Neuroprotective effect of insulin-like growth factor-1: Effects on tyrosine kinase receptor (Trk) expression in dorsal root ganglion neurons with glutamate-induced excitotoxicity in vitro

Brain Research Bulletin 97 (2013) 86–95

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Neuroprotective effect of insulin-like growth factor-1: Effects on tyrosine kinase receptor (Trk) expression in dorsal root ganglion neurons with glutamate-induced excitotoxicity in vitro Hao Li a,b , Haixia Dong c,d , Jianmin Li b , Huaxiang Liu e , Zhen Liu a , Zhenzhong Li a,∗ a

Department of Anatomy, Shandong University School of Medicine, Jinan 250012, China Department of Orthopaedics, Shandong University Qilu Hospital, Jinan 250012, China c Department of Computer Tomography and Magnetic Resonance Imaging, Weifang Medical College Affiliated Yidu Central Hospital, Qingzhou 262500, China d Shandong Provincial Key Laboratory of Mental Disorders, Shandong University School of Medicine, Jinan 250012, China e Department of Rheumatology, Shandong University Qilu Hospital, Jinan 250012, China b

a r t i c l e

i n f o

Article history: Received 22 March 2013 Received in revised form 24 May 2013 Accepted 28 May 2013 Available online xxx Keywords: Insulin-like growth factor-1 Glutamate Neurotoxicity Tyrosine kinase receptor Dorsal root ganglion

a b s t r a c t Insulin-like growth factor-1 (IGF-1) may play an important role in regulating the expression of distinct tyrosine kinase receptor (Trk) in primary sensory dorsal root ganglion (DRG) neurons. Glutamate (Glu) is the main excitatory neurotransmitter and induces neuronal excitotoxicity for primary sensory neurons. It is not known whether IGF-1 influences expression of TrkA, TrkB, and TrkC in DRG neurons with excitotoxicity induced by Glu. In the present study, primary cultured DRG neurons with Glu-induced excitotoxicity were used to determine the effects of IGF-1 on TrkA, TrkB, and TrkC expression. The results showed that IGF-1 increased the expression of TrkA and TrkB and their mRNAs, but not TrkC and its mRNA, in primary cultured DRG neurons with excitotoxicity induced by Glu. Interestingly, neither the extracellular signal-regulated protein kinase (ERK1/2) inhibitor PD98059 nor the phosphatidylinositol 3kinase (PI3K) inhibitor LY294002 blocked the effect of IGF-1, but both inhibitors together were effective. IGF-1 may play an important role in regulating different Trk receptor expression in DRG neurons through ERK1/2 and PI3K/Akt signaling pathways. The contribution of distinct Trk receptors might be one of the mechanisms that IGF-1 rescues dying neurons from Glu excitotoxic injury. These data imply that IGF-1 signaling might be a potential target on modifying distinct Trk receptor-mediated biological effects of primary sensory neurons with excitotoxicity. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Insulin-like growth factor-1 (IGF-1) is a basic peptide composed of 70 amino acids with rather a ubiquitous distribution in various tissues and cells that mediates the growth-promoting actions of growth hormone and plays an important role in postnatal and adolescent growth (Harada et al., 2010). IGF-1 is a polypeptide growth factor with a variety of functions in both neuronal and nonneuronal cells (Zheng and Quirion, 2006; Wood et al., 2007; Croci et al., 2011). IGF-1 has been shown to act as a neuroprotectant in both in vitro studies and in vivo animal models (Miltiadous et al.,

∗ Corresponding author at: Department of Anatomy, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong Province 250012, China. Tel.: +86 158 6379 3602. E-mail addresses: [email protected] (H. Li), [email protected] (H. Dong), [email protected] (J. Li), [email protected] (H. Liu), [email protected] (Z. Liu), [email protected] (Z. Li). 0361-9230/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2013.05.014

2010; Kitiyanant et al., 2012). IGF-1 influences neuronal survival, axonal growth, differentiation, and maintenance of synaptic connections (Lunetta et al., 2012; Tran et al., 2012). IGF-1 supports to a population of predominantly nociceptive neurons, which may contribute to neuropathic pain (Craner et al., 2002; Miura et al., 2011). It has been shown that local administration of IGF-1 increases the rate of axon regeneration in sciatic nerve injury rats (Emel et al., 2011). IGF-1 and its receptor (IGF-1R) are expressed in small dorsal root ganglion (DRG) neurons (Takayama et al., 2011; Chirivella et al., 2012). IGF-1 promoted neuronal survival by activating its tyrosine kinase receptor IGF-1R (Zhong et al., 2004; McCusker et al., 2006). IGF-1 binds to the IGF-1R in neurons and activates mitogenactivated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling to produce various important biological effects (Vincent et al., 2004; Chen and Russo-Neustadt, 2007; Sullivan et al., 2008; Lawton et al., 2010; Arboleda et al., 2010; Zhang et al., 2011). Glutamate (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

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N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5methyl-4-isoazolepropionic acid (AMPA) receptors to stimulate Ca2+ influx (Marshall et al., 2003; Sanelli et al., 2007). This excitotoxicity can cause the death of neurons due to its high permeability to Ca2+ (Del Río et al., 2008). Interestingly, in response to hypoxia, the periventricular white matter tissue concentration of Glu and IGF-1 as well as expression of AMPA receptors was upregulated (Sivakumar et al., 2010). Activation of TrkB expressed by primary afferent fibers could enhance the release of sensory neurotransmitters Glu, substance P (SP), and calcitonin gene-related peptide (CGRP) (Merighi et al., 2008). Neurotrophin (NT) receptor trafficking plays an important role in directing cellular communication in developing as well as mature neurons (Yano and Chao, 2005). During embryonic development, expression of NT receptor tyrosine kinases (Trks) by sensory ganglia is continuously and dynamically regulated (Genc et al., 2005). Three members of tyrosine kinase receptor (Trk) family have been identified: TrkA, TrkB, and TrkC. It has been identified that DRG neurons express these three Trk receptors that are restricted to subpopulations of mature neurons and only minimally overlap. These transmembrane receptors exhibit considerable ligand specificity: Nerve growth factor (NGF) principally with TrkA, brain-derived neurotrophic factor (BDNF) with TrkB, neurotrophin 3 (NT-3) principally (although not exclusively) with TrkC. Neurotrophins (NTs) acting through high-affinity Trk receptors play a crucial role in regulating survival and maintenance of specific neuronal functions after injury (Jang et al., 2007; Morcuende et al., 2011). A downregulation of mRNA and protein levels was observed for all the Trk receptors (TrkA, TrkB and TrkC) after axotomy injury in rats (Bergman et al., 1999). Our latest research has shown that IGF1 plays an important role in regulating the expression of distinct Trk receptors in DRG neurons through the extracellular signalregulated protein kinase (ERK1/2) and PI3K/Akt signaling pathways (Li et al., 2013). However, whether IGF-1 could influence expression of TrkA, TrkB, and TrkC of cultured DRG neurons with excitotoxicity induced by Glu is still unlear. It is hypothesized that IGF-1 may have different effects on distinct Trk receptor expression in cultured DRG neurons with Glu-induced excitotoxicity. In the present study, primary cultured DRG neurons were used to determine the effects of IGF-1 on the expression of TrkA, TrkB, and TrkC in cultured DRG neurons with excitotoxicity induced by Glu and ERK1/2 and PI3K/Akt pathways involved in IGF-1 effects. 2. Materials and methods 2.1. Preparations of DRG cell culture Embryonic rats on embryonic day 15 (E15) used in the present experiment were taken from the breeding colony of Wistar rats and obtained from the Experimental Animal Center of Shandong University. Procedures involving animals were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (revised 1996). All animal experiments 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. Under aseptic conditions, the bilateral dorsal root ganglia (DRGs) were removed from each embryo, placed in culture media, and digested with 0.25% trypsin (Sigma, St. Louis, MO, USA) in D-Hanks solution at 37 ◦ C for 10 min. Following digestion, fetal bovine serum was added to 10% to stop digestion and the DRG cells were dissociated by trituration. Then, the suspension of DRG cells was centrifuged at 1 × 103 rpm for 5 min. The supernatants were removed and the pellets were resuspended in Dulbecco’s Modified Eagle Medium with F-12 supplement (DMEM/F-12) media (Gibco, Grand Island, NY, USA) and

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triturated using a sterile modified Pasteur’s glass pipette. Cells were then filtered using a 130 ␮m filter followed by counting. Dissociated DRG cells were cultured in 24-well clusters (Costar, Corning, NY, USA) at 37 ◦ C with 5% CO2 for 24 h and then maintained in culture media containing cytosine arabinoside (5 ␮g/mL) for another 24 h to inhibit growth of non-neuronal cells, and then cultured in culture media for an additional 24 h in different experimental conditions before observation. DRG cells for double 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. The composition of the culture media is D-MEM/F-12 (1:1) supplemented with 5% fetal bovine serum, 2% B-27 supplement (Gibco, Grand Island, NY, USA), l-glutamine (0.1 mg/mL, Sigma, St. Louis, MO, USA), penicillin (100 U/mL), and streptomycin (100 ␮g/mL). 2.2. Exposure of different agents on DRG neurons The DRG neurons at 48 h post-culture were exposed to Glu (0.2 mmol/L, Sigma, St. Louis, MO, USA), Glu (0.2 mmol/L) plus IGF1 (20 nmol/L, Peprotech), ERK1/2 inhibitor PD98059 (10 ␮mol/L, Cell Signaling Technology, Danvers, MA, USA) 30 min before treatment with Glu (0.2 mmol/L) plus IGF-1 (20 nmol/L), PI3K inhibitor LY294002 (10 ␮mol/L, Invitrogen) 30 min before treatment with Glu (0.2 mmol/L) plus IGF-1 (20 nmol/L), PD98059 (10 ␮mol/L) plus LY294002 (10 ␮mol/L) 30 min before treatment with Glu (0.2 mmol/L) plus IGF-1 (20 nmol/L) for an additional 24 h. The DRG neurons were continuously exposed to culture media as a control. All aforementioned cultures were incubated at 37 ◦ C in a humidified 5% CO2 -air atmosphere. 2.3. Real-time PCR analysis of mRNA levels of TrkA, TrkB, and TrkC After treatment with different agents for 24 h, the mRNA levels of TrkA, TrkB, and TrkC in DRG cultures at different experimental conditions were analyzed by real-time PCR. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was determined as an internal control. Total DRG cell RNA of each well of the clusters was isolated by TRIzol (TakaRa). cDNA was synthesized using cDNA synthesis kit (Fermentas) according to the manufacturer’s instructions. The synthetic oligonucleotide primer sequences for TrkA, TrkB, TrkC, and GAPDH were as follows: TrkA 5 -GAG TTG AGA AGC CTA ACC ATC G-3 (coding sense) and 5 -AAG CAT TGG AGG AGA GAT TCA G-3 (coding antisense). TrkB 5 -GAA GGG AAG TCT GTG ACC ATT T-3 (coding sense) and 5 -GTG TGT GGC TTG TTT CAT TCA T-3 (coding antisense). TrkC 5 -CCC ACT ACA ACA ATG GCA ACT A-3 (coding sense) and 5 -CCA AAA GTG TCT TCC TCT GGT T-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) according to the manufacture’s instructions. PCR were performed at 50 ◦ C for 2 min, 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 transcript 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. 2.4. Western blot assay for protein levels of the Trks After treatment with different agents for 24 h, the protein levels of TrkA, TrkB, and TrkC, were analyzed by Western blot assay, with

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␤-actin protein levels as an internal control. Fresh cultured DRG neurons after treatment with different agents 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 supernatant collected for Western blot assay. After determining the protein concentrations of the supernatants (BCA method, standard: BSA), 50 ␮g protein of each sample was loaded onto the 12% SDS gel, separated by electrophoresis and transferred to 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-TrkA monoclonal IgG (1:500, Abcam, Cambridge, MA, USA), rabbit anti-TrkB polyclonal IgG (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-TrkC polyclonal IgG (1:1500, Abcam, Cambridge, 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 (1:4000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat anti-mouse IgG-HRP (1:3000, Jackson Immuno). The immunoreactive bands were visualized by an ECL Western blotting detection kit (Millipore Corporation, Billerica, MA, USA) on light sensitive film. The films were scanned and the images were analyzed quantitatively by using an ImagJ 1.39u image analysis software. The expression of the protein levels of TrkA, TrkB, and TrkC was expressed as a ratio with respect to the ␤-actin protein levels. The final results of the Western blot assay are expressed as a ratio of the expression of the protein of interest to that of the control.

2.5. Double fluorescent labeling of MAP2 and TrkA, TrkB, or TrkC At 24 h of the treatment with different agents, DRG cultures were processed for double immunofluorescent labeling of microtubule-associated protein 2 (MAP2) and TrkA, TrkB, or TrkC. The cells on coverslips were quickly rinsed once in 0.1 mol/L phosphate buffer saline (PBS) to remove media. 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 by 2% normal goat serum in 0.6% Triton PBS to block non-specific sites and permeabilize cells. The samples were incubated with rabbit monoclonal anti-TrkA (1:250, Abcam, Cambridge, MA, USA), rabbit polyclonal anti-TrkB (1:200, Abcam, Cambridge, MA, USA), or rabbit polyclonal anti-TrkC (1:400, Abcam, Cambridge, MA, USA) overnight at 4 ◦ C, respectively. After washing in 0.1 mol/L PBS 3 times, the samples were incubated by goat anti-rabbit conjugated to Cy3 (1:500, Abcam, Cambridge, MA, USA) for 45 min in the dark. 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 the dark. 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) for 45 min in the dark. After washing in 0.1 mol/L PBS, the cover slips were placed on glass slides immediately with anti-fade mounting media (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and stored at 4 ◦ C until observation by fluorescent microscope.

2.6. Quantitative analysis of the percentage of TrkA, TrkB, and TrkC-expressing neurons TrkA-immunoreactive (IR), TrkB-IR, or TrkC-IR neurons were observed under a fluorescent microscope (Nikon) with 20× objective lens. TrkA-IR, TrkB-IR, or TrkC-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 percentage of TrkA-IR, TrkB-IR, or TrkC-IR neurons could be obtained. 2.7. Statistical analysis All experiments were performed in triplicate for each condition as one experiment. Five experiments (n = 5) were finished for final analysis. Data are presented as mean ± SD. 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 t-test for significance to compare the difference between two groups or one sample t-test to compare the difference between experimental data and control value for mRNA and protein levels. Values of P < 0.05 were considered to be significant. 3. Results 3.1. The mRNA levels of TrkA, TrkB, and TrkC in DRG neurons The DRG neurons at 48 h post-culture were treated with different agents for an additional 24 h. Then, the mRNA levels of TrkA, TrkB, and TrkC were detected by real-time PCR analysis. The TrkA mRNA levels in Glu, Glu + IGF-1, PD98059 + Glu + IGF-1, LY294002 + Glu + IGF-1, PD98059 + LY294002 + Glu + IGF-1 treated samples were 0.65 ± 0.05, 1.15 ± 0.04, 1.14 ± 0.07, 1.07 ± 0.07, and 0.82 ± 0.08 fold of control, respectively. Glu treatment decreased TrkA mRNA levels as compared with those observed in control group (P < 0.001). IGF-1 treatment increased TrkA mRNA expression in the presence of Glu as compared with that in Glu alone treated cultures (P < 0.001). PD98059 and LY294002 together blocked the effect of IGF-1 (P < 0.01) (Fig. 1). The TrkB mRNA levels in Glu, Glu + IGF-1, PD98059 + Glu + IGF-1, LY294002 + Glu + IGF-1, PD98059 + LY294002 + Glu + IGF-1 treated samples were 0.60 ± 0.06, 0.90 ± 0.04, 0.86 ± 0.04, 0.84 ± 0.04, and 0.71 ± 0.03 fold of control, respectively. Glu treatment decreased TrkB mRNA levels as compared with those observed in control group (P < 0.001). IGF-1 treatment increased TrkB mRNA expression in the presence of Glu as compared with that in Glu alone treated cultures (P < 0.001). PD98059 and LY294002 together blocked the effect of IGF-1 (P < 0.001) (Fig. 1). The TrkC mRNA levels in Glu, Glu + IGF-1, PD98059 + Glu + IGF-1, LY294002 + Glu + IGF-1, PD98059 + LY294002 + Glu + IGF-1 treated samples are 0.76 ± 0.06, 0.87 ± 0.05, 0.82 ± 0.05, 0.80 ± 0.06, and 0.79 ± 0.07 fold of control, respectively. Glu treatment decreased TrkC mRNA levels as compared with those observed in control group (P < 0.01). IGF-1 did not have significant effects on TrkC mRNA expression in the presence of Glu (Fig. 1). 3.2. The protein levels of TrkA, TrkB, and TrkC in DRG neurons The DRG neurons at 48 h post-culture were treated with different agents for an additional 24 h. Then, the protein levels of TrkA, TrkB, and TrkC were detected by Western blot assay. The TrkA protein levels in Glu, Glu + IGF-1, PD98059 + Glu + IGF1, LY294002 + Glu + IGF-1, PD98059 + LY294002 + Glu + IGF-1 incubated DRG neurons are 0.35 ± 0.11, 0.86 ± 0.07, 0.76 ± 0.06, 0.75 ± 0.10, and 0.48 ± 0.08 fold of control, respectively. Glu treatment decreased TrkA protein levels (P < 0.001) compared to those seen in the control group. IGF-1 could partially rescue the decreased TrkA protein levels (P < 0.001) caused by Glu exposure. PD98059 and LY294002 together blocked the effect of IGF-1 (P < 0.001) (Fig. 2). The TrkB protein levels in Glu, Glu + IGF-1, PD98059 + Glu + IGF1, LY294002 + Glu + IGF-1, PD98059 + LY294002 + Glu + IGF-1 incubated DRG neurons are 0.62 ± 0.06, 0.88 ± 0.08, 0.79 ± 0.05,

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Fig. 1. Real-time PCR analysis of mRNA levels of TrkA, TrkB, and TrkC in DRG neurons in different experimental conditions. Panel A: TrkA. Panel B: TrkB. Panel C: TrkC. Following Glu stimulation, the mRNA levels of TrkA, TrkB, and TrkC decreased. IGF1 treatment increased TrkA and TrkB, but not TrkC, mRNA levels in the presence of Glu as compared with that in Glu alone treated cultures. PD98059 and LY294002 together blocked the effect of IGF-1 on the mRNA expression. Bar graphs with error bars represent mean ± SD (n = 5). *P < 0.01, **P < 0.001.

0.77 ± 0.05, and 0.67 ± 0.09 fold of control, respectively. Glu treatment decreased TrkB protein levels (P < 0.01) compared to those seen in the control group. IGF-1 could partially rescue the decreased TrkB protein levels (P < 0.01) caused by Glu exposure. PD98059 and LY294002 together blocked the effect of IGF-1 (P < 0.001) (Fig. 2). The TrkC protein levels in Glu, Glu + IGF-1, PD98059 + Glu + IGF1, LY294002 + Glu + IGF-1, PD98059 + LY294002 + Glu + IGF-1 incubated DRG neurons are 0.59 ± 0.07, 0.68 ± 0.12, 0.61 ± 0.07, 0.67 ± 0.09, and 0.65 ± 0.06 fold of control, respectively. Glu treatment decreased TrkC protein levels (P < 0.001) as compared with

Fig. 2. Western blot assay of TrkA, TrkB, and TrkC protein expression in DRG neurons in the different experimental conditions. Panel A: immunoblotting bands of TrkA, TrkB, and TrkC protein. Panel B: quantification of TrkA, TrkB, and TrkC protein levels. The protein levels of TrkA, TrkB, and TrkC decreased after Glu stimulation. IGF1 administration partially rescued TrkA and TrkB, but not TrkC, expression in the presence of Glu. PD98059 and LY294002 together blocked the effect of IGF-1 on the protein expression. Bar graphs with error bars represent mean ± SD (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.

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that in control group. IGF-1 did not significantly affect TrkC protein levels in the presence of Glu (Fig. 2). 3.3. The expression of TrkA, TrkB, and TrkC in situ in DRG neurons To determine the expression of TrkA, TrkB, and TrkC in situ in DRG neuronal cultures, the DRG neurons at 48 h post-culture were treated with different agents for an additional 24 h. Then, the samples were processed for double fluorescent labeling. The percentage of TrkA-IR neurons in Glu, Glu + IGF-1, LY294002 + Glu + IGF-1, PD98059 + PD98059 + Glu + IGF-1, LY294002 + Glu + IGF-1 incubated DRG neurons is 29.61% ± 1.37%, 33.19% ± 1.37%, 32.87% ± 0.54%, 32.26% ± 0.77%, and 30.67% ± 1.21%, respectively. The percentage of TrkA-IR neurons in control group is 34.24% ± 2.23%. Glu treatment decreased the percentage of TrkA-IR neurons as compared with that in control group (P < 0.001). IGF-1 treatment elevated the percentage of TrkA-IR neurons in the presence of Glu as compared with the neurons stimulated with Glu alone (P < 0.01). PD98059 and LY294002 together blocked the effect of IGF-1 (P < 0.05) (Fig. 3). The percentage of TrkB-IR neurons in Glu, Glu + IGF-1, LY294002 + Glu + IGF-1, PD98059 + PD98059 + Glu + IGF-1, LY294002 + Glu + IGF-1 incubated DRG neurons is 12.27% ± 0.52%, 15.58% ± 0.67%, 15.08% ± 1.07%, 14.76% ± 0.77%, and 12.78% ± 0.52%, respectively. The percentage of TrkB-IR neurons in control group is 16.90% ± 1.05%. Glu treatment decreased the percentage of TrkB-IR neurons as compared with that in control group (P < 0.001). IGF-1 treatment elevated the percentage of TrkB-IR neurons in the presence of Glu as compared with the neurons stimulated with Glu alone (P < 0.001). PD98059 and LY294002 together blocked the effect of IGF-1 (P < 0.001) (Fig. 4). The percentage of TrkC-IR neurons in Glu, Glu + IGF-1, LY294002 + Glu + IGF-1, PD98059 + PD98059 + Glu + IGF-1, LY294002 + Glu + IGF-1 incubated DRG neurons is 20.45% ± 0.72%, 22.01% ± 0.98%, 21.64% ± 1.89%, 21.12% ± 0.59%, and 21.17% ± 1.18%, respectively. The percentage of TrkC-IR neurons in control group is 23.80% ± 1.67%. Glu treatment decreased the percentage of TrkC-IR neurons as compared with that in control group (P < 0.01). IGF-1 did not significantly affect the percentage of TrkC-IR neurons in the presence of Glu (Fig. 5). 4. Discussion IGF-1 plays various important roles in cellular proliferation, differentiation, potent antiapoptotic activity, survival, plasticity, anabolic effect and functions of the cell, thereby contributing to the maintenance of tissue integrity (Carroll, 2001; Okajima and Harada, 2008; Aberg, 2010). IGF-1 is a neuroprotective hormone, and neurodegenerative disorders have been associated with decreased serum IGF-1 concentration (Svensson et al., 2006). The efficacy of IGF-1 is specific to particular cellular phenotypes and its neuroprotective effects are mediated by IGF-1R and binding proteins. It has been indicated that DRG in cultures is commonly used to assay NT effects on developing sensory neurons and represent in vivo conditions in terms of Trk expression patterns (Genc et al., 2005). The activation of ERK1/2 and Akt has been associated with specific outcomes, such as differentiation or proliferation, and is neuroprotective in various settings (Areshkov et al., 2012; Akundi et al., 2012). In the present study, we found that (1) IGF-1 increased the percentage of DRG neuron profiles expressing TrkA and TrkB, did not modify the percentage of DRG neuron profiles expressing TrkC in primary DRG neuronal cultures in the presence of Glu; (2) Neither the ERK1/2 inhibitor PD98059 nor the PI3K inhibitor LY294002 blocked the effect of IGF-1, but both inhibitors together could inhibit the increasing protein and mRNA levels of TrkA and

TrkB and the percentage of TrkA-, and TrkB-expressing neurons induced by IGF-1. It has been identified that TrkA-expressing neurons are mostly small neurons, TrkC-expressing neurons are mostly large neurons, and TrkB-expressing cells vary from small to large and are generally medium-sized neurons (Ernsberger, 2009). It has been shown that TrkA and TrkB are also receptors for IGF-1. The expression patterns of TrkA and TrkB receptors paralleled the expression of IGF-1R in the hippocampus or cerebellum in mice (Chung et al., 2004). Both IGF-1 and TrkA levels were paralleled lower in DRG from type 1 diabetes after diabetic polyneuropathy (Pierson et al., 2003). IGF-1 and its receptor signaling is required for the proper development of DRG neurons involved in the perception of pain (Chirivella et al., 2012). In cultures of hypothalamic neurons taken from E16 male embryos, treatment with estradiol increased the levels of TrkB and IGF-1R, but not TrkC receptors (Carrer and Cambiasso, 2002). IGF-1 enhanced expression of BDNF receptors (TrkB) in cerebrocortical neurons suggested that IGF-1 could induce an increase of BDNF responsiveness (McCusker et al., 2006). In the present study, IGF-1 only increased the expression of TrkA and TrkB, but not TrkC, in primary cultured DRG neurons with excitotoxicity induced by Glu. The actions of IGF-1 might be through activating its receptor IGF1R which is expressed in small DRG neurons. TrkA and TrkB can be activated by their ligands NGF and BDNF, respectively, and play an important role in regulating survival and maintenance of specific neuronal functions after injury. The up-regulation of TrkA and TrkB as revealed by IGF-1 administration in primary cultured DRG neurons with Glu-induced excitotoxicity suggested that IGF-1 partially compensated for Glu-induced down-regulation of these receptors. In this situation, the biological or clinical significance might be that the up-regulated TrkA and TrkB may play a beneficial role in regulating neuronal survival or maintaining neuronal functions after Glu-induced injury. Interestingly, activation of TrkB expressed by primary afferent fibers could enhance the release of neurotransmitter Glu and the effect of TrkB could be blocked by Trk antagonist or anti-TrkB antibody suggested the closely relationship between Trk activation and Glu release (Merighi et al., 2008). DRG contains large proprioceptive neurons that are in a direct contact with the skeletal muscle target (i.e., muscle spindles) (Stephens et al., 2005). These neurons might be TrkC-expressing neurons but not are IGF-1-expressing neurons because TrkCexpressing neurons are mostly large cells. In the present study, IGF-1 did not have effects on TrkC expression in the presence of Glu. The reason might be that IGF-1R is not expressed in large DRG neurons. It has been suggested that differential expression of TrkC receptor is correlate of the preferential effect of NT-3 (Simon et al., 2002). The results of the present study suggested that TrkC expression is not dependent on the presence of IGF-1 in DRG cultures in the presence of Glu. PI3K is a downstream target of IGF1-R (Scolnick et al., 2008). IGF-1 mediates the sustained phosphorylation of Akt (a downstream target of PI3K), which is essential for long term survival and protection of glial progenitors from Glu toxicity (Romanelli et al., 2007). It has been suggested that IGF-1 can maintain long-term Akt activation and prevent Glu-induced apoptosis in cultured rat prooligodendroblasts (Ness et al., 2002; Ness and Wood, 2002) and in cultured rat cerebrocortical neurons (Digicaylioglu et al., 2004). IGF-1, through the generation of the lipidic second messenger phosphatidylinositol 3-phosphate by PI3K, activates Akt which acts as a node, playing a critical role in controlling cell survival (Aburto et al., 2012). The effects of IGF-1 on neurite outgrowth were similar to NGF and were blocked by inhibiting the PI3K pathway with LY294002 as well as the MAPK/ERK pathway with PD98059 in cultured DRG sensory neurons (Kimpinski and Mearow, 2001). Both IGF-1 and BDNF can activate ERK and Akt to promote the survival of hippocampal neurons (Johnson-Farley et al., 2007). Interestingly,

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Fig. 3. Double fluorescent labeling of MAP2 and TrkA and the percentage of TrkA-IR neurons. Panel A (Glu treatment): A1, MAP2-IR neurons; A2, TrkA-IR neurons; A3, overlay of A1 and A2. Panel B (Glu + IGF-1): B1, MAP2-IR neurons; B2, TrkA-IR neurons; B3, overlay of B1 and B2. Panel C (PD98059 + Glu + IGF-1): C1, MAP2-IR neurons; C2, TrkA-IR neurons; C3, overlay of C1 and C2. Panel D (LY294002 + Glu + IGF-1): D1, MAP2-IR neurons; D2, TrkA-IR neurons; D3, overlay of D1 and D2. Panel E (PD98059 + LY294002 + Glu + IGF-1): E1, MAP2-IR neurons; E2, TrkA-IR neurons; E3, overlay of E1 and E2. Panel F (control): F1, MAP2-IR neurons; F2, TrkA-IR neurons; F3, overlay of F1 and F2. Panel G: quantification of the percentage of TrkA-IR neurons. Glu stimulation decreased the percentage of TrkA-IR neurons. IGF-1 elevated the percentage of TrkA-IR neurons in the presence of Glu. PD98059 and LY294002 together blocked the effect of IGF-1 on TrkA expression. Bar graphs with error bars represent mean ± SD (n = 5). Scale bar = 50 ␮m. *P < 0.05, **P < 0.01, ***P < 0.001.

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Fig. 4. Double fluorescent labeling of MAP2 and TrkB and the percentage of TrkB-IR neurons. Panel A (Glu treatment): A1, MAP2-IR neurons; A2, TrkB-IR neurons; A3, overlay of A1 and A2. Panel B (Glu + IGF-1): B1, MAP2-IR neurons; B2, TrkB-IR neurons; B3, overlay of B1 and B2. Panel C (PD98059 + Glu + IGF-1): C1, MAP2-IR neurons; C2, TrkB-IR neurons; C3, overlay of C1 and C2. Panel D (LY294002 + Glu + IGF-1): D1, MAP2-IR neurons; D2, TrkB-IR neurons; D3, overlay of D1 and D2. Panel E (PD98059 + LY294002 + Glu + IGF-1): E1, MAP2-IR neurons; E2, TrkB-IR neurons; E3, overlay of E1 and E2. Panel F (control): F1, MAP2-IR neurons; F2, TrkB-IR neurons; F3, overlay of F1 and F2. Panel G: quantification of the percentage of TrkB-IR neurons. Glu stimulation decreased the percentage of TrkB-IR neurons. IGF-1 treatment increased the percentage of TrkB-IR neurons in the presence of Glu. PD98059 and LY294002 together blocked the effect of IGF-1 on TrkB expression. Bar graphs with error bars represent mean ± SD (n = 5). Scale bar = 50 ␮m. *P < 0.001.

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Fig. 5. Double fluorescent labeling of MAP2 and TrkC and the percentage of TrkC-IR neurons. Panel A (Glu treatment): A1, MAP2-IR neurons; A2, TrkC-IR neurons; A3, overlay of A1 and A2. Panel B (Glu + IGF-1): B1, MAP2-IR neurons; B2, TrkC-IR neurons; B3, overlay of B1 and B2. Panel C (PD98059 + Glu + IGF-1): C1, MAP2-IR neurons; C2, TrkC-IR neurons; C3, overlay of C1 and C2. Panel D (LY294002 + Glu + IGF-1): D1, MAP2-IR neurons; D2, TrkC-IR neurons; D3, overlay of D1 and D2. Panel E (PD98059 + LY294002 + Glu + IGF-1): E1, MAP2-IR neurons; E2, TrkC-IR neurons; E3, overlay of E1 and E2. Panel F (control): F1, MAP2-IR neurons; F2, TrkC-IR neurons; F3, overlay of F1 and F2. Panel G: quantification of the percentage of TrkC-IR neurons. Glu stimulation decreased the percentage of TrkC-IR neurons. IGF-1 administration did not modify the TrkC-expressing neuron profiles in the presence of Glu. Bar graphs with error bars represent mean ± SD (n = 5). Scale bar = 50 ␮m. *P < 0.01.

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neither the ERK1/2 inhibitor PD98059 nor the PI3K inhibitor LY294002 blocked the effect of IGF-1, but both inhibitors together were effective in the present experiment. This result is consistent with the previous research observed in motor, olfactory, and cerebellar granule neurons (Vincent et al., 2004; Scolnick et al., 2008). Interestingly, Glu at excitotoxic doses antagonizes Akt activation by IGF-1 and inhibit the neuroprotective effects of this growth factor on cultured neurons (Garcia-Galloway et al., 2003). It has been shown that one of the IGF-1 prosurvival signaling pathways is the autophosphorylation of IGF-1 receptors through activating NMDA receptors (NMDARs) (Sun et al., 2012). Synaptic and extrasynaptic NMDARs are composed of different subunits (GluN2A and GluN2B) that demonstrate opposing effects. Synaptic NMDAR activation is involved in neuroprotection, the stimulation of extrasynaptic NMDARs, which are composed of GluN2B subunits, triggers cell destruction pathways and may play a key role in the neurodegeneration associated with Glu-induced excitotoxicity (Vizi et al., 2013). The neuroprotective signaling of IGF-1 might be through activating different subunits of NMDARs in the presence of Glu. IGF-1 promoted expression of sensory neuropeptides SP and CGRP in cultured DRG neurons with excitotoxicity induced by Glu (Liu et al., 2010). The activation of Trk receptors seems to be essential in most cases for the biological effects of NTs (Pitts et al., 2006; Lykissas et al., 2007; Hennigan et al., 2007). The data of the present study suggested that IGF-1 not only acts as a neuroprotective molecule but also contributes distinct Trk receptor expression of primary sensory neurons with Glu-induced excitotoxicity. And also, the contribution of activation or promotion of distinct Trk receptors might be one of the mechanisms that IGF-1 rescues dying neurons from Glu excitotoxic injury. In conclusion, IGF-1 could promote expression of TrkA and TrkB, but not TrkC in primary cultured DRG neurons with excitotoxicity induced by Glu. IGF-1 may play an important role in regulating different Trk receptor expression of DRG neurons through ERK1/2 and PI3K/Akt signaling pathways. These data imply that IGF-1 signaling might be a potential target on modifying distinct Trk receptor-mediated biological effects of primary sensory neurons with excitotoxicity. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81071006), the Natural Science Foundation of Shandong Province of China (No. ZR2011HQ011). References Aberg, D., 2010. Role of the growth hormone/insulin-like growth factor 1 axis in neurogenesis. Endocrine Development 17, 63–76. ˜ Aburto, M.R., Magarinos, M., Leon, Y., Varela-Nieto, I., Sanchez-Calderon, H., 2012. AKT signaling mediates IGF-I survival actions on otic neural progenitors. PLoS ONE 7, e30790. Akundi, R.S., Zhi, L., Büeler, H., 2012. PINK1 enhances insulin-like growth factor1-dependent Akt signaling and protection against apoptosis. Neurobiology of Disease 45, 469–478. Arboleda, G., Cárdenas, Y., Rodríguez, Y., Morales, L.C., Matheus, L., Arboleda, H., 2010. Differential regulation of AKT, MAPK and GSK3␤ during C2-ceramide-induced neuronal death. Neurotoxicology 31, 687–693. Areshkov, P.O., Avdieiev, S.S., Balynska, O.V., Leroith, D., Kavsan, V.M., 2012. Two closely related human members of chitinase-like family, CHI3L1 and CHI3L2, activate ERK1/2 in 293 and U373 cells but have the different influence on cell proliferation. International Journal of Biological Science 8, 39–48. Bergman, E., Fundin, B.T., Ulfhake, B., 1999. Effects of aging and axotomy on the expression of neurotrophin receptors in primary sensory neurons. Journal of Comparative Neurology 410, 368–386.

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