Neurochemical Research

, Volume 34, Issue 5, pp 942–951

Neuroprotection by NGF and BDNF Against Neurotoxin-Exerted Apoptotic Death in Neural Stem Cells Are Mediated Through Trk Receptors, Activating PI3-Kinase and MAPK Pathways

Authors

  • Nga Nguyen
    • Department of Molecular Cell Biology, Samsung Biomedical Research InstituteSungkyunkwan University School of Medicine
  • Sang Bae Lee
    • Department of Molecular Cell Biology, Samsung Biomedical Research InstituteSungkyunkwan University School of Medicine
  • Yung Song Lee
    • Department of Molecular Cell Biology, Samsung Biomedical Research InstituteSungkyunkwan University School of Medicine
    • Department of Anatomy, Samsung Biomedical Research InstituteSungkyunkwan University School of Medicine
    • Department of Molecular Cell Biology, Samsung Biomedical Research InstituteSungkyunkwan University School of Medicine
OriginalPaper

DOI: 10.1007/s11064-008-9848-9

Cite this article as:
Nguyen, N., Lee, S.B., Lee, Y.S. et al. Neurochem Res (2009) 34: 942. doi:10.1007/s11064-008-9848-9

Abstract

Neural stem cells (NSC) undergo apoptotic cell death during development of nervous system and in adult. However, little is known about the biochemical regulation of neuroprotection by neurotrophin in these cells. In this report, we demonstrate that Staurosporine (STS) and Etoposide (ETS) induced apoptotic cell death of NSC by a mechanism requiring Caspase 3 activation, poly (ADP-ribose) polymerase and Lamin A/C cleavage. Although C17.2 cells revealed higher mRNA level of p75 neurotrophin receptor (p75NTR) compared with TrkA or TrkB receptor, neuroprotective effect of both nerve growth factor (NGF) and brain-derived growth factor (BDNF) mediated through the activation of tropomyosin receptor kinase (Trk) receptors. Moreover, both NGF and BDNF induced the activation of the phosphatidylinositide 3 kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) pathway. Inhibition of Trk receptor by K252a reduced PARP cleavage as well as cell viability, whereas inhibition of p75NTR did not affect the effect of neurotrophin on neurotoxic insults. Thus our studies indicate that the protective effect of NGF and BDNF in NSC against apoptotic stimuli is mediated by the PI3K/Akt and MAPK signaling pathway via Trk receptors.

Keywords

Neural stem cellsStaurosporineEtoposideApoptosisBDNFNGF

Introduction

Neural stem cells (NSC) can give rise to proliferate, differentiate into both neurons and glial cells during development of nervous system [1]. C17.2, a murine-derived multipotent NSC line was generated from the external granule layer cells of normal mouse cerebellum, has the ability to self-renew and differentiate into neurons, astrocytes and oligodendrocytes [2, 3]. The mechanisms that control the proliferation and differentiation of these cells are rapidly accumulating data including neurotrophic factor and cytokine signaling. In despite of the considerable death of NSC that rises during development of the nervous system and in the adult neuron [1, 47], the paucity of information is known about the cell survival signaling. Recent studies suggest that NSC may undergo an apoptotic cell death with the activation of Caspase 3 cascade under neurotoxic insults such as oxidative stress, DMNQ (2,3-dimethoxy-1,4-naphthoquinone) and STS [8], nitric oxide [9], manganese [10].

Neurotrophins such as NGF and BDNF play vital roles not only in neuronal growth, survival and differentiation but also in neuronal death [1117]. The neurotrophins manifest their effects by binding to two discrete receptor subtypes: the tropomyosin receptor kinase (Trk) family and the p75 neurotrophin receptor (p75NTR) [11, 18]. NGF and BDNF bind to TrkA and TrkB [19, 20], respectively and they can also interact with the p75NTR. Binding of neurotrophins to members of Trk family receptors leads to phosphorylation of tyrosine residues and activation of the signaling molecule by a variety of mechanisms: phosphatidylinositide 3 kinase (PI3K) binding to SH2 domain [21], phosphorylation of PLC-γ and STATs [22], targeting of Sos to the membrane and activation of Ras pathway [23].

Phosphatidylinositide 3 kinase /Akt and the mitogen-activated protein kinase (MAPK) pathway are shown to be involved in signal transduction from receptor tyrosine kinase [2426]. Activation of Akt phosphorylates and inhibits the pro-apoptotic Bcl-2 family member BAD, inhibiting BAD pro-apoptotic functions [27, 28] and Caspase 9 [29], and Akt controls a major class of transcription factors—the Forkhead box transcription factor by phosphorylating FOXOs (Forkhead box, group O) and inhibiting their ability to induce the expression of death genes [3032]. Moreover, Akt has also been shown to promote survival in hippocampal neurons by inhibiting the activity of p53 [33, 34]. Very recently, we have shown nuclear Akt prevents DNA fragmentation via inhibition of CAD (Caspase-activated DNase), associating with Ebp1(ErbB3 binding protein) in PC12 cells [35]. By comparison, the activation of MAPK mediates mitogenesis, differentiation and cell death depending on the length of activation and nuclear translocation [36, 37] through the phosphorylation of its downstream targets including MAPK-activated kinase, Rsks, which phosphorylates BAD and CREB. The phosphorylation of BAD prevents cell death, while the phosphorylation of CREB promotes cell survival [38, 39].

C17.2 NSC employed in the present study are derived from the external germinal layer of neonatal murine cerebellum previously immortalized by the retrovirus-mediated transduction of avian myc (v-myc) [2]. Although both NGF and BDNF are effective in enhancing survival in many neuronal cells, the underlying signaling mechanism and receptors involved in the survival of NSC have not been clarified yet. In this study, we determined the effect of NGF and BDNF in inhibiting apoptotic cell death and intracellular signaling pathway responsible for this protection under staurosporine (STS) and etoposide (ETS) insult in C17.2 cells. Our results show that both NGF and BDNF protect NSC from both STS and ETS induced apoptosis trough Trk receptor, up-regulating PI3K/Akt and MAPK pathway.

Materials and Methods

Cell Cultures and Experimental Treatments

The murine neural stem cell lines, C17.2 cell line was kindly provided by Dr. Jong Sun Kang (Sungkyunkwan Univ. School of medicine, Korea) and maintained in DMEM (Cellgro, Herndon, VA) containing 10% fetal bovine serum, 5% horse serum, and penicillin-streptomycin in a humidified atmosphere of 5% CO2, 95% air at 37°C. For experimental analyses, cells were grown in either cell culture dishes or on glass coverslips coated with poly-L-lysine (Sigma-Aldrich, Milwaukee, WI). Cells were treated with various drugs at the exponential phase of growth after 24 h of culture.

Reagents and Antibodies

Chemicals used were nerve growth factor (NGF), which was from Chemicon (Temecula, CA). STS, in Solution TM K-252a and p75NTR Signaling Inhibitor were obtained from Calbiochem (San Diego, CA). ETS and dimethyl sulphoxide (DMSO) were supported by Sigma and Brain-derived neurotrophic factor (BDNF) by R&D Systems (Minneapolis, MN). TRIzol and SuperScriptTM II Reverse Transcriptase kit were purchased from Invitrogen (Carlsbad, CA). Go Taq® Green Master Mix were purchased from Promega (Madison, WI). The anti-PARP and anti-LaminA/C utilized in this study were purchased from Cell signaling. The anti-tubulin was supplied by Santa Cruz (Santa Cruz, CA). The anti-p75NTR, anti-TrkA, anti-TrkB, and anti-Caspase 3 were acquired from Upstate (Lake Placid, NY). Alexa Fluor 488 goat anti rabbit IgG was acquired from Molecular Probes (Eugene, OR). All chemicals not referenced above were obtained from Sigma-Aldrich (Milwaukee, WI).

Immunoblotting

The cells were rinsed and harvested with lysis buffer (Tris 50 mM, pH 7.4, NaCl 40 mM, EDTA 1 mM, Triton X-100 0.5%, Na3VO4 1.5 mM, NaF 50 mM, sodium pyrophosphate 10 mM, glycerolphosphate 10 mM, PMSF 1 mM, Protease inhibitor cocktail 10 mM), vortexed, and centrifuged for 10 min at 13,000 rpm at 4°C. The protein concentration in each sample was determined using a Bio-Rad protein assay kit with bovine serum albumin (BSA) (Amresco, Solon, OH) as the standard. Aliquots (60 μg) of the proteins were analyzed via Western blotting using a 1:1000 dilution of monoclonal anti-PARP, anti-Caspase 3, anti-p75NTR, anti-TrkA, anti-TrkB, anti-LaminA/C and anti-tubulin. The immunocomplexes were visualized with enhanced chemiluminescence reagent (Amersham, UK), in accordance with the manufacturer’s instructions.

Immunocytochemistry

All steps were performed at room temperature. First, cells were fixed with 4% paraformaldehyde (MP Biomedicals, Aurora, OH) for 20 min and then washed with phosphate-buffered saline (PBS, pH 7.4). The cells were then brought to be permeable by being kept in 0.5% Triton X 100 in PBS for 20 min. The cells had been washed again before the blocking step was done with 5% BSA in PBS for 1 h. Primary antibodies were diluted 1:1000 in 2.5% BSA in PBS and added to the cells for 1 h. The following primary rabbit-derived antibodies were used: anti-p75NTR, anti-TrkA and anti-TrkB. The cells were then washed and stained with the appropriate fluorescent dye-conjugated antibody, Alexa Fluor 488 goat anti rabbit IgG (1:1000) for another 1 h. Finally, the cells were introduced to DAPI (0.5 ng/ml) for 7 min and the coverslips were mounted and examined by fluorescent microscope.

Reverse Transcriptase Polymerase Chain Reaction

Total RNA was isolated from C17.2 cells with TRIzol as described by the supplier (Invitrogen). After DNase treatment, cDNA was synthesized from 1 to 2 μg of total RNA using the SuperScriptTM II Reverse Transcriptase kit. PCR was carried out for 35 cycles (94°C for 30 s, 57°C for 30 s, 72°C for 30 s) using 1 μl of the RT product, the Go Taq® Green Master Mix and the following primers (200 nM, final concentration) specific for murine: p75NTR-intracellular domain (ICD) sense, 5′-CCAGCAGACCCACACACAGACTG; p75NTR-ICD antisense, 5′-CCCTACACAGAGATGCTCGGTTC; TrkA sense, 5′-AGGTGGCTGCTGGTATGGT; TrkA antisense, 5′-TCGCCTCAGTGTTGGAGAG; TrkB sense, 5′-TGGGAAGGATGAGAGACAGA; TrkB antisense, 5′-ATCACCACCACGGCATAGA.

MTT Assay

104 cells were seeded in 96-well plate. After growing for 1 day, they were exposed to 100 ng/ml of NGF or BDNF for 12 h following by pretreatment with 100 nM of K-252a or p75NTR inhibitor for 1 h. The cells were then treated with 30 nM of STS for 1 h or 25 μM of ETS for 2 h. DMSO was used as a vehicle control. MTT assay was utilized by the Cell Proliferation Kit (Roche). 10 μl of MTT labeling reagent was added to the well and the cells were allowed to incubate for 4 h at 37°C. 100 μl of solubilization buffer was then added to each well. The plates were finally incubated over night at 37°C and the absorption was measured at 570 nm by microplate-reader (TECAN, Männedorf, Switzerland).

Results

Staurosporine and Etoposide Induce Apoptotic Cell Death and Caspase Activation in C17.2 Cells

To determine the effect of apoptotic insults on NSC, we exposed C17.2 cells to increasing concentrations of the mycotoxin STS, which has been shown to induce apoptosis in a wide variety of cell type, and topoisomerase II inhibitor, ETS, which has been known to induce DNA-damaging agent, is over varying period of time and assessed caspase activity levels by performing immunoblot analyses of cell lysates. The blots were probed using antibodies against Caspase 3 and the best characterized proteolytic substrate of Caspase 3, such as PARP or LaminA/C. Both STS and ETS induced cleavage of procaspase 3 into the active form of Caspase 3. Essentially, no cleavage of Caspase 3 occurred in control cells. However, at concentrations from 15 to 120 nM of STS and from 6.25 to 100 μM ETS revealed Caspase 3 activation and PARP cleavage in a concentration dependent manner (Fig. 1a). In addition, cleavage of Caspase 3 and cleavage of PARP and Lamin A/C were observed with 4 h of exposure to 60 nM STS or 50 μM ETS in time dependent manner (Fig. 1b). We also quantified the percentage of cells exhibiting apoptotic morphological alteration and chromatin condensation (Fig. 1c).
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Fig. 1

Staurosporine (STS) and Etoposide (ETS) induce apoptotic cell death and caspase activation in C17.2 Cells. a Both STS and ETS induce Caspase 3 activation and PARP cleavage in C17.2 cells in a dose dependent manner (STS; 15–120 nM, ETS; 6.25–100 μM) for 6 and 8 h, respectively. Immunoblots are probed using antibodies against Caspase 3 and PARP. Tubulin is used for loading control. b Both STS (60 nM) and ETS (50 μM) induce Caspase 3 activation, PARP and Lamin A/C cleavage in C17.2 cells in a time dependent manner (0–8 h). Immunoblots are probed using antibodies against Caspase 3, PARP, and Lamin A/C. Tubulin is used for loading control. c Apoptotic morphological alteration and chromatin condensation in C17.2 cells by STS (60 nM) and ETS (50 μM) in a time dependent manner (0–10 h) are quantified using DAPI staining by percentage versus non-stimulated control. Quantification shown is mean (±S.E.M.) of triplicate of each experiments from three independent experiments. Statistical analysis by student’s t-test versus control. All data has a P-value under 0.005

NSCs Express TrkA/B and p75NTR Receptors

The expression of diverse neurotrophins by NSCs is consistent with the role of these factors in the differentiation and development of the central nervous system [40]. Recent studies have proved that C17.2 cells constitutively secret neurotrophic factors, including NGF and BDNF [40, 41]. Therefore, we attempted to test the presence of their correlative receptors in NSC. We performed RT-PCR analysis of p75NTR, TrkA or Trk B mRNA with the murine specific primers (as described in Materials and Methods) (Fig. 2a). Interestingly, the mRNA level of P75NTR receptor was robustly higher than the expression of TrkA and TrkB mRNA. However, the protein expression level of these receptors seemed to look similar by immunoblotting (Fig. 2b). PC12 cells which express endogenous p75NTR and TrkA receptors, but not the related TrkB receptor [42, 43] and A549 (derived from a human lung adenocarcinoma) cells which express TrkA, TrkB, and p75NTR [44] were used for control (Fig. 2b). Immunostaining with antibodies against p75NTR, TrkA or TrkB, we confirmed the expression of these receptors in NSC (data not shown).
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Fig. 2

NSCs express TrkA, TrkB, and p75NTR receptors. a Expression levels of TrkA, TrkB, and p75NTR are revealed by RT-PCR. Murine specific primers for TrkA, TrkB, and p75NTR are used for RT-PCR. GAPDH is used for control. b Expression levels of receptors are probed using antibodies against TrkA, TrkB, and p75NTR. Tubulin is used for loading control. Expression levels of receptors in C17.2 cells are compared with PC12 and A549 cell lines

Both NGF and BDNF Protect Apoptotic Cell Death of NSC Through Trk Receptors

Because the NSC expresses both NGF and BDNF, and because NGF and BDNF specifically binds to TrkA and TrkB receptor and also binds to p75NTR receptor, we investigated which receptor is responsible for NGF or BDNF mediated neuroprotection effect of NSC. To show the clear effect of growth factors, we choose the mild apoptotic condition such as 30 nM STS for 1 h or 25 μM ETS for 2 h that might be initiated to produce cleaved PARP. To test whether NGF and BDNF provide neuroprotection effect in STS or ETS induced apoptosis, we exposed cells to 100 ng/ml of NGF or 100 ng/ml of BDNF for 12 h before insult with 30 nM STS or 25 μM ETS, respectively and to test the respective role of the Trk receptors and p75NTR in survival promoting effect of NGF and BDNF, we conducted the experiment with pretreatment of C17.2 cells with either tyrosine kinase inhibitor, K252a or p75NTR inhibitor in the presence of NGF or BDNF. Our immunoblotting data demonstrated that NGF efficiently prevents PARP cleavage under both STS and ETS induced apoptotic cell death condition (Fig. 3a, top) as well as BDNF does (Fig. 3a, bottom). Stimulation of C17.2 cells with 100 nM K252a for 1 h abolished neuroprotection effect of both NGF and BDNF, revealing clear cleavage of PARP, whereas pretreatment of p75NTR inhibitor showed intact form of PARP in the presence of either NGF or BDNF against STS mediated cell death (Fig. 3a, right). Similar results were observed with ETS induced apoptosis (Fig. 3a, left).
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Fig. 3

Both NGF and BDNF protect apoptotic cell death of NSC through Trk receptors. a K252a, the specific inhibitor for Trk abolishes the protective effect of both NGF and BDNF, but not inhibitor for p75NTR. C17.2 cells are pretreated with 100 nM of K252 and/or p75NTR inhibitor (p75NTR Inh.) for 1 h prior to growth factor treatment. And then, STS (30 nM) or ETS (25 μM) is treated for 1 or 2 h, respectively prior to growth factor treatment for 12 h (100 ng/ml of NGF or BDNF). Immunoblot is probed using antibody against PARP. Tubulin is used for loading control. b Trk receptor mediated protective effect of NGF and BDNF is revealed by MTT assay. C17.2 cells are treated with inhibitors, growth factors, and apoptosis inducible chemicals, sequentially same as above. Quantitative analysis of cell viability is validated by MTT assay and presented by fold change compared with non-treated control. Statistical analysis by student’s t-test versus data of STS or ETS with growth factor; **: P < 0.005. Quantification shown is mean (±S.E.M.) of triplicate of each experiments from three independent experiments

After sequential drug treatment, we applied cells for MTT assay to quantify the cell viability. Pretreatment of K252a significantly blocked the protective effect of both NGF and BDNF during STS insults while the p75NTR inhibitor had no effect. Combination of K252a and p75NTR inhibitor did not increase the rate of cell death under NGF treatment condition (Fig. 3b, right). Parallel studies on the BDNF in NSC revealed similar patterns of cell viability (Fig. 3b, left). Taken together, these results suggested that both NGF and BDNF protect NSC against STS or ETS induced apoptosis through Trk receptor, but not p75NTR.

Neuroprotection by NGF and BDNF Against Neurotxic Insult Induced Apoptotsis is Mediated by PI3K and MAPK Pathways

Since phosphorylation of Trk receptors by neurotrophins was required for neuroprotection, we examined the phosphorylation of Trk receptor using phospho-TrkA/B antibody following by either NGF or BDNF exposure. Enhanced phospho-Trk immunoreactivity was observed by both NGF and BDNF treatment up to 90 min, although BDNF induced to relatively less extent of tyrosine phosphorylation (Fig. 4a). To establish the intracellular mechanism involved in NGF and BDNF mediated neuroprotection, the activation/phosphorylation of the PI3K/Akt and MAPK (MEK/Erk) pathways was studied. C17.2 cells were treated with 100 nM NGF or BDNF at different time points and phosphorylation levels of Akt and Erk1/2 were determined. Figure 4b shows that both NGF and BDNF time dependently induced the phsohpryaltion of Akt and Erk1/2 in C17.2 cells though the level of total Akt or Erk1/2 remained unchanged. However, JNK1 phsophosrylation has not been altered in any of this growth factor stimulation in NSC (Fig. 4c), suggesting its effect on Akt and Erk is specific.
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Fig. 4

NGF and BDNF promote cell survival by the activation of the PI3K/Akt and MAPK pathway. a 100 ng/ml of NGF and BDNF stimulate tyrosine phosphorylation of TrkA/B in a time dependent manner (10–90 min). Immunoblot is probed using antibody against p-TrkA/B Y490. Tubulin is used for loading control. b 100 ng/ml of NGF and BDNF stimulate the Akt and Erk1/2 in a time dependent manner (10–90 min). Immunoblots are probed using antibodies against p-Akt S473, Akt, p-Erk1/2, and Erk1/2. Tubulin is used for loading control. c Immunoblots are probed using antibody against p-JNK1 in the same condition with (B). Tubulin is used for loading control. d C17.2 cells are pretreated with inhibitors (1 μM of Wortmannine; PI3K inhibitor, 50 μM of PD98059; MAPK inhibitor, and 10 μM of GF109203X; PKC inhibitor) for 1 h prior to treat STS (30 nM, 1 h) and growth factors (100 ng/ml of NGF and BDNF, 12 h), sequentially same as Fig. 3. Immunoblot is probed using antibody against PARP. Tubulin is used for loading control. The graph represents the desitometric analysis of western blot

To further extend the role of the PI3K/Akt and MAPK pathways in neuronal survival effect of NGF and BDNF, C17.2 cells were pretreated with the PI3K inhibitor, wortmannin, MEK inhibitor, PD98059, or protein kinase C (PKC) inhibitor, GF109203X. In addition, we have also chosen the mild cleavage condition of PARP to verify the effect of inhibitor molecules. Pretreatment of Wortmannin or PD98059 dramatically declined the protective effect of both NGF and BDNF, revealing substantial cleavage of PARP, while PKC inhibitor, GF109203X had no effect under STS stimulation (Fig. 4d). The quantification data was represented the densitometric analysis of PARP cleavage in Fig. 4d. Similar data were obtained with another neurotoxic insult, ETS stimulation (data not shown). Thus these finding suggest that PI3K/Akt and MAPK pathways are responsible for preventing neuronal cell death by both NGF and BDNF.

Discussion

Death of NSC occurs during development of the nervous system, and is also believed to occur in dentate gyrus of the hippocampus [4547] and the subventricular zone in the adult brain [7]. In contrast to the considerable data available on the cellular and molecular mechanisms that regulate the stem cell differentiation, the mechanisms that regulate survival of stem cells are largely unknown. In this study, we provide evidence that NGF and BDNF protect C17.2 NSC from apoptotic cell death induced by exposure of neurotoxic agents, by acting at the level or upstream of the activation Caspase 3 (Fig. 1). The survival promoting effect of both NGF and BDNF are dependent on the activation TrkA/B receptors, which was detected by the phosphorylation of TrkA/B receptors. Blocking of TrkA/B receptor with specific pharmacological inhibitors prevented the neuroprotective effect of NGF and BDNF, whereas p75NTR inhibitor failed to inhibit the survival effect of both neurotrophins (Fig. 3). Our results also show that activation of PI3K/Akt and MEK/Erk1/2 pathways, which was detected by the phosphorylation of its mediators Erk1/2 and Akt, respectively, are required for neuroprotection by NGF and BDNF. The effect of NGF and BDNF was deduced when C17.2 cells were preincubated with specific pharmacological inhibitor of PI3K or MAPK, but not with inhibitor of PKC (Fig. 4).

Strict control of apoptosis via caspase activation by growth factor secretion in NSC presumably functions as a safely brake that would prevent apoptosis from occurring in the event of accidental mitochondrial damage. However, in situations where death is inevitable, such as during development or irreparable DNA damage, the efficient removal of this brake is important as it would allow apoptosis thereby avoiding necrosis. Indeed both STS, general kinase inhibitor, and ETS, DNA-damaging agent treatment mediates Caspase-3 dependent apoptotic cell death (Fig. 1a). However, we observed that significant decrease of the expression levels of PARP and Caspase-3 in higher concentrations of either STS or ETS treatment. It is possible that the ability of growth factor secretion in C17.2 cells [41] is influenced to prevent apoptosis and maintain protein synthesis in the low concentrations of drugs. In contrast, higher dose of STS or ETS treatment allow cells readily undergo apoptosis, possibly suppressing the natural production of growth factor or even protein synthesis in cells. Further study will be required to verify whether apoptotic induction in cells is specifically regulated by the secretion of growth factor in this cell line. In addition, accumulation of cleaved PARP under the STS treatment as early as 4 h but no change in intact PARP levels (Fig. 1b) seems to be shown fasting process of Caspase-3 activation and be implied that STS is more effective apoptotic inducer in C17.2 cells compared with ETS.

The PI3K/Akt and the MAPK pathways are two major intracellular signaling network activated by growth factors involved in cell survival [38, 48]. NGF mediated neuroprotective signaling has been shown most likely depends on PI3K/Akt rather than MEK/Erk pathway in PC12 cells, cerebellar cortex, sympathetic, sensory and motor neurons, [4956], while the mechanism by which BDNF exert its neuroprotective effect is still controversial, may depend on the insult. In the present study, we have found that the pharmacological inhibitor of both PI3K and MEK, Wortmannin and PD098059, dramatically exert Caspase 3 activity on PARP cleavage against STS or ETS insults under NGF treatment condition, and similar results were obtained with BDNF treatment. The MAPK pathway stimulated by NGF inhibits sympathetic neuronal apoptosis by cytosine arabinoside [54], suggesting the MEK/Erk pathway might have a role to protect neuron from injury or toxicity. It has also shown that PI3K and MAPK pathway contribute neuronal protection in hippocampal neurons by BDNF from glutamate toxicity [57]. It is possible that the simultaneous contribution of the PI3K and MEK/Erk to protection of NSC by NGF and BDNF from both STS and ETS evoked apoptotic death may be due to the cross talk between two pathways. Besides the cross talk between these pathways, it is also possible explanation that common mechanism acting downstream of PI3K/Akt and MAPK pathways devotes to NSC from apoptotic death. Indeed, PI3K/Akt and MAPK pathways are known to facilitate the serum response factor (SRF), which plays essential role in neuronal survival [58].

Although neurotrophic factors protect NSC from neurotoxic insult, the regulation of specific neurotrophin signaling and their effect on the survival of NSC in the developing and adult nervous system remain to be elucidated. Further work will be required to establish the specific roles of neurotrophins and their downstream signaling in determining NSC fate in vivo.

Acknowledgment

This work was supported by grants from the Korea Research Foundation (KRF-2005-204-E00015) to J.-Y. Ahn. We thank Dr. Jong sun Kang for her generous gift of C17.2 cells.

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