Koji Shimoke

Differences in survival‐promoting effects and intracellular signaling properties of BDNF and IGF‐1 in cultured cerebral cortical neurons

Brain‐derived neurotrophic factor (BDNF) and insulin‐like growth factor‐1 (IGF‐1) act on various neurons of the CNS as neurotrophic factors promoting neuronal differentiation and survival. We examined the survival‐promoting effects of BDNF and IGF‐1 on serum deprivation‐induced death in cultured cerebral cortical neurons, and compared the intracellular signaling pathways stimulated by BDNF and IGF‐1 in the neurons. We found that the survival‐promoting effect of BDNF was much weaker than that of IGF‐1 in serum deprivation‐induced death of cultured cortical neurons. We found no differences in the levels of phosphatidylinositol 3‐kinase (PtdIns3‐K) activity or Akt (also called PKB) phosphorylation induced by BDNF and IGF‐1 in the cultured cortical neurons, although many reports suggest that PtdIns3‐K and Akt are involved in survival promotion. In addition, phosphorylation signals of mitogen‐activated protein kinase (MAPK) and cAMP responsive element‐binding protein (CREB), which have also been reported to be involved in survival promotion, were stimulated by BDNF much more potently than by IGF‐1. These results show that there may be, as yet unidentified, intracellular signaling pathways other than the PtdIns3‐K‐Akt, MAPK and CREB signaling, to regulate survival promotion. These unidentified signaling pathways may be responsible for the distinct strengths of the survival‐promoting effects of BDNF and IGF‐1.

Inhibition of phosphatidylinositol 3-kinase activity elevates c-Jun N-terminal kinase activity in apoptosis of cultured cerebellar granule neurons.

Abstract Cerebellar granule neurons maintained in medium containing 26 mM potassium or in medium (5 mM potassium) with 50 ng/ml brain-derived neurotrophic factor (BDNF) undergo an apoptotic cell death when exposed to 10 μM LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). To investigate the intracellular signaling mechanism of LY294002-induced apoptosis, the activities of Akt and c-Jun N-terminal kinase (JNK) were measured in cells in HK (26 mM potassium) medium or LK+ (5 mM potassium) medium containing BDNF, with or without 10 μM LY294002. Akt activity decreased following the addition of 10 μM LY294002. In addition, we found that LY294002 increased the JNK activity, which is known to mediate some types of cell death in cultured PNS neurons. We also observed elevated expression of c-Jun by LY294002 in HK+ or LK++BDNF. These findings demonstrated that apoptosis induced by inhibition of PI3-K activity involves suppression of the Akt activity and elevation of the JNK activity in cultured cerebellar granule neurons. Our results suggested that the PI3-K-Akt pathway suppresses the activation of JNK and c-Jun expression, and as a result prevents the neuronal cell death in cerebellar granule neurons.

Involvement of phosphatidylinositol-3 kinase in prevention of low K+-induced apoptosis of cerebellar granule neurons

Cerebellar granule neurons obtained from 9-day-old rats die in an apoptotic manner when cultured in serum-free medium containing a low concentration of potassium (5 mM). A high concentration of potassium (26 mM) in the culture medium and BDNF can effectively prevent this apoptosis. The survival effects of high potassium and BDNF were additive, and the effect of high potassium was not blocked by addition of anti-BDNF antibody. These observations indicated that these survival effects were independent. To examine which molecules are involved in the survival pathway induced by BDNF or high K+, we used wortmannin, a specific inhibitor of PI-3 kinase. Wortmannin blocked the survival effects of both BDNF and high K+ on cerebellar granule neurons. Furthermore, in vitro PI-3 kinase assay showed that treatment with BDNF or high K+ induced PI-3 kinase activity, which was diminished by addition of wortmannin. These results indicate that different survival-promoting agents, BDNF and high K+, can prevent apoptosis in cerebellar granule neurons via a common enzyme, PI-3 kinase.

Prevention of endoplasmic reticulum stress-induced cell death by brain-derived neurotrophic factor in cultured cerebral cortical neurons

Brain-derived neurotrophic factor (BDNF), one of the neurotrophic factors acting in the central nervous system (CNS), prevents ordinary types of neuronal cell death induced by various stimulants. On the other hand, an accumulation of unfolded proteins in the endoplasmic reticulum (ER) leads to ER stress and then induces ER stress-mediated cell death. The ER stress-mediated cell death is distinctive because the caspase-12 activity plays a crucial role in the progression of cell death. We previously showed that nerve growth factor (NGF) attenuated ER stress-mediated cell death in non-neuronal PC12 cells. Here, we report that BDNF suppressed the ER stress-mediated cell death in tunicamycin (Tm)-treated cerebral cortical neurons. An analysis using a specific inhibitor of phosphatidylinositol 3-kinase (PI3-K), LY294002, revealed that BDNF prevented this cell death via the PI3-K signaling pathway. We found that the number of NeuN/TUNEL-double positive cells and the activity of caspase-3 suppressed by BDNF were increased by LY294002. We also discovered that LY294002 diminished the effect of BDNF on the activation of caspase-12, indicating that BDNF prevents ER stress-mediated cell death via a PI3-K-dependent mechanism by suppressing the activation of caspase-12 in cultured CNS neurons.

Nerve growth factor attenuates 2-deoxy-D-glucose-triggered endoplasmic reticulum stress-mediated apoptosis via enhanced expression of GRP78

The glucose analog 2-deoxy-d-glucose (2DG) depletes cells of glucose. Inhibition of glycosylation caused by glucose depletion induces endoplasmic reticulum (ER) stress with subsequent apoptosis. Glucose-regulated protein 78 (GRP78) is a molecular chaperone that acts within the ER. During ER stress, GRP78 expression is induced as part of the unfolded protein response (UPR). We found that nerve growth factor (NGF) prevented 2DG-triggered ER stress-mediated apoptosis, but not the induction of GRP78 expression, in PC12 cells. Surprisingly, GRP78 expression was further up-regulated when NGF was added to 2DG-treated PC12 cells. When a specific inhibitor of phosphatidylinositol 3-kinase (PI3-K), LY294002, was added to 2DG plus NGF-treated cells, both the effects of NGF on 2DG-induced apoptosis and GRP78 expression were significantly diminished. In addition, versipelostatin (VST), a specific inhibitor of GRP78 expression, and small interfering RNA (siRNA) against GRP78 mRNA also decreased both the effects of NGF on 2DG-induced apoptosis and GRP78 expression. RT-PCR and Western blot analyses revealed that enhanced production of nuclear p50 ATF6, but not spliced XBP1, mainly contributed to the NGF-induced enhancement of GRP78 expression in 2DG-treated cells. These results suggest that the NGF-activated PI3-K/Akt signaling pathway plays a protective role against ER stress-mediated apoptosis via enhanced expression of GRP78 in PC12 cells.

Dibutyryl-cAMP up-regulates nur77 expression via histone modification during neurite outgrowth in PC12 cells

An elevated level of cyclic AMP (cAMP) within cells activates gene expression through the cAMP-PKA-CREB pathway. Among the CREB target genes, some immediate early genes exist that are responsive to cAMP including the nur77 and c-fos genes. Treatment with dibutyryl-cAMP (dbcAMP) as well as nerve growth factor (NGF) induces neurite outgrowth in PC12 cells. Here, we report that acetylation of histone H3 was gradually stimulated after treatment with dbcAMP in PC12 cells and peaked 1 h after treatment. As the result of reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) experiments, both nur77 and c-fos gene expression were found to have peak 1 h after treatment. Knock-down with siRNA against nur77 mRNA inhibited the neurite outgrowth induced by dbcAMP, whereas knock-down with siRNA against c-fos mRNA did not inhibit the dbcAMP-induced neurite outgrowth. A chromatin immunoprecipitation (ChIP) assay revealed that the nur77 gene was associated with the acetylated Lys14 of histone H3 after treatment with dbcAMP. However, the amount of c-fos gene associated with acetylated histone H3 was not changed after treatment with dbcAMP. These results suggest that the expression of nur77, which is essential for the neuronal differentiation induced by dbcAMP, is up-regulated via dbcAMP-induced acetylation of the Lys14 of histone H3 in PC12 cells.

Nicotine Suppresses Tunicamycin-Induced, But Not Thapsigargin-Induced, Expression of GRP78 during ER Stress-Mediated Apoptosis in PC12 Cells

We previously reported that nicotine protected against tunicamycin (Tm)-induced ER stress-mediated apoptosis, but not thapsigargin (Tg)-induced apoptosis in PC12 cells. In the present study, we report that the expression of glucose-regulated protein 78 (GRP78) was suppressed by nicotine in Tm-treated PC12 cells. Interestingly, the GRP78 expression was not changed by nicotine in Tg-treated cells. Moreover, nicotine reduced the activation of caspase-12 in Tm-treated cells, but not in Tg-treated cells. These results suggest that nicotine prevented Tm-induced ER stress-mediated apoptosis by attenuating an early stage of Tm-induced ER stress. It was possible that the suppression of GRP78 expression by nicotine was achieved through the suppression of the Ire1-XBP1 and/or ATF6 pathways. We observed that nicotine suppressed the Tm-induced, but not Tg-induced, splicing of XBP1 mRNA, and also suppressed the Tm-induced, but not Tg-induced, production of cleaved ATF6 in PC12 cells. These results indicate that the suppression of Ire1-XBP1 and ATF6 pathways contributes to the suppression of GRP78 expression by nicotine in Tm-treated PC12 cells, suggesting that nicotine suppresses a common step upstream of both the Ire1-XBP1 and ATF6 pathways which are required for the expression of GRP78 during Tm-induced ER stress.

NGF-induced phosphatidylinositol 3-kinase signaling pathway prevents thapsigargin-triggered ER stress-mediated apoptosis in PC12 cells

Tunicamycin, an inhibitor of the glycosylation of newly biosynthesized proteins, induces endoplasmic reticulum (ER) stress and subsequent apoptosis, and caspase family proteases are activated during the process of ER stress-mediated apoptosis. In the present study, we showed that thapsigargin (Th), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA), also induced ER stress-mediated apoptosis, and nerve growth factor (NGF) prevented the apoptosis in PC12 cells. We also found that LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI 3-K), reduced the survival of cells treated with NGF for 24h in the presence of Th. We discovered that the activities of caspase-3, -9 and -12 were increased time-dependently after the treatment with Th, and NGF suppressed the Th-triggered activation of caspase-3, -9 and -12. LY 294002 diminished the effect of NGF on the inactivation of all these caspases. These results indicate that the NGF-induced PI 3-K signaling pathway prevents Th-triggered ER stress-specific apoptosis via inhibition of caspase-mediated apoptotic signal.

Synthetic lipid products of PI3‐kinase which are added to culture medium prevent low K+‐induced apoptosis of cerebellar granule neurons via Akt kinase activation

To examine which lipid product of phosphatidylinositol 3‐kinase (PI3‐K) is essential for the survival‐promoting pathway in cultured cerebellar granule neurons, three synthetic derivatives of lipid products of PI3‐K were added to culture medium containing a low concentration (5 mM) of potassium (LK+) which induces apoptotic cell death. We found that dipalmitoylphosphatidylinositol 3,4‐bisphosphate and dipalmitoylphosphatidylinositol 3,4,5‐trisphosphate, but not dipalmitoylphosphatidylinositol 3‐monophosphate, effectively blocked the LK+‐induced apoptosis. These two synthetic phospholipids increased Akt activity but not that of PI3‐K. These findings demonstrated that specific lipid products of PI3‐K which are added to culture medium activate Akt/PKB without modulating PI3‐K itself, and as a result prevent neuronal cell death in cerebellar granule neurons.

Subtypes of neuronal nicotinic acetylcholine receptors involved in nicotine-induced phosphorylation of extracellular signal-regulated protein kinase in PC12h cells

Although many kinds of nicotinic acetylcholine receptor (nAChR) subtypes have been reported in the neuronal tissues, subtype differences in the nAChR-mediated intracellular signaling remains obscure. Using nAChR agonists and antagonists, the involvement of nAChRs in extracellular signal-regulated protein kinase (ERK) phosphorylation in PC12h cells was investigated. Cytisine and nicotine induced the phosphorylation of ERKs in a dose-dependent manner, whereas RJR-2403 had no effect. Cytisine, but not RJR-2403, also induced phosphorylation of CREB. Mecamylamine, dextromethorphan and 18-methoxycoronaridine inhibited nicotine-induced ERK phosphorylation with much higher affinity than dihydro-beta-erythroidine and alpha-conotoxin MII. These results suggest the involvement of alpha3beta4 nAChRs in ERK phosphorylation in PC12h cells.