Cha-Gyun Jung

FTY720 modulates human oligodendrocyte progenitor process extension and survival

FTY720, a sphingosine‐1‐phosphate (S1P) receptor agonist that crosses the blood–brain barrier, is a potential immuno‐therapy for multiple sclerosis. Our objective was to assess the effect of FTY720 on process extension, differentiation, and survival of human oligodendrocyte progenitor cells (OPCs), and link the functional effects with S1P receptor expression and signaling.

Functional consequences of S1P receptor modulation in rat oligodendroglial lineage cells

Fingolimod (FTY720) and its phosphorylated form FTY720P are modulators of sphingosine‐1‐phosphate (S1P) receptors, which are G‐protein coupled receptors linked to cell migration and vascular maturation. The efficacy of FTY720 in autoimmune diseases such as multiple sclerosis and its animal models has been attributed to its inhibition of lymphocyte trafficking to target organs. In this study, we examined the role of S1P receptors in cultured rat oligodendrocytes (OLGs) and OLG progenitor cells (OPCs) using the active phosphorylated form of FTY720. We found that (1) FTY720P improves the survival of neonatal rat OLGs during serum withdrawal, which is associated with the phosphorylation of extracellular signal regulated kinases (ERK1/2) and Akt; (2) FTY720P regulates OPC differentiation into OLGs in a concentration‐dependent manner; and (3) S1P receptors are differentially modulated by platelet‐derived growth factor (PDGF) resulting in downregulation of S1P5 and upregulation of S1P1 in OPCs. In addition, siRNA studies revealed that S1P1 participates in PDGF‐induced OPC mitogenesis. We conclude that S1P1 and S1P5 serve different functions during oligodendroglial development, and possibly during remyelination.

Glial cell line-derived neurotrophic factor-induced signaling in Schwann cells.

Glial cell line‐derived neurotrophic factor (GDNF), a known survival factor for neurons, has recently been shown to stimulate the migration of Schwann cells (SCs) and to enhance myelination. GDNF exerts its biological effects by activating the Ret tyrosine kinase in the presence of glycosylphosphatidylinositol‐linked receptor, GDNF family receptor (GFR) α1. In Ret‐negative cells, the alternative transmembrane coreceptor is the 140‐kDa isoform of neural cell adhesion molecule (NCAM) associated with a non‐receptor tyrosine kinase Fyn. We confirmed that GDNF, GFRα1 and NCAM are expressed in neonatal rat SCs. We found that GDNF induces an increase in the partitioning of NCAM and heparan sulfate proteoglycan agrin into lipid rafts and that heparinase inhibits GDNF‐signaling in SCs. In addition to activation of extracellular signal‐regulated kinases, and phosphorylation of cAMP response element binding protein, we found that cAMP‐dependent protein kinase A and protein kinase C are involved in GDNF‐mediated signaling in SCs. Although GDNF did not promote the differentiation of purified SCs into the myelinating phenotype, it enhanced myelination in neuron–SC cocultures. We conclude that GDNF utilizes NCAM signaling pathways to regulate SC function prior to myelination and at early stages of myelin formation.

Pleiotrophin exhibits a trophic effect on survival of dopaminergic neurons in vitro

To understand what kind of trophic factors are up‐regulated in dopamine (DA)‐depleted striatum, we first analysed the up‐regulation of mRNAs using a DNA microarray in DA‐depleted striatum where DAergic inputs were denervated by 6‐OHDA. We then investigated whether or not such trophic factors had an effect on cultured dopaminergic neurons. The microarray analysis revealed that pleiotrophin (PTN), glial‐derived neurotopic factor (GDNF) and others were up‐regulated in DA‐depleted striatum. As PTN has been reported to have a wide range of trophic effects on neurons, we focused on the functional role of PTN in the present study. The increase in PTN mRNA was confirmed by Northern blotting at 1–3 weeks after the lesion, reaching a peak at 1 week. In embryonic day 15 mesencephalic neuron culture, PTN increased the number of tyrosine hydroxylase (TH) ‐positive neurons in a dose‐dependent manner (125.2 ± 2.0% of the control at 50 ng/mL), while a family protein, midkine (10 ng/mL) did not show any trophic effect (99.3 ± 0.7%). In addition, the PTN effect on DAergic neurons was additive to the GDNF effect. As PTN did not increase the number of microtubule‐associated protein‐2 (MAP 2)‐positive neurons or promote the proliferation of dopaminergic progenitors in a bromodeoxyuridine (BrdU) labelling study, the effect appeared to enhance the specific survival of dopaminergic neurons. Expression of PTN receptors (syndecan‐3, PTP‐ζ) was detected on the cultured mesencephalic neurons, and also up‐regulated in DA‐depleted striatum. The data indicate that PTN is up‐regulated in DA‐depleted striatum and exhibits a trophic effect specifically on the survival of cultured dopaminergic neurons.

Pleiotrophin mRNA is highly expressed in neural stem (progenitor) cells of mouse ventral mesencephalon and the product promotes production of dopaminergic neurons from embryonic stem cell-derived nestin-positive cells

Neural stem cells are promising candidates for donor cells in neural transplantation. However, the mechanism by which neural stem cells differentiate into neurons is not well understood. In the present study, a serial analysis of gene expression (SAGE) was carried out to generate a gene file of neural stem (progenitor) cells from the mouse ventral mesencephalon. Among the 15,815 tags investigated, the mRNA of the housekeeping genes (elongation factor 1‐α, ATPase subunit 6, GAPDH, actin), laminin receptor 1, HSP 70, pleiotrophin, and nestin were highly expressed. Because pleiotrophin (PTN) exhibits mitogenic and trophic effects on neural development and exhibits trophic effects on survival of dopaminergic (DAergic) neurons, we investigated the role of PTN in neurogenesis, especially to DAergic neurons. Here, we show that PTN increased the production of tyrosine hydroxylase (TH)‐positive neurons from embryonic stem (ES) cell‐ derived nestin‐positive cells. The expression of Nurr1 mRNA was enhanced by PTN. L‐dopa in the culture medium was increased by PTN. This effect was as strong as with sonic hedgehog. Data suggest that PTN mRNA is highly expressed in neural stem (progenitor) cells of mouse ventral mesencephalon, and PTN promotes the production of DAergic neurons from ES cell‐ derived nestin‐positive cells.

Homeotic factor ATBF1 induces the cell cycle arrest associated with neuronal differentiation

The present study aimed to elucidate the function of AT motif-binding factor 1 (ATBF1) during neurogenesis in the developing brain and in primary cultures of neuroepithelial cells and cell lines (Neuro 2A and P19 cells). Here, we show that ATBF1 is expressed in the differentiating field in association with the neuronal differentiation markers β-tubulin and MAP2 in the day E14.5 embryo rat brain, suggesting that it promotes neuronal differentiation. In support of this, we show that ATBF1 suppresses nestin expression, a neural stem cell marker, and activates the promoter of Neurod1 gene, a marker for neuronal differentiation. Furthermore, we show that in Neuro 2A cells, overexpressed ATBF1 localizes predominantly in the nucleus and causes cell cycle arrest. In P19 cells, which formed embryonic bodies in the floating condition, ATBF1 is mainly cytoplasmic and has no effect on the cell cycle. However, the cell cycle was arrested when ATBF1 became nuclear after transfer of P19 cells onto adhesive surfaces or in isolated single cells. The nuclear localization of ATBF1 was suppressed by treatment with caffeine, an inhibitor of PI(3)K-related kinase activity of ataxa-telangiectasia mutated (ATM) gene product. The cytoplasmic localization of ATBF1 in floating/nonadherent cells is due to CRM1-dependent nuclear export of ATBF1. Moreover, in the embryonic brain ATBF1 was expressed in the cytoplasm of proliferating stem cells on the ventricular zone, where cells are present at high density and interact through cell-to-cell contact. Conversely, in the differentiating field, where cell density is low and extracellular matrix is dense, the cell-to-matrix interaction triggered nuclear localization of ATBF1, resulting in the cell cycle arrest. We propose that ATBF1 plays an important role in the nucleus by organizing the neuronal differentiation associated with the cell cycle arrest.

Increase in dopaminergic neurons from mouse embryonic stem cell‐derived neural progenitor/stem cells is mediated by hypoxia inducible factor‐1α

A reliable method to induce neural progenitor/stem cells (NPCs) into dopaminergic (DAergic) neurons has not yet been established. As well, the mechanism involved remains to be elucidated. To induce DAergic differentiation from NPCs, a cytokine mixture (C‐Mix) of interleukin (IL)‐1β, IL‐11, leukemia‐inhibitory factor (LIF), and glial‐derived neurotrophic factor or low oxygen (3.5% O2: L‐Oxy) was used to treat embryonic stem (ES) cell‐derived NPCs. Treatment with C‐Mix increased the number of tyrosine hydroxylase (TH)‐positive cells compared with controls (2.20‐fold of control). The C‐Mix effect was induced by mainly LIF or IL‐1β treatment. Although L‐Oxy caused an increase in TH‐positive cells (1.34‐fold), the combination of L‐Oxy with C‐Mix did not show an additive effect. Increases in DA in the medium were shown in the presence of C‐Mix, LIF, and L‐Oxy by high‐performance liquid chromatography. Gene expression patterns of neural markers [tryptophan hydroxylase (TPH), GAD67, GluT1, β‐tubulin III, glial fibrillary acidc protein, and TH] were different in C‐Mix and L‐Oxy treatments. Because increases in hypoxia‐inducible factor (HIF)‐1α protein were found in both treatments, we investigated the effect of HIF‐1α on differentiation of NPCs to DAergic neurons. Inhibition of HIF‐1α by the application of antisense oligodeoxynucleotides (ODNs) to NPCs caused a decrease in TH‐positive cells induced by LIF treatment. Gene expressions of TH, GAD67, and GluT1 were decreased, and those of TPH, β‐tubulin III, and S‐100β were increased by treatment with just ODNs, indicating the importance of the endogenous effect of HIF‐1α on neuronal differentiation. These data suggest that enhanced differentiation into DAergic neurons from ES cell‐derived NPCs was induced by C‐Mix or L‐Oxy mediated by HIF‐1α.

The ZFHX3 (ATBF1) transcription factor induces PDGFRB, which activates ATM in the cytoplasm to protect cerebellar neurons from oxidative stress

SUMMARY Ataxia telangiectasia (A-T) is a neurodegenerative disease caused by mutations in the large serine-threonine kinase ATM. A-T patients suffer from degeneration of the cerebellum and show abnormal elevation of serum alpha-fetoprotein. Here, we report a novel signaling pathway that links ATM via cAMP-responsive-element-binding protein (CREB) to the transcription factor ZFHX3 (also known as ATBF1), which in turn promotes survival of neurons by inducing expression of platelet-derived growth factor receptor β (PDGFRB). Notably, AG1433, an inhibitor of PDGFRB, suppressed the activation of ATM under oxidative stress, whereas AG1433 did not inhibit the response of ATM to genotoxic stress by X-ray irradiation. Thus, the activity of a membrane-bound tyrosine kinase is required to trigger the activation of ATM in oxidative stress, independent of the response to genotoxic stress. Kainic acid stimulation induced activation of ATM in the cerebral cortex, hippocampus and deep cerebellar nuclei (DCN), predominately in the cytoplasm in the absence of induction of γ-H2AX (a marker of DNA double-strand breaks). The activation of ATM in the cytoplasm might play a role in autophagy in protection of neurons against oxidative stress. It is important to consider DCN of the cerebellum in the etiology of A-T, because these neurons are directly innervated by Purkinje cells, which are progressively lost in A-T.

Enhanced neurogenesis from neural progenitor cells with G1/S-phase cell cycle arrest is mediated by transforming growth factor β1

We have previously demonstrated that a G1/S‐phase cell cycle blocker, deferoxamine (DFO), increased the number of new neurons from rat neurosphere cultures, which correlated with prolonged expression of cyclin‐dependent kinase (cdk) inhibitor p27kip1 [ H. J. Kim et al. (2006)Brain Research, 1092, 1–15]. The present study focuses on neuronal differentiation mechanisms following treatment of neural stem/progenitor cells (NPCs) with a G1/S‐phase cell cycle blocker. The addition of DFO (0.5 mm) or aphidicolin (Aph) (1.5 μm) to neurospheres for 8 h, followed by 3 days of differentiation, resulted in an increased number of neurons and neurite outgrowth. DFO induced enhanced expression of transforming growth factor (TGF)‐β1 and cdk5 at 24 h after differentiation, whereas Aph only increased TGF‐β1 expression. DFO‐induced neurogenesis and neurite outgrowth were attenuated by administration of a cdk5 inhibitor, roscovitine, suggesting that the neurogenic mechanisms differ between DFO and Aph. TGF‐β1 (10 ng/mL) did not increase neurite outgrowth but rather the number of β‐tubulin III‐positive cells, which was accompanied by enhanced p27kip1 mRNA expression. In addition, TGF‐β receptor type II expression was observed in nestin‐positive NPCs. Results indicated that DFO‐induced TGF‐β1 signaling activated smad3 translocation from the cytoplasm to the nucleus. In contrast, TGF‐β1 signaling inhibition, via a TGF‐β receptor type I inhibitor (SB‐505124), resulted in decreased DFO‐induced neurogenesis, in conjunction with decreased p27kip1 protein expression and smad3 translocation to the nucleus. These results suggest that cell cycle arrest during G1/S‐phase induces TGF‐β1 expression. This, in turn, prompts enhanced neuronal differentiation via smad3 translocation to the nucleus and subsequent p27kip1 activation in NPCs.

Targeting of Myelin Protein Zero in a Spontaneous Autoimmune Polyneuropathy

Elimination of the costimulatory molecule B7-2 prevents autoimmune diabetes in NOD mice, but leads to the development of a spontaneous autoimmune polyneuropathy (SAP), which resembles the human disease chronic inflammatory demyelinating polyneuropathy (CIDP). In this study, we examined the immunopathogenic mechanisms in this model, including identification of SAP Ags. We found that B7-2-deficient NOD mice exhibit changes in cytokine and chemokine gene expression in spleens over time. There was an increase in IL-17 and a decrease in IL-10 transcript levels at 4 mo (preclinical phase), whereas IFN-γ expression peaked at 8 mo (clinical phase). There was also an increase in transcript levels of Th1 cytokines, CXCL10, and RANTES in sciatic nerves of mice that developed SAP. Splenocytes from SAP mice exhibited proliferative and Th1 cytokine responses to myelin P0 (180–199), but not to other P0 peptides or P2 (53–78). Adoptive transfer of P0-reactive T cells generated from SAP mice induced neuropathy in four of six NOD.SCID mice. Data from i.v. tolerance studies indicate that myelin P0 is one of the autoantigens targeted by T cells in SAP in this model. The expression of P0 by peri-islet Schwann cells provides a potential mechanism linking islet autoimmunity and inflammatory neuropathy.