The Japan-Multimodal Intervention Trial for Prevention of Dementia (J-MINT): The Study Protocol for an 18-Month, Multicenter, Randomized, Controlled Trial.

BACKGROUND/OBJECTIVES: The Japan-multimodal intervention trial for prevention of dementia (J-MINT) is intended to verify the effectiveness of multi-domain interventions and to clarify the mechanism of cognitive improvement and deterioration by carrying out assessment of dementia-related biomarkers, omics analysis and brain imaging analysis among older adults at high risk of dementia. Moreover, the J-MINT trial collaborates with partnering private enterprises in the implementation of relevant interventional measures. This manuscript describes the study protocol. DESIGN/SETTING: Eighteen-month, multi-centered, randomized controlled trial. PARTICIPANTS: We plan to recruit 500 older adults aged 65-85 years with mild cognitive impairment. Subjects will be centrally randomized into intervention and control groups at a 1:1 allocation ratio using the dynamic allocation method with all subjects stratified by age, sex, and cognition. INTERVENTION: The multi-domain intervention program includes: (1) management of vascular risk factors; (2) group-based physical exercise and self-monitoring of physical activity; (3) nutritional counseling; and (4) cognitive training. Health-related information will be provided to the control group every two months. MEASUREMENTS: The primary and secondary outcomes will be assessed at baseline, 6-, 12-, and 18-month follow-up. The primary outcome is the change from baseline to 18 months in a global composite score combining several neuropsychological domains. Secondary outcomes include: cognitive change in each neuropsychological test, incident dementia, changes in blood and dementia-related biomarkers, changes in geriatric assessment including activities of daily living, frailty status and neuroimaging, and number of medications taken. CONCLUSIONS: This trial that enlist the support of private enterprises will lead to the creation of new services for dementia prevention as well as to verify the effectiveness of multi-domain interventions for dementia prevention.

Lysophosphatidylcholine increases the neurotoxicity of Alzheimer's amyloid ß1-42 peptide: role of oligomer formation.

Oligomer formation is considered as a critical process for the neurotoxic effects of Alzheimer's amyloid ß (Aß) peptide. Previously we have demonstrated that lysophosphatidylcholine (LPC) increases the oligomer formation of Aß1-42, the major Aß peptide found Alzheimer's disease (AD) lesions. In this study, we have investigated whether LPC affects the neurotoxic effects of Aß1-42 in a neuronal cell line (A1) culture. Dimethyl thiazolyl diphenyl tetrazolium (MTT) assay revealed that up to 10µM concentration, LPC did not affect A1 cell viability. Aß1-42 decreased the cell viability, and such effect was dose dependently enhanced by LPC. However, neither LPC nor Aß1-42, alone or in combination increased lactate dehydrogenase (LDH) release from A1 cells after 24-h treatment. Terminal deoxynucleotidyl transferase dUTP-biotin nick-end-labeling (TUNEL) assay showed that LPC increased Aß1-42-induced apoptotic cell number. To determine the underlying mechanisms, the proteins implicated in apoptosis pathways including Bcl-2- and caspase-family were analyzed by Western blotting. The results demonstrated that Aß1-42 decreased Bcl-2 in A1 cells at 24h, whereas LPC had no effect at any time point. Both LPC and Aß1-42 increased Bax level at 24h, and their combined stimulation showed a synergistic effect. Similar synergistic effect of LPC and Aß1-42 on caspase9 activation was observed. Dot blot immunoassay and Western blotting showed that LPC augmented Aß1-42 oligomer formation in cell culture medium. Removing LPC-induced early-formed Aß1-42 oligomer from the culture medium by immunoprecipitation decreased active caspase9 level and neurotoxicity, as revealed by Western blotting and MTT assay. Furthermore, dihydroethidium (DHE) assay showed that Aß1-42 increased reactive oxygen species level in A1 cells, such effect was further enhanced by LPC. Thus, our results demonstrated that LPC increased the oligomer formation process of Aß1-42 peptide in culture condition, and consequently increased apoptotic neuronal death. Such process might be important for the pathogenesis of AD, and inhibition of LPC generation could be a therapeutic target for the disease.

Role of cholesterol in amyloid cascade: cholesterol-dependent modulation of tau phosphorylation and mitochondrial function.

Apolipoprotein E (apoE) alleles are important genetic risk factors for Alzheimer's disease (AD), with the epsilon4 allele increasing and the epsilon2 allele decreasing the risk of developing AD. ApoE is the major apolipoprotein that modulates cholesterol transport in the central nervous system, cholesterol being an essential component of membranes for maintaining their structure and functions. Epidemiological studies have suggested a link between serum cholesterol levels and AD development and the potential therapeutic effectiveness of statins for AD; and furthermore, biological studies have shown that amyloid beta-protein (Abeta) secretion is modulated by cellular cholesterol level. However, other lines of evidence show controversial results. In addition to the role of cholesterol in Abeta generation, different interactions of cholesterol with Abeta and its role in AD pathogenesis have been shown, i.e. Abeta affects cholesterol dynamics in neurons, and altered cholesterol metabolism in turn leads to neurodegeneration with abnormally phosphorylated tau (tauopathy). In this review, the reciprocal interactions between cholesterol and Abeta, and the role of cholesterol in tauopathy are discussed. The isoform-specific involvement of apoE in this cascade, in which high-density lipoprotein-like particles are generated and supplied to neurons to maintain cholesterol homeostasis, is also discussed.

A novel action of alzheimer's amyloid beta-protein (Abeta): oligomeric Abeta promotes lipid release.

Interactions between amyloid beta-protein (Abeta) and lipids have been suggested to play important roles in the pathogenesis of Alzheimer's disease. However, the molecular mechanism underlying these interactions has not been fully understood. We examined the effect of Abeta on lipid metabolism in cultured neurons and astrocytes and found that oligomeric Abeta, but not monomeric or fibrillar Abeta, promoted lipid release from both types of cells in a dose- and time-dependent manner. The main components of lipids released after the addition of Abeta were cholesterol, phospholipids, and monosialoganglioside (GM1). Density-gradient and electron microscopic analyses of the conditioned media demonstrated that these Abeta and lipids formed particles and were recovered from the fractions at densities of approximately 1.08-1.18 g/ml, which were similar to those of high-density lipoprotein (HDL) generated by apolipoproteins. The lipid release mediated by Abeta was abolished by concomitant treatment with Congo red and the PKC inhibitor, H7, whereas it was not inhibited with N-acetyl-l-cysteine. These Abeta-lipid particles were not internalized into neurons, whereas HDL-like particles produced by apolipoprotein E were internalized. Our findings indicate that oligomeric Abeta promotes lipid release from neuronal membrane, which may lead to the disruption of neuronal lipid homeostasis and the loss of neuronal function.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) causes Akt phosphorylation and morphological changes in intracellular organellae in cultured rat astrocytes.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is widely used for cell viability and cytotoxicity assays, but cell biological effects of MTT itself have not been investigated. In this paper we show that MTT induces a morphological change in an intracellular membranous compartment labeled with anti-Rab5 antibody, dissociation of early endosomal auto-antigen (EEA1) from the membrane fraction, and phosphorylation of Akt probably through a phosphatidylinositol-3-OH kinase [PI(3)K] pathway in cultured rat astrocytes. These findings suggest that MTT affects cellular functions and conditions to some extent, and such effects of MTT may cause some discrepancies of measurement of cell viability using MTT assay and other assays. That is, the effects of MTT on cells could influence the results of cell viability assay. Moreover, MTT or other tetrazolium salts could be used as interesting activators of Akt to investigate the mechanism by which Akt or PI(3)K is activated.

Cholesterol-dependent modulation of tau phosphorylation in cultured neurons.

One of the hallmarks of Alzheimer's disease (AD) is the abnormal state of tau. It is both highly phosphorylated and aggregated into paired helical filaments (PHFs) in neurofibrillary tangles (NFTs). However, the mechanism underlying the hyperphosphorylation of tau in NFTs and neuronal degeneration in AD remains to be elucidated. The fact that hyperphosphorylation of tau in NFTs are also found in the patients with Niemann-Pick disease, type C (NPC), which is a cholesterol storage disease associated with defective intracellular trafficking of exogenous cholesterol, implies that perturbation of cholesterol metabolism may be involved in tau phosphorylation and neurodegeneration. Here, we report that cholesterol deficiency induced by inhibition of cholesterol biosynthesis in cultured neurons results in hyperphosphorylation of tau, accompanied by axonal degeneration associated with microtubule depolymerization. These changes were prevented by concurrent treatment with beta-migrating very low-density lipoprotein (beta-VLDL) or cholesterol. We propose that intracellular cholesterol plays an essential role in the modulation of tau phosphorylation and the maintenance of microtubule stability.

Site-specific phosphorylation of tau accompanied by activation of mitogen-activated protein kinase (MAPK) in brains of Niemann-Pick type C mice.

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol in most tissues and progressive neurodegeneration with the formation of neurofibrillary tangles. Neurofibrillary tangles are composed of paired helical filaments (PHF), a major component of which is the hyperphosphorylated tau. In this study we used NPC heterozygous and NPC homozygous mouse brains to investigate the molecular mechanism responsible for tauopathy in NPC. Immunoblot analysis using anti-tau antibodies (Tau-1, PHF-1, AT-180, and AT-100) revealed site-specific phosphorylation of tau at Ser-396 and Ser-404 in the brains of NPC homozygous mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases such as glycogen synthase kinase-3beta and cyclin-dependent kinase 5 were inactive. Morphological examination demonstrated that a number of neurons, the perikarya of which strongly immunostained with PHF-1, exhibited polymorphorous cytoplasmic inclusion bodies and multi-concentric lamellar-like bodies. Importantly, the accumulation of intracellular cholesterol in NPC mouse brains was determined to be a function of age. From these results we conclude that abnormal cholesterol metabolism due to the genetic mutation in NPC1 may be responsible for activation of the mitogen-activated protein kinase-signaling pathway and site-specific phosphorylation of tau in vivo, leading to tauopathy in NPC.

Expression and regulation of apolipoprotein E receptors in the cells of the central nervous system in culture: A review.

The importance of apolipoprotein E (apoE) in the central nervous system (CNS) became increasingly clear since the descovery that apoE ε4 allele is a major risk factor for Alzheimer's disease. ApoE is one of the major apolipoproteins that acts as a ligand for the cellular uptake of lipoproteins via apoE receptors, members of low-density lipoprotein receptor (LDLR) family, in the CNS. Recently, LDLR family has been shown to have new functions that modulate intracellular signalling and affect neuronal and glial functions, survival and regeneration. However, the pattern of expression of apoE receptors in the CNS has not been fully clarified yet. The LDLR, very low density lipoprotein receptor (VLDLR), LDLR-related protein (LRP), and apolipoprotein E receptor 2 (apoER2) are known to bind to and internalize apoE-containing lipoproteins. Here we summarize the expression of apoE receptors in the CNS and demonstrate additional our original data on cell type specific expression and regulation of those receptors in the CNS, using in situ hybridization and RT-PCR. The cells used in our study were highly enriched cultures of neurons, astrocytes, microglia and oligodendrocytes isolated from rat brain and neuroblastoma cell line, Neuro2a. All of these four types of receptors were shown to be expressed in neurons, astrocytes, microglia and oligodendrocytes, while LDLR and LRP were expressed in Neuro2a cells. We further examined the regulation of the expression of these receptors by altering the cholesterol content of the cells, and found that only the LDLR expression was downregulated following internalization of lipoprotein cholesterol and upregulated by cholesterol deprivation, in neuronal and astroglial cells. These data together with previous studies suggest that LDLR, VLDL, LRP, and apoER2 may be involved in apoE-mediated lipid uptake and/or intracellualr signalling in the cells of the CNS cells, i.e., neurons, astrocytes, microglia, and oligodendrocytes.

A possible model of senile plaques using synthetic amyloid beta-protein and rat glial culture.

The senile plaque (SP) is one of the pathological hallmarks in the brains of patients with Alzheimer's disease (AD), but the mechanism of its formation and its role in AD progression are not yet fully understood. Synthetic amyloid beta-protein (Abeta)1-40 is known to aggregate in vitro, and the aggregated Abeta has been widely used for in vitro experiments, in which its peculiar effects on neuronal and glial cells have been shown. To date, however, the formation of a SP-like structure in a culture system using synthetic Abeta has not been demonstrated. In this study, we established a possible SP model using synthetic Abeta1-40 and rat glial cultures as follows: (1) large spherical aggregates of synthetic Abeta (sAmys) were produced from synthetic Abeta1-40 (10-50 microm in diameter), (2) the sAmys were added to a glial culture, and (3) the characteristics of the sAmys and the reactions of glial cells (microglia and astrocytes) around the sAmys were analyzed. We found that the sAmys exhibited the same features as the dense amyloid core in SPs, including the intense green birefringence under polarized light with Congo red, and induced reactive features in glial cells, including induction of major histocompatibility complex class II antigen in the microglia and interleukin-1beta in the astrocytes, similar to those seen in SPs in the brain in AD. Given our findings, we consider that this glial culture system with the sAmys is a possible in vitro SP model and useful for investigating the effects of massive amyloid deposition on neuronal and glial cells.

Apolipoprotein E exhibits isoform-specific promotion of lipid efflux from astrocytes and neurons in culture.

Many studies have shown that apolipoprotein E (apoE) plays important roles in maintaining intracellular lipid homeostasis in nonneuronal cells. However, little is known about the extracellular transport of lipids in the CNS. In this study, we determined whether and to what degree lipid efflux from astrocytes and neurons depended on apoE. Our results showed that exogenously added apoE promoted the efflux of cholesterol and phosphatidylcholine from both astrocytes and neurons in culture, resulting in the generation of high-density lipoprotein-like particles. The order of potency of the apoE isoforms as lipid acceptors was apoE2 > apoE3 = apoE4 in astrocytes and apoE2 > apoE3 > apoE4 in neurons. Treatment with brefeldin A, monensin, and a protein kinase C inhibitor, H7, abolished the ability of apoE to promote cholesterol efflux from cultured astrocytes, without altering apoE-mediated phosphatidylcholine efflux. In contrast, the efflux of both cholesterol and phosphatidylcholine promoted by apoE was abolished following treatment with heparinase or lactoferrin, which block the interaction of apoE with heparan sulfate proteoglycans (HSPGs) or low-density lipoprotein receptor-related protein (LRP), respectively. This study suggests that apoE promotes lipid efflux from astrocytes and neurons in an isoform-specific manner and that cell surface HSPGs and/or HSPG-LRP pathway may mediate this apoE-promoted lipid efflux.

Amyloid precursor protein in unique cholesterol-rich microdomains different from caveolae-like domains.

To determine the localization of the amyloid precursor protein (APP) on the cellular membrane, we performed membrane fractionation of cultured cells including that of Madin-Darby canine kidney (MDCK) and P19 cells transfected with human APP cDNA, non-transfected SH-SY5Y cells, and rat cerebral cortices. In MDCK cells, APP was exclusively present in abundance in the supernatant following solubilization of the plasma membranes using Triton X-100, and in high-density fractions of sucrose density gradient fractionation (SDGF) following Triton X-100 solubilization of whole cellular membranes. Caveolin-1 was not cofractionated with APP. In experiments using P19 cells and rat cerebral cortices, we detected two isoforms of APP. The APP with the apparently lower molecular weight (immature type) coexisted in abundance with integrin in the high-density fractions, whereas the APP with the apparently higher molecular weight (mature type) was recovered predominantly in the low-density fractions with cholesterol and GM1 gangliosides, the concentrations of which were higher than those in the bulk plasma membranes, but lower than those in caveolae-like domains (CLDs), following SDGF of Triton X-100-solubilized cellular membranes. The results of this study suggest the following; first, APP is not present in abundance in caveolae or CLDs, but is in unique cholesterol-rich microdomains; second, the targeting of APP to these unique microdomains may be linked to the maturation of APP in some cells.

Midkine inhibits caspase-dependent apoptosis via the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase in cultured neurons.

Midkine (MK) is a new member of the heparin-binding neurotrophic factor family. MK plays important roles in development and carcinogenesis and has several important biological effects, including promotion of neurite extension and neuronal survival. However, the mechanism by which MK exerts its neurotrophic actions on neurons has not been elucidated to date. We have established an apoptosis induction system by serum deprivation in primary neuronal cultures isolated from mouse cerebral cortices. Neuronal apoptosis induced by serum deprivation was accompanied by the activation of caspase-3. MK, when added into the culture medium, inhibited the induction of apoptosis and activation of caspase-3 in a dose-dependent manner. Extracellular signal-regulated kinase (ERK) and Akt were not activated by serum deprivation, whereas ERK and Akt were rapidly activated by addition of MK. In addition, the trophic actions of MK of suppressing apoptosis and suppressing the activation of caspase-3 were abolished by concomitant treatment with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, and with wort-mannin or LY294002, specific inhibitors of phosphatidyl-inositol 3-kinase (PI 3-kinase). These PI 3-kinase inhibitors also inhibited the activation of ERK in response to MK, demonstrating a link between ERK and the caspase-3 pathway that is modulated by the PI 3-kinase activation. These results indicate that the ERK cascade plays a central role in MK-mediated neuronal survival via inhibition of caspase-3 activation.

[cDNA cloning of ju-myo protein (JP) and elucidation of the molecular mechanisms by which it exerts neurotrophic effects on neurons].

The adult life span in inbred strains of Drosophila melanogaster (Dm) has been found to be controlled by a few major genes (Hereditas 111:207, 1989; Hereditas 117: 251, 1992). A 77 kDa protein, which we named ju-myo (life-span) protein (JP) and is supposed to be the product of the gene on autosomal locus JmA on adult Dm was shown to have life-span prolonging effect when it was supplied in food. However, the knowledge of its structure and molecular mechanisms by which JP exerts its effects on cells is still unclear. Here we show that JP can exert neurotrophic activities on postmitotic fetal rat neurons isolated from cerebral cortical and dopaminergic neurons isolated from the midbrain: it enhanced survival of MAP2-positive cells and tyrosine hydroxylase immunoreactive neurons by approximately 2-fold over the control group. JP did not increase the density of astrocytes nor expression of glial fibrillary acidic protein (GFAP) in the mesencephalic neuron cultures. Amino acid analysis of JP protein showed that JP is identical to the larval serum protein 2, of which sequence and structure were determined in 1997. Our work provides basis for defining the physiological role of JP at the molecular level and for exploring its potential utility as an alternative approach to study mechanisms of aging.

Apolipoprotein E4 isoform-specific actions on neuronal cells in culture.

Apolipoprotein E (apoE) allele epsilon4 is a major risk factor for Alzheimer's disease (AD); however, the molecular mechanism underlying the acceleration of the development of AD in patients possessing epsilon4 remains to be determined. To investigate the isoform-specific effects of apoE on neurons, primary neuron cultures were prepared from fetal rat cerebral cortices. Inhibition of de novo cholesterol synthesis by compactin, a 3-hydroxyl-3-methylglutaryl CoA reductase inhibitor, induced neuronal cell death in a dose dependent manner. In the presence of a sublethal dose of compactin, apoE4 with beta-migrating very low density lipoproteins (beta-VLDL) caused apoptotic cell death in neuronal cultures. The same results were obtained with inhibition of de novo cholesterol synthesis by sublethal doses of squalestatin, an inhibitor of squalene synthase. The de novo cholesterol synthesis was suppressed to a higher degree by apoE4 than by apoE3, administered with beta-VLDL in the presence or absence of compactin. Mevalonate and squalene, which are metabolites of the cholesterol synthesis pathway, protected neuronal cells from apoE4-induced cell death. These results may suggest that apoE4 may exhibit neurotoxic action when de novo cholesterol synthesis is suppressed to a certain level, and that apoE4 induces neuronal cell death through the suppression of de novo cholesterol synthesis via an undetermined isoform-specific mechanism.

Inhibition of cholesterol production but not of nonsterol isoprenoid products induces neuronal cell death.

Deficiency of nonsterol isoprenoids, intermediate metabolites of the cholesterol biosynthetic pathway, has been known to cause an inhibition of DNA synthesis and cell growth, and to induce apoptosis in nonneuronal cells. To investigate whether this is also the case in neurons, we examined the effect of a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor on the viability of neuronal cultures prepared from fetal rat brains. Treatment with compactin, a competitive inhibitor of HMG-CoA reductase, induced neuronal death in a dose-dependent manner. Concurrent treatment with cholesterol, beta-migrating very low density lipoprotein, mevalonate, or squalene substantially inhibited the induction of neuronal death by compactin. Cell death was also induced by treatment with squalestatin, which specifically inhibits cholesterol biosynthesis at a site downstream from the generation of nonsterol metabolites. Furthermore, squalestatin-induced neuronal death was inhibited by concurrent incubation with squalene but not mevalonate. In contrast, cell growth of proliferating cells such as NIH 3T3 and PC12 cells was exclusively dependent on the level of nonsterol isoprenoid products and not that of cholesterol. The results of this study clearly indicate that the viability of neurons, different from that of nonneuronal cells, depends on the intracellular cholesterol content and not on the intermediate nonsterol isoprenoid products.

Enhancement of MTT, a tetrazolium salt, exocytosis by amyloid beta-protein and chloroquine in cultured rat astrocytes.

The effect of amyloid beta-protein (Abeta) on the cellular reducing activity has been a controversial issue. We determined the cellular reducing activity in cultured astrocytes using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl )-2H-tetrazolium (WST-8) reduction assays following Abeta treatment. MTT reduction was inhibited whereas WST-8 reduction was unaffected by the Abeta treatment. Furthermore, the early extracellular appearance of MTT formazan, a reduced product of MTT, was observed in association with the rapid disappearance of intracellular formazan granules. Notably, similar results were obtained in cultures treated with chloroquine, a perturbant of membrane trafficking. Our results suggest that MTT formazan exocytosis is enhanced in a similar manner by Abeta and chloroquine and that this biological activity of Abeta may underlie the pathogenesis of Alzheimer's disease.

Cholesterol-dependent generation of a seeding amyloid beta-protein in cell culture.

Deposition of aggregated amyloid beta-protein (Abeta), a proteolytic cleavage product of the amyloid precursor protein (Abeta ), is a critical step in the development of Alzheimer's disease(Abeta++). However, we are far from understanding the molecular mechanisms underlying the initiation of Abeta polymerization in vivo. Here, we report that a seeding Abeta, which catalyzes the fibrillogenesis of soluble Abeta, is generated from the apically missorted amyloid precursor protein in cultured epithelial cells. Furthermore, the generation of this Abeta depends exclusively on the presence of cholesterol in the cells. Taken together with mass spectrometric analysis of this novel Abeta and our recent study (3), it is suggested that a conformationally altered form of Abeta, which acts as a "seed" for amyloid fibril formation, is generated in intracellular cholesterol-rich microdomains.

Expression of the mRNA for two isoforms of neural plakophilin-related arm-repeat protein/delta-catenin in rodent neurons and glial cells.

Neural plakophilin-related arm-repeat protein (NPRAP) is a mammalian brain protein of the armadillo family. Here, in an attempt to elucidate its function, we determined the mouse brain regions and cell types expressing the mRNAs for two NPRAP isoforms (the longer and the shorter isoforms), and examined the regulation of expression of the NPRAP mRNAs during the differentiation of P19 cells into neurons. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that both variants were expressed in various mouse brain regions, but the mRNA for the short isoform was predominant in most regions. Primary cultures of both neurons and glial cells exhibited high expression levels of both the mRNAs, indicating that NPRAP mRNA expression is not neuron-specific. The expression of the two NPRAP mRNA variants was dramatically induced even prior to the terminal neuronal and glial differentiation of P19 cells after retinoic acid treatment. These data suggest that the two NPRAP isoforms function not only in neurons and glial cells in the brain, but also play a role in the differentiation of precursor cells into neurons and glial cells.

Midkine inhibits apoptosis via extracellular signal regulated kinase (ERK) activation in PC12 cells.

Midkine (MK) is a new member of the family of heparin-binding neurotrophic factors. MK has several important biological effects and plays an important role in the development and survival of neurons. The mechanism by which MK exerts its neurotrophic actions, however has not been sufficiently clarified. To understand the intracellular pathway activated by MK, we established an apoptosis-induction system with the neuronal cell line PC12 and studied the involvement of the mitogen-activated protein kinase (MAPK) cascade in neuroprotective actions of MK. We demonstrate here that MK rescued PC12 cells from apoptosis induced by serum deprivation in a dose-dependent manner. MK also activated extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), which are well known as signal transducer acting downstream several receptors. PD98059, an inhibitor of MAPK kinase (MAPKK), inhibited ERK activation and also prevented the trophic effect of MK. These results indicate that MK exerts its neuroprotective actions mainly via ERK activation.