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Neuroscience Research 76 (2013) 22–30

Contents lists available at SciVerse ScienceDirect

Neuroscience Research

jo ur nal home p age: www.elsev ier .com/ locate /neures

ffects of neuregulin-1� on growth-associated protein 43 expression in dorsaloot ganglion neurons with excitotoxicity induced by glutamate in vitro

unfeng Lia, Hao Lib, Guixiang Liuc, Zhen Liud,∗

Faculty of Clinical Medicine, Shandong University School of Medicine, Jinan 250012, ChinaDepartment of Orthopaedics, Shandong University Qilu hospital, Jinan 250012, ChinaDepartment of Histology and Embryology, Binzhou Medical College, Binzhou 256603, ChinaDepartment of Anatomy, Shandong University School of Medicine, Jinan 250012, China

r t i c l e i n f o

rticle history:eceived 7 October 2012eceived in revised form 18 February 2013ccepted 19 February 2013vailable online 22 March 2013

eywords:

a b s t r a c t

Neuregulin-1� (NRG-1�) is a growth factor with potent neuroprotective capacity. Growth-associatedprotein 43 (GAP-43) is expressed in dorsal root ganglion (DRG) neurons and an indicator of neuronalsurvival in vitro. The purpose of present study is to evaluate the effects of NRG-1� on GAP-43 expressionin 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 inthese effects were also determined. Embryonic rat DRG neurons were treated with Glu in the absence or

euregulin-1�lutamaterowth-associated protein-43euronorsal root ganglion

presence of NRG-1� and PI3K inhibitor LY294002 and/or ERK1/2 inhibitor PD98059. After that, GAP-43mRNA 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 thedecreased 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 providenew insights of the actions of NRG-1� in sensory neurons.

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. Introduction

Neuregulin-1� (NRG-1�) is a growth factor with potent neu-oprotective capacity (Li et al., 2007). NRG-1 is a neuron-derivedrophic molecule and plays an important role in neural devel-pment, synapse formation, and synaptic plasticity by activatingpidermal growth factor receptor (ErbB) receptor tyrosine kinasesLiu et al., 2007; Fenster et al., 2012). Both NRG-1 and its ErbB recep-ors are expressed in dorsal root ganglion (DRG) of embryonic ratnd postnatal rat (Reinhard et al., 2009; Kanzaki et al., 2012). NRG-

plays a crucial role in axoglial signaling during the developmentf the peripheral nervous system (PNS) (Fricker et al., 2011). Bothhosphatidylinositol 3-kinase (PI3K)/Akt (Croslan et al., 2008; Kimt al., 2012) and extracellular signal-regulated protein kinase (ERK)Higa-Nakamine et al., 2012) signaling pathways are involved in theffects of NRG-1 (Calvo et al., 2011).

Growth-associated protein-43 (GAP-43), an axonally localizedeuronal protein, plays a major role in many aspects of neu-onal function in vertebrates (Gupta et al., 2009). GAP-43 is an

∗ Corresponding author at: Department of Anatomy, Shandong University Schoolf Medicine, 44 West Wenhua Xi Road, Jinan, Shandong Province 250012, China.el.: +86 135 8906 9972.

E-mail addresses: zhen@sdu.edu.cn, liuzhen0502@yahoo.com.cn (Z. Liu).

168-0102/$ – see front matter © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Sttp://dx.doi.org/10.1016/j.neures.2013.02.012

sevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

intracellular growth-associated protein that appears to assistneuronal path finding and branching during development andregeneration (Denny, 2006). Increases of GAP-43 are a frequentlyused marker of nerve regeneration or active sprouting of axonsafter 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 largeDRG neurons (Hukkanen et al., 2002; Kato et al., 2003). Accumu-lation of neuronal protein GAP-43 in nerve endings participatesin control of neurotransmitter release and signal transduction(Zakharov and Mosevitsky, 2001). GAP-43 is involved in the releaseor in the regulation of exocytosis of neurotransmitter glutamate(Glu) (Hens et al., 1998).

Glu is the main excitatory neurotransmitter in the ner-vous system, including in primary afferent neurons (Brumovskyet al., 2011) and induces neuronal excitotoxicity by activatingN-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) receptors (Sanelli et al.,2007; Finn et al., 2010). NRG-1� acts as an extracellular signalingligand in neurons, rapidly regulating currents through ionotropicGlu receptors (Schapansky et al., 2009).

Mounting evidence supports the notion that NRG-1s are a fam-ily of growth factors with multiple roles in the development andfunction in the nervous system (Edrey et al., 2012) and exhibitpotent neuroprotective properties (Woo et al., 2012). GAP-43

ociety. All rights reserved.

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s a presynaptic protein implicated in axonal growth, neuronalifferentiation, plasticity, regeneration and remyelination duringevelopment or following nerve injury (Chakravarthy et al., 2008;ricker and Bennett, 2011). It has been demonstrated that NRG-1ffects plasticity at glutamatergic synapses in principal glutamater-ic neurons (Fenster et al., 2012). The purpose of present study is tovaluate the effects of NRG-1� on GAP-43 expression in DRG neu-ons with excitotoxicity induced by Glu in vitro. We hypothesizedhat NRG-1� could regulate GAP-43 expression in DRG neuronsith Glu-induced excitotoxicity. In the present study, primary cul-

ured DRG neurons were used to determine the effects of NRG-1�n GAP-43 expression of DRG sensory neurons with excitotoxicitynduced by Glu and the signaling pathways involved in this process

ere also evaluated.

. Materials and methods

.1. Preparation of DRG cultures

All culture preparations utilized rats taken from the breedingolony of Wistar rats maintained in the Experimental Animal Cen-er at Shandong University of China. All animals were cared for inccordance with the National Institute of Health Guide for the Carend Use of Laboratory Animals (NIH Publications No. 80-23) revised996. All procedures described herein were reviewed by and hadrior approval by the Ethical Committee for Animal Experimenta-ion of the Shandong University. All surgery was performed undernesthesia, and all efforts were made to minimize the suffering ofhese animals. One hundred and forty embryonic rats at embryonicay 15 (E15) were used for DRG culture preparations. Under asep-ic conditions, about 40 dorsal root ganglia (DRGs) were removedrom each embryo, placed in culture medium, and digested with.25% trypsin (Sigma) in D-Hanks solution at 37 ◦C for 10 min.he suspension of DRG cells were centrifuged at 1 × 103 rpm for

min. The supernatants were removed and the pellets were resus-ended in Glu-free neurobasal medium (Invitrogen Corporation,rand Island, NY, USA) consisting of 2% N-2 Supplement. Dissoci-ted DRG cells were then filtered using a 130 �m filter followedy counting and cultured in 24-well clusters (Costar, Corning, NY,SA) at 37 ◦C with 5% CO2 for 24 h and then maintained in cul-

ure medium containing cytarabine (ara-C) (5 �g/mL) for another4 h to inhibit growth of non-neuronal cells, and then culturedor an additional 24 h in the different experimental conditionsefore observation. DRG cells for fluorescent labeling were plated at

× 105 cells/well which would contain a coverslip precoated witholy-l-lysine (0.1 mg/mL) in each well. DRG cells for real time-PCRnd Western blot assay were plated at a density of 5 × 105 cells/mL.

.2. Exposure of different agents on DRG neurons

DRG neurons at 48 h post-culture were exposed to Glu2 mmol/L, Sigma, St. Louis, MO, USA), Glu (2 mmol/L) plus NRG-� (20 nmol/L, Peprotech), PI3K inhibitor LY294002 (10 �mol/L,

nvitrogen) 30 min before treatment with Glu (2 mmol/L) plusRG-1� (20 nmol/L), ERK1/2 inhibitor PD98059 (10 �mol/L,ell Signaling Technology) 30 min before treatment with Glu2 mmol/L) plus NRG-1� (20 nmol/L), LY294002 (10 �mol/L) plusD98059 (10 �mol/L) 30 min before treatment with Glu (2 mmol/L)lus NRG-1� (20 nmol/L), respectively, for an additional 24 h. DRGeurons were continuously exposed to culture medium as a control.ll aforementioned cultures were incubated at 37 ◦C in a humidified

% CO2–air atmosphere.

To test the dose-response effects of Glu on neurite outgrowth,RG neurons at 48 h post-culture were exposed to differentoses of Glu (0.1 mmol/L, 0.5 mmol/L, 2.0 mmol/L, and 4.0 mmol/L,

arch 76 (2013) 22–30 23

respectively) for an additional 24 h. After that, the total neuritelength was measured by fluorescent labeling of microtubuleassociated protein 2 (MAP2).

2.3. Living cell observation in different experimental conditions

All cultures were observed by using an inverted phase contrastmicroscope at different culture age. The pictures were taken formonitoring morphological characteristics of DRG neurons with dif-ferent agents exposure.

2.4. Total neurite length measurement

Total neurite length with different doses of Glu treatment andin each experimental group was measured by fluorescent label-ing of MAP2. The cells on coverslips were quickly rinsed once in0.1 mol/L phosphate buffer saline (PBS) to remove medium. Thecells 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 blockedwith 2% normal goat serum in 0.6% Triton PBS to block non-specificsites and permeabilize the cells. The samples were incubated withmouse 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. Afterwashing in 0.1 mol/L PBS, the coverslips were placed on glassslides immediately with anti-fade mounting medium (Santa CruzBiotechnology, Santa Cruz, CA, USA) and stored at 4 ◦C prior toobservation 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 mRNAlevels of GAP-43 in DRG cultures in the different experimental con-ditions were analyzed using real time-PCR. The expression level ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA wasalso determined as an internal control. The total DRG cell RNA ofeach well of the clusters was isolated with TRIzol (TakaRa). cDNAwas synthesized using a cDNA synthesis kit (Fermentas).

The synthetic oligonucleotide primer sequences for GAP-43 andGAPDH were as follows: GAP-43 5′-AAG AAG GAG GGA GAT GGCTCT-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′ (codingantisense).

Real time-PCR was performed by using SYBR Green dye (Fer-mantas). 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 for30 s.

A comparative cycle of threshold fluorescence (Ct) method wasused and the relative transcription level amount of the target genewas normalized to that of GAPDH using the 2−��Ct method. Thefinal results of the real time-PCR are expressed as a ratio of theexpression 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 phos-phorylated ERK1/2 (pERK1/2) was analyzed by Western blot assay.Fresh cultured DRG neurons in the different experimental condi-tions 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 werecollected for Western blot assay. After determining the proteinconcentrations of the supernatants (BCA method, standard: BSA),50 �g protein from each sample was loaded onto the 12% SDS

24 Y. Li et al. / Neuroscience Research 76 (2013) 22–30

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ig. 1. Phase contrast photomicrographs of cultured DRG neurons with different danel D: 4.0 mmol/L; panel E: control. Scale bar = 25 �m.

el, separated by electrophoresis and transferred to a PVDF mem-rane. The membranes were blocked in blocking buffer (5% nonfatilk) for 2 h at room temperature, and then were incubated with

abbit anti-GAP-43 monoclonal IgG (1:20,000, Abcam, Cambridge,A, USA), rabbit anti-pAkt monoclonal IgG (1:500, Cell Signaling

echnology, Danvers, MA, USA), rabbit anti-pERK1/2 monoclonalgG (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbitnti-Akt monoclonal IgG (1:500, Cell Signaling Technology, Dan-ers, MA, USA), rabbit anti-ERK1/2 monoclonal IgG (1:1000, Cellignaling Technology, Danvers, MA, USA), or mouse anti-�-actinonoclonal IgG (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA,SA) overnight at 4 ◦C. After being washed three times for 10 minith washing solution, the membranes were incubated with goat

nti-rabbit IgG-HRP (1:7000, Santa Cruz Biotechnology, Santa Cruz,A, USA) or goat anti-mouse IgG-HRP (1:3000, Santa Cruz Biotech-ology, Santa Cruz, CA, USA). The immunoreactive bands wereisualized with an ECL Western blotting detection kit (Millipore

orporation) on light sensitive film. The films were scanned andhe images were analyzed quantitatively by using an ImagJ 1.39umage analysis software. The levels of GAP-43 were expressed ashe ratio of the protein to �-actin. The levels of pAkt and pERK1/2

ig. 2. Phase contrast photomicrographs of cultured DRG neurons in different experimen�; panel D: PD98059 + Glu + NRG-1�; panel E: LY294002 + PD98059 + Glu + NRG-1�; pan

of Glu treatment. Panel A: 0.1 mmol/L; panel B: 0.5 mmol/L; panel C: 2.0 mmol/L;

were expressed as the ratio of the non-phosphorylated Akt andERK1/2, respectively.

2.7. Double fluorescent labeling of GAP-43 and MAP2

At 24 h post-treatment, DRG cultures were processed for dou-ble immunofluorescent labeling of GAP-43 and MAP2. The cells oncoverslips were quickly rinsed once in 0.1 mol/L PBS to removemedium. 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, thecells were blocked with 2% normal goat serum in 0.6% Triton PBSto block non-specific sites and permeabilize the cells. The sam-ples were incubated with rabbit monoclonal anti-GAP-43 (1:500,Abcam, Cambridge, MA, USA) overnight at 4 ◦C. After washing in0.1 mol/L PBS 3 times, the samples were incubated by goat anti-rabbit conjugated to Cy3 (1:500, Abcam, Cambridge, MA, USA) for45 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 wash-ing 3 times in 0.1 mol/L PBS, the cells were incubated with goatanti-mouse conjugated to Cy2 (1:100, Abcam, Cambridge, MA, USA)

tal conditions. Panel A: Glu; panel B: Glu + NRG-1�; panel C: LY294002 + Glu + NRG-el F: control. Scale bar = 25 �m.

Y. Li et al. / Neuroscience Research 76 (2013) 22–30 25

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ig. 3. Neurite outgrowth with Glu treatment. Panel A: 0.1 mmol/L; panel B: 0.5 mm: quantification of total neurite length with different doses of Glu treatment. Bar g

or 45 min in darkness. After washing in 0.1 mol/L PBS, the cov-rslips were placed on glass slides immediately with anti-fadeounting medium and stored at 4 ◦C prior to observation with a

uorescent microscope. The two primary antibodies applied to cellseparately, other than at the same time, would be preferably usedor getting a better background in this experiment.

.8. Quantitative analysis of the ratio of GAP-43-expressingeurons

GAP-43-imunoreactive (IR) neurons were observed under a flu-rescent microscope (Olympus) with 20× objective lens. GAP-43-IReurons in five visual fields in the central part of each coverslipere counted as the positive neurons in each sample. MAP2-IReurons in the same visual field were also counted as the total neu-ons in each sample. Then the ratio of GAP-43-IR neurons could bebtained.

.9. Statistical analysis

All experiments were performed in triplicate for each conditions one experiment. Five experiments (n = 5) were finished for finalnalysis and reported as mean ± SD. All the data were processedor verifying normality test for Variable. If normality test is fail,

anel C: 2.0 mmol/L; panel D: 4.0 mmol/L; panel E: control; Scale bar = 25 �m. Panel with error bars represent mean ± SD. *P < 0.01, **P < 0.001.

the data were analyzed with non-parametric test. If normality testis pass, statistical analysis was calculated with SPSS software byone-way ANOVA, followed by the Student–Newman–Keuls test forsignificance to compare the differences among various groups ortwo independent sample t-test for significance to compare the dif-ference between two groups. Values of P < 0.05 were considered tobe significant.

3. Results

3.1. Morphology of DRG neurons under inverted phase contrastmicroscope

Shrinkage of neuronal cell bodies and retraction of neurites wasobserved after 24 h exposure of Glu (Fig. 1). The neurons presentedan evidence of dense neurite outgrowth in Glu-treated cultures inthe presence of NRG-1� (Fig. 2).

3.2. Total neurite length with different doses of Glu treatment

DRG neurons at 48 h post-culture were exposed to differentdoses of Glu and then the total neurite length was measuredby fluorescent labeling of MAP2. The total neurite length in

26 Y. Li et al. / Neuroscience Research 76 (2013) 22–30

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ig. 4. Neurite outgrowth in different experimental conditions. Panel A: Glu; paneanel E: LY294002 + PD98059 + Glu + NRG-1�; panel F: control, scale bar = 25 �m.

raphs with error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

.1 mmol/L, 0.5 mmol/L, 2.0 mmol/L, 4.0 mmol/L Glu treated neu-ons and control group is 210.14 �m ± 7.6 �m, 197.4 �m ± 6.0 �m,78.0 �m ± 5.6 �m, 158.9 �m ± 3.2 �m, and 212.7 �m ± 9.0 �m,espectively (Fig. 3).

.3. Total neurite length in different experimental conditions

The DRG neurons at 48 h of culture age were treated withifferent agents for additional 24 h and then the total neurite

ength was measured by fluorescent labeling of MAP2. The totaleurite length in Glu, Glu + NRG-1�, LY294002 + Glu + NRG-1�,D98059 + Glu + NRG-1�, LY294002 + PD98059 + Glu + NRG-1�reated neurons, and control group is 175.4 �m ± 7.1 �m,02.6 �m ± 7.0 �m, 192.2 �m ± 4.4 �m, 194.8 �m ± 4.7 �m,86.0 �m ± 5.0 �m, and 217.08 �m ± 7.1 �m, respectively (Fig. 4).

.4. GAP-43 mRNA expression at different conditions

The DRG neurons at 48 h of culture age were treated withifferent agents for additional 24 h and then the levels of GAP-43RNA were detected by real time-PCR. GAP-43 mRNA levels in Glu,

u + NRG-1�; panel C: LY294002 + Glu + NRG-1�; panel D: PD98059 + Glu + NRG-1�;G: quantification of total neurite length in different experimental conditions. Bar

Glu + NRG-1�, LY294002 + Glu + NRG-1�, PD98059 + Glu + NRG-1�, LY294002 + PD98059 + Glu + NRG-1� treated samples are0.54 ± 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 treatmentdecreased GAP-43 mRNA of DRG neurons as compared with thatin the control group. The decreased GAP-43 mRNA levels inducedby Glu could be partially reversed by the presence of NRG-1�.Inhibitors (LY294002, PD98059) either alone or in combinationblocked 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 treatedwith different agents for additional 24 h and then the lev-els of GAP-43 protein were detected by Western blot assay.GAP-43 protein levels in Glu, Glu + NRG-1�, LY294002 + Glu + NRG-1�, PD98059 + Glu + NRG-1�, LY294002 + PD98059 + Glu + NRG-1�

treated samples are 0.56 ± 0.05-fold, 0.83 ± 0.04-fold, 0.75 ± 0.03-fold, 0.77 ± 0.03-fold, and 0.70 ± 0.03-fold of the control, respec-tively. The decreased GAP-43 protein levels induced by Glu could bepartially reversed by the presence of NRG-1�. Inhibitors (LY294002,

Y. Li et al. / Neuroscience Research 76 (2013) 22–30 27

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Fig. 6. Western blot assay of GAP-43 protein levels in different experimental condi-

ig. 5. Real time-PCR analysis of GAP-43 mRNA levels in different experimentalonditions. Bar graphs with error bars represent mean ± SD. *P < 0.001.

D98059) either alone or in combination blocked the effects ofRG-1� (Fig. 6).

.6. The expression of GAP-43 in situ in DRG neurons

After treatment with different agents for 24 h, the expressionf GAP-43 in situ in DRG neurons was determined by double flu-rescent labeling. The percentage of GAP-43-IR neurons in Glu,lu + NRG-1�, LY294002 + Glu + NRG-1�, PD98059 + Glu + NRG-1�,Y294002 + PD98059 + Glu + NRG-1� treated samples, and con-rol group is 15.06% ± 1.84%, 22.374% ± 2.38%, 19.31% ± 1.93%,8.36% ± 1.52%, 15.91% ± 1.05%, and 25.53% ± 2.35%, respectively.lu treatment decreased the percentage of GAP-43-IR neuronss compared with that in the control group. NRG-1� treatmentncreased the percentage of GAP-43-IR neurons in the presence oflu as compared with that in Glu alone treated cultures. Inhibitors

LY294002, PD98059) either alone or in combination blocked theffects of NRG-1� (Fig. 7).

.7. Determination of pAkt and pERK1/2 levels in DRG neurons

To test the effects of NRG-1� on activation of PI3K/Akt andRK1/2, DRG cultures were incubated with NRG-1� for 30 min.fter that, the levels of pAkt and pERK1/2 were investigated byestern blot assay. The levels of pAkt and pERK1/2 in NRG-1�

reated cultures are 1.56 ± 0.10-fold and 1.35 ± 0.06-fold of the con-rol, respectively. NRG-1� treatment increased pAkt (P < 0.001) andERK1/2 (P < 0.001) levels in DRG neurons as compared with that

n control cultures (Fig. 8).

. Discussion

The aim of the present study was to approach the question ofeuronal plasticity, which is implicated by the alterations of thexpression of GAP-43 both in mRNA and protein levels and GAP-43xpression in situ, dependence on the presence of NRG-1� duringevelopment in vitro of DRG sensory neurons with neurotoxicity

nduced by Glu. The results showed that the decreased GAP-43 lev-ls induced by Glu could be partially reversed by the presence ofRG-1�. Inhibitors (LY294002, PD98059) either alone or in combi-ation blocked the effects of NRG-1�.

tions. 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 barsrepresent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

GAP-43 plays a crucial role in growth and in branch forma-tion of neurites during development or neurogenesis (Yanagisawaet al., 2010). Glu (2 mmol/L) exposure decreased GAP-43 levels incultured DRG neurons, confirming the existence of severe exci-totoxicity in down-regulating GAP-43 expression thus inhibitingneurite outgrowth or axon regeneration at the dose of Glu used inthe present study. It has been suggested that the cultured DRG neu-rons exhibit pathologic changes similar to those found in injuredneurons (Aoki et al., 2007). GAP-43 has been shown to be a generalmarker, staining virtually all regenerating DRG neurons (Gavazziet al., 1999). However, in our present study, the ratio of GAP-43 IRneurons in control group is too low (about 25%). It might be theresult of poor neuronal regeneration because there were not anypotent neurotrophic factors in the culture medium in the controlgroup. In the other experiments of our recent research, the ratio ofGAP-43 IR neurons is more than 60% with different neurotrophin(NT) treatment which represents a good regeneration status of DRGneurons.

The recent research has shown that GAP-43 promotes axongrowth by multiple synergistic mechanisms that potentiate theintrinsic motility of the elongating processes (Foscarin et al., 2009).NMDA receptor activity has been directly associated with GAP-43expression and axonal growth during development and in adultmodels 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 cul-tures (McKinney et al., 1999). In the present study, we observedthat Glu treatment could decrease the protein levels of GAP-43and its mRNA and the ratio of GAP-43-IR neurons in primary DRG

28 Y. Li et al. / Neuroscience Research 76 (2013) 22–30

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 + NRG-1�): 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 ofC1 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.

Y. Li et al. / Neuroscience Rese

Fig. 8. Western blot assay of pAkt and pERK1/2 protein levels after NRG-1� treat-ment. 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 pAktam

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of Akt, Bcl-2, and Bad signaling after focal cerebral ischemia in rats. Biochem.

nd pERK1/2 increased after NRG-1� treatment. Bar graphs with error bars representean ± SD. *P < 0.001.

ultures. This result is consistent with that activation of NMDAeceptors suppresses GAP-43 expression. Administration of NRG-� could reverse the effects of GAP-43 and its mRNA decreases

nduced by Glu suggesting a regenerative activity promoted byRG-1� in injured DRG neurons in vitro. The ratio of GAP-43-IReurons in Glu-treated cultures, increased in the presence of NRG-� also suggesting a regenerative activity promoted by NRG-1�

n injured DRG neurons in vitro. The up-regulation of GAP-43 asevealed in mRNA and protein levels after NRG-1� administra-ion suggested that NRG-1� partially compensated for Glu-inducedown-regulation of GAP-43 expression. Increase of GAP-43 expres-ion indicates a role in axon sprouting as observed the denseeurite outgrowth in the presence of NRG-1� in the presenttudy.

Several recent studies, both in vitro and in vivo, provide newnsights that NRG-1 promotes neurite outgrowth or improvesxonal growth and recovery of sensory functions (Joung et al., 2010;iu et al., 2011; Audisio et al., 2012). Interestingly, expression ofAP-43 and NRG receptors erbB2 and erbB4 was paralleled aftererve transection suggesting the closely relationship between GAP-3 and NRG (Rueger et al., 2008). NRG-1’s cognate ErbB receptorctivation is evidenced by increased phosphorylation of Akt andRK (Luo et al., 2011). The protein kinase Akt acts as a node, play-ng a critical role in controlling cell survival (Aburto et al., 2012).t has been suggested that the neuroprotective effects of NRG-1�n ischemia-induced neuronal death were prevented by inhibitionf the PI3K/Akt pathway both in an in vitro rat ischemia modelCroslan et al., 2008) and in an in vivo rat ischemia model (Guot al., 2010). ERK can be activated by NRG-1 (Higa-Nakamine et al.,012). Increase of expression of the cytoprotective protein GAP-3 in the nervous system is associated the activation of the ERKathway which is involved in promotion of neurite growth andell survival (Fei et al., 2011). In the present study, the effects of

RG-1� on GAP-43 and its mRNA expression could be blocked by

he presence of inhibitors (LY294002, PD98059) either alone or inombination.

arch 76 (2013) 22–30 29

In conclusion, the decrease of GAP-43 expression could bepatially reversed by NRG-1� administration. The effects of NRG-1� were involved in PI3K/Akt and/or ERK1/2 activation. Thesedata provide new insights of the actions of NRG-1� in sensoryneurons.

Acknowledgements

This work was supported by the National Natural ScienceFoundation of China (No. 81000517), China Postdoctoral ScienceFoundation (No. 200904501203), and the Natural Science Founda-tion of Shandong Province of China (No. ZR2011HQ011).

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