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The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 2006 Mar 27;172(7):1081–1091. doi: 10.1083/jcb.200508156

Novel role of neuronal Ca2+ sensor-1 as a survival factor up-regulated in injured neurons

Tomoe Y Nakamura 1,2, Andreas Jeromin 4, George Smith 5, Hideaki Kurushima 3, Hitoshi Koga 2, Yusaku Nakabeppu 3, Shigeo Wakabayashi 1, Junichi Nabekura 2,6,7
PMCID: PMC2063765  PMID: 16549499

Abstract

A molecular basis of survival from neuronal injury is essential for the development of therapeutic strategy to remedy neurodegenerative disorders. In this study, we demonstrate that an EF-hand Ca2+-binding protein neuronal Ca2+ sensor-1 (NCS-1), one of the key proteins for various neuronal functions, also acts as an important survival factor. Overexpression of NCS-1 rendered cultured neurons more tolerant to cell death caused by several kinds of stressors, whereas the dominant-negative mutant (E120Q) accelerated it. In addition, NCS-1 proteins increased upon treatment with glial cell line–derived neurotrophic factor (GDNF) and mediated GDNF survival signal in an Akt (but not MAPK)-dependent manner. Furthermore, NCS-1 is significantly up-regulated in response to axotomy-induced injury in the dorsal motor nucleus of the vagus neurons of adult rats in vivo, and adenoviral overexpression of E120Q resulted in a significant loss of surviving neurons, suggesting that NCS-1 is involved in an antiapoptotic mechanism in adult motor neurons. We propose that NCS-1 is a novel survival-promoting factor up-regulated in injured neurons that mediates the GDNF survival signal via the phosphatidylinositol 3-kinase–Akt pathway.

Introduction

Neuronal apoptosis is induced by numerous stressors and underlies many human neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. Under such apoptotic conditions, several neurotrophic factors such as glial cell line–derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) can activate the antiapoptotic process to rescue neurons from death. However, the signaling pathway leading to cell survival is not yet completely understood. GDNF was reported to exert a potent survival-promoting activity in neurons (Henderson et al., 1994; Oppenheim et al., 1995; Yan et al., 1995) and to reduce neuronal death induced by various toxic challenges both in vitro (Nicole et al., 2001) and in vivo (Wang et al., 2002; Kirik et al., 2004). Recent evidence suggests that a part of molecular mechanisms for GDNF-induced cell survival relates to an increase in intracellular Ca2+ concentration, and it subsequently activates some survival pathways such as the phosphatidylinositol 3-kinase (PI3-K)–Akt pathway (Perez-Garcia et al., 2004).

Ca2+ is the most versatile and important intracellular messenger in neurons, regulating a variety of neuronal processes such as neurotransmission and signal transductions. The various actions of Ca2+ are mediated by a large family of EF-hand Ca2+-binding proteins, which may act as Ca2+ sensors or Ca2+ buffers. One of them, neuronal Ca2+ sensor-1 (NCS-1; mammalian homologue of frequenin), was originally identified in Drosophila melanogaster in a screen for neuronal hyperexcitability mutants (Mallart et al., 1991). Overexpression of NCS-1 has been shown to enhance evoked neurotransmitter release and exocytosis (Pongs et al., 1993; Olafsson et al., 1995). NCS-1 directly interacts with phosphatidylinositol 4-hydroxykinase (PI4-K; Hendricks et al., 1999; Weisz et al., 2000) and enhances neuronal secretion by modulating vesicular trafficking steps in a phosphoinositide-dependent manner (Koizumi et al., 2002). We have previously demonstrated that NCS-1 modulates the voltage-gated K+ channel Kv4 (Nakamura et al., 2001). Subsequently, certain voltage-gated Ca2+ channels have also been reported to be regulated by NCS-1 (Weiss et al., 2000; Wang et al., 2001; Tsujimoto et al., 2002). Furthermore, NCS-1 enhances the number of functional synapses (Chen et al., 2001), potentiates paired pulse facilitation (Sippy et al., 2003), and may be involved in associative learning and memory in Caenorhabditis elegans (Gomez et al., 2001). Despite the participation of NCS-1 in a wide range of biological functions, however, the role of NCS-1 in neuronal survival under pathophysiological conditions or the involvement of NCS-1 in neurotrophic factor–mediated neuroprotection are unknown.

Because we found that the expression levels of NCS-1 is significantly higher in immature brain (Nakamura et al., 2003) and a remarkable similarity exists between immature and injured neurons during the development and regeneration process, respectively (Nabekura et al., 2002b), these findings prompted us to study the expression level and the functional roles of NCS-1 in damaged neurons.

In this study, we found that NCS-1 is a survival-promoting factor, which increases the resistance of neurons to several kinds of stressors. In addition, NCS-1 is up-regulated in response to axonal injury in adult motor neurons, and this protects cells from apoptosis. Furthermore, NCS-1 mediates GDNF-induced neuroprotection via activation of Akt pathways. This is the first study demonstrating a novel role of NCS-1 on neuronal survival.

Results

The expression level of NCS-1 protein increases with neuronal injury

To examine the expression level of NCS-1 in injured neurons, we performed unilateral vagal axotomy (transaction of nerves) on adult rats. 1 d to 2 mo after the in vivo axotomy, brainstems, including the bilateral dorsal motor nucleus of the vagus (DMV) neurons, were isolated. Immunohistochemical staining and computerized image analysis of frozen sections revealed that axotomy significantly (more than threefold) increased the expression level of NCS-1 in the DMV when compared with those on the control side at 1 wk after the surgery (Fig. 1, A–C). NCS-1 immunoreactivity was mainly expressed in cell bodies of neurons, as shown using hematoxylin counterstaining to identify the nuclei (Fig. 1 B, brown staining accompanied with blue staining; depicted by arrows). The increase in NCS-1 level started at 1 d after axotomy, reached a peak at 1 wk, and gradually decreased to control levels over the next 2 mo (Fig. 1 D). We also conducted quantitative immunoblot analysis on tissue samples from DMV neurons 1 d and 1 wk after axotomy, expressing NCS-1 density relative to levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The results confirmed the immunohistochemistry experiments, with levels of NCS-1 protein in ipsilateral DMV being increased significantly (by about threefold) by 1 wk after axotomy (Fig. 1, E and F).

Figure 1.

Figure 1.

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NCS-1 is up-regulated in the DMV neurons after in vivo axotomy. The 10th cranial nerve on one side of the neck of adult (4–6-wk-old) rats was cut, and, 1 d to 2 mo later, the brainstem was excised and frozen sections were cut. The NCS-1 expression was examined by counterstaining with NCS-1 antibody (brown signal) and with hematoxylin to identify nuclei (blue signal). (A) Staining pattern 1 wk after in vivo axotomy. The amount of NCS-1 protein increased in the DMV neurons ipsilateral to axotomy (injured side) when compared with control neurons contralateral to axotomy. Positions of DMV neurons are represented by circles. (B) Magnified image of DMV neurons. Arrows indicate that some neurons have both NCS-1 and hematoxylin staining, indicating that NCS-1 is expressed in cell bodies. Bars (A), 200 μm; (B) 50 μm. (C) Summarized data obtained by computerized image analysis. The relative area stained using NCS-1 antibody on the injured side 1 wk after axotomy was normalized to that of the control side (means ± SEM [error bars]; n = 6). (D) Time course of the expression levels of NCS-1 in injured DMV neurons relative to uninjured DMV neurons. Immunohistochemical analysis of NCS-1 levels were performed in rats 1, 4, 7, 30, and 60 d after axotomy (means ± SEM; n = 6). (E) Immunoblots indicating the expression levels of NCS-1 in the tissue samples obtained from DMV regions in brainstem sections at control (C) and injured sites (I) 1 wk and 1 d after axotomy. 10 sections were used for each group. Similar amount of proteins were loaded on the gel as indicated by the similar amount of the internal control GAPDH. (F) The densities of NCS-1 bands were expressed relative to the density of GAPDH bands in each tissue sample, and the group data is summarized (means ± SEM; n = 3).

Up-regulation of NCS-1 protein was also observed using a different type of stressor. Continuous treatment of neurons with colchicine for 4 d, which disrupts tubulin polymerization and blocks axonal transport, also increased NCS-1 expression levels (1.40 ± 0.03-fold above control levels; P < 0.05; n = 4), indicating that NCS-1 is up-regulated in vivo in response to two different kinds of stressors—one being mechanical and the other being chemical injury.

Expression of NCS-1 renders PC-12 cells more tolerant to stressors

To study the physiological role of NCS-1 in damaged neurons, we next examined the effect of NCS-1 overexpression on the susceptibility of cells to several kinds of stressors. PC-12 cells stably transfected with either the NCS-1 expression vector (NCS-1/PC-12) or the vector alone (vector/PC-12) were differentiated into neuronlike cells, and the resistance to H2O2 toxicity was compared between these two groups. As shown in the immunofluorescent micrographs and immunoblot in Fig. 2 (A, B, and G), the expression level of NCS-1 was found to be significantly higher in NCS-1/PC-12 cells compared with vector-transfected cells, although these cells also had some endogenous NCS-1. Treatment with a relatively high dose (300 μM) of H2O2 for 3 d in the absence of pyruvate resulted in severe cellular damage in vector/PC-12 control cells; most cells were rounded up and detached from the substratum (Fig. 2 E). In contrast, the same treatment caused only a little damage to cells overexpressing NCS-1 (Fig. 2 F), indicating that the expression of NCS-1 rendered PC-12 cells more tolerant to H2O2 toxicity. The expression of NCS-1 reduced cell death caused by treatment with up to 1,000 μM H2O2 (Fig. 2 H). The aforementioned results were obtained from three cell lines transfected with NCS-1 and with corresponding vector-transfected control. A similar beneficial effect of NCS-1 on cell survival in response to 300 μM H2O2 was seen in two PC-12 cell lines that were not treated with NGF (not depicted).

Figure 2.

Figure 2.

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Effects of the overexpression of NCS-1 on the susceptibility of PC-12 cells to H2O2 toxicity. PC-12 cells stably transfected with NCS-1 (NCS-1/PC12) or vector alone (vector/PC-12) were differentiated into neuronlike cells by treatment with 100 ng/ml NGF and exposed to several concentrations (0–1 mM) of H2O2. (A and B) Immunofluorescent micrographs show that the expression level of NCS-1/PC12 cells is much higher than that in vector/PC12 cells. (C–F) Phase-contrast micrographs of PC-12 cells exposed to 0 (C and D) or 300 μM H2O2 for 3 d (E and F). Bar, 40 μm. (G) Representative immunoblots showing expression levels of NCS-1 in control vector and NCS-1–transfected cells. Also shown is the expression levels of the control protein GADPH obtained from immunoblots from the same cell samples. Unlike for NCS-1 levels, GADPH levels were not markedly different in control and NCS-1–transfected cells. (H) Bar graph shows the cell viability evaluated by trypan blue exclusion assay (means ± SEM [error bars]; n = 8). *, P < 0.05 versus vector/PC-12 cells.

NCS-1 promotes the long-term survival of primary cultured cortical neurons under stress and normal conditions

To further confirm the involvement of NCS-1 in neuronal survival, we overexpressed NCS-1 or its mutant E120Q in primary cultured embryonic rat cortical neurons that express endogenous NCS-1. The E120Q mutant possesses an amino acid substitution within the third EF-hand Ca2+-binding motif, which impairs Ca2+ binding (Jeromin et al., 2004) but preserves the interaction with target proteins and, thereby, exerts a dominant-negative effect by disrupting the function of endogenous NCS-1 (Weiss et al., 2000). We used an adenoviral transfer system to transiently deliver the cDNA encoding NCS-1 together with EGFP (using an internal ribosome entry site–containing vector) and its E120Q mutant form into neurons cultured for 5 d in neurobasal medium containing B27 trophic supplements. As indicated by cells with EGFP fluorescence and nuclei stained with Hoechst 33258, nearly 70% of neurons were successfully infected with each virus at 3 d after infection (Fig. 3 A). We examined the effects of overexpression of wild-type and dominant-negative NCS-1 on neuronal survival under stress caused by B27 withdrawal, which has been reported to induce neuronal apoptosis (Brewer, 1995; Cheng et al., 2003). As shown in Fig. 3 B, B27 withdrawal promoted cell death in vector-treated control neurons (left; also compare the vector groups with and without B27 in Fig. 3 D). Overexpression of NCS-1, on the other hand, significantly rescued cells from death (Fig. 3 B, middle). In contrast, the expression of E120Q resulted in more severe cell death accompanying bleb formation (Fig. 3 B, right). To quantitatively analyze the time course for the changes in cell viability, the total number of surviving cells from the same field was counted daily by phase-contrast microscopy during 9 d (see Materials and methods). The results show that high cell viability was preserved upon expression of the wild-type NCS-1, whereas cell viability was reduced after the expression of E120Q; i.e., the number of days required to reach 70% cell viability were 5, 8, and 3 d for vector, NCS-1, and E120Q groups, respectively (Fig. 3 C). The expression levels of NCS-1 in each group of neurons before and after adenovirus infection in the absence of B27 trophic supplements are shown in the immunoblot (Fig. 3 E). Essentially the same results were obtained by counting neurons with condensed nuclei using Hoechst staining (Fig. 3, F and G), thus reinforcing the finding that the expression of NCS-1 protects neurons from cell death under apoptotic conditions. Furthermore, when B27 trophic supplement was kept in the culture medium (which is a less stress condition), the dominant-negative effect of E120Q was more clearly observed when compared with the vector control group (Fig. 3 D; also see A, where some blebs were observed in the neurons infected with E120Q mutant). In other preliminary experiments (not depicted), although the time course of the loss in cell viability was variable, overexpression of NCS-1 consistently delayed the loss of cell viability when B27 supplements were omitted, and the expression of E120Q always increased the rate of cell death when B27 was present. These results suggest that endogenous NCS-1 is playing an important role in keeping the long-term survival of cultured neurons under normal conditions in addition to the protective role from stress under apoptotic conditions.

Figure 3.

Figure 3.

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Survival-promoting effect of NCS-1 in primary cultured cortical neurons. Neurons were infected with adenovirus carrying EGFP vector alone, NCS-1, and its EF-hand mutant (E120Q) together with EGFP in the same internal ribosome entry site vector in culture medium containing neurobasal medium plus B27 trophic supplements, and they were further cultured in the presence or absence of B27 supplements (B27 supplements were withdrawn 2 d after the virus infections). (A) Fluorescent micrographs show the cultured neurons treated with adenovirus for 3 d (exhibiting strong EGFP signals) followed by treatment with a DNA-binding dye Hoechst 33258 to label their nuclei (red signals, pseudo-colored). (B) Phase-contrast micrographs show the cultured neurons treated with adenovirus for 5 d in the absence of B27 trophic supplements. Bars, 40 μm. (C) Time course of cell viability for neurons infected with adenovirus in the absence of B27 trophic supplements. Living neurons were counted daily by phase-contrast microscopy and plotted as a percentage of the initial number of neurons present on day 0 (n = 4). The number of days required to reach 70% cell viability is shown by the dotted lines. (D) Summary of cell viability data obtained from neurons cultured in the absence and presence of B27 trophic supplements at 5 d after virus infection. Error bars represent SEM. *, P < 0.05 versus the vector-controlled group. (E) Expression levels of NCS-1 and GAPDH in cultured neurons infected with each adenovirus indicated in the absence of B27 trophic supplements. (F and G) Staining patterns (light blue and white signals) of nuclei with Hoechst 33258 (F) and normalized numbers of cells having condensed nuclei (G). The dark blue color was changed to light blue or white to visualize signals more clearly. Bar, 15 μm.

NCS-1 mediates GDNF-induced cell survival

A large body of evidence suggests that neuronal survival is promoted by neurotrophic factors such as BDNF and GDNF (Boyd and Gordon, 2003). Because the long-term application of GDNF has been reported to enhance the expression of NCS-1 in Xenopus laevis motor neurons (Wang et al., 2001), we attempted to clarify the role of NCS-1 as a downstream mechanism of GDNF-induced cell survival in rat cortical neurons. When primary cultured cortical neurons were treated with 10 ng/ml GDNF for 2 d after the withdrawal of B27 supplements, neuronal survival was significantly enhanced when compared with time-matched control (Fig. 4, A [top] and B). Interestingly, the expression of NCS-1 mimicked the survival-promoting effects of GDNF; i.e., NCS-1 exerted a robust survival effect even in the absence of GDNF (Fig. 4, A [middle] and B). Most strikingly, the expression of the dominant-negative NCS-1 mutant E120Q largely prevented cell survival induced by GDNF (Fig. 4, A [bottom] and B). Immunoblot analysis revealed that the application of 10 ng/ml GDNF resulted in a significant increase in the expression level of endogenous NCS-1 within 10 min, which further increased at 40 min in these neurons (Fig. 4 C). The amount of NCS-1 remained elevated through 2 d of exposure to GDNF (not depicted). These results show that the treatment of GDNF increases the expression level of NCS-1, which subsequently promotes neuronal survival, suggesting that GDNF-induced neuroprotection is at least in part mediated by NCS-1.

Figure 4.

Figure 4.

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NCS-1 mediates GDNF-induced neuronal survival. (A) Primary cultured cortical neurons infected with adenovirus carrying vector alone (top), NCS-1 (middle), or E120Q mutant (bottom) were treated for 2 d with or without 10 ng/ml GDNF under the condition where B27 trophic supplement was depleted. Bar, 40 μm. (B) Summary of cell viability data. Viable cells were counted 2 d after GDNF treatment (+GDNF) or no treatment (control) and plotted as the percentage of the initial number of neurons present at day 0 in the same visual field (mean ± SEM [error bars]; n = 4). All neurons, not just transfected cells, were included in the cell viability counts. Note that treatment with E120Q largely prevented the GDNF-induced neuronal survival effect. *, P < 0.05 versus the data without exposure to GDNF. (C) Expression levels of NCS-1 in cultured neurons treated with 10 ng/ml GNDF for the indicated times.

Activation of the PI3-K–Akt pathway is involved in NCS-1–induced neuronal survival

In neurons, GDNF has been reported to promote cell survival via activation of signaling cascades involving the PI3-K–Akt pathway (Soler et al., 1999; Takahashi, 2001). In accordance with these studies, we also observed that exposure of primary cultured cortical neurons to GDNF resulted in a large increase in phospho-Akt levels (Fig. 5 A, left). Therefore, it was of interest for us to test whether NCS-1 also activates this kinase. We examined the effect of NCS-1 expression or its dominant-negative form on Akt phosphorylation in the presence or absence of B27 trophic supplements. When B27 trophic supplements were absent, the expression of NCS-1 significantly enhanced the phosphorylation of Akt, whereas expression of the dominant-negative mutant E120Q had little effect when compared with control vector-infected neurons (Fig. 5, A [middle] and B). On the other hand, when B27 supplements were present, a relatively high level of phosphorylated Akt was observed in the vector-treated control group (Fig. 5, A [right] and B). Additional expression of exogenous NCS-1 further increased the Akt phosphorylation level, whereas expression of the dominant-negative mutant suppressed phosphorylation (Fig. 5, A [right] and B). Thus, the phosphorylation levels of Akt in each group of neurons were well correlated with their viabilities, as shown in Fig. 3 D.

Figure 5.

Figure 5.

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NCS-1 promotes neuronal survival via activation of the PI3-K–Akt pathway. (A and B) Both NCS-1 expression and GDNF treatment increase the phosphorylation of Akt kinase. Primary cultured cortical neurons were treated with 10 ng/ml GDNF for the indicated time periods or were infected with adenovirus carrying vector, NCS-1, or E120Q mutant and further incubated for 7 d in the presence or absence of B27 trophic supplements. They were then subjected to immunoblot analysis to detect protein levels of total (Akt) and phosphorylated form (p-Akt, the mixture of anti–P-Ser-473 and anti–P-Thr-308 antibodies, was used; A). The densities of phosphorylated Akt were normalized by those of total Akt levels and summarized in the bar graph (B). *, P < 0.05 versus vector control. (C and D) Both GDNF- and NCS-1–induced neuronal survival were abolished by PI3-K inhibitor but not by MEK inhibitor. Primary cultured cortical neurons were infected with adenovirus carrying NCS-1 or vector alone in medium lacking B27 trophic supplements. 3 d later, cultures were treated with 20 μM LY294002 or PD98059. For the vector-treated group, some cultures were further treated with 10 ng/ml GDNF. (C) Adenovirus-infected viable neurons treated or untreated with GDNF for 3 d. Bar, 40 μm. (D) Viable cells were counted and plotted as the percentage of the initial number of neurons present on day 0 (means ± SEM [error bars]; n = 4). *, P < 0.05 versus the data with no inhibitors. (E and F) NCS-1 activates Akt kinase by increasing the plasma membrane PtdIns(3,4)P2 and PtdIns(3,4,5)P3 levels in both CCL39 cells (E) and cultured neurons (F). CCL39 cells and primary cultured rat cortical neurons were transiently transfected with EGFP-Akt/PKB-PH together with either pCDNA3, NCS-1, or E120Q (1:3 ratios). 2 d later, cells were fixed, and immunocytochemistry was performed to detect NCS-1 proteins. Both EGFP distribution (E, top; and F, green signals) and NCS-1 expression (E, bottom; and F, red signals) were visualized using a laser confocal microscope. Laser confocal sections through the middle of representative cells in each treatment are shown. Arrows indicate the peripheral redistribution of the EGFP–PKB–PH in NCS-1–expressing cells. Bars, 10 μm.

In addition, pretreatment of cultured cortical neurons with LY294002, an inhibitor of PI3-K, completely abolished both GDNF- and NCS-1–induced neuronal survival, whereas PD98059, an inhibitor of MAPK kinase (MEK), did not (Fig. 5, C and D). These results suggest that the NCS-1–induced survival-promoting effect is mediated via the PI3-K–Akt pathway but not the MAPK pathway in cultured cortical neurons.

To further understand the upstream mechanism of the NCS-1–induced activation of Akt, we next examined the effect of overexpression of NCS-1 and E120Q on the subcellular localization of Akt/PKB in living cells. Akt/PKB is known to be translocated to the plasma membrane when it is fully activated upon phosphorylation and bound with its substrates PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (Alessi et al., 1996). We constructed the GFP-tagged pleckstrin homology (PH) domain of Akt/PKBα (EGFP-Akt/PKB-PH) and transiently cotransfected it into CCL39 cells, which express a small amount of endogenous NCS-1, together with NCS-1, E120Q, or empty vector. 2 d later, the subcellular localization of EGFP-tagged Akt/PKB-PH was assessed on a confocal microscope. Akt/PKB-PH was diffusely localized in the cytosol of vector-transfected control cells (Fig. 5 E). Interestingly, Akt/PKB-PH became localized in the peripheral region of cells when NCS-1 was coexpressed, but this peripheral localization was abolished when E120Q was coexpressed (Fig. 5 E). Qualitatively similar results were also obtained when primary cultured cortical neurons were treated with the same vectors; i.e., Akt/PKB-PH was localized in the peripheral regions of neurons when NCS-1 was overexpressed, but a more diffuse localization pattern was observed when E120Q was overexpressed (Fig. 5 F). The distribution pattern of Akt/PKB-PH in vector-transfected neurons was similar to that of NCS-1–overexpressing cells (not depicted). These results strongly demonstrate that NCS-1 increases the levels of plasma membrane PtdIns(3,4)P2 and PtdIns(3,4,5)P3 and, thus, activates Akt/PKB in living cells.

Dominant-negative NCS-1 accelerates the in vivo axotomy-induced loss of neurons

We examined the effects of the overexpression of NCS-1 and its dominant-negative mutant on the survival of these neurons to clarify the physiological role of NCS-1 in injured motor neurons in vivo. One side of vagus nerves of adult rats were axotomized as previously described (Fig. 1) and infected with adenoviral vectors encoding NCS-1, E120Q, or EGFP vector alone, and neuronal degeneration was evaluated by histological analysis. 1 wk after axotomy, nearly 30% of nerve cells were found to be EGFP positive in the injured side (Fig. 6 A). There were clear differences in the staining pattern between control and injured sides for all groups, probably because the regeneration process, such as activation of the surrounding glial cells, was ongoing on injured sides. However, the number of surviving motor neurons stained with hematoxylin were not significantly decreased at the injured side for vector-treated DMV sections (Fig. 6, B and D; examples of counted neurons are indicated by black arrows in C). This would probably be the result of natural antiapoptotic mechanisms induced by injury, which exist in mature neurons as previously reported (Benn and Woolf, 2004). Because the expression level of NCS-1 was significantly increased in response to in vivo axotomy (Fig. 1), we hypothesized that NCS-1 may be involved in this antiapoptotic mechanism. If so, blocking of endogenous NCS-1 would reduce this beneficial effect. As expected, the dominant-negative E120Q mutant resulted in a significant loss of neurons in the injured side (Fig. 6, B–D), and some TUNEL-positive nuclei were also detected only in this group (Fig. 6 E, arrows; and its magnified image in F). Considering that the infection efficiency was only ∼30% in these experiments, a large majority of neurons successfully infected with E120Q appear to have undergone apoptosis. Infection of neurons with the functional NCS-1 adenovirus only had a modest effect on neuronal survival. This probably results from both the low infection efficiency and the high levels of endogenous NCS-1 expression in axotomized neurons (Fig. 1) because the NCS-1 effects are already close to maximum. Thus, the dominant-negative mutant E120Q inhibited the survival of adult DMV neurons from axotomy-induced injury, strongly suggesting that NCS-1 is one of the important factors mediating neuronal survival after in vivo axotomy.

Figure 6.

Figure 6.

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Dominant-negative NCS-1 mutant E120Q promotes the axotomy-induced degeneration of DMV neurons. After the axotomy of vagus motor neurons were performed as described in Fig.1, adenoviral vectors carrying NCS-1, E120Q, or EGFP alone were injected from the stump of the nerve. 1 wk after the treatment, the brainstem was excised, and serial sections were cut. (A) Representative EGFP fluorescence image showing that EGFP signals were detected in some cells on the injured side. Positions of DMV neurons are indicated by circles in A, B, and E. (B) Histological evaluation of DMV neurons in adenovirus-treated animals by hematoxylin/eosin staining. (C) Magnified image of the boxed area in B for E120Q-treated DMV neurons. Only neurons (indicated by black arrows), not nuclei, of glial or endothelial cells (indicated by red arrows) were counted. (D) Summarized data obtained from B. *, P < 0.05 versus the control side of the same section. #, P < 0.05 versus the injured side of vector-treated animals (means ± SEM [error bars]; n = 6 from three animals). (E) An example of the TUNEL-staining pattern obtained from an E120Q-treated animal. (F) The magnified image of the boxed area in E. TUNEL-positive nuclei are indicated by arrows. Bar (B), 100 μm; (F) 25 μm.

Discussion

Numerous stressors, including physical or chemical injury and genetic abnormalities, lead to neuronal degeneration by programmed cell death along an apoptotic pathway. Under these conditions, some intrinsic and extrinsic factors, including neurotrophic factors, are known to activate the antiapoptotic process to rescue neurons from death. However, the signaling pathway leading to cell survival is not yet completely understood.

In this study, we identified a novel function for the Ca2+-binding protein NCS-1, which (1) promotes the long-term survival of cultured neurons via PI3-K–Akt signaling pathways; (2) mediates, at least in part, GDNF-induced neuroprotection; and (3) is up-regulated in response to axonal injury and plays an important role in the antiapoptotic mechanism in injured motor neurons.

NCS-1 is a novel survival-promoting factor in neuronal cells

We observed that the overexpression of NCS-1 rendered PC-12 cells and primary cultured cortical neurons more tolerant to several kinds of stressors, such as oxidative stress or trophic supplement withdrawal (Figs. 2 and 3), demonstrating that the expression of NCS-1 protects neurons from cell death under apoptotic conditions. In addition, overexpression of an EF-hand dominant-negative mutant E120Q significantly accelerated apoptosis when B27 trophic supplements were kept in the culture medium (Fig. 3 D), suggesting that endogenous NCS-1 is important for keeping the long-term survival of cultured neurons under normal (or less apoptotic) conditions. The latter finding also indicates that Ca2+ binding is required for NCS-1–mediated cell survival. On the basis of these findings, we propose that NCS-1 is a novel member of survival-promoting factors in cultured neurons.

NCS-1 mediates GDNF-induced cell survival via activation of the PI3-K–Akt survival pathway

We found that treatment of cultured cortical neurons with a neurotrophic factor GDNF increased the expression level of NCS-1 (Fig. 4 C) and enhanced neuronal survival (Fig. 4, A and B), which is consistent with a previous study reporting that GDNF enhanced the expression of frequenin/NCS-1 in Xenopus motor neurons (Wang et al., 2001). GDNF-induced increase in the NCS-1 level appeared to be caused by the synthesis of protein and/or mRNA but not by the prevention of NCS-1 degradation because GDNF did not raise the expression level of NCS-1 in the presence of the inhibitor of protein synthesis cycloheximide (10 μg/ml for 20 h; not depicted). In contrast to the vector-treated control neurons, GDNF did not further enhance the survival effect in neurons overexpressing NCS-1 (Fig. 4, A and B), suggesting that cell viability was already sufficiently high under this condition. Strikingly, the survival-promoting effect of GDNF was largely prevented by overexpression of the dominant-negative mutant E120Q (Fig. 4, A and B), suggesting that NCS-1 mediates the GDNF survival signal.

GDNF activates at least two intracellular pathways in neurons: one involving the PI3-K–Akt pathway and another involving the MAPK (p42 and p44, also called ERK1 and ERK2) pathway. However, PI3-K but not the MAPK pathway has been reported to be responsible for GDNF-mediated neuronal survival in motor neurons (Soler et al., 1999). In accordance with this study, we observed that exposure of primary cultured cortical neurons to GDNF resulted in a large increase in the phospho-Akt level (Fig. 5 A). In the same way, the overexpression of NCS-1 also dramatically enhanced the phosphorylation levels of Akt both in the presence and absence of B27 trophic supplements, whereas the overexpression of dominant-negative mutant E120Q did not (Fig. 5 A). In addition, the NCS-1–nduced survival-promoting effect was largely inhibited by the PI3-K inhibitor LY294002 but not the MEK inhibitor PD98059 (Fig. 5, C and D). Furthermore, NCS-1 increased the plasma membrane PtdIns(3,4)P2 and PtdIns(3,4,5)P3 levels, which indicates the activation of Akt in intact cells (Fig. 5, E and F). These results strongly suggest that NCS-1 is a novel downstream target that mediates GDNF survival signal through activation of the PI3-K–Akt pathway.

Possible mechanisms of the action of NCS-1

Several possible mechanisms may underlie the survival action of NCS-1. We observed that Akt/PKB-PH was recruited to the plasma membrane when NCS-1 was coexpressed, suggesting that NCS-1 acts upstream of the Akt pathway. NCS-1 was previously reported to activate PI4-K (Hendricks et al., 1999), which increases the level of plasma membrane PtdIns(4)P, the substrate of PI3-K, as well as PI5-K. Therefore, upon activation of these kinases, other phosphoinositides would be produced. Indeed, it has been reported that the overexpression of NCS-1 significantly increased both PtdIns(4)P and PtdIns(4,5)P2 levels in PC-12 cells (Koizumi et al., 2002). Furthermore, PtdIns(3,4)P2 and PtdIns(3,4,5)P3, the substrates of Akt/PKB, would also be produced, which, in turn, would activate the Akt pathway (Cantley, 2002). As expected, the overexpression of NCS-1 increased the plasma membrane PtdIns(3,4)P2 and PtdIns(3,4,5)P3 levels both in CCL39 cells and neuronal cells (Fig. 5, E and F), enhanced the phosphorylation of Akt (Fig. 5 A), and promoted neuronal survival (Fig. 3). Therefore, we propose that activation of such a phosphatidylinositol pathway is a mechanism for the survival action of NCS-1. In addition, we observed that in contrast to GDNF, our preliminary data show that BDNF did not increase the expression level of NCS-1 (unpublished data) despite the reported survival-promoting effect of BDNF in cultured cortical neurons (Cheng et al., 2003). Interestingly, BDNF-induced survival signaling has been reported to be mediated by CaM, another Ca2+-binding protein in cortical neurons (Cheng et al., 2003), and CaM has been reported to directly activate PI3-K (Perez-Garcia et al., 2004). Therefore, NCS-1 mediates the GDNF signal by activating PI4-K, whereas CaM mediates the BDNF signal by activating PI3-K. These two signals would lead the survival signal to the Akt pathway.

On the other hand, it is also possible that NCS-1 promotes neuronal survival by some other mechanisms in addition to activation of the Akt pathway. For example, the survival-promoting effect of NCS-1 appears to be analogous to that of the recently characterized antiapoptotic protein family called inhibitors of apoptosis, which suppress apoptosis through the direct inhibition of caspases (Liston et al., 2003). Some of these proteins, such as neuronal apoptosis inhibitory protein and X-linked inhibitors of apoptosis protein, have been reported to be essential for GDNF-mediated neuroprotective effects in injured motor neurons in vivo (Perrelet et al., 2002). Furthermore, recent evidence demonstrates that neuronal apoptosis inhibitory protein interacts with hippocalcin, another closely related Ca2+-binding protein that affects caspase-12 activity (Korhonen et al., 2005) and protects neurons against Ca2+-induced cell death (Mercer et al., 2000). Therefore, we do not exclude the possibility that like these proteins, NCS-1 also exerts a more direct effect on some caspases. We are currently investigating the possible interaction of these proteins.

As NCS-1 is known to interact with voltage-gated K+ channels (Kv4; Nakamura et al., 2001), it might also increase the resistance of neurons to excitotoxic apoptosis through the activation of K+ channels. Increased outward K+ current would prevent neurons from reaching firing threshold and, thereby, prevent cells from Ca2+ overload leading to cell death.

NCS-1 is a novel survival-promoting factor up-regulated in injured neurons

In this study, we found that the expression level of NCS-1 was significantly increased in response to axonal injuries (transection of the vagus nerve as well as treatment of nerves with colchicine) in the DMV neurons of adult rats (Fig. 1). The behavior of NCS-1 appears to be analogous to that of the recently identified protein damage-induced neuronal endopeptidase, which is expressed in response to neuronal damages induced by nerve transection and colchicine treatment in both the central and peripheral nervous systems (Kiryu-Seo et al., 2000). Because antiapoptotic mechanisms are activated in mature neurons in response to stress to protect against accidental apoptotic cell death, it has been described that peripheral axotomy in adult neurons does not result in extensive cell death (Benn and Woolf, 2004). In accordance with this, we also observed that little loss of motor neurons was evident by in vivo axotomy in vector-treated control neurons (Fig. 6, B and D). The expression of exogenous NCS-1 did not exert the further beneficial effect (Fig. 6, B and D). This marginal effect of exogenous NCS-1 (compared with the vector control group) would be the result of the increased expression level of endogenous NCS-1 in axotomized neurons, which occurs for all groups. In contrast, overexpression of dominant-negative E120Q significantly decreased the number of surviving neurons (Fig. 6, B–D) and produced TUNEL-positive apoptotic neurons at the injured side (Fig. 6, E and F), indicating that disruption of NCS-1 function increased the vulnerability of DMV neurons to axotomy.

Overexpression of NCS-1 rendered PC-12 cells resistant to H2O2 toxicity even in the absence of GDNF (Fig. 2), suggesting that NCS-1 itself is enough to promote cell survival. This is consistent with our view that NCS-1 is the downstream target for GDNF. Because growing evidence indicates that nerve injury leads to the up-regulation of multiple antiapoptotic molecules, including GDNF (Liberatore et al., 1997; Yamamoto et al., 1998; Wang et al., 2002), it is possible that neuronal damages induced by in vivo axotomy enhance the synthesis and/or secretion of GDNF, which, in turn, up-regulates NCS-1 expression and promotes neuronal survival in injured neurons. Although an underlying mechanism would be different, the up-regulation of NCS-1 has also been reported in the cortex of schizophrenic and bipolar patients, demonstrating the involvement of NCS-1 in neurological disease (Koh et al., 2003).

In conclusion, we characterized a novel function of NCS-1 mediating a GDNF-induced neuroprotective effect via activations of Akt kinase. Furthermore, we found that NCS-1 is up-regulated in response to nerve injury and plays an important role in the antiapoptotic mechanism in adult motor neurons. Our present findings would provide new and basic insights into the mechanism of neuronal regeneration.

Materials and methods

Plasmids and viral vectors

E120Q NCS-1 point mutant was generated with a conventional PCR protocol using the wild-type rat NCS-1 (GenBank/EMBL/DDBJ accession no. L27421) as a template and was sequenced to confirm the mutation.

Akt/PKBα cDNA was cloned from the human kidney cDNA library (CLONTECH Laboratories, Inc.), and NH2-terminally tagged fluorescent protein EGFP-Akt/PKB-PH was constructed incorporating a fragment of 750 bp, encoding the first 250 amino acids of PKBα (containing the PH domain) into EGFP-vector as described previously (Currie et al., 1999).

Adenovirus containing wild-type NCS-1 and the E120Q mutant inserts were generated by cotransfecting either of these plasmids and pBHG11 (Microbix Biosystems, Inc.) into HEK 293 cells. Viral DNA was isolated from the supernatant in the wells displaying the cytopathic effect. Replication-incompetent virus containing DNA inserts were plaque-purified twice and grown on HEK 293 cells to produce large amounts of adenovirus. Tissue culture supernatant containing adenovirus was concentrated by centrifugation over cesium chloride. The titers of viral stocks were 2.2 × 1010 pfu/ml for EGFP–NCS-1, 1.1 × 1010 pfu/ml for EGFP-E120Q, and 2.2 × 1010 pfu/ml for EGFP-vector.

Cell cultures

PC-12 cells stably transfected with vector alone or vector containing cDNA coding for the wild-type NCS-1 (several clones) were grown onto collagen-coated (500 μg/ml of type I; Sigma-Aldrich) culture dishes in growth medium (DME containing 10% horse serum, 5% FBS, and 400 μg/ml geneticin and gentamicin) as described previously (Koizumi et al., 2002). When cells became 80% confluent, they were switched to the differentiation medium (growth medium with half serum) supplemented with 100 ng/ml NGF-7S (Invitrogen).

Primary culture of cortical neurons was performed using the cortex from Sprague-Dawley rats at embryonic day 18. In brief, cortical tissues were isolated from whole brain, minced into small pieces, and digested for 10 min at 37°C in a 20-U/ml papain solution containing 0.002% DNase I (Worthington Biochemical Corp.). After titration of the enzymatic activity, cells were mechanically dissociated by several passages through pipette tips. After centrifugation, cells were resuspended in neurobasal medium supplemented with B27 trophic factors (both from Invitrogen), whose compositions were reported previously (Brewer et al., 1993). They were then plated onto culture dishes coated with 0.1% polyethylenimine at a density of 2.5–5 × 104 cells/cm2 for cell survival assay and 105 cells/cm2 for immunoblot analysis.

Fluorescent microscopy

CCL39 cells and primary cultured rat cortical neurons were plated onto collagen-coated glass coverslips and cultivated for 1 d. They were then transiently transfected with the EGFP-Akt/PKB-PH construct together with either NCS-1, E120Q, or pCDNA3 (1:3 ratio) using LipofectAMINE 2000 (Invitrogen) and were subjected to immunocytochemistry. In brief, cells were fixed with 4% PFA, permeabilized with 0.2% Triton X-100, and blocked with 5% BSA. They were then incubated for 1 h with anti–NCS-1 antibody (1:200) followed by incubation with secondary antibodies (FITC- or rhodamine-conjugated goat anti–rabbit IgG; 1:200; Jackson ImmunoResearch Laboratories). After extensive wash with PBS, cell images were scanned on a laser confocal microscope (MRC-1024K; Bio-Rad Laboratories) or obtained with conventional epifluorescence illumination (BX50WI; Olympus) with a cooled CCD camera (CoolSNAP; Photometrics) using a 0.9-W 60× water immersion objective lens. Immunocytochemistry for PC-12 cells were also performed in the same way.

Evaluation of neuronal survival

The primary cultured neurons were infected with viruses at a multiplicity of infection of 100 pfu/cell at 5 d after plating. Under this condition, we found that nearly 70% of the neurons were infected by monitoring EGFP fluorescence (Fig. 3 A). The number of living neurons was counted within the fixed area of images taken by a digital camera (Coolpix 4500; Nikon). To count the number of cells always within the same area, a grid seal with numbering (Asahi Techno glass) was stuck on the bottom of each culture dish. The number of living neurons remaining at each day was expressed as a percentage of the initial number. Neurons showing the degenerating stage characterized by nuclear condensation, membrane blebbing, or extensive neurite fragmentation were excluded. Four different regions were selected from one dish, and six separate experiments were performed for each condition.

To identify and quantify apoptotic neurons, cells were fixed with 4% PFA and were stained with Hoechst 33258. Coverslips were mounted onto glass slides, and cells were observed under epifluorescence illumination on an inverted microscope (1X71; Olympus) using a 40× NA 1.35 oil immersion objective lens (Olympus). Cells were considered apoptotic if their nuclear chromatin was condensed or fragmented, whereas cells were considered viable if their chromatin was diffusely and evenly distributed throughout the nucleus (Fig. 3 F).

Immunoblot analysis

DMV tissue samples were obtained by scratching the DMV neurons from several frozen sections of brainstem (described in the next section) using pulled glass capillary under the light microscope. These tissue samples or cultured cells (PC-12 cells and cortical neurons) were then solubilized in SDS-PAGE sample buffer containing protease and phosphatase inhibitors and subjected to immunoblot analysis using image density software (Scion Image; Scion Corp.) as previously described (Nakamura et al., 2001). Primary antibodies used were anti–NCS-1 antibody (1:1,000), which was previously described (Jeromin et al., 1999), and publicly available antibodies: monoclonal anti-GAPDH antibody (1:1,000) obtained from Chemicon as well as antiphospho-Akt antibodies (detectable for the phosphorylation of Thr308 and Ser473; 1:1,000) and anti-Akt antibody (1:1,000; both from Cell Signaling Technology). Secondary antibodies used were HRP-conjugated anti–rabbit and anti–mouse antibodies or a combination of biotinylated anti–rabbit (or mouse) antibodies (Zymed Laboratories) and HRP-conjugated streptavidin (Zymed Laboratories).

In vivo axotomy and colchicine treatment

The method of vagus axotomy was described previously (Nabekura et al., 2002a). In brief, 4–6-wk-old Sprague-Dawley rats were deeply anesthetized with 50 mg/kg pentobarbital, and axotomy of the vagus motor neurons was performed with fine scissors at the unilateral vagus nerve at the neck. Injured neurons were confirmed by detecting the fluorescence of Di-I in the DMV, which had been placed at the proximal cut site of the nerve bundle (Nabekura et al., 2002a).

To test the effects of colchicine, an implantable polymer containing 10% (wt/vol) colchicine was made by mixing colchicine with ethylene-vinyl acetate copolymer (Elvax) followed by drying as described previously (Kakizawa et al., 2000). Solid slices (∼1 mm2) were placed around the unilateral vagal nerve to allow the continuous release of colchicine from slices. The skin incision was closed, and rats were returned to the cage after awaking from the anesthetic.

Histology

1 d to 2 mo (usually 1 wk) after receiving ipsilateral vagal axotomy, brainstems were quickly removed, and 8–10-μm-thick frozen sections were cut. Immunohistochemistry was performed using the labeled biotin–streptavidin method. In brief, after fixation and blocking, the sections were incubated at 4°C overnight with a rabbit polyclonal antibody against NCS-1 at a dilution of 1:15,000 and were sequentially incubated with a biotinylated anti–rabbit secondary antibody and a HRP-conjugated streptavidin–biotin complex (GE Healthcare). The colored reaction product was developed with DAB solution. The sections were lightly counterstained with hematoxylin to visualize nuclei. Images were acquired using a digital camera (FX380; Olympus) equipped with an image filing software (FLVFS-LS; Flovel).

Comparison of the expression level of NCS-1 between injured and control sides were performed using computerized image analysis (Win Roof; Mitani Corp.). In brief, the DMV region from the injured side was at first selected, and the image was converted to binary images by thresholding so that only the area highly stained with anti–NCS-1 antibody could be detected. The same threshold level was used for both the control and injured DMV in each tissue section. The highly stained area was summated and represented as normalized values.

Neuronal degeneration was evaluated by counting surviving neurons as described previously (Rothstein et al., 2005) as well as by TUNEL staining using the apoptag peroxidase in situ Apoptosis Detection Kit (Chemicon). In brief, in vivo axotomy was performed as described above, and, at the same time, adenoviral vectors carrying EGFP only, EGFP plus NCS-1, or E120Q (109 pfu each) was injected into the stump of the nerve using a 34-gauge needle. 1 wk after axotomy, paraffin-embedded serial sections (3–4 μm) were made from the brainstem. After they were deparaffinized, sections were directly stained with hematoxylin/eosin to visualize the structure of the DMV region. TUNEL staining was performed in accordance with the manufacturer's method. The sections were lightly counterstained with methyl green. Control sections were treated similarly but incubated in the absence of TdT enzyme. To confirm whether the adenoviral vectors were transferred to the DMV neurons, another set of animals were treated in the same way. 8-μm frozen sections were cut 1 wk after operation, and EGFP signals were viewed under a fluorescence microscope (1X71; Olympus). All image acquisitions were performed at room temperature, and images were subsequently processed using Adobe Photoshop (version 7) and Adobe Illustrator (version 10) software. All experiments conformed to the Guiding Principles for the Care and Use of Animals approved by the Council of the Physiological Society of Japan. All efforts were made to minimize the number of animals used and their suffering.

Statistics

Comparisons between two groups were performed using the paired or unpaired t test. Values of P < 0.05 were considered statistically significant. All summarized data are expressed as means ± SEM.

Acknowledgments

We thank Dr. Andrew Moorhouse (The University of New South Wales, Sydney, Australia) for critical reading of this manuscript. We also thank Dr. Takeharu Nishimoto and Dr. Hideo Nishitani (Kyushu University, Fukuoka, Japan) for useful discussions of this study.

This work was supported, in part, by a Grant-in-Aid for Scientific Research (17590196) and Priority Areas (13142210) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Japan Heart Foundation and by a grant from the National Institutes of Health/National Institute of Neurological Disorder and Stroke (NS38126).

Abbreviations used in this paper: BDNF, brain-derived neurotrophic factor; DMV, dorsal motor nucleus of the vagus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GDNF, glial cell line–derived neurotrophic factor; MEK, MAPK kinase; NCS-1, neuronal Ca2+ sensor-1; PH, pleckstrin homology; PI3-K, phosphatidylinositol 3-kinase; PI4-K, phosphatidylinositol 4-hydroxykinase.

References

  1. Alessi, D.R., M. Andjelkovic, B. Caudwell, P. Cron, N. Morrice, P. Cohen, and B.A. Hemmings. 1996. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 15:6541–6551. [PMC free article] [PubMed] [Google Scholar]
  2. Benn, S.C., and C.J. Woolf. 2004. Adult neuron survival strategies–slamming on the brakes. Nat. Rev. Neurosci. 5:686–700. [DOI] [PubMed] [Google Scholar]
  3. Boyd, J.G., and T. Gordon. 2003. Neurotrophic factors and their receptors in axonal regeneration and functional recovery after peripheral nerve injury. Mol. Neurobiol. 27:277–324. [DOI] [PubMed] [Google Scholar]
  4. Brewer, G.J. 1995. Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J. Neurosci. Res. 42:674–683. [DOI] [PubMed] [Google Scholar]
  5. Brewer, G.J., J.R. Torricelli, E.K. Evege, and P.J. Price. 1993. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35:567–576. [DOI] [PubMed] [Google Scholar]
  6. Cantley, L.C. 2002. The phosphoinositide 3-kinase pathway. Science. 296:1655–1657. [DOI] [PubMed] [Google Scholar]
  7. Chen, X.L., Z.G. Zhong, S. Yokoyama, C. Bark, B. Meister, P.O. Berggren, J. Roder, H. Higashida, and A. Jeromin. 2001. Overexpression of rat neuronal calcium sensor-1 in rodent NG108-15 cells enhances synapse formation and transmission. J. Physiol. 532:649–659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheng, A., S. Wang, D. Yang, R. Xiao, and M.P. Mattson. 2003. Calmodulin mediates brain-derived neurotrophic factor cell survival signaling upstream of Akt kinase in embryonic neocortical neurons. J. Biol. Chem. 278:7591–7599. [DOI] [PubMed] [Google Scholar]
  9. Currie, R.A., K.S. Walker, A. Gray, M. Deak, A. Casamayor, C.P. Downes, P. Cohen, D.R. Alessi, and J. Lucocq. 1999. Role of phosphatidylinositol 3,4,5-trisphosphate in regulating the activity and localization of 3-phosphoinositide-dependent protein kinase-1. Biochem. J. 337:575–583. [PMC free article] [PubMed] [Google Scholar]
  10. Gomez, M., E. De Castro, E. Guarin, H. Sasakura, A. Kuhara, I. Mori, T. Bartfai, C.I. Bargmann, and P. Nef. 2001. Ca2+ signaling via the neuronal calcium sensor-1 regulates associative learning and memory in C. elegans. Neuron. 30:241–248. [DOI] [PubMed] [Google Scholar]
  11. Henderson, C.E., H.S. Phillips, R.A. Pollock, A.M. Davies, C. Lemeulle, M. Armanini, L. Simmons, B. Moffet, R.A. Vandlen, L.C. Simpson, et al. 1994. GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science. 266:1062–1064. [DOI] [PubMed] [Google Scholar]
  12. Hendricks, K.B., B.Q. Wang, E.A. Schnieders, and J. Thorner. 1999. Yeast homologue of neuronal frequenin is a regulator of phosphatidylinositol-4-OH kinase. Nat. Cell Biol. 1:234–241. [DOI] [PubMed] [Google Scholar]
  13. Jeromin, A., A.J. Shayan, M. Msghina, J. Roder, and H.L. Atwood. 1999. Crustacean frequenins: molecular cloning and differential localization at neuromuscular junctions. J. Neurobiol. 41:165–175. [PubMed] [Google Scholar]
  14. Jeromin, A., D. Muralidhar, M.N. Parameswaran, J. Roder, T. Fairwell, S. Scarlata, L. Dowal, S.M. Mustafi, K.V. Chary, and Y. Sharma. 2004. N-terminal myristoylation regulates calcium-induced conformational changes in neuronal calcium sensor-1. J. Biol. Chem. 279:27158–27167. [DOI] [PubMed] [Google Scholar]
  15. Kakizawa, S., M. Yamasaki, M. Watanabe, and M. Kano. 2000. Critical period for activity-dependent synapse elimination in developing cerebellum. J. Neurosci. 20:4954–4961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kirik, D., B. Georgievska, and A. Bjorklund. 2004. Localized striatal delivery of GDNF as a treatment for Parkinson disease. Nat. Neurosci. 7:105–110. [DOI] [PubMed] [Google Scholar]
  17. Kiryu-Seo, S., M. Sasaki, H. Yokohama, S. Nakagomi, T. Hirayama, S. Aoki, K. Wada, and H. Kiyama. 2000. Damage-induced neuronal endopeptidase (DINE) is a unique metallopeptidase expressed in response to neuronal damage and activates superoxide scavengers. Proc. Natl. Acad. Sci. USA. 97:4345–4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Koh, P.O., A.S. Undie, N. Kabbani, R. Levenson, P.S. Goldman-Rakic, and M.S. Lidow. 2003. Up-regulation of neuronal calcium sensor-1 (NCS-1) in the prefrontal cortex of schizophrenic and bipolar patients. Proc. Natl. Acad. Sci. USA. 100:313–317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Koizumi, S., P. Rosa, G.B. Willars, R.A. Challiss, E. Taverna, M. Francolini, M.D. Bootman, P. Lipp, K. Inoue, J. Roder, and A. Jeromin. 2002. Mechanisms underlying the neuronal calcium sensor-1-evoked enhancement of exocytosis in PC12 cells. J. Biol. Chem. 277:30315–30324. [DOI] [PubMed] [Google Scholar]
  20. Korhonen, L., I. Hansson, J.P. Kukkonen, K. Brannvall, M. Kobayashi, K. Takamatsu, and D. Lindholm. 2005. Hippocalcin protects against caspase-12-induced and age-dependent neuronal degeneration. Mol. Cell. Neurosci. 28:85–95. [DOI] [PubMed] [Google Scholar]
  21. Liberatore, G.T., J.Y. Wong, M.J. Porritt, G.A. Donnan, and D.W. Howells. 1997. Expression of glial cell line-derived neurotrophic factor (GDNF) mRNA following mechanical injury to mouse striatum. Neuroreport. 8:3097–3101. [DOI] [PubMed] [Google Scholar]
  22. Liston, P., W.G. Fong, and R.G. Korneluk. 2003. The inhibitors of apoptosis: there is more to life than Bcl2. Oncogene. 22:8568–8580. [DOI] [PubMed] [Google Scholar]
  23. Mallart, A., D. Angaut-Petit, C. Bourret-Poulain, and A. Ferrus. 1991. Nerve terminal excitability and neuromuscular transmission in T(X;Y)V7 and Shaker mutants of Drosophila melanogaster. J. Neurogenet. 7:75–84. [DOI] [PubMed] [Google Scholar]
  24. Mercer, E.A., L. Korhonen, Y. Skoglosa, P.A. Olsson, J.P. Kukkonen, and D. Lindholm. 2000. NAIP interacts with hippocalcin and protects neurons against calcium-induced cell death through caspase-3-dependent and -independent pathways. EMBO J. 19:3597–3607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nabekura, J., T. Ueno, S. Katsurabayashi, A. Furuta, N. Akaike, and M. Okada. 2002. a. Reduced NR2A expression and prolonged decay of NMDA receptor-mediated synaptic current in rat vagal motoneurons following axotomy. J. Physiol. 539:735–741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nabekura, J., T. Ueno, A. Okabe, A. Furuta, T. Iwaki, C. Shimizu-Okabe, A. Fukuda, and N. Akaike. 2002. b. Reduction of KCC2 expression and GABAA receptor-mediated excitation after in vivo axonal injury. J. Neurosci. 22:4412–4417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nakamura, T.Y., D.J. Pountney, A. Ozaita, S. Nandi, S. Ueda, B. Rudy, and W.A. Coetzee. 2001. A role for frequenin, a Ca2+-binding protein, as a regulator of Kv4 K+-currents. Proc. Natl. Acad. Sci. USA. 98:12808–12813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nakamura, T.Y., E. Sturm, D.J. Pountney, B. Orenzoff, M. Artman, and W.A. Coetzee. 2003. Developmental expression of NCS-1 (frequenin), a regulator of Kv4 K+ channels, in mouse heart. Pediatr. Res. 53:554–557. [DOI] [PubMed] [Google Scholar]
  29. Nicole, O., C. Ali, F. Docagne, L. Plawinski, E.T. MacKenzie, D. Vivien, and A. Buisson. 2001. Neuroprotection mediated by glial cell line-derived neurotrophic factor: involvement of a reduction of NMDA-induced calcium influx by the mitogen-activated protein kinase pathway. J. Neurosci. 21:3024–3033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Olafsson, P., T. Wang, and B. Lu. 1995. Molecular cloning and functional characterization of the Xenopus Ca2+-binding protein frequenin. Proc. Natl. Acad. Sci. USA. 92:8001–8005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Oppenheim, R.W., L.J. Houenou, J.E. Johnson, L.F. Lin, L. Li, A.C. Lo, A.L. Newsome, D.M. Prevette, and S. Wang. 1995. Developing motor neurons rescued from programmed and axotomy-induced cell death by GDNF. Nature. 373:344–346. [DOI] [PubMed] [Google Scholar]
  32. Perez-Garcia, M.J., V. Cena, Y. de Pablo, M. Llovera, J.X. Comella, and R.M. Soler. 2004. Glial cell line-derived neurotrophic factor increases intracellular calcium concentration. Role of calcium/calmodulin in the activation of the phosphatidylinositol 3-kinase pathway. J. Biol. Chem. 279:6132–6142. [DOI] [PubMed] [Google Scholar]
  33. Perrelet, D., A. Ferri, P. Liston, P. Muzzin, R.G. Korneluk, and A.C. Kato. 2002. IAPs are essential for GDNF-mediated neuroprotective effects in injured motor neurons in vivo. Nat. Cell Biol. 4:175–179. [DOI] [PubMed] [Google Scholar]
  34. Pongs, O., J. Lindemeier, X.R. Zhu, T. Theil, D. Engelkamp, I. Krah-Jentgens, H.G. Lambrecht, K.W. Koch, J. Schwemer, R. Rivosecchi, et al. 1993. Frequenin–a novel calcium-binding protein that modulates synaptic efficacy in the Drosophila nervous system. Neuron. 11:15–28. [DOI] [PubMed] [Google Scholar]
  35. Rothstein, J.D., S. Patel, M.R. Regan, C. Haenggeli, Y.H. Huang, D.E. Bergles, L. Jin, M. Dykes Hoberg, S. Vidensky, D.S. Chung, et al. 2005. Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature. 433:73–77. [DOI] [PubMed] [Google Scholar]
  36. Sippy, T., A. Cruz-Martin, A. Jeromin, and F.E. Schweizer. 2003. Acute changes in short-term plasticity at synapses with elevated levels of neuronal calcium sensor-1. Nat. Neurosci. 6:1031–1038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Soler, R.M., X. Dolcet, M. Encinas, J. Egea, J.R. Bayascas, and J.X. Comella. 1999. Receptors of the glial cell line-derived neurotrophic factor family of neurotrophic factors signal cell survival through the phosphatidylinositol 3-kinase pathway in spinal cord motoneurons. J. Neurosci. 19:9160–9169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Takahashi, M. 2001. The GDNF/RET signaling pathway and human diseases. Cytokine Growth Factor Rev. 12:361–373. [DOI] [PubMed] [Google Scholar]
  39. Tsujimoto, T., A. Jeromin, N. Saitoh, J.C. Roder, and T. Takahashi. 2002. Neuronal calcium sensor 1 and activity-dependent facilitation of P/Q-type calcium currents at presynaptic nerve terminals. Science. 295:2276–2279. [DOI] [PubMed] [Google Scholar]
  40. Wang, C.Y., F. Yang, X. He, A. Chow, J. Du, J.T. Russell, and B. Lu. 2001. Ca2+ binding protein frequenin mediates GDNF-induced potentiation of Ca2+ channels and transmitter release. Neuron. 32:99–112. [DOI] [PubMed] [Google Scholar]
  41. Wang, Y., C.F. Chang, M. Morales, Y.H. Chiang, and J. Hoffer. 2002. Protective effects of glial cell line-derived neurotrophic factor in ischemic brain injury. Ann. NY Acad. Sci. 962:423–437. [DOI] [PubMed] [Google Scholar]
  42. Weiss, J.L., D.A. Archer, and R.D. Burgoyne. 2000. Neuronal Ca2+ sensor-1/frequenin functions in an autocrine pathway regulating Ca2+ channels in bovine adrenal chromaffin cells. J. Biol. Chem. 275:40082–40087. [DOI] [PubMed] [Google Scholar]
  43. Weisz, O.A., G.A. Gibson, S.M. Leung, J. Roder, and A. Jeromin. 2000. Overexpression of frequenin, a modulator of phosphatidylinositol 4-kinase, inhibits biosynthetic delivery of an apical protein in polarized madin-darby canine kidney cells. J. Biol. Chem. 275:24341–24347. [DOI] [PubMed] [Google Scholar]
  44. Yamamoto, M., N. Mitsuma, Y. Ito, N. Hattori, M. Nagamatsu, M. Li, T. Mitsuma, and G. Sobue. 1998. Expression of glial cell line-derived neurotrophic factor and GDNFR-alpha mRNAs in human peripheral neuropathies. Brain Res. 809:175–181. [DOI] [PubMed] [Google Scholar]
  45. Yan, Q., C. Matheson, and O.T. Lopez. 1995. In vivo neurotrophic effects of GDNF on neonatal and adult facial motor neurons. Nature. 373:341–344. [DOI] [PubMed] [Google Scholar]

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