Modelling Takenouchi-Kosaki syndrome using disease-specific iPSCs.

Takenouchi-Kosaki Syndrome (TKS) is a congenital multi-organ disorder caused by the de novo missense mutation c.191A > G p. Tyr64Cys (Y64C) in the CDC42 gene. We previously elucidated the functional abnormalities and thrombopoietic effects of Y64C using HEK293 and MEG01 cells. In the present study, we used iPSCs derived from TKS patients to model the disease and successfully recapitulated macrothrombocytopenia, a prominent TKS phenotype. The megakaryopoietic differentiation potential of TKS-iPSCs and platelet production capacity were examined using an efficient platelet production method redesigned from existing protocols. The results obtained showed that TKS-iPSCs produced fewer hematopoietic progenitor cells, exhibited defective megakaryopoiesis, and released platelets with an abnormally low count and giant morphology. We herein report the first analysis of TKS-iPSC-derived megakaryocytes and platelets, and currently utilize this model to perform drug evaluations for TKS. Therefore, our simple yet effective differentiation method, which mimics the disease in a dish, is a feasible strategy for studying hematopoiesis and related diseases.

C-Mannosylated tryptophan-containing WSPW peptide binds to actinin-4 and alters E-cadherin subcellular localization in lung epithelial-like A549 cells.

The Trp-x-x-Trp (W-x-x-W) peptide motif, a consensus site for C-mannosylation, is the functional motif in cytokine type I receptors or thrombospondin type I repeat (TSR) superfamily proteins. W-x-x-W motifs are important for physiological and pathological functions of their parental proteins, but effects of C-mannosylation on protein functions remain to be elucidated. By using chemically synthesized WSPW peptides and C-mannosylated WSPW peptides (C-Man-WSPW), we herein investigated whether C-mannosylation of WSPW peptides confer additional biological functions to WSPW peptides. C-Man-WSPW peptide, but not non-mannosylated WSPW, reduced E-cadherin levels in A549 cells. Via peptide mass fingerprinting analysis, we identified actinin-4 as a C-Man-WSPW-binding protein in A549 cells. Actinin-4 partly co-localized with E-cadherin or ß-catenin, despite no direct interaction between actinin-4 and E-cadherin. C-Man-WSPW reduced co-localization of E-cadherin and actinin-4; non-mannosylated WSPW had no effect on localization. In actinin-4-knockdown cells, E-cadherin was upregulated and demonstrated a punctate staining pattern in the cytoplasm, which suggests that actinin-4 regulated cell-surface E-cadherin localization. Thus, C-mannosylation of WSPW peptides is required for interaction with actinin-4 that subsequently alters expression and subcellular localization of E-cadherin and morphology of epithelial-like cells. Our results therefore suggest a regulatory role of C-mannosylation of the W-x-x-W motif in interactions between the motif and its binding partner and will thereby enhance understanding of protein C-mannosylation.

Macrothrombocytopenia of Takenouchi-Kosaki syndrome is ameliorated by CDC42 specific- and lipidation inhibitors in MEG-01 cells.

Macrothrombocytopenia is a common pathology of missense mutations in genes regulating actin dynamics. Takenouchi-Kosaki syndrome (TKS) harboring the c.191A > G, Tyr64Cys (Y64C) variant in Cdc42 exhibits a variety of clinical manifestations, including immunological and hematological anomalies. In the present study, we investigated the functional abnormalities of the Y64C mutant in HEK293 cells and elucidated the mechanism of macrothrombocytopenia, one of the symptoms of TKS patients, by monitoring the production of platelet-like particles (PLP) using MEG-01 cells. We found that the Y64C mutant was concentrated at the membrane compartment due to impaired binding to Rho-GDI and more active than the wild-type. The Y64C mutant also had lower association with its effectors Pak1/2 and N-WASP. Y64C mutant-expressing MEG-01 cells demonstrated short cytoplasmic protrusions with aberrant F-actin and microtubules, and reduced PLP production. This suggested that the Y64C mutant facilitates its activity and membrane localization, resulting in impaired F-actin dynamics for proplatelet extension, which is necessary for platelet production. Furthermore, such dysfunction was ameliorated by either suppression of Cdc42 activity or prenylation using chemical inhibitors. Our study may lead to pharmacological treatments for TKS patients.

Neuropathophysiological significance of the c.1449T>C/p.(Tyr64Cys) mutation in the CDC42 gene responsible for Takenouchi-Kosaki syndrome.

Takenouchi-Kosaki syndrome (TKS) is an autosomal dominant congenital syndrome, of which pathogenesis is not well understood. Recently, a heterozygous mutation c.1449T > C/p.(Tyr64Cys) in the CDC42 gene, encoding a Rho family small GTPase, has been demonstrated to contribute to the TKS clinical features, including developmental delay with intellectual disability (ID). However, specific molecular mechanisms underlying the neuronal pathophysiology of TKS remain largely unknown. In this study, biochemical analyses revealed that the mutation moderately activates Cdc42. In utero electroporation-based acute expression of Cdc42-Y64C in ventricular zone progenitor cells in embryonic mice cerebral cortex resulted in migration defects and cluster formation of excitatory neurons. Expression the mutant in primary cultured hippocampal neurons caused impaired axon elongation. These data suggest that the c.1449T > C/p.(Tyr64Cys) mutation causes altered CDC42 function and results in defects in neuronal morphology and migration during brain development, which is likely to be responsible for pathophysiology of psychomotor delay and ID in TKS.

Chondroitin sulfate proteoglycan tenascin-R regulates glutamate uptake by adult brain astrocytes.

In our previous study, the CS-56 antibody, which recognizes a chondroitin sulfate moiety, labeled a subset of adult brain astrocytes, yielding a patchy extracellular matrix pattern. To explore the molecular nature of CS-56-labeled glycoproteins, we purified glycoproteins of the adult mouse cerebral cortex using a combination of anion-exchange, charge-transfer, and size-exclusion chromatographies. One of the purified proteins was identified as tenascin-R (TNR) by mass spectrometric analysis. When we compared TNR mRNA expression patterns with the distribution patterns of CS-56-positive cells, TNR mRNA was detected in CS-56-positive astrocytes. To examine the functions of TNR in astrocytes, we first confirmed that cultured astrocytes also expressed TNR protein. TNR knockdown by siRNA expression significantly reduced glutamate uptake in cultured astrocytes. Furthermore, expression of mRNA and protein of excitatory amino acid transporter 1 (GLAST), which is a major component of astrocytic glutamate transporters, was reduced by TNR knockdown. Our results suggest that TNR is expressed in a subset of astrocytes and contributes to glutamate homeostasis by regulating astrocytic GLAST expression.

Transglutaminase 2-dependent deamidation of glyceraldehyde-3-phosphate dehydrogenase promotes trophoblastic cell fusion.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein as well as a classic glycolytic enzyme, and its pleiotropic functions are achieved by various post-translational modifications and the resulting translocations to intracellular compartments. In the present study, GAPDH in the plasma membrane of BeWo choriocarcinoma cells displayed a striking acidic shift in two-dimensional electrophoresis after cell-cell fusion induction by forskolin. This post-translational modification was deamidation of multiple glutaminyl residues, as determined by molecular mass measurement and tandem mass spectrometry of acidic GAPDH isoforms. Transglutaminase (TG) inhibitors prevented this acidic shift and reduced cell fusion. Knockdown of the TG2 gene by short hairpin RNA reproduced these effects of TG inhibitors. Various GAPDH mutants with replacement of different numbers (one to seven) of Gln by Glu were expressed in BeWo cells. These deamidated mutants reversed the suppressive effect of wild-type GAPDH overexpression on cell fusion. Interestingly, the mutants accumulated in the plasma membrane, and this accumulation was increased according to the number of Gln/Glu substitutions. Considering that GAPDH binds F-actin via an electrostatic interaction and that the cytoskeleton is rearranged in trophoblastic cell fusion, TG2-dependent GAPDH deamidation was suggested to participate in actin cytoskeletal remodeling.

Calponin 3 regulates stress fiber formation in dermal fibroblasts during wound healing.

Skin wound healing is an intricate process involving various cell types and molecules. In granulation tissue, fibroblasts proliferate and differentiate into myofibroblasts and generate mechanical tension for wound closure and contraction. Actin stress fibers formed in these cells, especially those containing α-smooth muscle actin (α-SMA), are the central machinery for contractile force generation. In the present study, calponin 3 (CNN3), which has a phosphorylation-dependent actin-binding property, was identified in the molecular mechanism underlying stress fiber formation. CNN3 was expressed by fibroblasts/myofibroblasts in the proliferation phase of wound healing, and was associated with α-SMA in stress fibers formed by cultured dermal fibroblasts. CNN3 expression was post-transcriptionally regulated by tension, as demonstrated by disruption of actin filament organization under floating culture or blebbistatin treatment. CNN3 knockdown in primary fibroblasts impaired stress fiber formation, resulting in a phenotype of decreased cellular dynamics such as cell motility and contractile ability. These findings indicate that CNN3 participates in actin stress fiber remodeling, which is required for cell motility and contraction of dermal fibroblasts in the wound healing process.

Rock-dependent calponin 3 phosphorylation regulates myoblast fusion.

Myogenesis occurs during embryonic development as well as regeneration following postnatal muscle fiber damage. Herein, we show that acidic calponin or calponin 3 (CNN3) regulates both myoblast cell fusion and muscle-specific gene expressions. Overexpression of CNN3 impaired C2C12 cell fusion, whereas CNN3 gene knockdown promoted skeletal myosin expression and fusion. CNN3 was phosphorylated at Ser293/296 in the C-terminal region. The basal inhibitory property of CNN3 against myoblast differentiation was enhanced by Ser293/296Ala mutation or deletion of the C-terminal region, and this inhibition was reversed by Ser293/296Asp mutation. Ser293/296 phosphorylation was required for CNN3 to bind actin and was dependent on Rho-associated kinases 1/2 (ROCK 1/2). Gene knockdown of ROCK1/2 suppressed CNN3 phosphorylation and impaired myoblast fusion, and these effects were partially attenuated by additional CNN3 overexpression of Ser293/296Asp CNN3. These findings indicated that CNN3 phosphorylation by ROCK blunts CNN3's inhibitory effects on muscle cell differentiation and fusion. In muscle tissues, satellite cells, but not mature myofibrils, expressed CNN3. CNN3 was also expressed and phosphorylated during myotube induction in isolated muscle satellite cells. Taken together, these results indicate that CNN3 is a downstream regulator of the ROCK signaling pathway for myogenesis.

Brd2 is required for cell cycle exit and neuronal differentiation through the E2F1 pathway in mouse neuroepithelial cells.

To understand genetic programs controlling mammalian central nervous system (CNS) development, we have identified one transgene-inserted mutation, which showed embryonic lethality during neurulation. Determination of the transgene integration site and rescue experiments revealed that the Brd2 gene, whose products specifically bind acetylated histone H4 and can mediate transcription, was the cause of this mutation. Expression studies with specific markers demonstrated that cell cycle progression was accelerated and neuronal differentiation as well as cell cycle exit were impaired in Brd2-deficient neruoepithelial cells. To investigate whether Brd2 regulates neuronal differentiation through a E2F1 transcriptional factor, which directly binds Brd2 and controls genes expression for cell cycle progression and exit, we analyzed Brd2;E2F1 double mutant phenotypes and, consequently found that abnormalities in neuronal differentiation and cell cycle progression due to Brd2-deficiency were restored by removing the E2F1 gene. These findings suggest that Brd2 is required for cell cycle exit and neuronal differentiation of neuroepithelial cells through the E2F1 pathway during mouse CNS development.

N-glycans of SREC-I (scavenger receptor expressed by endothelial cells): essential role for ligand binding, trafficking and stability.

Scavenger receptor expressed by endothelial cells (SREC-I) mediates the endocytosis of chemically modified lipoproteins such as acetylated low-density lipoprotein (Ac-LDL) and oxidized LDL and is implicated in atherogenesis. We produced recombinant SREC-I in Chinese hamster ovary-K1 cells and identified three potential glycosylation sites, Asn(289), Asn(382) and Asn(393), which were all glycosylated. To determine the function of N-glycans in SREC-I, we characterized SREC-I mutant proteins by intracellular distribution and the cellular incorporation rate of Ac-LDL. N382Q/N393Q and N289Q/N382Q/N393Q were sequestered in the endoplasmic reticulum, resulting in a severe reduction in the cellular incorporation of Ac-LDL. N382Q showed a normal cell surface residency and an enhanced affinity for Ac-LDL, resulting in an elevated Ac-LDL cellular incorporation. These results indicate that the N-glycan of Asn(393) regulates the intracellular sorting of SREC-I and that the N-glycan of Asn(382) controls ligand-binding affinity. Furthermore, we detected an enhanced trypsin sensitivity of the N289Q. Glycan structure analyses revealed that the core-fucosylated bi-antennary is the common major structure at all glycosylation sites. In addition, tri- and tetra-antennary were detected as minor constituents at Asn(289). A bisecting GlcNAc was also detected at Asn(382) and Asn(393). Structural analyses and homology modeling of SREC-I suggest that the N-glycan bearing a ß1-6GlcNAc branch at Asn(289) protects from proteinase attack and thus confers a higher stability on SREC-I. These data indicate that Asn(289)-, Asn(382)- and Asn(393)-linked N-glycans of SREC-I have distinct functions in regulating proteolytic resistance, ligand-binding affinity and subcellular localization, all of which might be involved in the development of atherogenesis.

Calponin 3 regulates actin cytoskeleton rearrangement in trophoblastic cell fusion.

Cell-cell fusion is an intriguing differentiation process, essential for placental development and maturation. A proteomic approach identified a cytoplasmic protein, calponin 3 (CNN3), related to the fusion of BeWo choriocarcinoma cells. CNN3 was expressed in cytotrophoblasts in human placenta. CNN3 gene knockdown promoted actin cytoskeletal rearrangement and syncytium formation in BeWo cells, suggesting CNN3 to be a negative regulator of trophoblast fusion. Indeed, CNN3 depletion promoted BeWo cell fusion. CNN3 at the cytoplasmic face of cytoskeleton was dislocated from F-actin with forskolin treatment and diffused into the cytoplasm in a phosphorylation-dependent manner. Phosphorylation sites were located at Ser293/296 in the C-terminal region, and deletion of this region or site-specific disruption of Ser293/296 suppressed syncytium formation. These CNN3 mutants were colocalized with F-actin and remained there after forskolin treatment, suggesting that dissociation of CNN3 from F-actin is modulated by the phosphorylation status of the C-terminal region unique to CNN3 in the CNN family proteins. The mutant missing these phosphorylation sites displayed a dominant negative effect on cell fusion, while replacement of Ser293/296 with aspartic acid enhanced syncytium formation. These results indicated that CNN3 regulates actin cytoskeleton rearrangement which is required for the plasma membranes of trophoblasts to become fusion competent.

N-Glycosylation of laminin-332 regulates its biological functions. A novel function of the bisecting GlcNAc.

Laminin-332 (Lm332) is a large heterotrimeric glycoprotein that has been identified as a scattering factor, a regulator of cancer invasion as well as a prominent basement membrane component of the skin. Past studies have identified the functional domains of Lm332 and revealed the relationships between its activities and the processing of its subunits. However, there is little information available concerning the effects of N-glycosylation on Lm332 activities. In some cancer cells, an increase of beta1,6-GlcNAc catalyzed by N-acetylglucosaminyltransferase V (GnT-V) is related to the promotion of cancer cell motility. By contrast, bisecting GlcNAc catalyzed by N-acetylglucosaminyltransferase III (GnT-III) suppresses the further processing with branching enzymes, such as GnT-V, and the elongation of N-glycans. To examine the effects of those N-glycosylations to Lm332 on its activities, we purified Lm332s from the conditioned media of GnT-III- and GnT-V-overexpressing MKN45 cells. Lectin blotting and mass spectrometry analyses revealed that N-glycans containing the bisecting GlcNAc and beta1,6-GlcNAc structures were strongly expressed on Lm332 purified from GnT-III-overexpressing (GnT-III-Lm332) and GnT-V-overexpressing (GnT-V-Lm332) cells, respectively. Interestingly, the cell adhesion activity of GnT-III-Lm332 was apparently decreased compared with those of control Lm332 and GnT-V-Lm332. In addition, the introduction of bisecting GlcNAc to Lm332 resulted in a decrease in its cell scattering and migration activities. The weakened activities were most likely derived from the impaired alpha3beta1 integrin clustering and resultant focal adhesion formation. Taken together, our results clearly demonstrate for the first time that N-glycosylation may regulate the biological function of Lm332. This finding could introduce a new therapeutic strategy for cancer.

Cycloprodigiosin hydrochloride activates the Ras-PI3K-Akt pathway and suppresses protein synthesis inhibition-induced apoptosis in PC12 cells.

Cycloprodigiosin hydrochloride (cPrG-HCl), a member of the prodigiosin family of compounds, has been reported to act as an H(+)/Cl(-) symporter. This compound induces apoptosis in several cancer cells and acts as an antitumor drug in animal models. In this study, we found a novel function of cPrG-HCl; to suppress cell death in PC12 cells, which is caused by protein synthesis inhibitors cycloheximide and actinomycin D. cPrG-HCl activated Akt and suppressed apoptosis, and this was accompanied by inhibition of caspase-3 activity and DNA fragmentation independently of its H(+)/Cl(-) symporter activity. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and dominant-negative Ras attenuated the anti-apoptotic activity of cPrG-HCl, which indicates that cPrG-HCl activated the Ras-PI3K-Akt pathway suppressing apoptosis. On the other hand, serum-deprivation-induced apoptosis was not suppressed by cPrG-HCl.

Introduction of bisecting GlcNAc in N-glycans of adenylyl cyclase III enhances its activity.

Adenylyl cyclases (ACs) catalyze the synthesis of cAMP in response to extracellular and intracellular signals and are responsible for a wide variety of biological activities including cell growth, differentiation, and metabolism. There are nine, currently known, isoforms of transmembrane ACs, and the primary structure of the catalytic unit and the potential N-glycosylation sites are highly conserved among them. The enzyme beta1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of a bisecting N-acetylglucosamine (GlcNAc) to N-glycans. We have been studying the function of GnT-III on signaling molecules. In this study, we report on the effects of a bisecting GlcNAc on AC signaling. We established GnT-III stable expressing cell lines of Neuro-2a mouse neuroblastoma cells and B16 mouse melanoma cells. Forskolin-induced AC activation and downstream signaling, such as the synthesis of cAMP and the phosphorylation of transcriptional factor CRE-binding protein were upregulated in the GnT-III transfectants compared with mock transfectants or a dominant negative mutant of GnT-III-transfected cells. Since endogenous AC expression levels in Neuro-2a and B16 cells were too low to permit the glycosylation status to be examined, AC type III (ACIII) was overexpressed in a stable expression system using Flp-In-293 cells. The N-glycans of ACIII in the GnT-III transfectants were confirmed to be modified by the introduction of a bisecting GlcNAc, and AC activity was found to be significantly up-regulated in the GnT-III transfectants. Thus, the structure of N-glycans of ACIII regulates its enzymatic activity and downstream signaling.

beta1,4-N-Acetylglucosaminyltransferase III potentiates beta1 integrin-mediated neuritogenesis induced by serum deprivation in Neuro2a cells.

Aspects of the biological significance of the bisecting N-acetylglucosamine (GlcNAc) structure on N-glycans introduced by beta1,4-N-acetylglucosaminyltransferase III (GnT-III) in Neuro2a cell differentiation are demonstrated. The overexpression of GnT-III in the cells led to the induction of axon-like processes with numerous neurites and swellings, in which beta1 integrin was localized, under conditions of serum starvation. This enhancement in neuritogenesis was suppressed by either the addition of a bisecting GlcNAc-containing N-glycan or erythroagglutinating phytohemagglutinin (E(4)-PHA), which preferentially recognizes the bisecting GlcNAc. GnT-III-promoted neuritogenesis was also significantly perturbed by treatment with a functional blocking anti-beta1 integrin antibody. In fact, beta1 integrin was found to be one of the target proteins of GnT-III, as confirmed by a pull-down assay with E(4)-PHA. These data suggest that N-glycans with a bisecting GlcNAc on target molecules, such as beta1 integrin, play important roles in the regulation of neuritogenesis.

Redox regulation of nerve growth factor-induced neuronal differentiation of PC12 cells through modulation of the nerve growth factor receptor, TrkA.

We investigated the effects of the cellular redox state on nerve growth factor (NGF)-induced neuronal differentiation and its signaling pathways. Treatment of PC12 cells with buthionine sulfoximine (BSO) reduced the levels of GSH, a major cellular reductant, and enhanced NGF-induced neuronal differentiation, activation of AP-1 and the NGF receptor tyrosine kinase, TrkA. Conversely, incubation of the cells with a reductant, N-acetyl-L-cysteine (NAC), inhibited NGF-induced neuronal differentiation and AP-1 activation. Consistent with the suppression, NAC inhibited NGF-induced activation of TrkA, formation of receptor complexes comprising TrkA, Shc, Grb2, and Sos, and activation of phospholipase Cgamma and phosphatidylinositol 3-kinase. Biochemical analysis suggested that the cellular redox state regulates TrkA activity through modulation of protein tyrosine phosphatases (PTPs). Thus, cellular redox state regulates signaling pathway of NGF through PTPs, and then modulates neuronal differentiation.

Beta1,4-N-Acetylglucosaminyltransferase III down-regulates neurite outgrowth induced by costimulation of epidermal growth factor and integrins through the Ras/ERK signaling pathway in PC12 cells.

A rat pheochromocytoma cell line (PC12), when transfected with beta1,4-N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulted in the suppression of neurite outgrowth induced by costimulation of epidermal growth factor (EGF) and integrins. The neurite outgrowth was restored by the overexpression of a constitutively activated mitogen- or extracellular signal-regulated kinase kinase-1 (MEK-1). Consistent with this, the EGF receptor (EGFR)-mediated ERK activation was blocked in GnT-III transfectants. Conversely, the overexpression of dominant negative MEK-1 or treatment with PD98059, a specific inhibitor of MEK-1, inhibited neurite outgrowth in controls transfected with mock. Furthermore GnT-III activity is required for these inhibitions, because the overexpression of a dominant negative GnT-III mutant (D321A) failed to reduce neurite outgrowth and EGFR-mediated ERK activation. Lectin blot analysis confirmed that EGFR from wild-type GnT-III transfectants had been modified by bisecting GlcNAc in its N-glycan structures. This modification led to a significant decrease in EGF binding and EGFR autophosphorylation. Collectively, the results constitute a comprehensive body of evidence to show clearly that the overexpression of GnT-III prevents neurite outgrowth induced by costimulation of EGF and integrins through the Ras/MAPK activation pathway and indicates that GnT-III may be an important regulator for cell differentiation in neural tissues.

Down-regulation of hydrogen peroxide-induced PKC delta activation in N-acetylglucosaminyltransferase III-transfected HeLaS3 cells.

N-acetylglucosaminyltransferase III (GnT-III) is a key enzyme that inhibits the extension of N-glycans by introducing a bisecting N-acetylglucosamine residue. Our previous studies have shown that modification of N-glycans by GnT-III affects a number of intracellular signaling pathways. In this study, the effects of GnT-III on the cellular response to reactive oxygen species (ROS) were examined. We found that an overexpression of GnT-III suppresses H(2)O(2)-induced apoptosis in HeLaS3 cells. In the case of GnT-III transfectants, activation of Jun N-terminal kinase (JNK) following H(2)O(2) treatment was markedly reduced compared with control cells. Either the depletion of protein kinase C (PKC) by prolonged treatment with phorbol 12-myristate 13-acetate or the inhibition of PKC by the specific inhibitor H7 attenuated the H(2)O(2)-induced activation of JNK1 and apoptosis in control cells but not in the GnT-III transfectants. Furthermore, we found that H(2)O(2)-induced phosphorylation of PKC delta was markedly suppressed in GnT-III transfectants. Rottlerin, a specific inhibitor of PKC delta, significantly inhibited H(2)O(2)-induced activation of JNK1 in control cells, indicating that PKC delta is involved in the pathway. These findings suggest that the overexpression of GnT-III suppresses H(2)O(2)-induced activation of PKC delta-JNK1 pathway, resulting in inhibition of apoptosis.

Apolipoprotein E activates Akt pathway in neuro-2a in an isoform-specific manner.

Apolipoprotein E (apoE) is a ligand for members of the low density lipoprotein (LDL) receptor family, receptors highly expressed in neurons. A study of one of the mechanisms by which apoE might affect neuronal cell metabolism is reported herein. ApoE can induce Akt/protein kinase B phosphorylation in Neuro-2a via two different pathways. Both pathways are mediated by phosphatidylinositol 3-kinase and cAMP-dependent protein kinase. The first pathway is stimulated by apoE3 and E4, but not by E2, after a 1-h incubation. The process requires the binding of apoE to the heparan sulfate proteoglycan/LDL receptor-related protein complex. The second pathway is activated after a 2-h incubation of the cells, in another isoform-dependent manner (E2 = E3 dbl greater-than sign E4) and is mediated by calcium. Our results suggest that apoE might affect cell metabolism and survival in neurons in an isoform-specific manner by inducing novel signaling pathways.