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Effects of neuregulin-1β on growth-associated protein 43 expression in dorsal root ganglion neurons with excitotoxicity induced by glutamate in vitro
Neuroscience Research 76 (2013) 22–30
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Effects of neuregulin-1 on growth-associated protein 43 expression in dorsal root ganglion neurons with excitotoxicity induced by glutamate in vitro Yunfeng Li a , Hao Li b , Guixiang Liu c , Zhen Liu d,∗ a
Faculty of Clinical Medicine, Shandong University School of Medicine, Jinan 250012, China Department of Orthopaedics, Shandong University Qilu hospital, Jinan 250012, China Department of Histology and Embryology, Binzhou Medical College, Binzhou 256603, China d Department of Anatomy, Shandong University School of Medicine, Jinan 250012, China b c
a r t i c l e
i n f o
Article history: Received 7 October 2012 Received in revised form 18 February 2013 Accepted 19 February 2013 Available online 22 March 2013 Keywords: Neuregulin-1 Glutamate Growth-associated protein-43 Neuron Dorsal root ganglion
a b s t r a c t Neuregulin-1 (NRG-1) is a growth factor with potent neuroprotective capacity. Growth-associated protein 43 (GAP-43) is expressed in dorsal root ganglion (DRG) neurons and an indicator of neuronal survival in vitro. The purpose of present study is to evaluate the effects of NRG-1 on GAP-43 expression in DRG neurons with excitotoxicity induced by glutamate (Glu) in vitro. The phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated protein kinase 1/2 (ERK1/2) signaling pathways involved in these effects were also determined. Embryonic rat DRG neurons were treated with Glu in the absence or presence of NRG-1 and PI3K inhibitor LY294002 and/or ERK1/2 inhibitor PD98059. After that, GAP-43 mRNA and GAP-43 protein levels were analyzed by real time-PCR and western blot assay, respectively. GAP-43 expression in situ was determined by immunofluorescent labeling. The results showed that the decreased GAP-43 levels induced by Glu could be partially reversed by the presence of NRG-1. Inhibitors (LY294002, PD98059) either alone or in combination blocked the effects of NRG-1. These data provide new insights of the actions of NRG-1 in sensory neurons. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
1. Introduction Neuregulin-1 (NRG-1) is a growth factor with potent neuroprotective capacity (Li et al., 2007). NRG-1 is a neuron-derived trophic molecule and plays an important role in neural development, synapse formation, and synaptic plasticity by activating epidermal growth factor receptor (ErbB) receptor tyrosine kinases (Liu et al., 2007; Fenster et al., 2012). Both NRG-1 and its ErbB receptors are expressed in dorsal root ganglion (DRG) of embryonic rat and postnatal rat (Reinhard et al., 2009; Kanzaki et al., 2012). NRG1 plays a crucial role in axoglial signaling during the development of the peripheral nervous system (PNS) (Fricker et al., 2011). Both phosphatidylinositol 3-kinase (PI3K)/Akt (Croslan et al., 2008; Kim et al., 2012) and extracellular signal-regulated protein kinase (ERK) (Higa-Nakamine et al., 2012) signaling pathways are involved in the effects of NRG-1 (Calvo et al., 2011). Growth-associated protein-43 (GAP-43), an axonally localized neuronal protein, plays a major role in many aspects of neuronal function in vertebrates (Gupta et al., 2009). GAP-43 is an
∗ Corresponding author at: Department of Anatomy, Shandong University School of Medicine, 44 West Wenhua Xi Road, Jinan, Shandong Province 250012, China. Tel.: +86 135 8906 9972. E-mail addresses: [email protected], [email protected] (Z. Liu).
intracellular growth-associated protein that appears to assist neuronal path finding and branching during development and regeneration (Denny, 2006). Increases of GAP-43 are a frequently used marker of nerve regeneration or active sprouting of axons after traumatic injury in vivo (Kaneda et al., 2008; Gravel et al., 2011) and an indicator of neuronal survival in vitro (Anand et al., 2008). GAP-43 is expressed in all subpopulations of small and large DRG neurons (Hukkanen et al., 2002; Kato et al., 2003). Accumulation of neuronal protein GAP-43 in nerve endings participates in control of neurotransmitter release and signal transduction (Zakharov and Mosevitsky, 2001). GAP-43 is involved in the release or in the regulation of exocytosis of neurotransmitter glutamate (Glu) (Hens et al., 1998). Glu is the main excitatory neurotransmitter in the nervous system, including in primary afferent neurons (Brumovsky et al., 2011) and induces neuronal excitotoxicity by activating N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5methyl-4-isoazolepropionic acid (AMPA) receptors (Sanelli et al., 2007; Finn et al., 2010). NRG-1 acts as an extracellular signaling ligand in neurons, rapidly regulating currents through ionotropic Glu receptors (Schapansky et al., 2009). Mounting evidence supports the notion that NRG-1s are a family of growth factors with multiple roles in the development and function in the nervous system (Edrey et al., 2012) and exhibit potent neuroprotective properties (Woo et al., 2012). GAP-43
0168-0102/$ – see front matter © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. http://dx.doi.org/10.1016/j.neures.2013.02.012
Y. Li et al. / Neuroscience Research 76 (2013) 22–30
is a presynaptic protein implicated in axonal growth, neuronal differentiation, plasticity, regeneration and remyelination during development or following nerve injury (Chakravarthy et al., 2008; Fricker and Bennett, 2011). It has been demonstrated that NRG-1 affects plasticity at glutamatergic synapses in principal glutamatergic neurons (Fenster et al., 2012). The purpose of present study is to evaluate the effects of NRG-1 on GAP-43 expression in DRG neurons with excitotoxicity induced by Glu in vitro. We hypothesized that NRG-1 could regulate GAP-43 expression in DRG neurons with Glu-induced excitotoxicity. In the present study, primary cultured DRG neurons were used to determine the effects of NRG-1 on GAP-43 expression of DRG sensory neurons with excitotoxicity induced by Glu and the signaling pathways involved in this process were also evaluated. 2. Materials and methods 2.1. Preparation of DRG cultures All culture preparations utilized rats taken from the breeding colony of Wistar rats maintained in the Experimental Animal Center at Shandong University of China. All animals were cared for in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996. All procedures described herein were reviewed by and had prior approval by the Ethical Committee for Animal Experimentation of the Shandong University. All surgery was performed under anesthesia, and all efforts were made to minimize the suffering of these animals. One hundred and forty embryonic rats at embryonic day 15 (E15) were used for DRG culture preparations. Under aseptic conditions, about 40 dorsal root ganglia (DRGs) were removed from each embryo, placed in culture medium, and digested with 0.25% trypsin (Sigma) in D-Hanks solution at 37 ◦ C for 10 min. The suspension of DRG cells were centrifuged at 1 × 103 rpm for 5 min. The supernatants were removed and the pellets were resuspended in Glu-free neurobasal medium (Invitrogen Corporation, Grand Island, NY, USA) consisting of 2% N-2 Supplement. Dissociated DRG cells were then filtered using a 130 m filter followed by counting and cultured in 24-well clusters (Costar, Corning, NY, USA) at 37 ◦ C with 5% CO2 for 24 h and then maintained in culture medium containing cytarabine (ara-C) (5 g/mL) for another 24 h to inhibit growth of non-neuronal cells, and then cultured for an additional 24 h in the different experimental conditions before observation. DRG cells for fluorescent labeling were plated at 1 × 105 cells/well which would contain a coverslip precoated with poly-l-lysine (0.1 mg/mL) in each well. DRG cells for real time-PCR and Western blot assay were plated at a density of 5 × 105 cells/mL. 2.2. Exposure of different agents on DRG neurons DRG neurons at 48 h post-culture were exposed to Glu (2 mmol/L, Sigma, St. Louis, MO, USA), Glu (2 mmol/L) plus NRG1 (20 nmol/L, Peprotech), PI3K inhibitor LY294002 (10 mol/L, Invitrogen) 30 min before treatment with Glu (2 mmol/L) plus NRG-1 (20 nmol/L), ERK1/2 inhibitor PD98059 (10 mol/L, Cell Signaling Technology) 30 min before treatment with Glu (2 mmol/L) plus NRG-1 (20 nmol/L), LY294002 (10 mol/L) plus PD98059 (10 mol/L) 30 min before treatment with Glu (2 mmol/L) plus NRG-1 (20 nmol/L), respectively, for an additional 24 h. DRG neurons were continuously exposed to culture medium as a control. All aforementioned cultures were incubated at 37 ◦ C in a humidified 5% CO2 –air atmosphere. To test the dose-response effects of Glu on neurite outgrowth, DRG neurons at 48 h post-culture were exposed to different doses of Glu (0.1 mmol/L, 0.5 mmol/L, 2.0 mmol/L, and 4.0 mmol/L,
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respectively) for an additional 24 h. After that, the total neurite length was measured by fluorescent labeling of microtubule associated protein 2 (MAP2). 2.3. Living cell observation in different experimental conditions All cultures were observed by using an inverted phase contrast microscope at different culture age. The pictures were taken for monitoring morphological characteristics of DRG neurons with different agents exposure. 2.4. Total neurite length measurement Total neurite length with different doses of Glu treatment and in each experimental group was measured by fluorescent labeling of MAP2. The cells on coverslips were quickly rinsed once in 0.1 mol/L phosphate buffer saline (PBS) to remove medium. The cells were fixed in 4% paraformaldehyde, pH 7.4, for 20 min at 4 ◦ C. After washing in 0.1 mol/L PBS for 3 times, the cells were blocked with 2% normal goat serum in 0.6% Triton PBS to block non-specific sites and permeabilize the cells. The samples were incubated with mouse monoclonal anti-MAP2 (1:400, Abcam, Cambridge, MA, USA) overnight at 4 ◦ C. After washing in 0.1 mol/L PBS 3 times, the cells were incubated with goat anti-mouse conjugated to Cy2 (1:100, Abcam, Cambridge, MA, USA) for 45 min in darkness. After washing in 0.1 mol/L PBS, the coverslips were placed on glass slides immediately with anti-fade mounting medium (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and stored at 4 ◦ C prior to observation with a fluorescent microscope. 2.5. Real time-PCR for detection of the mRNA levels of GAP-43 After treatment with the different agents for 24 h, the mRNA levels of GAP-43 in DRG cultures in the different experimental conditions were analyzed using real time-PCR. The expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was also determined as an internal control. The total DRG cell RNA of each well of the clusters was isolated with TRIzol (TakaRa). cDNA was synthesized using a cDNA synthesis kit (Fermentas). The synthetic oligonucleotide primer sequences for GAP-43 and GAPDH were as follows: GAP-43 5 -AAG AAG GAG GGA GAT GGC TCT-3 (coding sense) and 5 -GAG GAC GGC GAG TTA TCA GTG-3 (coding antisense). GAPDH 5 -GGC ACA GTC AAG GCT GAG AAT G-3 (coding sense) and 5 -ATG GTG GTG AAG ACG CCA GTA-3 (coding antisense). Real time-PCR was performed by using SYBR Green dye (Fermantas). PCR was performed at 50 ◦ C for 2 min and 94 ◦ C for 15 min, followed by 40 cycles at 94 ◦ C for 15 s, 58 ◦ C for 30 s, and 72 ◦ C for 30 s. A comparative cycle of threshold fluorescence (Ct) method was used and the relative transcription level amount of the target gene was normalized to that of GAPDH using the 2−Ct method. The final results of the real time-PCR are expressed as a ratio of the expression of the mRNA of interest to that of the control. 2.6. Western blot assay for detection of the protein levels Expression of GAP-43, phosphorylated Akt (pAkt), and phosphorylated ERK1/2 (pERK1/2) was analyzed by Western blot assay. Fresh cultured DRG neurons in the different experimental conditions were homogenized in 10 mmol/L Tris homogenization buffer (pH 7.4) with protease inhibitors (Amersco). The samples were centrifuged at 10,000 × g for 20 min and the supernatants were collected for Western blot assay. After determining the protein concentrations of the supernatants (BCA method, standard: BSA), 50 g protein from each sample was loaded onto the 12% SDS
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Fig. 1. Phase contrast photomicrographs of cultured DRG neurons with different doses of Glu treatment. Panel A: 0.1 mmol/L; panel B: 0.5 mmol/L; panel C: 2.0 mmol/L; panel D: 4.0 mmol/L; panel E: control. Scale bar = 25 m.
gel, separated by electrophoresis and transferred to a PVDF membrane. The membranes were blocked in blocking buffer (5% nonfat milk) for 2 h at room temperature, and then were incubated with rabbit anti-GAP-43 monoclonal IgG (1:20,000, Abcam, Cambridge, MA, USA), rabbit anti-pAkt monoclonal IgG (1:500, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pERK1/2 monoclonal IgG (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Akt monoclonal IgG (1:500, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ERK1/2 monoclonal IgG (1:1000, Cell Signaling Technology, Danvers, MA, USA), or mouse anti--actin monoclonal IgG (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 ◦ C. After being washed three times for 10 min with washing solution, the membranes were incubated with goat anti-rabbit IgG-HRP (1:7000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat anti-mouse IgG-HRP (1:3000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunoreactive bands were visualized with an ECL Western blotting detection kit (Millipore Corporation) on light sensitive film. The films were scanned and the images were analyzed quantitatively by using an ImagJ 1.39u image analysis software. The levels of GAP-43 were expressed as the ratio of the protein to -actin. The levels of pAkt and pERK1/2
were expressed as the ratio of the non-phosphorylated Akt and ERK1/2, respectively. 2.7. Double fluorescent labeling of GAP-43 and MAP2 At 24 h post-treatment, DRG cultures were processed for double immunofluorescent labeling of GAP-43 and MAP2. The cells on coverslips were quickly rinsed once in 0.1 mol/L PBS to remove medium. The cells were fixed in 4% paraformaldehyde, pH 7.4, for 20 min at 4 ◦ C. After washing in 0.1 mol/L PBS for 3 times, the cells were blocked with 2% normal goat serum in 0.6% Triton PBS to block non-specific sites and permeabilize the cells. The samples were incubated with rabbit monoclonal anti-GAP-43 (1:500, Abcam, Cambridge, MA, USA) overnight at 4 ◦ C. After washing in 0.1 mol/L PBS 3 times, the samples were incubated by goat antirabbit conjugated to Cy3 (1:500, Abcam, Cambridge, MA, USA) for 45 min in darkness. After washing 3 times in 0.1 mol/L PBS, the cells were incubated with mouse monoclonal anti-MAP2 (1:400, Abcam, Cambridge, MA, USA) for 60 min in darkness. After washing 3 times in 0.1 mol/L PBS, the cells were incubated with goat anti-mouse conjugated to Cy2 (1:100, Abcam, Cambridge, MA, USA)
Fig. 2. Phase contrast photomicrographs of cultured DRG neurons in different experimental conditions. Panel A: Glu; panel B: Glu + NRG-1; panel C: LY294002 + Glu + NRG1; panel D: PD98059 + Glu + NRG-1; panel E: LY294002 + PD98059 + Glu + NRG-1; panel F: control. Scale bar = 25 m.
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Fig. 3. Neurite outgrowth with Glu treatment. Panel A: 0.1 mmol/L; panel B: 0.5 mmol/L; panel C: 2.0 mmol/L; panel D: 4.0 mmol/L; panel E: control; Scale bar = 25 m. Panel F: quantification of total neurite length with different doses of Glu treatment. Bar graphs with error bars represent mean ± SD. *P < 0.01, **P < 0.001.
for 45 min in darkness. After washing in 0.1 mol/L PBS, the coverslips were placed on glass slides immediately with anti-fade mounting medium and stored at 4 ◦ C prior to observation with a fluorescent microscope. The two primary antibodies applied to cells separately, other than at the same time, would be preferably used for getting a better background in this experiment. 2.8. Quantitative analysis of the ratio of GAP-43-expressing neurons GAP-43-imunoreactive (IR) neurons were observed under a fluorescent microscope (Olympus) with 20× objective lens. GAP-43-IR neurons in five visual fields in the central part of each coverslip were counted as the positive neurons in each sample. MAP2-IR neurons in the same visual field were also counted as the total neurons in each sample. Then the ratio of GAP-43-IR neurons could be obtained.
the data were analyzed with non-parametric test. If normality test is pass, statistical analysis was calculated with SPSS software by one-way ANOVA, followed by the Student–Newman–Keuls test for significance to compare the differences among various groups or two independent sample t-test for significance to compare the difference between two groups. Values of P < 0.05 were considered to be significant. 3. Results 3.1. Morphology of DRG neurons under inverted phase contrast microscope Shrinkage of neuronal cell bodies and retraction of neurites was observed after 24 h exposure of Glu (Fig. 1). The neurons presented an evidence of dense neurite outgrowth in Glu-treated cultures in the presence of NRG-1 (Fig. 2).
2.9. Statistical analysis 3.2. Total neurite length with different doses of Glu treatment All experiments were performed in triplicate for each condition as one experiment. Five experiments (n = 5) were finished for final analysis and reported as mean ± SD. All the data were processed for verifying normality test for Variable. If normality test is fail,
DRG neurons at 48 h post-culture were exposed to different doses of Glu and then the total neurite length was measured by fluorescent labeling of MAP2. The total neurite length in
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Fig. 4. Neurite outgrowth in different experimental conditions. Panel A: Glu; panel B: Glu + NRG-1; panel C: LY294002 + Glu + NRG-1; panel D: PD98059 + Glu + NRG-1; panel E: LY294002 + PD98059 + Glu + NRG-1; panel F: control, scale bar = 25 m. Panel G: quantification of total neurite length in different experimental conditions. Bar graphs with error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
0.1 mmol/L, 0.5 mmol/L, 2.0 mmol/L, 4.0 mmol/L Glu treated neurons and control group is 210.14 m ± 7.6 m, 197.4 m ± 6.0 m, 178.0 m ± 5.6 m, 158.9 m ± 3.2 m, and 212.7 m ± 9.0 m, respectively (Fig. 3). 3.3. Total neurite length in different experimental conditions The DRG neurons at 48 h of culture age were treated with different agents for additional 24 h and then the total neurite length was measured by fluorescent labeling of MAP2. The total neurite length in Glu, Glu + NRG-1, LY294002 + Glu + NRG-1, PD98059 + Glu + NRG-1, LY294002 + PD98059 + Glu + NRG-1 treated neurons, and control group is 175.4 m ± 7.1 m, 202.6 m ± 7.0 m, 192.2 m ± 4.4 m, 194.8 m ± 4.7 m, 186.0 m ± 5.0 m, and 217.08 m ± 7.1 m, respectively (Fig. 4). 3.4. GAP-43 mRNA expression at different conditions The DRG neurons at 48 h of culture age were treated with different agents for additional 24 h and then the levels of GAP-43 mRNA were detected by real time-PCR. GAP-43 mRNA levels in Glu,
Glu + NRG-1, LY294002 + Glu + NRG-1, PD98059 + Glu + NRG1, LY294002 + PD98059 + Glu + NRG-1 treated samples are 0.54 ± 0.06-fold, 0.80 ± 0.04-fold, 0.73 ± 0.03-fold, 0.72 ± 0.03-fold, and 0.66 ± 0.03-fold of the control, respectively. Glu treatment decreased GAP-43 mRNA of DRG neurons as compared with that in the control group. The decreased GAP-43 mRNA levels induced by Glu could be partially reversed by the presence of NRG-1. Inhibitors (LY294002, PD98059) either alone or in combination blocked the effects of NRG-1 (Fig. 5). 3.5. GAP-43 protein expression at different conditions The DRG neurons at 48 h of culture age were treated with different agents for additional 24 h and then the levels of GAP-43 protein were detected by Western blot assay. GAP-43 protein levels in Glu, Glu + NRG-1, LY294002 + Glu + NRG1, PD98059 + Glu + NRG-1, LY294002 + PD98059 + Glu + NRG-1 treated samples are 0.56 ± 0.05-fold, 0.83 ± 0.04-fold, 0.75 ± 0.03fold, 0.77 ± 0.03-fold, and 0.70 ± 0.03-fold of the control, respectively. The decreased GAP-43 protein levels induced by Glu could be partially reversed by the presence of NRG-1. Inhibitors (LY294002,
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Fig. 5. Real time-PCR analysis of GAP-43 mRNA levels in different experimental conditions. Bar graphs with error bars represent mean ± SD. *P < 0.001.
PD98059) either alone or in combination blocked the effects of NRG-1 (Fig. 6). 3.6. The expression of GAP-43 in situ in DRG neurons After treatment with different agents for 24 h, the expression of GAP-43 in situ in DRG neurons was determined by double fluorescent labeling. The percentage of GAP-43-IR neurons in Glu, Glu + NRG-1, LY294002 + Glu + NRG-1, PD98059 + Glu + NRG-1, LY294002 + PD98059 + Glu + NRG-1 treated samples, and control group is 15.06% ± 1.84%, 22.374% ± 2.38%, 19.31% ± 1.93%, 18.36% ± 1.52%, 15.91% ± 1.05%, and 25.53% ± 2.35%, respectively. Glu treatment decreased the percentage of GAP-43-IR neurons as compared with that in the control group. NRG-1 treatment increased the percentage of GAP-43-IR neurons in the presence of Glu as compared with that in Glu alone treated cultures. Inhibitors (LY294002, PD98059) either alone or in combination blocked the effects of NRG-1 (Fig. 7). 3.7. Determination of pAkt and pERK1/2 levels in DRG neurons To test the effects of NRG-1 on activation of PI3K/Akt and ERK1/2, DRG cultures were incubated with NRG-1 for 30 min. After that, the levels of pAkt and pERK1/2 were investigated by Western blot assay. The levels of pAkt and pERK1/2 in NRG-1 treated cultures are 1.56 ± 0.10-fold and 1.35 ± 0.06-fold of the control, respectively. NRG-1 treatment increased pAkt (P < 0.001) and pERK1/2 (P < 0.001) levels in DRG neurons as compared with that in control cultures (Fig. 8). 4. Discussion The aim of the present study was to approach the question of neuronal plasticity, which is implicated by the alterations of the expression of GAP-43 both in mRNA and protein levels and GAP-43 expression in situ, dependence on the presence of NRG-1 during development in vitro of DRG sensory neurons with neurotoxicity induced by Glu. The results showed that the decreased GAP-43 levels induced by Glu could be partially reversed by the presence of NRG-1. Inhibitors (LY294002, PD98059) either alone or in combination blocked the effects of NRG-1.
Fig. 6. Western blot assay of GAP-43 protein levels in different experimental conditions. Panel A: immunoblotting bands of GAP-43 protein expression in DRG neurons. Panel B: quantitative analysis of GAP-43 protein levels. Bar graphs with error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
GAP-43 plays a crucial role in growth and in branch formation of neurites during development or neurogenesis (Yanagisawa et al., 2010). Glu (2 mmol/L) exposure decreased GAP-43 levels in cultured DRG neurons, confirming the existence of severe excitotoxicity in down-regulating GAP-43 expression thus inhibiting neurite outgrowth or axon regeneration at the dose of Glu used in the present study. It has been suggested that the cultured DRG neurons exhibit pathologic changes similar to those found in injured neurons (Aoki et al., 2007). GAP-43 has been shown to be a general marker, staining virtually all regenerating DRG neurons (Gavazzi et al., 1999). However, in our present study, the ratio of GAP-43 IR neurons in control group is too low (about 25%). It might be the result of poor neuronal regeneration because there were not any potent neurotrophic factors in the culture medium in the control group. In the other experiments of our recent research, the ratio of GAP-43 IR neurons is more than 60% with different neurotrophin (NT) treatment which represents a good regeneration status of DRG neurons. The recent research has shown that GAP-43 promotes axon growth by multiple synergistic mechanisms that potentiate the intrinsic motility of the elongating processes (Foscarin et al., 2009). NMDA receptor activity has been directly associated with GAP-43 expression and axonal growth during development and in adult models of synaptic plasticity (Cantallops and Routtenberg, 1999). It has been suggested that activation of NMDA receptors suppresses GAP-43 expression and axonal outgrowth in hippocampal slice cultures (McKinney et al., 1999). In the present study, we observed that Glu treatment could decrease the protein levels of GAP-43 and its mRNA and the ratio of GAP-43-IR neurons in primary DRG
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Fig. 7. Double fluorescent labeling of GAP-43 and MAP2. Panel A (Glu treatment): A1, MAP2-IR neurons; A2, GAP-43-IR neurons; A3, overlay of A1 and A2. Panel B (Glu + NRG1): B1, MAP2-IR neurons; B2, GAP-43-IR neurons; B3, overlay of B1 and B2. Panel C (LY294002 + Glu + NRG-1): C1, MAP2-IR neurons; C2, GAP-43-IR neurons; C3, overlay of C1 and C2. Panel D (PD98059 + Glu + NRG-1): D1, MAP2-IR neurons; D2, GAP-43-IR neurons; D3, overlay of D1 and D2. Panel E (LY294002 + PD98059 + Glu + NRG-1): E1, E2, GAP-43-IR neurons; E3, overlay of E1 and E2. Panel F (control): F1, MAP2-IR neurons; GAP-43-IR neurons; F2, MAP2-IR neurons; F3, overlay of F1 and F2. Scale bar = 25 m. Panel G, The ratio of GAP-43-IR neurons in different experimental conditions. Bar graphs with error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
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In conclusion, the decrease of GAP-43 expression could be patially reversed by NRG-1 administration. The effects of NRG1 were involved in PI3K/Akt and/or ERK1/2 activation. These data provide new insights of the actions of NRG-1 in sensory neurons. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81000517), China Postdoctoral Science Foundation (No. 200904501203), and the Natural Science Foundation of Shandong Province of China (No. ZR2011HQ011). References
Fig. 8. Western blot assay of pAkt and pERK1/2 protein levels after NRG-1 treatment. Panel A: immunoblotting bands of pAkt and pERK1/2 after NRG-1 treatment. Panel B: quantitative analysis of pAkt and pERK1/2 protein levels. The levels of pAkt and pERK1/2 increased after NRG-1 treatment. Bar graphs with error bars represent mean ± SD. *P < 0.001.
cultures. This result is consistent with that activation of NMDA receptors suppresses GAP-43 expression. Administration of NRG1 could reverse the effects of GAP-43 and its mRNA decreases induced by Glu suggesting a regenerative activity promoted by NRG-1 in injured DRG neurons in vitro. The ratio of GAP-43-IR neurons in Glu-treated cultures, increased in the presence of NRG1 also suggesting a regenerative activity promoted by NRG-1 in injured DRG neurons in vitro. The up-regulation of GAP-43 as revealed in mRNA and protein levels after NRG-1 administration suggested that NRG-1 partially compensated for Glu-induced down-regulation of GAP-43 expression. Increase of GAP-43 expression indicates a role in axon sprouting as observed the dense neurite outgrowth in the presence of NRG-1 in the present study. Several recent studies, both in vitro and in vivo, provide new insights that NRG-1 promotes neurite outgrowth or improves axonal growth and recovery of sensory functions (Joung et al., 2010; Liu et al., 2011; Audisio et al., 2012). Interestingly, expression of GAP-43 and NRG receptors erbB2 and erbB4 was paralleled after nerve transection suggesting the closely relationship between GAP43 and NRG (Rueger et al., 2008). NRG-1’s cognate ErbB receptor activation is evidenced by increased phosphorylation of Akt and ERK (Luo et al., 2011). The protein kinase Akt acts as a node, playing a critical role in controlling cell survival (Aburto et al., 2012). It has been suggested that the neuroprotective effects of NRG-1 on ischemia-induced neuronal death were prevented by inhibition of the PI3K/Akt pathway both in an in vitro rat ischemia model (Croslan et al., 2008) and in an in vivo rat ischemia model (Guo et al., 2010). ERK can be activated by NRG-1 (Higa-Nakamine et al., 2012). Increase of expression of the cytoprotective protein GAP43 in the nervous system is associated the activation of the ERK pathway which is involved in promotion of neurite growth and cell survival (Fei et al., 2011). In the present study, the effects of NRG-1 on GAP-43 and its mRNA expression could be blocked by the presence of inhibitors (LY294002, PD98059) either alone or in combination.
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Effects of neuregulin-1 on growth-associated protein 43 expression in dorsal root ganglion neurons with excitotoxicity induced by glutamate in vitro Yunfeng Li a , Hao Li b , Guixiang Liu c , Zhen Liu d,∗ a
Faculty of Clinical Medicine, Shandong University School of Medicine, Jinan 250012, China Department of Orthopaedics, Shandong University Qilu hospital, Jinan 250012, China Department of Histology and Embryology, Binzhou Medical College, Binzhou 256603, China d Department of Anatomy, Shandong University School of Medicine, Jinan 250012, China b c
a r t i c l e
i n f o
Article history: Received 7 October 2012 Received in revised form 18 February 2013 Accepted 19 February 2013 Available online 22 March 2013 Keywords: Neuregulin-1 Glutamate Growth-associated protein-43 Neuron Dorsal root ganglion
a b s t r a c t Neuregulin-1 (NRG-1) is a growth factor with potent neuroprotective capacity. Growth-associated protein 43 (GAP-43) is expressed in dorsal root ganglion (DRG) neurons and an indicator of neuronal survival in vitro. The purpose of present study is to evaluate the effects of NRG-1 on GAP-43 expression in DRG neurons with excitotoxicity induced by glutamate (Glu) in vitro. The phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated protein kinase 1/2 (ERK1/2) signaling pathways involved in these effects were also determined. Embryonic rat DRG neurons were treated with Glu in the absence or presence of NRG-1 and PI3K inhibitor LY294002 and/or ERK1/2 inhibitor PD98059. After that, GAP-43 mRNA and GAP-43 protein levels were analyzed by real time-PCR and western blot assay, respectively. GAP-43 expression in situ was determined by immunofluorescent labeling. The results showed that the decreased GAP-43 levels induced by Glu could be partially reversed by the presence of NRG-1. Inhibitors (LY294002, PD98059) either alone or in combination blocked the effects of NRG-1. These data provide new insights of the actions of NRG-1 in sensory neurons. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
1. Introduction Neuregulin-1 (NRG-1) is a growth factor with potent neuroprotective capacity (Li et al., 2007). NRG-1 is a neuron-derived trophic molecule and plays an important role in neural development, synapse formation, and synaptic plasticity by activating epidermal growth factor receptor (ErbB) receptor tyrosine kinases (Liu et al., 2007; Fenster et al., 2012). Both NRG-1 and its ErbB receptors are expressed in dorsal root ganglion (DRG) of embryonic rat and postnatal rat (Reinhard et al., 2009; Kanzaki et al., 2012). NRG1 plays a crucial role in axoglial signaling during the development of the peripheral nervous system (PNS) (Fricker et al., 2011). Both phosphatidylinositol 3-kinase (PI3K)/Akt (Croslan et al., 2008; Kim et al., 2012) and extracellular signal-regulated protein kinase (ERK) (Higa-Nakamine et al., 2012) signaling pathways are involved in the effects of NRG-1 (Calvo et al., 2011). Growth-associated protein-43 (GAP-43), an axonally localized neuronal protein, plays a major role in many aspects of neuronal function in vertebrates (Gupta et al., 2009). GAP-43 is an
∗ Corresponding author at: Department of Anatomy, Shandong University School of Medicine, 44 West Wenhua Xi Road, Jinan, Shandong Province 250012, China. Tel.: +86 135 8906 9972. E-mail addresses: [email protected], [email protected] (Z. Liu).
intracellular growth-associated protein that appears to assist neuronal path finding and branching during development and regeneration (Denny, 2006). Increases of GAP-43 are a frequently used marker of nerve regeneration or active sprouting of axons after traumatic injury in vivo (Kaneda et al., 2008; Gravel et al., 2011) and an indicator of neuronal survival in vitro (Anand et al., 2008). GAP-43 is expressed in all subpopulations of small and large DRG neurons (Hukkanen et al., 2002; Kato et al., 2003). Accumulation of neuronal protein GAP-43 in nerve endings participates in control of neurotransmitter release and signal transduction (Zakharov and Mosevitsky, 2001). GAP-43 is involved in the release or in the regulation of exocytosis of neurotransmitter glutamate (Glu) (Hens et al., 1998). Glu is the main excitatory neurotransmitter in the nervous system, including in primary afferent neurons (Brumovsky et al., 2011) and induces neuronal excitotoxicity by activating N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5methyl-4-isoazolepropionic acid (AMPA) receptors (Sanelli et al., 2007; Finn et al., 2010). NRG-1 acts as an extracellular signaling ligand in neurons, rapidly regulating currents through ionotropic Glu receptors (Schapansky et al., 2009). Mounting evidence supports the notion that NRG-1s are a family of growth factors with multiple roles in the development and function in the nervous system (Edrey et al., 2012) and exhibit potent neuroprotective properties (Woo et al., 2012). GAP-43
0168-0102/$ – see front matter © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. http://dx.doi.org/10.1016/j.neures.2013.02.012
Y. Li et al. / Neuroscience Research 76 (2013) 22–30
is a presynaptic protein implicated in axonal growth, neuronal differentiation, plasticity, regeneration and remyelination during development or following nerve injury (Chakravarthy et al., 2008; Fricker and Bennett, 2011). It has been demonstrated that NRG-1 affects plasticity at glutamatergic synapses in principal glutamatergic neurons (Fenster et al., 2012). The purpose of present study is to evaluate the effects of NRG-1 on GAP-43 expression in DRG neurons with excitotoxicity induced by Glu in vitro. We hypothesized that NRG-1 could regulate GAP-43 expression in DRG neurons with Glu-induced excitotoxicity. In the present study, primary cultured DRG neurons were used to determine the effects of NRG-1 on GAP-43 expression of DRG sensory neurons with excitotoxicity induced by Glu and the signaling pathways involved in this process were also evaluated. 2. Materials and methods 2.1. Preparation of DRG cultures All culture preparations utilized rats taken from the breeding colony of Wistar rats maintained in the Experimental Animal Center at Shandong University of China. All animals were cared for in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996. All procedures described herein were reviewed by and had prior approval by the Ethical Committee for Animal Experimentation of the Shandong University. All surgery was performed under anesthesia, and all efforts were made to minimize the suffering of these animals. One hundred and forty embryonic rats at embryonic day 15 (E15) were used for DRG culture preparations. Under aseptic conditions, about 40 dorsal root ganglia (DRGs) were removed from each embryo, placed in culture medium, and digested with 0.25% trypsin (Sigma) in D-Hanks solution at 37 ◦ C for 10 min. The suspension of DRG cells were centrifuged at 1 × 103 rpm for 5 min. The supernatants were removed and the pellets were resuspended in Glu-free neurobasal medium (Invitrogen Corporation, Grand Island, NY, USA) consisting of 2% N-2 Supplement. Dissociated DRG cells were then filtered using a 130 m filter followed by counting and cultured in 24-well clusters (Costar, Corning, NY, USA) at 37 ◦ C with 5% CO2 for 24 h and then maintained in culture medium containing cytarabine (ara-C) (5 g/mL) for another 24 h to inhibit growth of non-neuronal cells, and then cultured for an additional 24 h in the different experimental conditions before observation. DRG cells for fluorescent labeling were plated at 1 × 105 cells/well which would contain a coverslip precoated with poly-l-lysine (0.1 mg/mL) in each well. DRG cells for real time-PCR and Western blot assay were plated at a density of 5 × 105 cells/mL. 2.2. Exposure of different agents on DRG neurons DRG neurons at 48 h post-culture were exposed to Glu (2 mmol/L, Sigma, St. Louis, MO, USA), Glu (2 mmol/L) plus NRG1 (20 nmol/L, Peprotech), PI3K inhibitor LY294002 (10 mol/L, Invitrogen) 30 min before treatment with Glu (2 mmol/L) plus NRG-1 (20 nmol/L), ERK1/2 inhibitor PD98059 (10 mol/L, Cell Signaling Technology) 30 min before treatment with Glu (2 mmol/L) plus NRG-1 (20 nmol/L), LY294002 (10 mol/L) plus PD98059 (10 mol/L) 30 min before treatment with Glu (2 mmol/L) plus NRG-1 (20 nmol/L), respectively, for an additional 24 h. DRG neurons were continuously exposed to culture medium as a control. All aforementioned cultures were incubated at 37 ◦ C in a humidified 5% CO2 –air atmosphere. To test the dose-response effects of Glu on neurite outgrowth, DRG neurons at 48 h post-culture were exposed to different doses of Glu (0.1 mmol/L, 0.5 mmol/L, 2.0 mmol/L, and 4.0 mmol/L,
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respectively) for an additional 24 h. After that, the total neurite length was measured by fluorescent labeling of microtubule associated protein 2 (MAP2). 2.3. Living cell observation in different experimental conditions All cultures were observed by using an inverted phase contrast microscope at different culture age. The pictures were taken for monitoring morphological characteristics of DRG neurons with different agents exposure. 2.4. Total neurite length measurement Total neurite length with different doses of Glu treatment and in each experimental group was measured by fluorescent labeling of MAP2. The cells on coverslips were quickly rinsed once in 0.1 mol/L phosphate buffer saline (PBS) to remove medium. The cells were fixed in 4% paraformaldehyde, pH 7.4, for 20 min at 4 ◦ C. After washing in 0.1 mol/L PBS for 3 times, the cells were blocked with 2% normal goat serum in 0.6% Triton PBS to block non-specific sites and permeabilize the cells. The samples were incubated with mouse monoclonal anti-MAP2 (1:400, Abcam, Cambridge, MA, USA) overnight at 4 ◦ C. After washing in 0.1 mol/L PBS 3 times, the cells were incubated with goat anti-mouse conjugated to Cy2 (1:100, Abcam, Cambridge, MA, USA) for 45 min in darkness. After washing in 0.1 mol/L PBS, the coverslips were placed on glass slides immediately with anti-fade mounting medium (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and stored at 4 ◦ C prior to observation with a fluorescent microscope. 2.5. Real time-PCR for detection of the mRNA levels of GAP-43 After treatment with the different agents for 24 h, the mRNA levels of GAP-43 in DRG cultures in the different experimental conditions were analyzed using real time-PCR. The expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was also determined as an internal control. The total DRG cell RNA of each well of the clusters was isolated with TRIzol (TakaRa). cDNA was synthesized using a cDNA synthesis kit (Fermentas). The synthetic oligonucleotide primer sequences for GAP-43 and GAPDH were as follows: GAP-43 5 -AAG AAG GAG GGA GAT GGC TCT-3 (coding sense) and 5 -GAG GAC GGC GAG TTA TCA GTG-3 (coding antisense). GAPDH 5 -GGC ACA GTC AAG GCT GAG AAT G-3 (coding sense) and 5 -ATG GTG GTG AAG ACG CCA GTA-3 (coding antisense). Real time-PCR was performed by using SYBR Green dye (Fermantas). PCR was performed at 50 ◦ C for 2 min and 94 ◦ C for 15 min, followed by 40 cycles at 94 ◦ C for 15 s, 58 ◦ C for 30 s, and 72 ◦ C for 30 s. A comparative cycle of threshold fluorescence (Ct) method was used and the relative transcription level amount of the target gene was normalized to that of GAPDH using the 2−Ct method. The final results of the real time-PCR are expressed as a ratio of the expression of the mRNA of interest to that of the control. 2.6. Western blot assay for detection of the protein levels Expression of GAP-43, phosphorylated Akt (pAkt), and phosphorylated ERK1/2 (pERK1/2) was analyzed by Western blot assay. Fresh cultured DRG neurons in the different experimental conditions were homogenized in 10 mmol/L Tris homogenization buffer (pH 7.4) with protease inhibitors (Amersco). The samples were centrifuged at 10,000 × g for 20 min and the supernatants were collected for Western blot assay. After determining the protein concentrations of the supernatants (BCA method, standard: BSA), 50 g protein from each sample was loaded onto the 12% SDS
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Fig. 1. Phase contrast photomicrographs of cultured DRG neurons with different doses of Glu treatment. Panel A: 0.1 mmol/L; panel B: 0.5 mmol/L; panel C: 2.0 mmol/L; panel D: 4.0 mmol/L; panel E: control. Scale bar = 25 m.
gel, separated by electrophoresis and transferred to a PVDF membrane. The membranes were blocked in blocking buffer (5% nonfat milk) for 2 h at room temperature, and then were incubated with rabbit anti-GAP-43 monoclonal IgG (1:20,000, Abcam, Cambridge, MA, USA), rabbit anti-pAkt monoclonal IgG (1:500, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pERK1/2 monoclonal IgG (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Akt monoclonal IgG (1:500, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ERK1/2 monoclonal IgG (1:1000, Cell Signaling Technology, Danvers, MA, USA), or mouse anti--actin monoclonal IgG (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 ◦ C. After being washed three times for 10 min with washing solution, the membranes were incubated with goat anti-rabbit IgG-HRP (1:7000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat anti-mouse IgG-HRP (1:3000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunoreactive bands were visualized with an ECL Western blotting detection kit (Millipore Corporation) on light sensitive film. The films were scanned and the images were analyzed quantitatively by using an ImagJ 1.39u image analysis software. The levels of GAP-43 were expressed as the ratio of the protein to -actin. The levels of pAkt and pERK1/2
were expressed as the ratio of the non-phosphorylated Akt and ERK1/2, respectively. 2.7. Double fluorescent labeling of GAP-43 and MAP2 At 24 h post-treatment, DRG cultures were processed for double immunofluorescent labeling of GAP-43 and MAP2. The cells on coverslips were quickly rinsed once in 0.1 mol/L PBS to remove medium. The cells were fixed in 4% paraformaldehyde, pH 7.4, for 20 min at 4 ◦ C. After washing in 0.1 mol/L PBS for 3 times, the cells were blocked with 2% normal goat serum in 0.6% Triton PBS to block non-specific sites and permeabilize the cells. The samples were incubated with rabbit monoclonal anti-GAP-43 (1:500, Abcam, Cambridge, MA, USA) overnight at 4 ◦ C. After washing in 0.1 mol/L PBS 3 times, the samples were incubated by goat antirabbit conjugated to Cy3 (1:500, Abcam, Cambridge, MA, USA) for 45 min in darkness. After washing 3 times in 0.1 mol/L PBS, the cells were incubated with mouse monoclonal anti-MAP2 (1:400, Abcam, Cambridge, MA, USA) for 60 min in darkness. After washing 3 times in 0.1 mol/L PBS, the cells were incubated with goat anti-mouse conjugated to Cy2 (1:100, Abcam, Cambridge, MA, USA)
Fig. 2. Phase contrast photomicrographs of cultured DRG neurons in different experimental conditions. Panel A: Glu; panel B: Glu + NRG-1; panel C: LY294002 + Glu + NRG1; panel D: PD98059 + Glu + NRG-1; panel E: LY294002 + PD98059 + Glu + NRG-1; panel F: control. Scale bar = 25 m.
Y. Li et al. / Neuroscience Research 76 (2013) 22–30
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Fig. 3. Neurite outgrowth with Glu treatment. Panel A: 0.1 mmol/L; panel B: 0.5 mmol/L; panel C: 2.0 mmol/L; panel D: 4.0 mmol/L; panel E: control; Scale bar = 25 m. Panel F: quantification of total neurite length with different doses of Glu treatment. Bar graphs with error bars represent mean ± SD. *P < 0.01, **P < 0.001.
for 45 min in darkness. After washing in 0.1 mol/L PBS, the coverslips were placed on glass slides immediately with anti-fade mounting medium and stored at 4 ◦ C prior to observation with a fluorescent microscope. The two primary antibodies applied to cells separately, other than at the same time, would be preferably used for getting a better background in this experiment. 2.8. Quantitative analysis of the ratio of GAP-43-expressing neurons GAP-43-imunoreactive (IR) neurons were observed under a fluorescent microscope (Olympus) with 20× objective lens. GAP-43-IR neurons in five visual fields in the central part of each coverslip were counted as the positive neurons in each sample. MAP2-IR neurons in the same visual field were also counted as the total neurons in each sample. Then the ratio of GAP-43-IR neurons could be obtained.
the data were analyzed with non-parametric test. If normality test is pass, statistical analysis was calculated with SPSS software by one-way ANOVA, followed by the Student–Newman–Keuls test for significance to compare the differences among various groups or two independent sample t-test for significance to compare the difference between two groups. Values of P < 0.05 were considered to be significant. 3. Results 3.1. Morphology of DRG neurons under inverted phase contrast microscope Shrinkage of neuronal cell bodies and retraction of neurites was observed after 24 h exposure of Glu (Fig. 1). The neurons presented an evidence of dense neurite outgrowth in Glu-treated cultures in the presence of NRG-1 (Fig. 2).
2.9. Statistical analysis 3.2. Total neurite length with different doses of Glu treatment All experiments were performed in triplicate for each condition as one experiment. Five experiments (n = 5) were finished for final analysis and reported as mean ± SD. All the data were processed for verifying normality test for Variable. If normality test is fail,
DRG neurons at 48 h post-culture were exposed to different doses of Glu and then the total neurite length was measured by fluorescent labeling of MAP2. The total neurite length in
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Fig. 4. Neurite outgrowth in different experimental conditions. Panel A: Glu; panel B: Glu + NRG-1; panel C: LY294002 + Glu + NRG-1; panel D: PD98059 + Glu + NRG-1; panel E: LY294002 + PD98059 + Glu + NRG-1; panel F: control, scale bar = 25 m. Panel G: quantification of total neurite length in different experimental conditions. Bar graphs with error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
0.1 mmol/L, 0.5 mmol/L, 2.0 mmol/L, 4.0 mmol/L Glu treated neurons and control group is 210.14 m ± 7.6 m, 197.4 m ± 6.0 m, 178.0 m ± 5.6 m, 158.9 m ± 3.2 m, and 212.7 m ± 9.0 m, respectively (Fig. 3). 3.3. Total neurite length in different experimental conditions The DRG neurons at 48 h of culture age were treated with different agents for additional 24 h and then the total neurite length was measured by fluorescent labeling of MAP2. The total neurite length in Glu, Glu + NRG-1, LY294002 + Glu + NRG-1, PD98059 + Glu + NRG-1, LY294002 + PD98059 + Glu + NRG-1 treated neurons, and control group is 175.4 m ± 7.1 m, 202.6 m ± 7.0 m, 192.2 m ± 4.4 m, 194.8 m ± 4.7 m, 186.0 m ± 5.0 m, and 217.08 m ± 7.1 m, respectively (Fig. 4). 3.4. GAP-43 mRNA expression at different conditions The DRG neurons at 48 h of culture age were treated with different agents for additional 24 h and then the levels of GAP-43 mRNA were detected by real time-PCR. GAP-43 mRNA levels in Glu,
Glu + NRG-1, LY294002 + Glu + NRG-1, PD98059 + Glu + NRG1, LY294002 + PD98059 + Glu + NRG-1 treated samples are 0.54 ± 0.06-fold, 0.80 ± 0.04-fold, 0.73 ± 0.03-fold, 0.72 ± 0.03-fold, and 0.66 ± 0.03-fold of the control, respectively. Glu treatment decreased GAP-43 mRNA of DRG neurons as compared with that in the control group. The decreased GAP-43 mRNA levels induced by Glu could be partially reversed by the presence of NRG-1. Inhibitors (LY294002, PD98059) either alone or in combination blocked the effects of NRG-1 (Fig. 5). 3.5. GAP-43 protein expression at different conditions The DRG neurons at 48 h of culture age were treated with different agents for additional 24 h and then the levels of GAP-43 protein were detected by Western blot assay. GAP-43 protein levels in Glu, Glu + NRG-1, LY294002 + Glu + NRG1, PD98059 + Glu + NRG-1, LY294002 + PD98059 + Glu + NRG-1 treated samples are 0.56 ± 0.05-fold, 0.83 ± 0.04-fold, 0.75 ± 0.03fold, 0.77 ± 0.03-fold, and 0.70 ± 0.03-fold of the control, respectively. The decreased GAP-43 protein levels induced by Glu could be partially reversed by the presence of NRG-1. Inhibitors (LY294002,
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Fig. 5. Real time-PCR analysis of GAP-43 mRNA levels in different experimental conditions. Bar graphs with error bars represent mean ± SD. *P < 0.001.
PD98059) either alone or in combination blocked the effects of NRG-1 (Fig. 6). 3.6. The expression of GAP-43 in situ in DRG neurons After treatment with different agents for 24 h, the expression of GAP-43 in situ in DRG neurons was determined by double fluorescent labeling. The percentage of GAP-43-IR neurons in Glu, Glu + NRG-1, LY294002 + Glu + NRG-1, PD98059 + Glu + NRG-1, LY294002 + PD98059 + Glu + NRG-1 treated samples, and control group is 15.06% ± 1.84%, 22.374% ± 2.38%, 19.31% ± 1.93%, 18.36% ± 1.52%, 15.91% ± 1.05%, and 25.53% ± 2.35%, respectively. Glu treatment decreased the percentage of GAP-43-IR neurons as compared with that in the control group. NRG-1 treatment increased the percentage of GAP-43-IR neurons in the presence of Glu as compared with that in Glu alone treated cultures. Inhibitors (LY294002, PD98059) either alone or in combination blocked the effects of NRG-1 (Fig. 7). 3.7. Determination of pAkt and pERK1/2 levels in DRG neurons To test the effects of NRG-1 on activation of PI3K/Akt and ERK1/2, DRG cultures were incubated with NRG-1 for 30 min. After that, the levels of pAkt and pERK1/2 were investigated by Western blot assay. The levels of pAkt and pERK1/2 in NRG-1 treated cultures are 1.56 ± 0.10-fold and 1.35 ± 0.06-fold of the control, respectively. NRG-1 treatment increased pAkt (P < 0.001) and pERK1/2 (P < 0.001) levels in DRG neurons as compared with that in control cultures (Fig. 8). 4. Discussion The aim of the present study was to approach the question of neuronal plasticity, which is implicated by the alterations of the expression of GAP-43 both in mRNA and protein levels and GAP-43 expression in situ, dependence on the presence of NRG-1 during development in vitro of DRG sensory neurons with neurotoxicity induced by Glu. The results showed that the decreased GAP-43 levels induced by Glu could be partially reversed by the presence of NRG-1. Inhibitors (LY294002, PD98059) either alone or in combination blocked the effects of NRG-1.
Fig. 6. Western blot assay of GAP-43 protein levels in different experimental conditions. Panel A: immunoblotting bands of GAP-43 protein expression in DRG neurons. Panel B: quantitative analysis of GAP-43 protein levels. Bar graphs with error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
GAP-43 plays a crucial role in growth and in branch formation of neurites during development or neurogenesis (Yanagisawa et al., 2010). Glu (2 mmol/L) exposure decreased GAP-43 levels in cultured DRG neurons, confirming the existence of severe excitotoxicity in down-regulating GAP-43 expression thus inhibiting neurite outgrowth or axon regeneration at the dose of Glu used in the present study. It has been suggested that the cultured DRG neurons exhibit pathologic changes similar to those found in injured neurons (Aoki et al., 2007). GAP-43 has been shown to be a general marker, staining virtually all regenerating DRG neurons (Gavazzi et al., 1999). However, in our present study, the ratio of GAP-43 IR neurons in control group is too low (about 25%). It might be the result of poor neuronal regeneration because there were not any potent neurotrophic factors in the culture medium in the control group. In the other experiments of our recent research, the ratio of GAP-43 IR neurons is more than 60% with different neurotrophin (NT) treatment which represents a good regeneration status of DRG neurons. The recent research has shown that GAP-43 promotes axon growth by multiple synergistic mechanisms that potentiate the intrinsic motility of the elongating processes (Foscarin et al., 2009). NMDA receptor activity has been directly associated with GAP-43 expression and axonal growth during development and in adult models of synaptic plasticity (Cantallops and Routtenberg, 1999). It has been suggested that activation of NMDA receptors suppresses GAP-43 expression and axonal outgrowth in hippocampal slice cultures (McKinney et al., 1999). In the present study, we observed that Glu treatment could decrease the protein levels of GAP-43 and its mRNA and the ratio of GAP-43-IR neurons in primary DRG
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Fig. 7. Double fluorescent labeling of GAP-43 and MAP2. Panel A (Glu treatment): A1, MAP2-IR neurons; A2, GAP-43-IR neurons; A3, overlay of A1 and A2. Panel B (Glu + NRG1): B1, MAP2-IR neurons; B2, GAP-43-IR neurons; B3, overlay of B1 and B2. Panel C (LY294002 + Glu + NRG-1): C1, MAP2-IR neurons; C2, GAP-43-IR neurons; C3, overlay of C1 and C2. Panel D (PD98059 + Glu + NRG-1): D1, MAP2-IR neurons; D2, GAP-43-IR neurons; D3, overlay of D1 and D2. Panel E (LY294002 + PD98059 + Glu + NRG-1): E1, E2, GAP-43-IR neurons; E3, overlay of E1 and E2. Panel F (control): F1, MAP2-IR neurons; GAP-43-IR neurons; F2, MAP2-IR neurons; F3, overlay of F1 and F2. Scale bar = 25 m. Panel G, The ratio of GAP-43-IR neurons in different experimental conditions. Bar graphs with error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
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In conclusion, the decrease of GAP-43 expression could be patially reversed by NRG-1 administration. The effects of NRG1 were involved in PI3K/Akt and/or ERK1/2 activation. These data provide new insights of the actions of NRG-1 in sensory neurons. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81000517), China Postdoctoral Science Foundation (No. 200904501203), and the Natural Science Foundation of Shandong Province of China (No. ZR2011HQ011). References
Fig. 8. Western blot assay of pAkt and pERK1/2 protein levels after NRG-1 treatment. Panel A: immunoblotting bands of pAkt and pERK1/2 after NRG-1 treatment. Panel B: quantitative analysis of pAkt and pERK1/2 protein levels. The levels of pAkt and pERK1/2 increased after NRG-1 treatment. Bar graphs with error bars represent mean ± SD. *P < 0.001.
cultures. This result is consistent with that activation of NMDA receptors suppresses GAP-43 expression. Administration of NRG1 could reverse the effects of GAP-43 and its mRNA decreases induced by Glu suggesting a regenerative activity promoted by NRG-1 in injured DRG neurons in vitro. The ratio of GAP-43-IR neurons in Glu-treated cultures, increased in the presence of NRG1 also suggesting a regenerative activity promoted by NRG-1 in injured DRG neurons in vitro. The up-regulation of GAP-43 as revealed in mRNA and protein levels after NRG-1 administration suggested that NRG-1 partially compensated for Glu-induced down-regulation of GAP-43 expression. Increase of GAP-43 expression indicates a role in axon sprouting as observed the dense neurite outgrowth in the presence of NRG-1 in the present study. Several recent studies, both in vitro and in vivo, provide new insights that NRG-1 promotes neurite outgrowth or improves axonal growth and recovery of sensory functions (Joung et al., 2010; Liu et al., 2011; Audisio et al., 2012). Interestingly, expression of GAP-43 and NRG receptors erbB2 and erbB4 was paralleled after nerve transection suggesting the closely relationship between GAP43 and NRG (Rueger et al., 2008). NRG-1’s cognate ErbB receptor activation is evidenced by increased phosphorylation of Akt and ERK (Luo et al., 2011). The protein kinase Akt acts as a node, playing a critical role in controlling cell survival (Aburto et al., 2012). It has been suggested that the neuroprotective effects of NRG-1 on ischemia-induced neuronal death were prevented by inhibition of the PI3K/Akt pathway both in an in vitro rat ischemia model (Croslan et al., 2008) and in an in vivo rat ischemia model (Guo et al., 2010). ERK can be activated by NRG-1 (Higa-Nakamine et al., 2012). Increase of expression of the cytoprotective protein GAP43 in the nervous system is associated the activation of the ERK pathway which is involved in promotion of neurite growth and cell survival (Fei et al., 2011). In the present study, the effects of NRG-1 on GAP-43 and its mRNA expression could be blocked by the presence of inhibitors (LY294002, PD98059) either alone or in combination.
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