin) in the murine ovary by immunohistochemical study using specific antibody. S100A6 was localized substantially in the cytoplasm of luteal cells, with concomitant expression of S100A11, another S100 protein, but not in the other type of cells such as oocytes, follicle epithelial cells (granulosa cells), and cells of stroma including theca interna cells in the murine ovary. S100A6-immunoreactive corpora lutea (CLs) were divided into two types: homogeneously and heterogeneously stained CLs, and possibly they may represent differentiating and mature CL, respectively. Our regression analysis revealed that expression level of S100A6 positively correlated with that of cytochrome P450 11A, a steroidogenic enzyme in the heterogeously stained CL. These results suggested that S100A6 may contribute to differentiation of steroidogenic activity of luteal cells in a synergistic manner with S100A11 by facilitating some shared functions.

Dicalcin inhibits fertilization through its binding to a glycoprotein in the egg envelope in Xenopus laevis.

Fertilization comprises oligosaccharide-mediated sperm-egg interactions, including sperm binding to an extracellular egg envelope, sperm penetration through the envelope, and fusion with an egg plasma membrane. We show that Xenopus dicalcin, an S100-like Ca(2+)-binding protein, present in the extracellular egg envelope (vitelline envelope (VE)), is a suppressive mediator of sperm-egg interaction. Preincubation with specific antibody greatly increased the efficiency of in vitro fertilization, whereas prior application of exogenous dicalcin substantially inhibited fertilization as well as sperm binding to an egg and in vitro sperm penetration through the VE protein layer. Dicalcin showed binding to protein cores of gp41 and gp37, constituents of VE, in a Ca(2+)-dependent manner and increased in vivo reactivity of VE with a lectin, Ricinus communis agglutinin I, which was accounted for by increased binding ability of gp41 to the lectin and greater exposure of gp41 to an external environment. Our findings strongly suggest that dicalcin regulates the distribution of oligosaccharides within the VE through its binding to the protein core of gp41, probably by modulating configuration of oligosaccharides on gp41 and the three-dimensional structure of VE framework, and thereby plays a pivotal role in sperm-egg interactions during fertilization.

Characterization of S100A11, a suppressive factor of fertilization, in the mouse female reproductive tract.

We recently found that Xenopus dicalcin, present in the extracellular egg-coating envelope, suppresses the efficiency of fertilization in vitro through binding to envelope-constituent glycoproteins. In the present study, we explored the mouse counterpart of Xenopus dicalcin, specifically its localization in the female reproductive tract and its action on mouse fertilization. Our homology and phylogenetic analyses using known S100 proteins showed that S100A11 is most closely related to Xenopus dicalcin. S100A11 was localized in the cytosol of luteal cells, but not in the follicle, in the mouse ovary, and also in the cytosol of the oviductal epithelial cells. In addition, our quantitative analyses revealed preferential expression of S100A11 in the ampullary region of the oviduct and at the estrus stage during the mouse estrous cycle. In the cumulus cell-oocyte complex dissected from the oviduct following ovulation, S100A11 was present in the plasma membrane of cumulus cells, but not in the zona pellucida, which is comparable with Ca(2+) -dependent binding of exogenously applied S100A11 to the plasma membrane of cumulus cells. Pretreatment of the cumulus cell-oocyte complex with recombinant S100A11 substantially reduced the efficiency of in vitro fertilization, but S100A10, the next closest S100 protein to Xenopus dicalcin, had no effect. These results suggested that S100A11 is the mouse counterpart of Xenopus dicalcin, suppresses the fertilization process through its action on cumulus cells, and thereby plays a key role in fertilization success in the mouse.

Cell surface N-glycans mediated isolation of mouse neural stem cells.

The isolation of neural stem cells (NSCs) from the brain has been hampered by the lack of valid cell surface markers and the requirement for long-term in vitro cultivation that may lead to phenotype deterioration. However, few suitable specific cell surface antigens are available on NSCs that could be used for their prospective isolation. The present study demonstrated that the expression of complex type asparagine-linked oligosaccharide (N-glycans) was detected on brain cells dissociated from embryonic and adult brain using Phaseolus vulgaris erythroagglutinating lectin (E-PHA) which binds to biantennary complex type N-glycans, and demonstrated that E-PHA bound preferentially to purified NSCs, but not to neurons, microglia, or oligodendrocyte precursor cells. The labeling of dissociated mouse embryonic brain cells or adult brain cells with E-PHA enabled the enrichment of NSCs by 25-fold or 9-fold of the number of neurosphere-forming cells in comparison to that of unsorted cells, respectively. Furthermore, a lectin blot analysis revealed the presence of several glycoproteins which were recognized by E-PHA in the membrane fraction of the proliferating NSCs, but not in the differentiated cells. These results indicate that complex type N-glycans is a valuable cell surface marker for living mouse NSCs from both the embryonic and adult brain.

Developmental changes in the regulation of calcium-dependent neurite outgrowth.

Intracellular calcium ions (Ca(2+)) have an essential role in the regulation of neurite outgrowth, but how outgrowth is controlled remains largely unknown. In this study, we examined how the mechanisms of neurite outgrowth change during development in chick and mouse dorsal root ganglion neurons. 2APB, a potent inhibitor of inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R), inhibited neurite outgrowth at early developmental stages, but not at later stages. In contrast, pharmacological inhibition with Ni(2+), Cd(2+), or dantrolene revealed that ryanodine receptor (RyR)-mediated Ca(2+)-induced Ca(2+) release (CICR) was involved in neurite outgrowth at later stage, but not at early stages. The distribution of IP(3)R and RyR in growth cones also changed during development. Furthermore, pharmacological inhibition of the Ca(2+)-calmodulin-dependent phosphatase calcineurin with FK506 reduced neurite outgrowth only at early stages. These data suggest that the calcium signaling that regulates neurite outgrowth may change during development from an IP(3)R-mediated pathway to a RyR-mediated pathway.

Hippocalcin, new Ca(2+) sensor of a ROS-GC subfamily member, ONE-GC, membrane guanylate cyclase transduction system.

Hippocalcin is a member of the neuronal Ca(2+) sensor protein family. Among its many biochemical functions, its established physiological function is that via neuronal apoptosis inhibitory protein it protects the neurons from Ca(2+)-induced cell death. The precise biochemical mechanism/s, through which hippocalcin functions, is not clear. In the present study, a new mechanism by which it functions is defined. The bovine form of hippocalcin (BovHpca) native to the hippocampus has been purified, sequenced, cloned, and studied. The findings show that there is the evolutionary conservation of its structure. It is a Ca(2+)-sensor of a variant form of the ROS-GC subfamily of membrane guanylate cyclases, ONE-GC. It senses physiological increments of Ca(2+) with a K(1/2) of 0.5 microM and stimulates ONE-GC or ONE-GC-like membrane guanylate cyclase. The Hpca-modulated ONE-GC-like transduction system exists in the hippocampal neurons. And hippocalcin-modulated ONE-GC transduction system exists in the olfactory receptor neuroepithelium. The Hpca-gene knock out studies demonstrate that the portion of this is about 30% of the total membrane guanylate cyclase transduction system. The findings establish Hpca as a new Ca(2+) sensor modulator of the ROS-GC membrane guanylate cyclase transduction subfamily. They support the concept on universality of the presence and operation of the ROS-GC transduction system in the sensory and sensory-linked neurons. They validate that the ROS-GC transduction system exists in multiple forms. And they provide an additional mechanism by which ROS-GC subfamily acts as a transducer of the Ca(2+) signals originating in the neurons.

Cell-permeable p38 MAP kinase protects adult hippocampal neurons from cell death.

p38 mitogen-activated protein (MAP) kinase (p38) is a member of the MAP kinase family. Previous reports using p38 chemical inhibitors have suggested that its activation contributes to hippocampal neuronal cell death rather than cell survival. In this study, we used both a cell-permeable p38 protein containing the HIV protein transduction domain (PTD) and cultured adult hippocampal neurons, which were differentiated from cultured adult hippocampal neural stem/progenitor cells (NPCs), to evaluate the direct function of p38 on adult hippocampal neurons. Our immunocytochemical experiments demonstrated that wild-type cell-permeable p38 protein prevents cell death of adult hippocampal neurons induced by a low glucose condition. Our findings indicate that cell-permeable p38 protein may be useful in preventing the degeneration of higher brain function occurring through hippocampal neuronal cell death, and furthermore, that the maintenance of intracellular p38 levels could be another therapeutic target for neurodegenerative diseases such as Alzheimer's disease (AD).

Genome-wide DNA methylation analysis in obese women predicts an epigenetic signature for future endometrial cancer.

Aberrant DNA methylation is associated with the oncogenesis of a variety of human cancers, including endometrial cancer (EC), the seventh most common cancer among women. Obesity is known to be a high-risk factor for EC; however, whether obesity influences DNA methylation in the presymptomatic uterus and if this influences EC development remain unclear. Here, we performed genome-wide DNA methylation analysis of isolated endometrial epithelial cells obtained from obese presymptomatic participants. Using the Illumina MethylationEPIC array (850 K), we identified 592 differentially methylated regions (DMRs), most of which undergo hypomethylated changes. These DMRs were enriched for pyrimidine metabolism, Epstein-Barr virus infection, and B cell signaling pathways, indicating obesity-related dysregulation of certain metabolic processes in the presymptomatic uterus. Comparison of the DMRs with those in stage I EC revealed that 54 DMRs overlapped; additionally, B cell signaling and Epstein-Barr virus infection pathways were shared between the presymptomatic uterus of obese women and stage I EC with greater hypomethylation in women with EC than in presymptomatic obese women. These findings indicated that obesity influences DNA methylation in presymptomatic endometrial epithelial cells, and persistent dysregulation of DNA methylation in obese women may result in EC development.

The clock components Period2, Cryptochrome1a, and Cryptochrome2a function in establishing light-dependent behavioral rhythms and/or total activity levels in zebrafish.

The circadian clock generates behavioral rhythms to maximize an organism's physiological efficiency. Light induces the formation of these rhythms by synchronizing cellular clocks. In zebrafish, the circadian clock components Period2 (zPER2) and Cryptochrome1a (zCRY1a) are light-inducible, however their physiological functions are unclear. Here, we investigated the roles of zPER2 and zCRY1a in regulating locomotor activity and behavioral rhythms. zPer2/zCry1a double knockout (DKO) zebrafish displayed defects in total locomotor activity and in forming behavioral rhythms when briefly exposed to light for 3-h. Exposing DKO zebrafish to 12-h light improved behavioral rhythm formation, but not total activity. Our data suggest that the light-inducible circadian clock regulator zCRY2a supports rhythmicity in DKO animals exposed to 12-h light. Single cell imaging analysis revealed that zPER2, zCRY1a, and zCRY2a function in synchronizing cellular clocks. Furthermore, microarray analysis of DKO zebrafish showed aberrant expression of genes involved regulating cellular metabolism, including ATP production. Overall, our results suggest that zPER2, zCRY1a and zCRY2a help to synchronize cellular clocks in a light-dependent manner, thus contributing to behavioral rhythm formation in zebrafish. Further, zPER2 and zCRY1a regulate total physical activity, likely via regulating cellular energy metabolism. Therefore, these circadian clock components regulate the rhythmicity and amount of locomotor behavior.

Inhibitors of p38 mitogen-activated protein kinase enhance proliferation of mouse neural stem cells.

The p38 mitogen-activated protein kinase (MAPK) is induced in response to environmental stress. Although p38 MAPK has been implicated in diverse cellular processes, including cell proliferation, differentiation, and survival of differentiated cells in the central nervous system (CNS), the expression profile and roles of p38 MAPK in the developing brain remain largely unknown. In the present study, we demonstrate that p38 MAPK is expressed predominantly in nestin-positive cells in the cerebral cortex in embryonic day 10 (E10) brain and that expression of the protein decreases gradually during development. To investigate the roles of p38 MAPK in the embryonic brain, two selective p38 MAPK inhibitors, SB202190 and SB203580, were added to the primary neuronal cultures from E10-E14 brains. After 7 days of exposure to these inhibitors, but not SB202474, a negative analog of SB203580, numerous large neurospheres were present. MAPK inhibitors also selectively increased the growth rate of neural stem cells (NSCs) purified from secondary neurospheres and the number of bromodeoxyuridine-positive NSCs. Thus, p38 MAPK inhibitors are potent stimulators of NSC proliferation, and p38 MAPK may be an intrinsic negative regulator of NSC proliferation during early brain development.

Circadian modification network of a core clock driver BMAL1 to harmonize physiology from brain to peripheral tissues.

Circadian clocks dictate various physiological functions by brain SCN (a central clock) -orchestrating the temporal harmony of peripheral clocks of tissues/organs in the whole body, with adaptability to environments by resetting their timings. Dysfunction of this circadian adaptation system (CAS) occasionally causes/exacerbates diseases. CAS is based on cell-autonomous molecular clocks, which oscillate via a core transcriptional/translational feedback loop with clock genes/proteins, e.g., BMAL1: CLOCK circadian transcription driver and CRY1/2 and PER1/2 suppressors, and is modulated by various regulatory loops including clock protein modifications. Among mutants with a single clock gene, BMAL1-deficient mice exhibit the most drastic loss of circadian functions. Here, we highlight on numerous circadian protein modifications of mammalian BMAL1, e.g., multiple phosphorylations, SUMOylation, ubiquitination, acetylation, O-GlcNAcylation and S-nitrosylation, which mutually interplay to control molecular clocks and coordinate physiological functions from the brain to peripheral tissues through the input and output of the clocks.

Cooperative interaction among BMAL1, HSF1, and p53 protects mammalian cells from UV stress.

The circadian clock allows physiological systems to adapt to their changing environment by synchronizing their timings in response to external stimuli. Previously, we reported clock-controlled adaptive responses to heat-shock and oxidative stress and showed how the circadian clock interacts with BMAL1 and HSF1. Here, we present a similar clock-controlled adaptation to UV damage. In response to UV irradiation, HSF1 and tumor suppressor p53 regulate the expression of the clock gene Per2 in a time-dependent manner. UV irradiation first activates the HSF1 pathway, which subsequently activates the p53 pathway. Importantly, BMAL1 regulates both HSF1 and p53 through the BMAL1-HSF1 interaction to synchronize the cellular clock. Based on these findings and transcriptome analysis, we propose that the circadian clock protects cells against the UV stress through sequential and hierarchical interactions between the circadian clock, the heat shock response, and a tumor suppressive mechanism.

Inhibition of p38 mitogen-activated protein kinase-induced apoptosis in cultured mature oligodendrocytes using SB202190 and SB203580.

p38 Mitogen-activated protein kinase (p38 MAPK) is expressed in the oligodendrocyte lineage, and its activity has been implicated in the proliferation and transition of early progenitors into late progenitors. Although p38 MAPK expression has been found in the myelin sheath, however, its role in mature oligodendrocytes remains unknown. In the present study, in order to address the role of p38 MAPK in mature oligodendrocytes, selective inhibitors of p38 MAPK, SB202190, and SB203580 were added to primary cultures of mature oligodendrocytes. After 24h of exposure to the inhibitors, the appearance, and number of A2B5-positive progenitors were unchanged. However, the 2',3'-cyclic nucleotide-3'-phosphohydrolase-positive mature oligodendrocytes disappeared, and the numbers of living cells decreased in comparison to the control cells treated with SB202474, a negative analog of SB203580. Increases in the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive nuclei and in the activity of caspase-3/7 were detected 16 h after exposure to the inhibitors, thus causing the mature oligodendrocytes to die due to apoptosis. Similar results were obtained using a differentiated rat oligodendrocyte precursor cell (OPC) line, central glia-4 (CG-4). These findings indicate that p38 MAPK is vital for mature oligodendrocyte survival.