Amyloid-ß Reduces Exosome Release from Astrocytes by Enhancing JNK Phosphorylation.

Exosomes are small extracellular vesicles secreted by variety of cell types such as neurons, astrocytes, and oligodendrocytes. It is suggested that exosomes play essential role in the maintenance of the neuronal functions and also in the clearance of amyloid-ß (Aß) from the brain. Aß is well known to cause neuronal cell death, whereas little is known about its effect on astrocytes. In this study, we examined the effect of Aß on release of exosomes from astrocytes in culture. We analyzed release of exosomes and apoE, both of which are known to remove/clear Aß from the brain, in the culture medium of astrocytes. We found that exosome and apoE-HDL were successfully separated by density gradient ultracentrifugation demonstrated by distribution of their specific markers, flotillin and HSP90, and cholesterol, and morphological analysis using electron microscopy. Exosome release was significantly reduced by Aß1-42 treatment in cultured astrocytes accompanied by an increased JNK phosphorylation. Whereas, apoE-HDL release remained unchanged. A JNK inhibitor restored the decreased levels of exosome release induced by Aß treatment to levels similar to those of control, suggesting that Aß1-42 inhibits exosome release via stimulation of JNK signal pathway. Because exosomes are shown to remove Aß in the brain, our findings suggest that increased Aß levels in the brain may impair the exosome-mediated Aß clearance pathway.

Phosphatidylcholine protects neurons from toxic effects of amyloid ß-protein in culture.

Amyloid ß-protein (Aß) is the major component of extracellular plaques in the brains of patients with Alzheimer's disease. It has been suggested that the interaction of Aß with membrane cholesterol is essential for Aß to exert neurotoxicity; however, the effect of phospholipids, another major membrane lipid component, on Aß-induced neurotoxicity remains unclarified. Here we report the protective effect of phosphatidylcholine (PC) on primary cultured neurons against Aß1-42-induced damage. Aß1-42 caused neuronal death as demonstrated by lactose dehydrogenase (LDH) release, which was completely prevented by a pretreatment with PC in a dose-dependent manner. PC containing unsaturated long-chain acyl groups, 1,2-dioleoyl-PC (DOPC), also prevented neuronal death caused by Aß1-42. The oleic acid ethyl-ester (OAEE) partially prevented Aß1-42-induced neurotoxicity. Neurons that were pretreated with DOPC or OAEE for 24h, washed out, and exposed to Aß1-42 in the absence of either of these reagents, were still resistant to Aß1-42-induced neurotoxicity. In contrast, treatment with phosphotidylserine (PS) or docosahexaenoic acid etyl-ester (DHAEE) had no protective effect on neurons against Aß1-42-induced damage. These results suggest that the control of cellular PC content, not PS content, may prove useful in the prevention or treatment of Alzheimer's disease.

Feeder cells support the culture of induced pluripotent stem cells even after chemical fixation.

Chemically fixed mouse embryonic fibroblasts (MEFs), instead of live feeder cells, were applied to the maintenance of mouse induced pluripotent stem (miPS) cells. Formaldehyde and glutaraldehyde were used for chemical fixation. The chemically fixed MEF feeders maintained the pluripotency of miPS cells, as well as their undifferentiated state. Furthermore, the chemically fixed MEF feeders were reused several times without affecting their functions. These results indicate that chemical fixation can be applied to modify biological feeders chemically, without losing their original functions. Chemically fixed MEF feeders will be applicable to other stem cell cultures as a reusable extracellular matrix candidate that can be preserved on a long-term basis.

Bioactive TGF-beta can associate with lipoproteins and is enriched in those containing apolipoprotein E3.

Transforming growth factor-beta1 (TGF-beta1) has central functions in development, tissue maintenance, and repair and has been implicated in major diseases. We discovered that TGF-beta1 contains several amphipathic helices and hydrophobic domains similar to apolipoprotein E (apoE), a protein involved in lipoprotein metabolism. Indeed, TGF-beta1 associates with lipoproteins isolated from human plasma, cultured liver cells, or astrocytes, and its bioactivity was highest in high-density lipoprotein preparations. Importantly, lipoproteins containing the apoE3 isoform had higher TGF-beta levels and bioactivity than those containing apoE4, a major genetic risk factor for atherosclerosis and Alzheimer's disease. Because TGF-beta1 can be protective in these diseases an association with apoE3 may be beneficial. Association of TGF-beta with different types of lipoproteins may facilitate its diffusion, regulate signaling, and offer additional specificity for this important growth factor.

Angiotensin-converting enzyme converts amyloid beta-protein 1-42 (Abeta(1-42)) to Abeta(1-40), and its inhibition enhances brain Abeta deposition.

The abnormal deposition of the amyloid beta-protein (Abeta) in the brain appears crucial to the pathogenesis of Alzheimer's disease (AD). Recent studies have suggested that highly amyloidogenic Abeta(1-42) is a cause of neuronal damage leading to AD pathogenesis and that monomeric Abeta(1-40) has less neurotoxicity than Abeta(1-42). We found that mouse and human brain homogenates exhibit an enzyme activity converting Abeta(1-42) to Abeta(1-40) and that the major part of this converting activity is mediated by the angiotensin-converting enzyme (ACE). Purified human ACE converts Abeta(1-42) to Abeta(1-40) as well as decreases Abeta(1-42)/Abeta(1-40) ratio and degrades Abeta(1-42) and Abeta(1-40). Importantly, the treatment of Tg2576 mice with an ACE inhibitor, captopril, promotes predominant Abeta(1-42) deposition in the brain, suggesting that ACE regulates Abeta(1-42)/Abeta(1-40) ratio in vivo by converting secreted Abeta(1-42) to Abeta(1-40) and degrading Abetas. The upregulation of ACE activity can be a novel therapeutic strategy for AD.

Novel action of apolipoprotein E (ApoE): ApoE isoform specifically inhibits lipid-particle-mediated cholesterol release from neurons.

BACKGROUND: Since the majority of apolipoprotein E (apoE) existing in the cerebrospinal fluid is associated with high-density lipoprotein (HDL), one should focus on the role of the apoE-HDL complex rather than on that of free apoE in cholesterol metabolism in the central nervous system. However, the apoE-isoform-specific effect of apoE-HDL on cholesterol transport remains unclarified. RESULTS: Here we show that apoE3-HDL induced a marked cholesterol release from neurons, while apoE4-HDL induced little. To elucidate the mechanism underlying this phenomenon, we used a complex of lipid emulsion (EM) with recombinant apoE3 or apoE4 (apoE-EM) at various apoE concentrations. When a small number of apoE molecules were associated with EM, apoE3- and apoE4-EM, induced a marked cholesterol release to a level similar to that induced by EM alone. However, when apoE at given concentrations was incubated with EM, apoE3-EM induced a marked cholesterol release, while apoE4-EM induced little. Under these conditions, a greater number of apoE4 molecules were associated with EM than apoE3 molecules. When an increasing number of apoE molecules were associated with EM, both apoE3-EM and apoE4-EM induced little cholesterol release. Preincubation with beta-mercaptoethanol increased the number of apoE3 molecules associated with EM similar to that of apoE4 molecules, indicating that the presence (apoE3) or absence (apoE4) of intermolecular disulfide bond formation is responsible for the association of a greater number of apoE4 molecules to EM than apoE3 molecules. CONCLUSION: These results suggest that although apoE and a lipid particle are lipid acceptors, when apoE and a lipid particle form a complex, apoE on the particle surface inhibits the lipid particle-mediated cholesterol release from cells in an apoE-concentration-dependent manner.

Cholesterol-mediated neurite outgrowth is differently regulated between cortical and hippocampal neurons.

The acquisition of neuronal type-specific morphogenesis is a central feature of neuronal differentiation and has important consequences for region-specific nervous system functions. Here, we report that the cell type-specific cholesterol profile determines the differential modulation of axon and dendrite outgrowths in hippocampal and cerebral cortical neurons in culture. The extent of axon and dendrite outgrowths is greater and the polarity formation occurs earlier in cortical neurons than in hippocampal neurons. The cholesterol concentrations in total homogenate and the lipid rafts from hippocampal neurons are significantly higher than those from cortical neurons. Cholesterol depletion by beta-cyclodextrin markedly enhanced the neurite outgrowth and accelerated the establishment of neuronal polarity in hippocampal neurons, which were similarly observed in nontreated cortical neurons, whereas cholesterol loading had no effects. In contrast, both depletion and loading of cholesterol decreased the neurite outgrowths in cortical neurons. The stimulation of neurite outgrowth and polarity formation induced by cholesterol depletion was accompanied by an enhanced localization of Fyn, a Src kinase, in the lipid rafts of hippocampal neurons. A concomitant treatment with beta-cyclodextrin and a Src family kinase inhibitor, PP2, specifically blocked axon outgrowth but not dendrite outgrowth (both of which were enhanced by beta-cyclodextrin) in hippocampal neurons, suggesting that axon outgrowth modulated by cholesterol is induced in a Fyn-dependent manner. These results suggest that cellular cholesterol modulates axon and dendrite outgrowths and neuronal polarization under culture conditions and also that the difference in cholesterol profile between hippocampal and cortical neurons underlies the difference in neurite outgrowth between these two types of neurons.

Oligomerization of amyloid beta-protein occurs during the isolation of lipid rafts.

Cholesterol- and glycosphingolipid-rich microdomains, called "lipid rafts," are suggested to initiate and promote the pathophysiology of Alzheimer's disease by serving as a platform for generation, aggregation, or degradation of amyloid-beta protein (Abeta). However, methods for biochemical isolation of these microdomains may produce artifacts. In this study, when synthetic Abeta1- 40 monomers were added to the brain fragment at a final concentration of 2.1 microM, followed by homogenization and isolation of lipid rafts by an established method, Abeta1- 40 accumulated as oligomers in the lipid raft fraction. However, in the absence of a brain homogenate, synthetic Abeta1- 40 did not accumulate in the lipid raft fraction. When fractionation was performed in the absence of synthetic Abeta1-40 and synthetic Abeta1-40 was incubated in an aliquot of each fraction, a marked oligomerization of Abeta1- 40 was observed in the lipid raft aliquot. These results indicate that exogenous Abeta associates with lipid rafts, and Abeta bound to rafts forms oligomers during the isolation of lipid rafts. In addition, endogenous Abeta1-40 in a Triton X-100-insoluble fraction of a brain homogenate of the Tg2576 transgenic mouse model of Alzheimer's disease formed oligomers when the fraction was incubated at 4 degrees C for 20 hr. Thus, one should be careful when one discusses the role of lipid rafts in amyloid precursor protein processing and in the generation, aggregation, and degradation of Abeta.

Altered cholesterol metabolism in Niemann-Pick type C1 mouse brains affects mitochondrial function.

Niemann-Pick type C1 (NPC1) disease is a fatal hereditary disorder characterized by a defect in cholesterol trafficking and progressive neurodegeneration. Although the NPC1 gene has been identified, the molecular mechanism responsible for neuronal dysfunction in brains of patients with NPC1 disease remains unknown. This study demonstrates that the amount of cholesterol within mitochondria membranes is significantly elevated in NPC1 mouse brains and neural cells. In addition, the mitochondrial membrane potential, the activity of ATP synthase, and henceforth the level of ATP are markedly decreased in NPC1 mouse brains and neurons. Importantly, reducing the level of cholesterol within mitochondrial membranes using methyl-beta-cyclodextrin can restore the activity of ATP synthase. Finally, NPC1 neurons show an impaired neurite outgrowth, which can be rescued by exogenous ATP. These results suggest that mitochondrial dysfunctions and subsequent ATP deficiency, which are induced by altered cholesterol metabolism in mitochondria, may be responsible for neuronal impairment in NPC1 disease.

Association of a 5178C-->A (Leu237Met) polymorphism in the mitochondrial DNA with a low prevalence of myocardial infarction in Japanese individuals.

Because mitochondria are the major sources of reactive oxygen species (ROS) in cells, certain alterations in mitochondrial functions can lead to metabolic perturbation in vascular endothelial cells and smooth muscle cells, resulting in vascular dysfunction. We previously demonstrated that a C --> A transversion in mitochondrial DNA (mtDNA) at nucleotide 5178 of the NADH dehydrogenase subunit 2 (ND2) gene, which results in a Lue --> Met substitution at amino acid 237, was found more frequently in Japanese centenarians than in controls. We also demonstrated that this Mt5178C --> A polymorphism has anti-atherosclerotic effects in diabetic subjects. We have now examined whether the Mt5178C --> A (Leu237Met) polymorphism in the mitochondrial ND2 gene is associated with a low prevalence of myocardial infarction (MI) in a case-control study. The genotype of ND2 gene was determined either with a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or a colorimetry-based allele-specific DNA probe assay. Multivariate logistic regression analysis with adjustment for age, gender, body mass index, smoking status, hypertension, diabetes mellitus, hypercholesterolemia, and hyperuricemia revealed that the frequency of the Mt5178A genotype was significantly higher in controls than in subjects with MI. These results suggest that the 5178A genotype of mitochondrial ND2 gene polymorphism is protective against MI; and this effect would explain, at least in part, its contribution to longevity.

Modulation of amyloid precursor protein cleavage by cellular sphingolipids.

Lipid rafts and their component, cholesterol, modulate the processing of beta-amyloid precursor protein (APP). However, the role of sphingolipids, another major component of lipid rafts, in APP processing remains undetermined. Here we report the effect of sphingolipid deficiency on APP processing in Chinese hamster ovary cells treated with a specific inhibitor of serine palmitoyltransferase, which catalyzes the first step of sphingolipid biosynthesis, and in a mutant LY-B strain defective in the LCB1 subunit of serine palmitoyltransferase. We found that in sphingolipid-deficient cells, the secretion of soluble APPalpha (sAPPalpha) and the generation of C-terminal fragment cleaved at alpha-site dramatically increased, whereas beta-cleavage activity remained unchanged, and the epsilon-cleavage activity decreased without alteration of the total APP level. The secretion of amyloid beta-protein 42 increased in sphingolipid-deficient cells, whereas that of amyloid beta-protein 40 did not. All of these alterations were restored in sphingolipid-deficient cells by adding exogenous sphingosine and in LY-B cells by transfection with cLCB1. Sphingolipid deficiency increased MAPK/ERK activity and a specific inhibitor of MAPK kinase, PD98059, restored sAPPalpha level, indicating that sphingolipid deficiency enhances sAPPalpha secretion via activation of MAPK/ERK pathway. These results suggest that not only the cellular level of cholesterol but also that of sphingolipids may be involved in the pathological process of Alzheimer's disease by modulating APP cleavage.

Amyloid beta-protein (Abeta)1-40 protects neurons from damage induced by Abeta1-42 in culture and in rat brain.

Previously, we found that amyloid beta-protein (Abeta)1-42 exhibits neurotoxicity, while Abeta1-40 serves as an antioxidant molecule by quenching metal ions and inhibiting metal-mediated oxygen radical generation. Here, we show another neuroprotective action of nonamyloidogenic Abeta1-40 against Abeta1-42-induced neurotoxicity in culture and in vivo. Neuronal death was induced by Abeta1-42 at concentrations higher than 2 microm, which was prevented by concurrent treatment with Abeta1-40 in a dose-dependent manner. However, metal chelators did not prevent Abeta1-42-induced neuronal death. Circular dichroism spectroscopy showed that Abeta1-40 inhibited the beta-sheet transformation of Abeta1-42. Thioflavin-T assay and electron microscopy analysis revealed that Abeta1-40 inhibited the fibril formation of Abeta1-42. In contrast, Abeta1-16, Abeta25-35, and Abeta40-1 did not inhibit the fibril formation of Abeta1-42 nor prevent Abeta1-42-induced neuronal death. Abeta1-42 injection into the rat entorhinal cortex (EC) caused the hyperphosphorylation of tau on both sides of EC and hippocampus and increased the number of glial fibrillary acidic protein (GFAP)-positive astrocytes in the ipsilateral EC, which were prevented by the concurrent injection of Abeta1-40. These results indicate that Abeta1-40 protects neurons from Abeta1-42-induced neuronal damage in vitro and in vivo, not by sequestrating metals, but by inhibiting the beta-sheet transformation and fibril formation of Abeta1-42. Our data suggest a mechanism by which elevated Abeta1-42/Abeta1-40 ratio accelerates the development of Alzheimer's disease (AD) in familial AD.

Promotion of tau phosphorylation by MAP kinase Erk1/2 is accompanied by reduced cholesterol level in detergent-insoluble membrane fraction in Niemann-Pick C1-deficient cells.

Niemann-Pick type C (NPC) disease is a cholesterol-storage disease accompanied by neurodegeneration with the formation of neurofibrillary tangles, the major component of which is the hyperphosphorylated tau. Here, we examined the mechanism underlying hyperphosphorylation of tau using mutant Chinese hamster ovary (CHO) cell line defective in NPC1 (CT43) as a tool. Immunoblot analysis revealed that tau was hyperphosphorylated at multiple sites in CT43 cells, but not in their parental cells (25RA) or the wild-type CHO cells. In CT43 cells, mitogen-activated protein (MAP) kinase Erk1/2 was activated and the specific MAPK inhibitor, PD98059, attenuated the hyperphosphorylation of tau. The amount of protein phosphatase 2A not bound to microtubules was decreased in CT43 cells. CT43 cells but not 25RA cells were amphotericin B-resistant, indicating that cholesterol level in the plasma membrane of CT43 is decreased. In addition, the level of cholesterol in the detergent-insoluble, low-density membrane (LDM) fraction of CT43 cells was markedly reduced compared with the other two types of CHO cells. As LDM domain plays critical role in signaling pathways, these results suggest that the reduced cholesterol level in LDM domain due to the lack of NPC1 may activate MAPK, which subsequently promotes tau phosphorylation in NPC1-deficient cells.

Gene therapy for mitochondrial disease by delivering restriction endonuclease SmaI into mitochondria.

The restriction endonuclease SmaI has been used for the diagnosis of neurogenic muscle weakness, ataxia and retinitis pigmentosa disease or Leigh's disease, caused by the Mt8993T-->G mutation which results in a Leu156Arg replacement that blocks proton translocation activity of subunit a of F(0)F(1)-ATPase. Our ultimate goal is to apply SmaI to gene therapy for this disease, because the mutant mitochondrial DNA (mtDNA) coexists with the wild-type mtDNA (heteroplasmy), and because only the mutant mtDNA, but not the wild-type mtDNA, is selectively restricted by the enzyme. For this purpose, we transiently expressed the SmaI gene fused to a mitochondrial targeting sequence in cybrids carrying the mutant mtDNA. Here, we demonstrate that mitochondria targeted by the SmaI enzyme showed specific elimination of the mutant mtDNA. This elimination was followed with repopulation by the wild-type mtDNA, resulting in restoration of both the normal intracellular ATP level and normal mitochondrial membrane potential. Furthermore, in vivo electroporation of the plasmids expressing mitochondrion-targeted EcoRI induced a decrease in cytochrome c oxidase activity in hamster skeletal muscles while causing no degenerative changes in nuclei. Delivery of restriction enzymes into mitochondria is a novel strategy for gene therapy of a special form of mitochondrial diseases.

Amyloid beta-protein affects cholesterol metabolism in cultured neurons: implications for pivotal role of cholesterol in the amyloid cascade.

Recently, we have found that alterations in cellular cholesterol metabolism are involved in promotion of tau phosphorylation (Fan et al. [2001] J. Neurochem. 76: 391-400; Sawamura et al. [2001] J. Biol. Chem. 276:10314-10319). In addition, we have shown that amyloid beta-protein (A beta) promotes cholesterol release to form A beta-lipid particles (Michikawa et al. [2001] J. Neurosci. 21:7226-7235). These lines of evidence inspired us to conduct further studies on whether A beta affects cholesterol metabolism in neurons, which might lead to tau phosphorylation. Here, we report the effect of A beta1-40 on cholesterol metabolism in cultured neurons prepared from rat cerebral cortex. Oligomeric A beta1-40 inhibited cholesterol synthesis and reduced cellular cholesterol levels in a dose- and time-dependent manner, while freshly dissolved A beta had no effect on cholesterol metabolism. However, oligomeric A beta had no effect on the proteolysis of sterol regulatory element binding protein-2 (SREBP-2) or protein synthesis in cultured neurons. Oligomeric A beta did not enhance lactate dehydrogenase (LDH) release from neuronal cells or decrease signals in the cultures reactive to 3,3'-Bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein, hexaacetoxymethyl ester (calcein AM) staining, indicating that A beta used in this experiment did not cause neuronal death during the time course of our experiments. Since alterations in cholesterol metabolism induce tau phosphorylation, our findings that oligomeric A beta alters cellular cholesterol homeostasis may provide new insight into the mechanism underlying the amyloid cascade hypothesis.

Apolipoprotein E (ApoE) isoform-dependent lipid release from astrocytes prepared from human ApoE3 and ApoE4 knock-in mice.

We have reported previously (Michikawa, M., Fan, Q.-W., Isobe, I., and Yanagisawa, K. (2000) J. Neurochem. 74, 1008-1016) that exogenously added recombinant human apolipoprotein E (apoE) promotes cholesterol release in an isoform-dependent manner. However, the molecular mechanism underlying this isoform-dependent promotion of cholesterol release remains undetermined. In this study, we demonstrate that the cholesterol release is mediated by endogenously synthesized and secreted apoE isoforms and clarify the mechanism underlying this apoE isoform-dependent cholesterol release using cultured astrocytes prepared from human apoE3 and apoE4 knock-in mice. Cholesterol and phospholipids were released into the culture media, resulting in the generation of two types of high density lipoprotein (HDL)-like particles; one was associated with apoE and the other with apoJ. The amount of cholesterol released into the culture media from the apoE3-expressing astrocytes was approximately 2.5-fold greater than that from apoE4-expressing astrocytes. In contrast, the amount of apoE3 released in association with the HDL-like particles was similar to that of apoE4, and the sizes of the HDL-like particles released from apoE3- and apoE4-expressing astrocytes were similar. The molar ratios of cholesterol to apoE in the HDL fraction of the culture media of apoE3- and apoE4-expressing astrocytes were 250 +/- 6.0 and 119 +/- 5.1, respectively. These data indicate that apoE3 has an ability to generate similarly sized lipid particles with less number of apoE molecules than apoE4, suggesting that apoE3-expressing astrocytes can supply more cholesterol to neurons than apoE4-expressing astrocytes. These findings provide a new insight into the issue concerning the putative alteration of apoE-related cholesterol metabolism in Alzheimer's disease.

A novel function of monomeric amyloid beta-protein serving as an antioxidant molecule against metal-induced oxidative damage.

Aggregated and oligomeric amyloid beta-protein (Abeta) is known to exhibit neurotoxicity. However, the action of Abeta monomers on neurons is not fully understood. We have studied aggregation state-dependent actions of Abeta and found an oligomer-specific effect of Abeta on lipid metabolism in neurons (Michikawa et al., 2001). Here, we show a novel function of monomeric Abeta1-40, which is the major species found in physiological fluid, as a natural antioxidant molecule that prevents neuronal death caused by transition metal-induced oxidative damage. Monomeric Abeta1-40, which is demonstrated by SDS-PAGE after treatment with glutaraldehyde, protects neurons cultured in a medium containing 1.5 microm Fe(II) without antioxidant molecules. Metal ion chelators such as EDTA, CDTA (trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid), and DTPA (diethylenetriamine-N,N,N',N",N"-penta-acetic acid, an iron-binding protein, transferrin, and antioxidant scavengers such as catalase, glutathione, and vitamin E also inhibit neuronal death under the same conditions. Monomeric Abeta1-40 inhibits neuronal death caused by Cu(II), Fe(II), and Fe(III) but does not protect neurons against H2O2-induced damage. Monomeric Abeta1-40 inhibits the reduction of Fe(III) induced by vitamin C and the generation of superoxides and prevents lipid peroxidation induced by Fe(II). Abeta1-42 remaining as a monomer also exhibits antioxidant and neuroprotective effects. In contrast, oligomeric and aggregated Abeta1-40 and Abeta1-42 lose their neuroprotective activity. These results indicate that monomeric Abeta protects neurons by quenching metal-inducible oxygen radical generation and thereby inhibiting neurotoxicity. Because aggregated Abeta is known to be an oxygen radical generator, our results provide a novel concept that the aggregation-dependent biological effects of Abeta are dualistic, being either an oxygen radical generator or its inhibitor.

Cholesterol-dependent modulation of dendrite outgrowth and microtubule stability in cultured neurons.

Microtubule-associated protein 2 (MAP2) is a neuron-specific cytoskeletal protein enriched in dendrites and cell bodies. MAP2 regulates microtubule stability in a phosphorylation-dependent manner, which has been implicated in dendrite outgrowth and branching. We have previously reported that cholesterol deficiency causes tau phosphorylation and microtubule depolymerization in axons (Fan et al. 2001). To investigate whether cholesterol also modulates microtubule stability in dendrites by modulating MAP2 phosphorylation, we examined the effect of compactin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and TU-2078 (TU), a squalene epoxidase inhibitor, on these parameters using cultured neurons. We have found that cholesterol deficiency induced by compactin and TU, inhibited dendrite outgrowth, but not of axons, and attenuated axonal branching. Dephosphorylation of MAP2 and microtubule depolymerization accompanied these alterations. The amount of protein phosphatase 2 A (PP2A) and its activity in association with microtubules were decreased, while those unbound to microtubules were increased. The synthesized ceramide levels and the total ceramide content were increased in these cholesterol-deficient neurons. These alterations caused by compactin were prevented by concurrent treatment of cultured neurons with beta-migrating very-low-density lipoproteins (beta-VLDL) or cholesterol. Taken together, we propose that cholesterol-deficiency causes a selective inhibition of dendrite outgrowth due to the decreased stability of microtubules as a result of inhibition of MAP2 phosphorylation.