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Abnormal expression of netrin-G2 in temporal lobe epilepsy neurons in humans and a rat model
Experimental Neurology 224 (2010) 340–346
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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
Abnormal expression of netrin-G2 in temporal lobe epilepsy neurons in humans and a rat model Yumin Pan, Guangwei Liu, Min Fang, Lan Shen, Liang Wang, Yanbing Han, Dinglie Shen, Xuefeng Wang ⁎ Department of Neurology, the First Affiliated Hospital, Chongqing Medical University, 1 You Yi Road, Chongqing 400016, China
a r t i c l e
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Article history: Received 5 October 2009 Revised 31 March 2010 Accepted 1 April 2010 Available online 9 April 2010 Keywords: Intractable epilepsy (IE) Temporal lobe epileptic rats Netrin-g2
a b s t r a c t The membrane-bound axon guidance molecule netrin-g2 is preferentially expressed in the central nervous system and plays a role in synapse formation and maintenance. Using immunohistochemistry, immunofluorescence, and Western blotting, we investigated the possible correlation between netrin-g2 expression and intractable epilepsy (IE) using surgical samples from epilepsy patients. We used 35 samples of temporal neocortex from patients undergoing surgery for drug-refractory epilepsy and 15 autopsy samples from individuals who died in traffic accidents (i.e., samples of normal human brain). We also examined netrin-g2 expression in the hippocampus and adjacent cortex of rats with temporal lobe epilepsy (lithium chloridepilocarpine model). Netrin-g2 was expressed in the membrane and cytoplasm of neurons from control specimens, and expression was higher in tissue from patients with intractable epilepsy. Western blotting of rat brain tissue showed that netrin-g2 was upregulated starting at 6 h after kindling. Maximal expression was seen around 2 days, and relatively high expression was maintained until 30 days. Expression then returned to normal levels at 60 days, which was consistent with the immunohistochemical and immunofluorescence results. These data implicate netrin-g2 in the pathophysiology of epilepsy and are consistent with the hypothesis that this protein may participate in the abnormal development of synapses and in neuron migration. © 2010 Elsevier Inc. All rights reserved.
Introduction Epilepsy is a chronic brain dysfunction caused by repeated sudden abnormal neuronal discharges in the central nervous system. Data have shown that 30% of patients with recently diagnosed epilepsy will develop an intractable form of epilepsy. Epilepsy in patients with intractable epilepsy (IE) is resistant to a broad range of antiepileptic drugs (AEDs) with various modes of action (Mizrahi et al., 1990). The pathogenesis of IE is poorly understood. Temporal lobe epilepsy is one of the most common types of epilepsy. Neurophysiological studies have shown that the activation of thousands of neurons occurs primarily via the synapses between all neurons in the central nervous system. Precise arrangement of presynaptic and postsynaptic membrane proteins is critical for proper signal transduction. Reorganization of neural networks is a prominent pathological change in IE and can result in unusual self-excitation. This can lead to increased susceptibility to repeated epileptic seizures, synaptic reorganization,
⁎ Corresponding author. Fax: + 86 23 68811487. E-mail addresses: [email protected] (Y. Pan), [email protected] (X. Wang). 0014-4886/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2010.04.001
and axonal sprouting, which have been suggested as possible mechanisms for neural network reorganization. Netrin-g2 is a vertebrate-specific axon guidance molecule that belongs to the Netrin-G subfamily. Netrin-g2 binds to the plasma membrane via a glycosyl phosphatidylinositol (GPI) anchor (Nakashiba et al., 2002, 2000; Yin et al., 2002). Netrin-g2 is preferentially expressed in the central nervous system. Several studies have shown that netrin-g2 can regulate synapse formation and promote neurite outgrowth (Lin et al., 2003; Kim et al., 2006). Unlike classical secreted netrins, netrin-g2 does not bind to any known netrin receptors including deleted in colorectal cancer (DCC) and UNC-5. Abnormal expression of Netrin-g2 and its receptor are associated with impaired memory and learning, an abnormal acoustic startle response in transgenic mice (Zhang et al., 2008), and schizophrenia and bipolar disorder in human patients (Eastwood and Harrison, 2008). Here, we examined whether netrin-g2 protein expression is abnormal in IE tissue samples. We measured netrin-g2 protein expression in the temporal lobe of patients using immunohistochemistry, double label immunofluorescence, and Western blotting. We also examined the netrin-g2 protein expression pattern in the hippocampus and adjacent cortex of the temporal lobe epilepsy rat model.
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Materials and methods Patient selection In our study, all patients with IE had typical clinical manifestations and characteristic electroencephalograms. Samples from 35 patients (17 males; 18 females; mean age, 25.11 ± 8.77 years; range, 12– 44 years; mean disease course, 13.37 ± 7.05; range, 3–30 years) who had undergone resection surgery for IE were chosen at random from 200 specimens in our epilepsy brain tissue bank. Informed consent for the use of tissue in research was obtained before the surgery. Presurgical assessment consisted of obtaining a detailed history and neurological examination, interictal and ictal electroencephalogram studies, neuropsychological testing, and neuroradiological studies. Before surgery, each patient's epileptic lesion was localized by brain CT or magnetic resonance imaging (MRI), 24 h electroencephalogram or video electroencephalogram, sphenoidal electrode monitoring, and intraoperative electrocorticography. All patients were refractory to maximal doses of three or more AEDs, including Carbamazepine, Phenytoin, Valproic acid, Phenobarbital, Lamotrigine, and Topiramate. Table 1 summarizes the clinical features. There were no progressive lesions in the central nervous system as determined by cranial magnetic resonance imaging. In the IE group, surgical removal of the epileptogenic zone in the temporal neocortex was an alternative treatment option. After lesion resection, electrodes for intraoperative electrocorticography were placed on the remaining edge of the tissue to ensure that the lesion had been completely resected. For comparison, we obtained 15 histologically normal temporal neocortex samples from individuals treated for increased intracranial pressure Table 1 Clinical features of the 35 patients in the IE group. Patients Age Sex Course AEDs before surgery no. (y) (M/F) (y) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
22 40 41 29 32 27 29 12 13 38 20 36 18 24 22 17 22 38 13 25 12 22 18 21 33 24 35 26 22 28 22 17 15 22 44
M F M F F F F M F F M F F M M M F M M F F M M M M F F M M M F M F F F
20 20 20 24 20 14 26 6 4 15 10 16 16 20 16 7 6 19 8 10 9 12 13 14 14 19 12 20 9 3 4 5 3 4 30
VPA N 12y,PB N 3y,TPM N 5y*,GBP N 6 m* PHT N 15y,PB N 13y,CBZ N 10y*,TPM N 3y* VPA N 15y,CBZ N 10y*,PB N 8y,PHT N 5y* CBZ N 18y ,VPA N 10y, PB N 2y* ,TPM N 4y* CBZ N 10y*, VPA N 10y, TPM N 5y* CBZ N 3 m,TPM N 5 m, PHT N 2y*, VPA = 3y* PB N 10 m, VPA N 5 m*, CBZ N 1y PHT N 5 m, VPA N 5 m* ,CBZ N 1y, PB N 4y* CBZ N 4 m, VPA = 3y*, PB N 1y*, PHT = 9 m PHT N 5 m,TPM N 4 m,VPA N 10 m*, CBZ N 1y CBZ N 7y, VPA N 5y, PB N 3y*, TPM N 2y* PB N 10y,VPA N 10y*,CBZ N 5y*, TPMN2y* VPA N 10y, CBZ N 9y*, PB N 6y, PHT N 5y* CBZ N 8y*, VPA N 5y, TPM N 4y* CBZ N 10y,VPA N 8y, TPM N 2y*,LTG N 6 m* CBZ N 3 m, TPM N 5 m, PB = 3y*, VPA = 3y* PHTN 3 m,CBZN 5 m*,TPMN 1y*,PBN 10 m, VPAN 15 m PHT N 5 m, VPA N 5 m*, CBZ N 1y, PB N 4y* CBZ N 4 m, VPA = 2y*, PB N 1y*, PHT = 9 m CBZ N 1y*,VPA N 4 m*,PHT N 3 m, TPM N 7 m CBZ N 8 y*, VPA N 5 y, TPM N 2 y* CBZ N 6 y, VPA N 5 y, TPM N 2 y*, PB N 6 m* PHT N 10 y, PB N 2 y,VPA N 3 y*, LTG N 1 y* PB N 10 y, VPA N 5 y, TPM N 3 y, LTG N 2 y* CBZ N 10 y, TPM N 5 y*, PHT N 2 y* CBZ = 5 m, VPA N 7 m*, PB N 6 m, TPM N 1 y* PB N 4 m*, CBZ N 3 m, VPA = 5 m, TPM N 1 y* LTG N 5 m,CBZ N 14 m,PB N 8 m*, VPA N 9 m* CBZN 11 m*,VPAN 4 m*,PHTN 9 m, PBN 7 m, TPMN 1 y CBZ N 5 m*, VPA N 1 y,PB N 2 y, TPM N 1 y* CBZ N 3 y*,VPA N 2 y,TPM N 1 y*, LTG N 6 m* PHT N 3 y, PB N 2 y, CBZ N 2 y*, VPA N 1 y* VPA N 2 y, CBZ N 2 y*, TPM N 1 y* CBZ N 3 y*,VPA N 2 y,TPM N 1 y*, LTG N 6 m* CBZ N 20 y,VPA N 15 y*,PHT N 10 y*,TPM N 2 y*
F, female; M, male; m, month; y, year; *AEDs, antiepileptic drugs which taken before the operation; CBZ, carbamazepine; VPA, valproic acid; PB, phenobarbital; PHT, phenytoin; GBP, gabapentin; TPM, topamax; LTG, lamotrigine.
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due to head trauma from a traffic accident and who later died from these injuries. Conventional neuropathologic examination revealed no signs of central nervous system disease. The mean age of the control group was 27.08 ± 10.25 years (range, 15–50 years). These subjects had no history of epilepsy or exposure to AEDs. Our study protocol complied with the guidelines for the conduct of research involving human subjects as established by the National Institutes of Health and the committee on human research at Chongqing Medical University. Rat study The experimental procedures were approved by the Commission of Chongqing Medical University for ethics of experiments on animals and were conducted in accordance with international standards. Healthy adult male Sprague–Dawley rats (n= 64; from Chongqing Medical University Laboratory Animal Center) weighing 180–230 g were randomly divided into the normal control group (n = 8) or the experimental group (n =56). The experimental group was randomly divided into seven sub-groups: 6-h, 1-, 2-, 7-, 14-, 30-, and 60-day postkindling. In a sober state, the experimental rats were given an intraperitoneal injection of lithium chloride (127 mg/kg, i.p., Sigma, USA) and were pretreated with methyl-scopolamine-bromide (1 mg/ kg) 30 min prior to the first pilocarpine administration (30 mg/kg, i.p., Sigma). Pilocarpine was given repeatedly (10 mg/kg, i.p.) every 30 min until the rats developed seizures. Seizures were scored in each rat using Racine's scale in which stage 4 indicates rearing, and stage 5 indicates rearing plus loss of balance and falling. Rats in stages 4 or 5 were considered successfully kindled. Rats in the control group were intraperitoneally injected with normal saline. Animals were sacrificed 6 h, 1 day, 2 days, 7 days, 14 days, 30 days, or 60 days after kindling, and the hippocampus and adjacent cortex were removed. Tissue processing For both human and animal tissue, one portion of resected brain tissue was immediately fixed in 10% buffered formalin for 48 h. Tissues were then embedded in paraffin, sectioned at 5 μm thickness for immunohistochemistry and 10 μm thickness for double label immunofluorescence analysis, mounted on polylysine-coated slides, and stored at room temperature. One section of each specimen was processed for hematoxylin–eosin staining. Other portions of the resected brain tissues were stored in liquid nitrogen immediately and later used for Western blot analysis. Immunohistochemistry We used the Streptavidin biotin complex (SABC) method according to the manufacturer's protocol. Tissue sections were deparaffinized, rehydrated in a graded series of ethanol, and then incubated in H2O2 (0.3%, 15 min). For antigen retrieval, sections were treated with 10 mM sodium citrate buffer(pH 6.0) and heated with a microwave oven for 20 min at 92–98 °C, then blocked in goat serum (Zhongshan Golden Bridge Inc., Beijing, China) for 20 min at room temperature. Sections were then incubated in primary rabbit anti-human netrin-g2 (1:50, Uscnlife, Wuhan, China) overnight at 4 °C, followed by incubation in secondary goat anti-rabbit for 30 min at 37 °C. Sections were then treated with avidin–biotin complex (ABC) solution (Zhongshan Golden Bridge Inc.) for 30 min, washed with phosphate buffered saline (PBS), and incubated with 3,3′-diaminobenzidine (DAB) (Zhongshan Golden Bridge Inc.) for 5 min. Counterstaining was carried out with Harris's hematoxylin. For negative controls, the primary antibodies were replaced with PBS. Ten visual field images were obtained randomly from every section using an OLYMPUS PM20 automatic microscope (Olympus, Japan) and TCFY-2050 (Yuancheng Inc., China) pathology system. Immunopositivity was assessed semiquantitatively by automatically measuring the cumulative optical
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density and corresponding size of the yellow parts in photographs using the Motic Med 6.0 CMIAS pathology image analysis system (Beihang Motic Inc., China), then got the ratio of cumulative optical density and size, it was the optical densities (OD) value of every random visual field. Double label immunofluorescence Sections were deparaffinized, and antigen recovery was performed as above. Tissue was permeabilized with 0.5% Triton X-100 in 5% bovine serum albumin for 1 h, and then sections were incubated in 10% goat serum (Zhongshan Golden Bridge Inc.) for 5 h at room temperature. Sections were then incubated with a mixture of polyclonal rabbit anti-human netrin-g2 (1:25, Uscnlife) and antineuron-specific enolase (NSE) at 4 °C overnight. Sections were washed and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:200, Zhongshan Golden Bridge Inc.) and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat antimouse IgG (1:200, Zhongshan Golden Bridge Inc.) in the dark for 60 min at room temperature, and then mounted in 50% glycerol/PBS. Fluorescence was detected by laser scanning confocal microscopy (Leica Microsystems Heidelberg GmbH, Germany) on an Olympus IX 70 inverted microscope (Olympus) equipped with a Fluoview FVX confocal scanhead. Western blotting Tissues were snap-frozen in liquid nitrogen, and proteins were directly extracted using a protein extraction kit (Keygenbio, Nanjing, Wuhan, China). Protein concentrations were determined using a bicinchoninic acid protein (BCA) assay (MultiSciences Biotech, Beijing, China). Proteins (100 μg per lane) were then separated with SDS-PAGE (5% spacer gel, 80 V, 30 min; 10% separating gel, 100 V, 60 min). Proteins were then electrophoretically transferred to a polyvinylidene fluoride membrane at 250 mA for 1 h. Equivalent protein loading and transfer were confirmed by Ponceau S staining of the membranes. To block nonspecific binding, membranes were incubated at 37 °C for 1 h in 5% skim milk. Membranes were incubated with polyclonal rabbit anti-human netrin-g2 (1:200, Uscnlife) at 4 °C overnight, rinsed with Tween-20-Tris-buffered saline (TTBS) four times, and incubated with secondary antibody for 1 h at 37 °C. The membranes were washed as described. Anti-β-actin (1:5000, Zhongshan Golden Bridge Inc.) labeling was used to confirm equivalent
protein loading. Blots were visualized using chemiluminescence with an ECM kit and CCD camera in darkroom, stabilized the aperture of CCD camera, applied shading correction and unified the parameter settings before image acquisition, digitally scanned immune blots and analyzed them using Quantity One software (Bio-Rad Laboratories). Subtracted the value of background individually to each lane, band immune intensity ratio of netrin-g2 and corresponding β-actin at the same time of electrophoresis was analyzed (Gassmann et al., 2009), this ratio is the average optical densities (OD) value of netrin-g2 blot expression.
Statistical analysis All values were shown as mean ± standard deviation (x̄ ± s), and the analysis was carried out using t-test between epileptic groups and control groups and one-way ANOVA in rats study (SPSS11.5). Values of p b 0.05 were considered statistically significant.
Results Clinical characteristics In our study, 90% of patients had at least a 5-year history of seizure recurrence, and 66% had a clinical history of more than 10 years of seizures. There were no significant differences in age, sex, or topography of the studied tissues between IE and control tissue.
Elevated netrin-G2 protein staining in the temporal neocortex of patients with IE Thirty-five epileptic and 15 control tissue samples were immunohistochemically stained. Netrin-g2 protein was mainly expressed in the membrane and cytoplasm of neurons in the temporal neocortex of tissue from epilepsy patients, strong immunoreactivity for netrin-g2 was observed in epilepsy samples, whereas faint netrin-g2 staining was present in sections from the control group (Figs. 1 and 2A–C). No immunoreactivity was seen in negative controls in which the primary antibody had been omitted (data not shown). The mean OD value of Netrin-g2 protein was higher in the temporal lobe cortex of patients with IE compared to the control group; statistically significant between the IE group and control group (p b 0.05) (Fig. 3A).
Fig. 1. Immunohistochemistry for netrin-g2 in the temporal neocortex of patients with intractable epilepsy (IE) (A) and control subjects (B). (A) shows strongly positive neurons in the brains of IE patients. The black arrows indicate netrin-g2-positive cells, some of which had long processes. (B) shows scattered netrin-g2-positive cells in normal temporal neocortex tissue. Scale bar = 30 μm.
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Fig. 2. (A–C) Immunofluorescence shows many neurons that coexpressed netrin-g2 and neuron-specific enolase (NSE) in the temporal neocortex of patients with IE (netrin-g2, green; NSE, red). Scale bar = 94.05 μm. (D–F) show increased netrin-g2 and NSE coexpression in the hippocampus of TLE rats 2 days after kindling (netrin-g2, green; NSE, red) compared to control. Scale bar = 75 μm. The white arrows indicate netrin-g2-positive cells.
Higher netrin-G2 levels in the temporal neocortex of patients with IE Western blot analysis of homogenates from each brain was performed to further examine the elevated netrin-g2 immunostaining observed in epileptic brain sections. Netrin-g2 immunoreactive bands were seen at about 60 kDa, β-actin immunoreactive bands were 42 kDa. All IE temporal lobe samples showed higher netrin-g2 immunoreactivity compared to normal brain tissue (Fig. 4A.a). Netrin-g2 expression was normalized by calculating the ratio of the OD of the bands with corresponding β-actin. The mean OD value between IE group control group was statistically significant (P b 0.05)
(Fig. 4A.b). No staining was observed when the primary antibody was omitted (data not shown). Prominent netrin-G2 expression in the hippocampus and adjacent cortex in the TLE rat model In normal rats, the expression of netrin-g2 protein was examined in the hippocampus and adjacent cortex, and was mainly focused on the membrane and cytoplasm of neurons. In the epileptic groups, we observed stronger staining for netrin-g2 in neurons of the granule cell layer of the dentate gyrus, CA1 and CA3 pyramidal cell layers, and
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Fig. 3. (A) Comparison of the mean OD value indicates significantly higher expression of netrin-g2 in IE compared to controls (p b 0.05). (B) Comparison of the mean OD value between epileptic rats and the normal group at each time point after kindling (the blue bars are epileptic groups, the red bars are control groups). Statistically significant at 2and 7-day time points after kindling compared with the control group (*p b 0.05).
adjacent cortex. Many protruding nerve fibers were found around the immunoreactive cells in the CA1 region mainly at the 2-day time point after kindling (Figs. 2D–F and 5). Immunohistochemistry analysis showed the mean OD value of each time point after kindling were higher than control group except 60 days, reached a maximum around 2 days (Fig. 3B). Western blot analysis showed that netrin-g2 immunoreactive bands were seen at about 60 kDa, it was upregulated in kindled tissue starting at 6 h postkindling, reached a maximum around 2 days, and then was maintained at a relatively high level until 30 days. Levels then returned to normal around the 60-day time point, β-actin immunoreactive bands were 42 kDa (Fig. 4B.a). Netrin-g2 expression was normalized by calculating the ratio of the OD of the bands with corresponding βactin; statistically significant between each time point except the 60 days with the control group (Fig. 4B.b). Discussion The full-length human netrin-g2 cDNA is 2428 bp. The gene is located on chromosome 9q34 and encodes a protein of 530 amino acid residues with a molecular weight of about 60 kDa. Netrin-g2 is specifically expressed in the brain of human, rats, and other rodents, with little expression in other tissues (Eastwood and Harrison, 2008). Netrin-g2 is predominantly concentrated in excitatory neurons, with most protein located in the presynaptic membrane (Woo et al., 2009). The expression pattern of netrin-g2 suggests that it plays a role in development of the central nervous system, where it may function as a local guidance cue for axonal growth and may influence synapse formation and neuron migration (Nakashiba et al., 2002; Zhang et al., 2008). The formation of abnormal synapses is the anatomy basis for the recurrent excitatory loop. Abnormal synapses facilitate the emergence of epileptic discharges and spreading, and synaptic reorganization plays an important role in the development of the excitatory loop (Buckmaster et al., 2002). These synaptic changes can lead to frequent
Fig. 4. (A) (a) Proteins from individual brain homogenates from IE and control subjects were separated with gradient SDS-PAGE. Netrin-g2 levels were higher in patients with IE (lanes 1, 3, 5, 7) than in the control group (lanes 2, 4, 6). (b) Comparison of the mean OD value indicates significantly higher expression of netrin-g2 in IE compared to controls (P b 0.05). (B) (a) Western blotting of homogenates from TLE rats. Lane 1 shows netrin-g2 expression in a normal rat, and lanes 2–8 show the expression 6 h, 1 day, 2 days, 7 days, 14 days, 30 days, and 60 days after kindling, respectively. Overexpression of netrin-g2 was first observed at the 6 h time point, was maximal around 2 days, was maintained at a relatively high level until 30 days, and then returned to the normal level at about 60 days after kindling. (b) Comparison of the mean OD value between epileptic rats and the control group at each time point after kindling , statistically significant between each time point with the control except the 60-day time point (*p b 0.05).
seizures and promote the formation of IE. In addition, when neurons do not migrate normally, abnormal development of the nervous system occurs, which may be a partial explanation for the etiology of epilepsy (Barth, 1987; Gordon, 1996). Recent studies have suggested that the growth of axons and neuron migration may be mediated by the same ligand-receptor mechanism (Tanaka et al., 2008). Here we describe the expression pattern of netrin-g2 protein in the temporal neocortex of patients with IE and in the hippocampus and adjacent cortex of the TLE rat model. Immunohistochemistry and immunofluorescence analysis showed that netrin-g2 was mainly expressed in the neuronal membrane and cytoplasm in the normal human temporal lobe, and that the protein was widely expressed in the hippocampus and adjacent cortex in normal rats. Netrin-g2 expression
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Fig. 5. Immunohistochemistry for netrin-g2 in the CA1 region of the hippocampus (C, D) and adjacent cortex (A, B) in the TLE rat model. (A) shows strongly positive neurons in the cortex of TLE rats, some of which had long processes. (B) shows scattered netrin-g2-positive cells in the cortex of normal rats. (C) shows strongly positive cells in the CA1 region of the hippocampus of TLE rats 2 days after kindling. Greater numbers of protruding nerve fibers (black arrows) were found surrounding the immunoreactive cells. (D) shows scattered positive cells in the CA1 region of the hippocampus of normal rats. The black arrows indicate netrin-g2-positive cells. Scale bar = 40 μm.
was upregulated in the temporal neocortex of patients with IE compared to controls, and this was similar to what was observed in the brain of the TLE rat model. The abnormal expression of netrin-g2 in IE patients and epileptic rats supports the hypothesis that netrin-g2 plays a role in the pathogenesis of IE. In the rat model, there was a trend towards increased netrin-g2 expression with longer time since kindling, which is consistent with the results of Nadler et al. (1980) who reported that nascent axon sprouting and synapse reconstruction are usually completed 1 month after pilocarpine-induced seizures in rats. The reorganization of the neuronal network involves abnormal synapse formation and connectivity. Our animal study showed that the expression of netrin-g2 protein began to increase in the acute phase after kindling, and we observed many fibrous processes surrounding the immunoreactive cells in the pyramidal cell layers in particular, which provides further indications of altered synaptic protein expression in IE. Members of the netrin-G family play an important role in synapse formation and maintenance (Yin et al., 2002; Nishimura-Akiyoshi et al., 2007). Netrin-g2 may be primarily associated with the changes in synapses. In addition, our study found an increased number of immunoreactive neurons clustered in the cortex of both IE patients and epileptic rats compared to the control groups. Some neurons had longer processes, which may have guided the migration of neurons, thus participating in the reorganization of the neuronal network. Netrin-g2 may play a role in reorganization of the neuronal network, but the exact mechanisms are not clear. Netrin-g2 may play a role in maintaining the balance between positive and negative signals that are important during synapse formation. Kim et al. (2006) provided evidence that netrin-g2 and its
receptor play a role in development of cortical–cortical connections, in particular, between excitatory synapses. It is not clear whether the effect of netrin-g2 on the synapse is to attract or repel growth cones (or both). Recent studies have mainly focused on: (1) Netrin-g2 protein is bound to the plasma membrane via a GPI linkage and lacks affinity to all of the classical netrins receptors. The membrane localization of netrin-g2 and its expression pattern suggest that it may promote axonal growth and regulate synapse formation. GPI is a lipid structure that plays an important role in transmembrane signal transduction by participating in bi-directional signaling similar to ephrins (Davy et al., 1999). GPI also induces cellular responses and immune responses when linked to its associated proteins (Ikezawa, 2002). (2) Netrin-g2 binds to its specific ligand (named Netrin-G2 ligand (NGL-2)), which is a membrane-bound postsynaptic protein containing an intracellular postsynaptic density95/discs large/zona occludens-1 (PDZ) binding domain. Kim et al. (2006) reported that NGL-2 binds to the first two PDZ domains of PSD-95 and induces the aggregation of PSD-95 and associated proteins, including the NMDA receptor subunit NR2A. These interactions are thought to couple synaptic adhesion events to the assembly of synaptic proteins. A leucine-rich repeat domain in NGL-2 forms an extracellular horseshoeshaped hook that binds it to its pre-synaptic partner, netrin-g2 (Biederer, 2006). These data, together with the known importance of PSD-95 in receptor recruitment and glutamatergic synapse formation (Garner et al., 2000; Kim and Sheng, 2004), indicate that binding between netrin-g2 and NGL-2 is required for recruitment of NMDA receptors to the synapse and maintenance of excitatory synapses. In our study, the upregulation of netrin-g2 in both humans with IE and epileptic rats may due to NMDA receptor-mediated glutamatergic synaptic alterations.
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In summary, we observed overexpression of netrin-g2 protein in the temporal neocortex of patients with IE. Netrin-g2 was also upregulated concurrently with the time from seizure induction in the TLE rat model. We also observed many fibrous nerve processes surrounding the immunoreactive cells in the brains of TLE rats and more immunoreactive neurons clustered in the cortex of both IE patients and epileptic rats compared to controls. These findings indicated that netrin-g2 is expressed under physiological conditions and that its expression increased after seizures. The results in IE patients were consistent with those from the TLE rat model, which is consistent with the hypothesis that abnormal formation of the neuronal network is an important factor leading to IE. The putative physiological function of netrin-g2 suggests that the protein may play a role in IE and affect neuron migration during IE development. However, because of the limitations of our study using brain tissue from IE patients, it is difficult to determine the mechanism of netrin-g2 upregulation in IE patients. Another limitation is that we described the expression pattern of netring2 in humans and a rat model only. Future studies will need to focus on the specific mechanisms of how netrin-g2 influences abnormal synapse development and neuronal migration. These future studies may increase our understanding of neurogenesis in the epileptic brain. Acknowledgments This work is supported by the National Natural Science Foundation of China (No. 30870877). The authors sincerely thank the support of Beijing Tiantan Hospital, Xuanwu Hospital of the Capital University of Medical Sciences, Xin-Hua Hospital of Hubei for supplying the brain tissues, the patients and their families for their participation in this study, and the National Board of the Medical Affairs and the local ethics committee. References Barth, P.G., 1987. Disorders of neuronal migration. Can. J. Neurol. Sci. 14, 1–16. Biederer, T., 2006. Hooking up new synapses. Nat. Neurosci. 9, 1203–1204.
Buckmaster, P.S., Zhang, G.F., Yamawaki, R., 2002. Axon sprouting in a model of temporal lobe epilepsy creates a predominantly excitatory feedback circuit. J. Neurosci. 22, 6650–6658. Davy, A., Gale, N.W., Murray, E.W., Klinghoffer, R.A., Soriano, P., Feuerstein, C., Robbins, S.M., 1999. Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion. Genes Dev. 13, 3125–3135. Eastwood, S.L., Harrison, P.J., 2008. Decreased mRNA expression of netrin-G1 and netrin-G2 in the temporal lobe in schizophrenia and bipolar disorder. Neuropsychopharmacology 33, 933–945. Garner, C.C., Nash, J., Huganir, R.L., 2000. PDZ domains in synapse assembly and signaling. Trends Cell Biol. 10, 274–280. Gassmann, M., Grenacher, B., Rohde, B., Vogel, J., 2009. Quantifying Western blots: pitfalls of densitometry. Electrophoresis 30, 1845–1855. Gordon, N., 1996. Epilepsy and disorders of neuronal migration: II. Epilepsy as a symptom of neuronal migration defects. Dev. Med. Child Neurol. 38, 1131–1134. Ikezawa, H., 2002. Glycosylphosphatidylinositol (GPI)-anchored proteins. Biol. Pharm. Bull. 25, 409–417. Kim, E., Sheng, M., 2004. PDZ domain proteins of synapses. Nat. Rev. Neurosci. 5, 771–781. Kim, S., Burette, A., Chung, H.S., Kwon, S.K., Woo, J., Lee, H.W., Kim, K., Kim, H., Weinberg, R.J., Kim, E., 2006. NGL family PSD-95-interacting adhesion molecules regulate excitatory synapse formation. Nat. Neurosci. 9, 1294–1301. Lin, J.C., Ho, W.H., Gurney, A., Rosenthal, A., 2003. The netrin-G1 ligand NGL-1 promotes the outgrowth of thalamocortical axons. Nat. Neurosci. 6, 1270–1276. Mizrahi, E.M., Kellaway, P., Grossman, R.G., Rutecki, P.A., Armstrong, D., Rettig, G., Loewen, S., 1990. Anterior temporal lobectomy and medically refractory temporal lobe epilepsy of childhood. Epilepsia 31, 302–312. Nadler, J.V., Perry, B.W., Gentry, C., Cotman, C.W., 1980. Loss and reacquisition of hippocampal synapses after selective destruction of CA3–CA4 afferents with kainic acid. Brain Res. 191, 387–403. Nakashiba, T., Ikeda, T., Nishimura, S., Tashiro, K., Honjo, T., Culotti, J.G., Itohara, S., 2000. Netrin-G1: a novel glycosyl phosphatidylinositol-linked mammalian netrin that is functionally divergent from classical netrins. J. Neurosci. 20, 6540–6550. Nakashiba, T., Nishimura, S., Ikeda, T., Itohara, S., 2002. Complementary expression and neurite outgrowth activity of netrin-G subfamily members. Mech. Dev. 111, 47–60. Nishimura-Akiyoshi, S., Niimi, K., Nakashiba, T., Itohara, S., 2007. Axonal netrin-Gs transneuronally determine lamina-specific subdendritic segments. Proc. Natl. Acad. Sci. U. S. A. 104, 14801–14806. Tanaka, D.H., Yamauchi, K., Murakami, F., 2008. Guidance mechanisms in neuronal and axonal migration. Brain Nerve 60, 405–413. Woo, J., Kwon, S.K., Kim, E., 2009. The NGL family of leucine-rich repeat-containing synaptic adhesion molecules. Mol. Cell. Neurosci. 42, 1–10. Yin, Y., Miner, J.H., Sanes, J.R., 2002. Laminets: laminin- and netrin-related genes expressed in distinct neuronal subsets. Mol. Cell. Neurosci. 19, 344–358. Zhang, W., Rajan, I., Savelieva, K.V., Wang, C.Y., Vogel, P., Kelly, M., Xu, N., Hasson, B., Jarman, W., Lanthorn, T.H., 2008. Netrin-G2 and netrin-G2 ligand are both required for normal auditory responsiveness. Genes Brain Behav. 7, 385–392.
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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
Abnormal expression of netrin-G2 in temporal lobe epilepsy neurons in humans and a rat model Yumin Pan, Guangwei Liu, Min Fang, Lan Shen, Liang Wang, Yanbing Han, Dinglie Shen, Xuefeng Wang ⁎ Department of Neurology, the First Affiliated Hospital, Chongqing Medical University, 1 You Yi Road, Chongqing 400016, China
a r t i c l e
i n f o
Article history: Received 5 October 2009 Revised 31 March 2010 Accepted 1 April 2010 Available online 9 April 2010 Keywords: Intractable epilepsy (IE) Temporal lobe epileptic rats Netrin-g2
a b s t r a c t The membrane-bound axon guidance molecule netrin-g2 is preferentially expressed in the central nervous system and plays a role in synapse formation and maintenance. Using immunohistochemistry, immunofluorescence, and Western blotting, we investigated the possible correlation between netrin-g2 expression and intractable epilepsy (IE) using surgical samples from epilepsy patients. We used 35 samples of temporal neocortex from patients undergoing surgery for drug-refractory epilepsy and 15 autopsy samples from individuals who died in traffic accidents (i.e., samples of normal human brain). We also examined netrin-g2 expression in the hippocampus and adjacent cortex of rats with temporal lobe epilepsy (lithium chloridepilocarpine model). Netrin-g2 was expressed in the membrane and cytoplasm of neurons from control specimens, and expression was higher in tissue from patients with intractable epilepsy. Western blotting of rat brain tissue showed that netrin-g2 was upregulated starting at 6 h after kindling. Maximal expression was seen around 2 days, and relatively high expression was maintained until 30 days. Expression then returned to normal levels at 60 days, which was consistent with the immunohistochemical and immunofluorescence results. These data implicate netrin-g2 in the pathophysiology of epilepsy and are consistent with the hypothesis that this protein may participate in the abnormal development of synapses and in neuron migration. © 2010 Elsevier Inc. All rights reserved.
Introduction Epilepsy is a chronic brain dysfunction caused by repeated sudden abnormal neuronal discharges in the central nervous system. Data have shown that 30% of patients with recently diagnosed epilepsy will develop an intractable form of epilepsy. Epilepsy in patients with intractable epilepsy (IE) is resistant to a broad range of antiepileptic drugs (AEDs) with various modes of action (Mizrahi et al., 1990). The pathogenesis of IE is poorly understood. Temporal lobe epilepsy is one of the most common types of epilepsy. Neurophysiological studies have shown that the activation of thousands of neurons occurs primarily via the synapses between all neurons in the central nervous system. Precise arrangement of presynaptic and postsynaptic membrane proteins is critical for proper signal transduction. Reorganization of neural networks is a prominent pathological change in IE and can result in unusual self-excitation. This can lead to increased susceptibility to repeated epileptic seizures, synaptic reorganization,
⁎ Corresponding author. Fax: + 86 23 68811487. E-mail addresses: [email protected] (Y. Pan), [email protected] (X. Wang). 0014-4886/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2010.04.001
and axonal sprouting, which have been suggested as possible mechanisms for neural network reorganization. Netrin-g2 is a vertebrate-specific axon guidance molecule that belongs to the Netrin-G subfamily. Netrin-g2 binds to the plasma membrane via a glycosyl phosphatidylinositol (GPI) anchor (Nakashiba et al., 2002, 2000; Yin et al., 2002). Netrin-g2 is preferentially expressed in the central nervous system. Several studies have shown that netrin-g2 can regulate synapse formation and promote neurite outgrowth (Lin et al., 2003; Kim et al., 2006). Unlike classical secreted netrins, netrin-g2 does not bind to any known netrin receptors including deleted in colorectal cancer (DCC) and UNC-5. Abnormal expression of Netrin-g2 and its receptor are associated with impaired memory and learning, an abnormal acoustic startle response in transgenic mice (Zhang et al., 2008), and schizophrenia and bipolar disorder in human patients (Eastwood and Harrison, 2008). Here, we examined whether netrin-g2 protein expression is abnormal in IE tissue samples. We measured netrin-g2 protein expression in the temporal lobe of patients using immunohistochemistry, double label immunofluorescence, and Western blotting. We also examined the netrin-g2 protein expression pattern in the hippocampus and adjacent cortex of the temporal lobe epilepsy rat model.
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Materials and methods Patient selection In our study, all patients with IE had typical clinical manifestations and characteristic electroencephalograms. Samples from 35 patients (17 males; 18 females; mean age, 25.11 ± 8.77 years; range, 12– 44 years; mean disease course, 13.37 ± 7.05; range, 3–30 years) who had undergone resection surgery for IE were chosen at random from 200 specimens in our epilepsy brain tissue bank. Informed consent for the use of tissue in research was obtained before the surgery. Presurgical assessment consisted of obtaining a detailed history and neurological examination, interictal and ictal electroencephalogram studies, neuropsychological testing, and neuroradiological studies. Before surgery, each patient's epileptic lesion was localized by brain CT or magnetic resonance imaging (MRI), 24 h electroencephalogram or video electroencephalogram, sphenoidal electrode monitoring, and intraoperative electrocorticography. All patients were refractory to maximal doses of three or more AEDs, including Carbamazepine, Phenytoin, Valproic acid, Phenobarbital, Lamotrigine, and Topiramate. Table 1 summarizes the clinical features. There were no progressive lesions in the central nervous system as determined by cranial magnetic resonance imaging. In the IE group, surgical removal of the epileptogenic zone in the temporal neocortex was an alternative treatment option. After lesion resection, electrodes for intraoperative electrocorticography were placed on the remaining edge of the tissue to ensure that the lesion had been completely resected. For comparison, we obtained 15 histologically normal temporal neocortex samples from individuals treated for increased intracranial pressure Table 1 Clinical features of the 35 patients in the IE group. Patients Age Sex Course AEDs before surgery no. (y) (M/F) (y) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
22 40 41 29 32 27 29 12 13 38 20 36 18 24 22 17 22 38 13 25 12 22 18 21 33 24 35 26 22 28 22 17 15 22 44
M F M F F F F M F F M F F M M M F M M F F M M M M F F M M M F M F F F
20 20 20 24 20 14 26 6 4 15 10 16 16 20 16 7 6 19 8 10 9 12 13 14 14 19 12 20 9 3 4 5 3 4 30
VPA N 12y,PB N 3y,TPM N 5y*,GBP N 6 m* PHT N 15y,PB N 13y,CBZ N 10y*,TPM N 3y* VPA N 15y,CBZ N 10y*,PB N 8y,PHT N 5y* CBZ N 18y ,VPA N 10y, PB N 2y* ,TPM N 4y* CBZ N 10y*, VPA N 10y, TPM N 5y* CBZ N 3 m,TPM N 5 m, PHT N 2y*, VPA = 3y* PB N 10 m, VPA N 5 m*, CBZ N 1y PHT N 5 m, VPA N 5 m* ,CBZ N 1y, PB N 4y* CBZ N 4 m, VPA = 3y*, PB N 1y*, PHT = 9 m PHT N 5 m,TPM N 4 m,VPA N 10 m*, CBZ N 1y CBZ N 7y, VPA N 5y, PB N 3y*, TPM N 2y* PB N 10y,VPA N 10y*,CBZ N 5y*, TPMN2y* VPA N 10y, CBZ N 9y*, PB N 6y, PHT N 5y* CBZ N 8y*, VPA N 5y, TPM N 4y* CBZ N 10y,VPA N 8y, TPM N 2y*,LTG N 6 m* CBZ N 3 m, TPM N 5 m, PB = 3y*, VPA = 3y* PHTN 3 m,CBZN 5 m*,TPMN 1y*,PBN 10 m, VPAN 15 m PHT N 5 m, VPA N 5 m*, CBZ N 1y, PB N 4y* CBZ N 4 m, VPA = 2y*, PB N 1y*, PHT = 9 m CBZ N 1y*,VPA N 4 m*,PHT N 3 m, TPM N 7 m CBZ N 8 y*, VPA N 5 y, TPM N 2 y* CBZ N 6 y, VPA N 5 y, TPM N 2 y*, PB N 6 m* PHT N 10 y, PB N 2 y,VPA N 3 y*, LTG N 1 y* PB N 10 y, VPA N 5 y, TPM N 3 y, LTG N 2 y* CBZ N 10 y, TPM N 5 y*, PHT N 2 y* CBZ = 5 m, VPA N 7 m*, PB N 6 m, TPM N 1 y* PB N 4 m*, CBZ N 3 m, VPA = 5 m, TPM N 1 y* LTG N 5 m,CBZ N 14 m,PB N 8 m*, VPA N 9 m* CBZN 11 m*,VPAN 4 m*,PHTN 9 m, PBN 7 m, TPMN 1 y CBZ N 5 m*, VPA N 1 y,PB N 2 y, TPM N 1 y* CBZ N 3 y*,VPA N 2 y,TPM N 1 y*, LTG N 6 m* PHT N 3 y, PB N 2 y, CBZ N 2 y*, VPA N 1 y* VPA N 2 y, CBZ N 2 y*, TPM N 1 y* CBZ N 3 y*,VPA N 2 y,TPM N 1 y*, LTG N 6 m* CBZ N 20 y,VPA N 15 y*,PHT N 10 y*,TPM N 2 y*
F, female; M, male; m, month; y, year; *AEDs, antiepileptic drugs which taken before the operation; CBZ, carbamazepine; VPA, valproic acid; PB, phenobarbital; PHT, phenytoin; GBP, gabapentin; TPM, topamax; LTG, lamotrigine.
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due to head trauma from a traffic accident and who later died from these injuries. Conventional neuropathologic examination revealed no signs of central nervous system disease. The mean age of the control group was 27.08 ± 10.25 years (range, 15–50 years). These subjects had no history of epilepsy or exposure to AEDs. Our study protocol complied with the guidelines for the conduct of research involving human subjects as established by the National Institutes of Health and the committee on human research at Chongqing Medical University. Rat study The experimental procedures were approved by the Commission of Chongqing Medical University for ethics of experiments on animals and were conducted in accordance with international standards. Healthy adult male Sprague–Dawley rats (n= 64; from Chongqing Medical University Laboratory Animal Center) weighing 180–230 g were randomly divided into the normal control group (n = 8) or the experimental group (n =56). The experimental group was randomly divided into seven sub-groups: 6-h, 1-, 2-, 7-, 14-, 30-, and 60-day postkindling. In a sober state, the experimental rats were given an intraperitoneal injection of lithium chloride (127 mg/kg, i.p., Sigma, USA) and were pretreated with methyl-scopolamine-bromide (1 mg/ kg) 30 min prior to the first pilocarpine administration (30 mg/kg, i.p., Sigma). Pilocarpine was given repeatedly (10 mg/kg, i.p.) every 30 min until the rats developed seizures. Seizures were scored in each rat using Racine's scale in which stage 4 indicates rearing, and stage 5 indicates rearing plus loss of balance and falling. Rats in stages 4 or 5 were considered successfully kindled. Rats in the control group were intraperitoneally injected with normal saline. Animals were sacrificed 6 h, 1 day, 2 days, 7 days, 14 days, 30 days, or 60 days after kindling, and the hippocampus and adjacent cortex were removed. Tissue processing For both human and animal tissue, one portion of resected brain tissue was immediately fixed in 10% buffered formalin for 48 h. Tissues were then embedded in paraffin, sectioned at 5 μm thickness for immunohistochemistry and 10 μm thickness for double label immunofluorescence analysis, mounted on polylysine-coated slides, and stored at room temperature. One section of each specimen was processed for hematoxylin–eosin staining. Other portions of the resected brain tissues were stored in liquid nitrogen immediately and later used for Western blot analysis. Immunohistochemistry We used the Streptavidin biotin complex (SABC) method according to the manufacturer's protocol. Tissue sections were deparaffinized, rehydrated in a graded series of ethanol, and then incubated in H2O2 (0.3%, 15 min). For antigen retrieval, sections were treated with 10 mM sodium citrate buffer(pH 6.0) and heated with a microwave oven for 20 min at 92–98 °C, then blocked in goat serum (Zhongshan Golden Bridge Inc., Beijing, China) for 20 min at room temperature. Sections were then incubated in primary rabbit anti-human netrin-g2 (1:50, Uscnlife, Wuhan, China) overnight at 4 °C, followed by incubation in secondary goat anti-rabbit for 30 min at 37 °C. Sections were then treated with avidin–biotin complex (ABC) solution (Zhongshan Golden Bridge Inc.) for 30 min, washed with phosphate buffered saline (PBS), and incubated with 3,3′-diaminobenzidine (DAB) (Zhongshan Golden Bridge Inc.) for 5 min. Counterstaining was carried out with Harris's hematoxylin. For negative controls, the primary antibodies were replaced with PBS. Ten visual field images were obtained randomly from every section using an OLYMPUS PM20 automatic microscope (Olympus, Japan) and TCFY-2050 (Yuancheng Inc., China) pathology system. Immunopositivity was assessed semiquantitatively by automatically measuring the cumulative optical
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density and corresponding size of the yellow parts in photographs using the Motic Med 6.0 CMIAS pathology image analysis system (Beihang Motic Inc., China), then got the ratio of cumulative optical density and size, it was the optical densities (OD) value of every random visual field. Double label immunofluorescence Sections were deparaffinized, and antigen recovery was performed as above. Tissue was permeabilized with 0.5% Triton X-100 in 5% bovine serum albumin for 1 h, and then sections were incubated in 10% goat serum (Zhongshan Golden Bridge Inc.) for 5 h at room temperature. Sections were then incubated with a mixture of polyclonal rabbit anti-human netrin-g2 (1:25, Uscnlife) and antineuron-specific enolase (NSE) at 4 °C overnight. Sections were washed and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:200, Zhongshan Golden Bridge Inc.) and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat antimouse IgG (1:200, Zhongshan Golden Bridge Inc.) in the dark for 60 min at room temperature, and then mounted in 50% glycerol/PBS. Fluorescence was detected by laser scanning confocal microscopy (Leica Microsystems Heidelberg GmbH, Germany) on an Olympus IX 70 inverted microscope (Olympus) equipped with a Fluoview FVX confocal scanhead. Western blotting Tissues were snap-frozen in liquid nitrogen, and proteins were directly extracted using a protein extraction kit (Keygenbio, Nanjing, Wuhan, China). Protein concentrations were determined using a bicinchoninic acid protein (BCA) assay (MultiSciences Biotech, Beijing, China). Proteins (100 μg per lane) were then separated with SDS-PAGE (5% spacer gel, 80 V, 30 min; 10% separating gel, 100 V, 60 min). Proteins were then electrophoretically transferred to a polyvinylidene fluoride membrane at 250 mA for 1 h. Equivalent protein loading and transfer were confirmed by Ponceau S staining of the membranes. To block nonspecific binding, membranes were incubated at 37 °C for 1 h in 5% skim milk. Membranes were incubated with polyclonal rabbit anti-human netrin-g2 (1:200, Uscnlife) at 4 °C overnight, rinsed with Tween-20-Tris-buffered saline (TTBS) four times, and incubated with secondary antibody for 1 h at 37 °C. The membranes were washed as described. Anti-β-actin (1:5000, Zhongshan Golden Bridge Inc.) labeling was used to confirm equivalent
protein loading. Blots were visualized using chemiluminescence with an ECM kit and CCD camera in darkroom, stabilized the aperture of CCD camera, applied shading correction and unified the parameter settings before image acquisition, digitally scanned immune blots and analyzed them using Quantity One software (Bio-Rad Laboratories). Subtracted the value of background individually to each lane, band immune intensity ratio of netrin-g2 and corresponding β-actin at the same time of electrophoresis was analyzed (Gassmann et al., 2009), this ratio is the average optical densities (OD) value of netrin-g2 blot expression.
Statistical analysis All values were shown as mean ± standard deviation (x̄ ± s), and the analysis was carried out using t-test between epileptic groups and control groups and one-way ANOVA in rats study (SPSS11.5). Values of p b 0.05 were considered statistically significant.
Results Clinical characteristics In our study, 90% of patients had at least a 5-year history of seizure recurrence, and 66% had a clinical history of more than 10 years of seizures. There were no significant differences in age, sex, or topography of the studied tissues between IE and control tissue.
Elevated netrin-G2 protein staining in the temporal neocortex of patients with IE Thirty-five epileptic and 15 control tissue samples were immunohistochemically stained. Netrin-g2 protein was mainly expressed in the membrane and cytoplasm of neurons in the temporal neocortex of tissue from epilepsy patients, strong immunoreactivity for netrin-g2 was observed in epilepsy samples, whereas faint netrin-g2 staining was present in sections from the control group (Figs. 1 and 2A–C). No immunoreactivity was seen in negative controls in which the primary antibody had been omitted (data not shown). The mean OD value of Netrin-g2 protein was higher in the temporal lobe cortex of patients with IE compared to the control group; statistically significant between the IE group and control group (p b 0.05) (Fig. 3A).
Fig. 1. Immunohistochemistry for netrin-g2 in the temporal neocortex of patients with intractable epilepsy (IE) (A) and control subjects (B). (A) shows strongly positive neurons in the brains of IE patients. The black arrows indicate netrin-g2-positive cells, some of which had long processes. (B) shows scattered netrin-g2-positive cells in normal temporal neocortex tissue. Scale bar = 30 μm.
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Fig. 2. (A–C) Immunofluorescence shows many neurons that coexpressed netrin-g2 and neuron-specific enolase (NSE) in the temporal neocortex of patients with IE (netrin-g2, green; NSE, red). Scale bar = 94.05 μm. (D–F) show increased netrin-g2 and NSE coexpression in the hippocampus of TLE rats 2 days after kindling (netrin-g2, green; NSE, red) compared to control. Scale bar = 75 μm. The white arrows indicate netrin-g2-positive cells.
Higher netrin-G2 levels in the temporal neocortex of patients with IE Western blot analysis of homogenates from each brain was performed to further examine the elevated netrin-g2 immunostaining observed in epileptic brain sections. Netrin-g2 immunoreactive bands were seen at about 60 kDa, β-actin immunoreactive bands were 42 kDa. All IE temporal lobe samples showed higher netrin-g2 immunoreactivity compared to normal brain tissue (Fig. 4A.a). Netrin-g2 expression was normalized by calculating the ratio of the OD of the bands with corresponding β-actin. The mean OD value between IE group control group was statistically significant (P b 0.05)
(Fig. 4A.b). No staining was observed when the primary antibody was omitted (data not shown). Prominent netrin-G2 expression in the hippocampus and adjacent cortex in the TLE rat model In normal rats, the expression of netrin-g2 protein was examined in the hippocampus and adjacent cortex, and was mainly focused on the membrane and cytoplasm of neurons. In the epileptic groups, we observed stronger staining for netrin-g2 in neurons of the granule cell layer of the dentate gyrus, CA1 and CA3 pyramidal cell layers, and
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Fig. 3. (A) Comparison of the mean OD value indicates significantly higher expression of netrin-g2 in IE compared to controls (p b 0.05). (B) Comparison of the mean OD value between epileptic rats and the normal group at each time point after kindling (the blue bars are epileptic groups, the red bars are control groups). Statistically significant at 2and 7-day time points after kindling compared with the control group (*p b 0.05).
adjacent cortex. Many protruding nerve fibers were found around the immunoreactive cells in the CA1 region mainly at the 2-day time point after kindling (Figs. 2D–F and 5). Immunohistochemistry analysis showed the mean OD value of each time point after kindling were higher than control group except 60 days, reached a maximum around 2 days (Fig. 3B). Western blot analysis showed that netrin-g2 immunoreactive bands were seen at about 60 kDa, it was upregulated in kindled tissue starting at 6 h postkindling, reached a maximum around 2 days, and then was maintained at a relatively high level until 30 days. Levels then returned to normal around the 60-day time point, β-actin immunoreactive bands were 42 kDa (Fig. 4B.a). Netrin-g2 expression was normalized by calculating the ratio of the OD of the bands with corresponding βactin; statistically significant between each time point except the 60 days with the control group (Fig. 4B.b). Discussion The full-length human netrin-g2 cDNA is 2428 bp. The gene is located on chromosome 9q34 and encodes a protein of 530 amino acid residues with a molecular weight of about 60 kDa. Netrin-g2 is specifically expressed in the brain of human, rats, and other rodents, with little expression in other tissues (Eastwood and Harrison, 2008). Netrin-g2 is predominantly concentrated in excitatory neurons, with most protein located in the presynaptic membrane (Woo et al., 2009). The expression pattern of netrin-g2 suggests that it plays a role in development of the central nervous system, where it may function as a local guidance cue for axonal growth and may influence synapse formation and neuron migration (Nakashiba et al., 2002; Zhang et al., 2008). The formation of abnormal synapses is the anatomy basis for the recurrent excitatory loop. Abnormal synapses facilitate the emergence of epileptic discharges and spreading, and synaptic reorganization plays an important role in the development of the excitatory loop (Buckmaster et al., 2002). These synaptic changes can lead to frequent
Fig. 4. (A) (a) Proteins from individual brain homogenates from IE and control subjects were separated with gradient SDS-PAGE. Netrin-g2 levels were higher in patients with IE (lanes 1, 3, 5, 7) than in the control group (lanes 2, 4, 6). (b) Comparison of the mean OD value indicates significantly higher expression of netrin-g2 in IE compared to controls (P b 0.05). (B) (a) Western blotting of homogenates from TLE rats. Lane 1 shows netrin-g2 expression in a normal rat, and lanes 2–8 show the expression 6 h, 1 day, 2 days, 7 days, 14 days, 30 days, and 60 days after kindling, respectively. Overexpression of netrin-g2 was first observed at the 6 h time point, was maximal around 2 days, was maintained at a relatively high level until 30 days, and then returned to the normal level at about 60 days after kindling. (b) Comparison of the mean OD value between epileptic rats and the control group at each time point after kindling , statistically significant between each time point with the control except the 60-day time point (*p b 0.05).
seizures and promote the formation of IE. In addition, when neurons do not migrate normally, abnormal development of the nervous system occurs, which may be a partial explanation for the etiology of epilepsy (Barth, 1987; Gordon, 1996). Recent studies have suggested that the growth of axons and neuron migration may be mediated by the same ligand-receptor mechanism (Tanaka et al., 2008). Here we describe the expression pattern of netrin-g2 protein in the temporal neocortex of patients with IE and in the hippocampus and adjacent cortex of the TLE rat model. Immunohistochemistry and immunofluorescence analysis showed that netrin-g2 was mainly expressed in the neuronal membrane and cytoplasm in the normal human temporal lobe, and that the protein was widely expressed in the hippocampus and adjacent cortex in normal rats. Netrin-g2 expression
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Fig. 5. Immunohistochemistry for netrin-g2 in the CA1 region of the hippocampus (C, D) and adjacent cortex (A, B) in the TLE rat model. (A) shows strongly positive neurons in the cortex of TLE rats, some of which had long processes. (B) shows scattered netrin-g2-positive cells in the cortex of normal rats. (C) shows strongly positive cells in the CA1 region of the hippocampus of TLE rats 2 days after kindling. Greater numbers of protruding nerve fibers (black arrows) were found surrounding the immunoreactive cells. (D) shows scattered positive cells in the CA1 region of the hippocampus of normal rats. The black arrows indicate netrin-g2-positive cells. Scale bar = 40 μm.
was upregulated in the temporal neocortex of patients with IE compared to controls, and this was similar to what was observed in the brain of the TLE rat model. The abnormal expression of netrin-g2 in IE patients and epileptic rats supports the hypothesis that netrin-g2 plays a role in the pathogenesis of IE. In the rat model, there was a trend towards increased netrin-g2 expression with longer time since kindling, which is consistent with the results of Nadler et al. (1980) who reported that nascent axon sprouting and synapse reconstruction are usually completed 1 month after pilocarpine-induced seizures in rats. The reorganization of the neuronal network involves abnormal synapse formation and connectivity. Our animal study showed that the expression of netrin-g2 protein began to increase in the acute phase after kindling, and we observed many fibrous processes surrounding the immunoreactive cells in the pyramidal cell layers in particular, which provides further indications of altered synaptic protein expression in IE. Members of the netrin-G family play an important role in synapse formation and maintenance (Yin et al., 2002; Nishimura-Akiyoshi et al., 2007). Netrin-g2 may be primarily associated with the changes in synapses. In addition, our study found an increased number of immunoreactive neurons clustered in the cortex of both IE patients and epileptic rats compared to the control groups. Some neurons had longer processes, which may have guided the migration of neurons, thus participating in the reorganization of the neuronal network. Netrin-g2 may play a role in reorganization of the neuronal network, but the exact mechanisms are not clear. Netrin-g2 may play a role in maintaining the balance between positive and negative signals that are important during synapse formation. Kim et al. (2006) provided evidence that netrin-g2 and its
receptor play a role in development of cortical–cortical connections, in particular, between excitatory synapses. It is not clear whether the effect of netrin-g2 on the synapse is to attract or repel growth cones (or both). Recent studies have mainly focused on: (1) Netrin-g2 protein is bound to the plasma membrane via a GPI linkage and lacks affinity to all of the classical netrins receptors. The membrane localization of netrin-g2 and its expression pattern suggest that it may promote axonal growth and regulate synapse formation. GPI is a lipid structure that plays an important role in transmembrane signal transduction by participating in bi-directional signaling similar to ephrins (Davy et al., 1999). GPI also induces cellular responses and immune responses when linked to its associated proteins (Ikezawa, 2002). (2) Netrin-g2 binds to its specific ligand (named Netrin-G2 ligand (NGL-2)), which is a membrane-bound postsynaptic protein containing an intracellular postsynaptic density95/discs large/zona occludens-1 (PDZ) binding domain. Kim et al. (2006) reported that NGL-2 binds to the first two PDZ domains of PSD-95 and induces the aggregation of PSD-95 and associated proteins, including the NMDA receptor subunit NR2A. These interactions are thought to couple synaptic adhesion events to the assembly of synaptic proteins. A leucine-rich repeat domain in NGL-2 forms an extracellular horseshoeshaped hook that binds it to its pre-synaptic partner, netrin-g2 (Biederer, 2006). These data, together with the known importance of PSD-95 in receptor recruitment and glutamatergic synapse formation (Garner et al., 2000; Kim and Sheng, 2004), indicate that binding between netrin-g2 and NGL-2 is required for recruitment of NMDA receptors to the synapse and maintenance of excitatory synapses. In our study, the upregulation of netrin-g2 in both humans with IE and epileptic rats may due to NMDA receptor-mediated glutamatergic synaptic alterations.
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In summary, we observed overexpression of netrin-g2 protein in the temporal neocortex of patients with IE. Netrin-g2 was also upregulated concurrently with the time from seizure induction in the TLE rat model. We also observed many fibrous nerve processes surrounding the immunoreactive cells in the brains of TLE rats and more immunoreactive neurons clustered in the cortex of both IE patients and epileptic rats compared to controls. These findings indicated that netrin-g2 is expressed under physiological conditions and that its expression increased after seizures. The results in IE patients were consistent with those from the TLE rat model, which is consistent with the hypothesis that abnormal formation of the neuronal network is an important factor leading to IE. The putative physiological function of netrin-g2 suggests that the protein may play a role in IE and affect neuron migration during IE development. However, because of the limitations of our study using brain tissue from IE patients, it is difficult to determine the mechanism of netrin-g2 upregulation in IE patients. Another limitation is that we described the expression pattern of netring2 in humans and a rat model only. Future studies will need to focus on the specific mechanisms of how netrin-g2 influences abnormal synapse development and neuronal migration. These future studies may increase our understanding of neurogenesis in the epileptic brain. Acknowledgments This work is supported by the National Natural Science Foundation of China (No. 30870877). The authors sincerely thank the support of Beijing Tiantan Hospital, Xuanwu Hospital of the Capital University of Medical Sciences, Xin-Hua Hospital of Hubei for supplying the brain tissues, the patients and their families for their participation in this study, and the National Board of the Medical Affairs and the local ethics committee. References Barth, P.G., 1987. Disorders of neuronal migration. Can. J. Neurol. Sci. 14, 1–16. Biederer, T., 2006. Hooking up new synapses. Nat. Neurosci. 9, 1203–1204.
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