Glycogen synthase kinase-3beta regulates differentiation-induced apoptosis of human neural progenitor cells

Glycogen synthase kinase-3beta is a multifunctional key regulator enzyme in neural developmental processes and a main component of the canonical Wnt signaling pathway. It is already known that the Wnt-driven differentiation of neural progenitor cells is accompanied by an increase of apoptosis at which the pro-apoptotic function of GSK-3beta is still discussed. The aim of the present study was to investigate whether the phosphorylation level of GSK-3beta at serine 9 is the primary regulatory mechanism of differentiation-induced apoptosis.     

Dishevelled promotes neurite outgrowth in neuronal differentiating neuroblastoma 2A cells, via a DIX-domain dependent pathway

Dishevelled (Dvl) is a cytoplasmic protein involved in the Wnt-Frizzled signaling cascade, which has also been shown to interact with the cytoskeleton in part through inhibition of glycogen synthase kinase 3beta (GSK3beta). Using mouse neuroblastoma 2A (N2A) cells as a model system, we have found that overexpression of Dvl promotes the outgrowth of neurite-like processes, and leads to the induction of a striking, bipolar morphologic phenotype during neuronal differentiation. In contrast, suppression of Dvl expression using isoform-specific siRNAs led to an inhibition of neurite outgrowth in these cells. In order to further elucidate the mechanism(s) responsible for this effect, we overexpressed several mutant forms of Dvl in the N2A cells, including deletions in each of the three major functional subdomains of the protein (DeltaDIX, DeltaPDZ, DeltaDEP) and point mutations in the two well-defined interaction motifs within the DIX domain (the actin-binding and vesicle-association elements; K58A and K68A/E69A, respectively). These experiments revealed that the DIX domain (and its vesicle-binding subregion) was essential for Dvl's effect on neurite extension and morphogenesis in N2A cells. In contrast, direct overexpression of a degradation-resistant form of beta-catenin (S37A), or a dominant negative GSK3beta mutant (K85R), had no effect on neurite outgrowth or morphology in neuronally differentiating N2A cells; exposure of cells to a pharmacologic inhibitor of GSK3beta (lithium) also had no effect. Taken together, these results suggest that Dvl induces cytoskeletal and morphologic rearrangements in neuronal differentiating N2A cells through a mechanism that cannot be attributed exclusively to modulation of GSK3beta or beta-catenin activity, but which does depend upon a DIX-domain/vesicle-association-dependent signaling pathway.     

Carboxymethyl cellulose stimulation of neurite outgrowth of neuroblastoma cells in culture

The addition of 1% (w/v) carboxymethyl cellulose to the culture medium induces the formation of neurites of clone N18 neuroblastoma cells even in the presence of normal (5-10%) serum supplement concentrations, which rivals that previously observed by growth in low 0.1% serum. Heavy metal ions associated with the carboxymethyl cellulose were responsible for small increases in the sizes of cell bodies during treatment. Pretreatment of the PC12 pheochromocytoma line of neuroblasts with carboxymethyl cellulose for 1 day prior to their stimulation with nerve growth factor resulted in an acceleration in the rate, but not extent, of neurite outgrowth.     

Estrogen induces neurite outgrowth via Rho family GTPases in neuroblastoma cells

Estrogen (E2) has direct in vivo and in vitro effects, such as inducing neurite outgrowth, on neurons. We investigated the morphological changes and intracellular signaling pathway induced by E2 in neuroblastoma (SH-SY5Y) cells. The effect of medroxyprogesterone acetate (MPA) or progesterone (P4) on the E2-induced neurite outgrowth was also examined using SH-SY5Y cells. Neurite outgrowth was induced by E2 in association with the phosphorylation of Akt, and these effects of E2 were abolished by MPA but not by P4. Progesterone receptor antagonist RU486 blocked the inhibitory effects of MPA. Estrogen receptor antagonist ICI 182,780 and phosphatidylinositol 3-kinase inhibitor LY294002 inhibited the E2-induced neurite outgrowth. Because the Rho family of small GTPases has been shown to be involved in the regulation of neurite outgrowth, we examined the cross-talk among Rac1, Cdc42 and RhoA in the E2-induced neurite outgrowth. E2 immediately increased the Rac1 and Cdc42 activity and decreased the RhoA activity. E2-induced neurite outgrowth was attenuated in cells expressing dominant-negative mutants for Rac1 or Cdc42. These results suggest that regulation of Rho family GTPase activity by E2 is important for the neurite outgrowth in neuroblastoma cells, and that MPA may have an antagonistic effect against E2.     

Glycogen synthase kinase-3beta,mood stabilizers,and neuroprotection

Objectives: This paper reviews results of our studies examining the regulation of endoplasmic reticulum (ER) stress proteins by valproate (VPA), and discusses the possible implications in bipolar disorder.

Methods: Our previous studies in the field are reviewed along with relevant literature.

Results: Using differential display PCR, we identified GRP78 as a VPA-regulated gene in rat cerebral cortex. We also showed that other members of the ER stress proteins family, GRP94 and calreticulin, are also upregulated by VPA. Immunohistochemistry identified that ER stress proteins are increased in frontal and parietal cortex, as well as regions of the hippocampus in rat brain following chronic treatment with VPA.

Conclusions: Regulation of ER stress proteins by VPA may prove to be important to the mechanism of action of the drug. The neuroprotective role of these proteins may also prove to be involved in the pathophysiology of bipolar disorder.     

Glycogen synthase kinase-3 beta-mediated tau phosphorylation in cultured cell lines

To study further the role of glycogen synthase kinase-3beta on tau phosphorylation, glycogen synthase kinase-3beta and tau expression vectors were co-transfected into CHO-K1, COS-7 and SH-SY5Y cell. Tau phosphorylation was assessed by phosphorylation-dependent antibodies AT-8, AT-180, AT-270 and PHF-1. The AT-270 and AT-8 epitopes were consistently phosphorylated by glycogen synthase kinase-3beta in the three cell lines. Phosphorylation on AT-180 epitope was significant in CHO-K1 and SH-SY5Y cells while PHF-1 epitope was hyper-phosphorylated only in SH-SY5Y cells. We also found that lithium induces phosphorylation of the serine 9 residue of glycogen synthase kinase-3beta together with inhibition of tau phosphorylation on PHF-1 epitope in all the three cell lines. This suggests a novel mechanism whereby lithium-mediated inhibition of GSK-3beta activity influences tau phosphorylation.     

Glycogen synthase kinase-3 in neurodegeneration and neuroprotection: lessons from lithium

For over fifty years lithium has been a fundamental component of therapy for patients with bipolar disorders. Lithium has been considered recently for its potential to alleviate neuronal loss and other neurodegeneration processes. For instance, lithium reduces the severity of some behavioral complications of Alzheimer's disease (AD). And there are growing indications that lithium may be of benefit to the underlying pathology of AD, as well as an array of other common CNS disorders, including stroke, Parkinson's disease, and Huntington's disease. Despite these demonstrated and prospective therapeutic benefits, lithium's mechanism of action remains elusive, and opinions differ regarding the most relevant molecular targets. Lithium inhibits several enzymes; significant among these are inositol monophosphatase (IMPase), glycogen synthase kinase-3 (GSK-3), and the proteasome. Most recent publications discussing the medical application of lithium have converged on GSK-3, so this article reviews data and discussions regarding the roles and interactions of GSK-3 with other proteins and its proposed role in the pathogenesis of Alzheimer's disease.     

Glycogen synthase kinase-3 beta is highly activated in nuclei and mitochondria

Glycogen synthase kinase-3 beta (GSK3 beta) is located predominantly in the cytosol, but also is in nuclei and mitochondria. In SH-SY5Y cells, primary cortical neurons, and mouse brain, the portion of active GSK3 beta (not phosphorylated on serine-9) was 5- to 8-fold greater in nuclei and mitochondria than in cytosol. Correspondingly greater GSK3 beta activities were measured in nuclei and mitochondria compared with cytosol. Stimulation of apoptotic signaling by treatment with camptothecin or thapsigargin activated GSK3 beta in the nucleus and mitochondria, but not the cytosol, whereas inhibition of GSK3 beta by lithium treatment affected all three pools of GSK3 beta. Thus, the nuclei and mitochondria contain disproportionately high levels of active GSK3 beta, which is selectively further activated by some apoptotic stimuli.     

Gangliosides inhibit growth factor-stimulated neurite outgrowth in SH-SY5Y human neuroblastoma cells

Exogenously added gangliosides are known to promote neurite outgrowth in a variety of cell types, including some neuroblastoma cell lines. To study neuritogenesis in SH-SY5Y human neuroblastoma we serum starved the cells for 24 hr and exposed them to gangliosides (GM1, GM3, or GT1b), platelet-derived growth factor (PDGF), insulin, nerve growth factor (NGF), insulin-like growth factor I (IGF-I), or combinations of these for 3 days. We measured four parameters of neurite outgrowth using image analysis. PDGF induced neurite outgrowth in SH-SY5Y and GM1 inhibited this. Both phenomena were dose-dependent with neurites/cell and neurite length being below controls with 100 μM GM1, and percent of neurite-bearing cells being below controls with 25, 50, and 100 μM GM1. Similar but more inhibitory results were obtained with GM3 and GT1b. Insulin and IGF-I induced a neuritogenic response that was less potent than that of PDGF and was also inhibited by gangliosides. NGF had no effect on neurite outgrowth but gangliosides were still inhibitory even in cells not treated with growth factors. From this we conclude that gangliosides inhibit spontaneous and trophic factor-induced neurite outgrowth in SH-SY5Y cells. For GM1 and GT1b, but not GM3, this probably involves inhibition of trophic factor receptor function. J. Neurosci. Res. 47:617–625, 1997. © 1997 Wiley-Liss, Inc.     

Inhibition of neurite outgrowth of neuroblastoma neuro-2a cells by cholera toxin B-subunit and anti-GM1 antibody

The role of cell surface GM1 ganglioside in neurite outgrowth of Neuro-2a neuroblastoma cells was investigated by application of anti-GM1 antibody and the B subunit of cholera toxin (cholera B) to cultured cells stimulated to grow neurites in various ways. When the cells were simultaneously treated with stimulatory agent and cholera B, inhibition, as measured by percent of neurite-bearing cells, was observed with most stimuli: neuraminidase; GD1a ganglioside, retinoic acid, and low serum. However, with dibutyryl cyclic AMP the small reduction observed was not statistically significant. The inhibitory effect of cholera B on neurite outgrowth induced by low serum was dose-dependent, reaching a maximum at 200 ng/mL; 48 h after washout of cholera B the cells were released from inhibition and regrew neurites at nearly the previous rate in the presence of low serum. When the cells were exposed to stimulus for 6 h or more the inhibitory effect of subsequent addition of cholera B was reduced or eliminated; inhibition thus occurs during an early stage of neurite initiation. Anti-GM1 anti-body at dilutions of 1∶100–1∶400 had the same inhibitory effect as cholera B with cells stimulated by GD1a or retinoic acid, whereas anti-GM2 antibody had no effect at 1∶200 or 1∶400; inhibition by the latter antibody at 1∶100 dilution was similar to that attained with control ascites fluid. These results point to a pivotal role for cell surface GM1 in Neuro-2a differentiation induced by many (but not all) neuritogenic agents. ganglioside nomenclature follows that of Svennerholm (1963).     

Inhibition of neurite outgrowth of neuroblastoma neuro-2a cells by cholera toxin B-subunit and anti-GM1 antibody

The role of cell surface GM1 ganglioside in neurite outgrowth of Neuro-2a neuroblastoma cells was investigated by application of anti-GM1 antibody and the B subunit of cholera toxin (cholera B) to cultured cells stimulated to grow neurites in various ways. When the cells were simultaneously treated with stimulatory agent and cholera B, inhibition, as measured by percent of neurite-bearing cells, was observed with most stimuli: neuraminidase; GD1a ganglioside, retinoic acid, and low serum. However, with dibutyryl cyclic AMP the small reduction observed was not statistically significant. The inhibitory effect of cholera B on neurite outgrowth induced by low serum was dose-dependent, reaching a maximum at 200 ng/mL; 48 h after washout of cholera B the cells were released from inhibition and regrew neurites at nearly the previous rate in the presence of low serum. When the cells were exposed to stimulus for 6 h or more the inhibitory effect of subsequent addition of cholera B was reduced or eliminated; inhibition thus occurs during an early stage of neurite initiation. Anti-GM1 anti-body at dilutions of 1∶100–1∶400 had the same inhibitory effect as cholera B with cells stimulated by GD1a or retinoic acid, whereas anti-GM2 antibody had no effect at 1∶200 or 1∶400; inhibition by the latter antibody at 1∶100 dilution was similar to that attained with control ascites fluid. These results point to a pivotal role for cell surface GM1 in Neuro-2a differentiation induced by many (but not all) neuritogenic agents.     

FLRT3 is expressed in sensory neurons after peripheral nerve injury and regulates neurite outgrowth

We used a molecular screen to identify genes upregulated in regenerating adult rat dorsal root ganglion cells. FLRT3 mRNA and protein characterized by a fibronectin type III domain and a leucine-rich repeat motif was upregulated in damaged sensory neurons. The protein was then transported into their peripheral and central processes where the FLRT3 protein was localized to presynaptic axon terminals. In vitro, the FLRT3 protein was expressed at the cell surface, regulated neurite outgrowth in sensory neurons, but did not exhibit homophilic binding. FLRT3 was widely expressed in the developing embryo, particularly in the central nervous system and somites. However, in the adult, we found no evidence for accumulation or reexpression of the FLRT3 protein in damaged axons of the central nervous system. We conclude that FLRT3 codes for a putative cell surface receptor implicated in both the development of the nervous system and in the regeneration of the peripheral nervous system (PNS).     

Purkinje cell protein 4 positively regulates neurite outgrowth and neurotransmitter release

Purkinje cell protein 4 (PCP4), also called brain-specific polypeptide 19 (PEP19), is a neurospecific, small calmodulin-binding protein that binds both calcium-free and calcium-binding calmodulin to regulate the calmodulin-mediated signal. The expression level of this molecule is decreased in the brain in Alzheimer's disease, Huntington's disease, and alcoholism. However, little is known of the function of PCP4 regarding neuronal or neuroendocrine cell differentiation and neurotransmitter release. To address this, we established a PCP4 tetracycline-inducible rat chromaffin cell line, PC12. When PCP4 expression was induced with doxcycline, neurite outgrowth was significantly advanced in the presence of nerve growth factor (NGF) and dibutyryl cAMP, which was inhibited by W-7, a calmodulin inhibitor, and PD98059, an ERK inhibitor. In addition, size of the cell body also was increased by treatment with NGF in the PCP4-induced PC12 cells. Constitutive and potassium-evoked release of acetylcholine and dopamine was increased and apoptosis induced by hydrogen peroxide (H(2)O(2)) was inhibited in PCP4-induced PC12 cells. On the other hand, knockdown of PCP4 by siRNA transfection decreased neurite outgrowth and dopamine release and increased H(2)O(2)-induced apoptosis in PC12 cells. These results indicate that PCP4 promotes neuroendocrine cell differentiation and neurotransmitter release by activating calmodulin function.