SYNAPSE 68:306–316 (2014)
Altered Expression of c-Abl in Patients with Epilepsy and in a Rat Model LING CHEN,* ZHIHUA WANG, BO TANG, MIN FANG, JIE LI, GUOJUN CHEN, AND XUEFENG WANG* Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Neurology, Chongqing 400016, China
KEY WORDS
temporal lobe epilepsy; c-Abl; neurodegeneration
ABSTRACT c-Abl is an ubiquitous nonreceptor tyrosine kinase involved in signal transduction pathways that promote cytoskeleton remodeling and apoptosis. In brain, c-Abl plays important roles in neuronal development, neurogenesis, neuronal migration, neurite outgrowth, and synaptic plasticity. Neuronal death, gliosis and synaptic remodeling are thought to be involved in the development of epilepsy. Here we investigated the expression pattern and distribution of total and phosphorylated c-Abl in patients with temporal lobe epilepsy (TLE) and a rat model of epilepsy to explore the probable relationship between c-Abl expression and TLE. Double immunolabeling, Immunohistochemistry, and immunoblotting results showed that both total and phosphorylated c-Abl were upregulated in the temporal neocortex of 26 patients with TLE compared to nonepileptic controls. In the temporal neocortex of pilocarpine-treated rats, upregulation of total and phosphorylated c-Abl began 6 hours after seizures, with relatively high expression for 60 days. In the hippocampus of experimental rats, total unphosphorylated c-Abl elevated from 6 hours to 30 days after seizures, the expression then returned to normal levels at 60 days, while phosphorylated c-Abl increased along with the time and maintained at significant high levels for up to 60 days. These results indicate that c-Abl may play an important role in the development of TLE. Synapse 68:306–316, 2014. VC 2014 Wiley Periodicals, Inc. INTRODUCTION Epilepsy is a common, chronic neurological disorder characterized by recurrent spontaneous seizures that affects about 70 million people worldwide (Ngugi et al., 2010). Onethird of the patients treated with antiepileptic drugs continue to experience seizures. Furthermore, most current therapies for epilepsy are merely symptomatic treatments, which suppress seizures without affecting the underlying mechanisms of epileptogenesis (the process by which a normal brain becomes epileptic) and seizure-related brain injury (Pitk€ anen and Lukasiuk, 2011). Thus, understanding the molecular events underlying epileptogenesis is critical for devising novel therapeutic strategies. Temporal lobe epilepsy (TLE), the most common form of epilepsy, is often resistant to antiepileptic drugs. Neurodegeneration, gliosis, neurogenesis, axonal sprouting, dentritic plasticity are thought to be involved in the process of TLE development (McNamara et al., 2006; Pitk€ anen and Lukasiuk, 2009). Yet, to date, our understanding of molecular mechanisms underlying epileptogenesis is still incomplete. c-Abl is a nonreceptor tyrosine kinase belongs to Abl family and involved in the signal transduction pathways that promote apoptotic signaling and cytosÓ 2014 WILEY PERIODICALS, INC.
keletal rearrangement (Estrada et al., 2011). c-Abl is a homolog of the transforming element of the Abelson murine leukemia virus, and its genetic rearrangement causes chronic myelogenous leukemia (Wang et al.,1984). In brain, c-Abl kinase plays roles in different neuronal processes including neurogenesis, neuronal migration, axon path finding, and synaptic plasticity (Jones et al., 2004; Moresco and Koleske, 2003; Rhee et al., 2002; Zukerberg et al., 2000). Phosphorylation of tyrosine residue in the c-Abl catalytic domain (Y412) is necessary for activity of c-Abl (Brasher and Van Etten, 2000; Dorey et al., 2001). Growing evidence has suggested a role for c-Abl in neurodegeneration in culture neurons and the brains exposed to oxidative stress or fibrillar Ab (Alvarez et al., 2004a; Schlattere et al., 2011; Xiao et al., 2011). In addition, c-Abl has been implicated in several *Correspondence to: Ling Chen; Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, 1 You Yi Road, Chongqing 400016, China. E-mail: [email protected] or Xuefeng Wang; Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, 1 You Yi Road, Chongqing 400016, China. E-mail: [email protected] Received 12 November 2013; Revised 18 February 2014; Accepted 8 March 2014 DOI: 10.1002/syn.21741 Published online 13 (wileyonlinelibrary.com).
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ALTERED EXPRESSION OF C-ABL TABLE I. Clinical characteristics of TLE patients Patients 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
Gender (M/F)
Age (years)
Course (years)
AEDs before surgery
Resectiontissue
Pathology
M F M M F M F M M F M F M F M F M M F M M F M F M M
26 19 28 39 32 22 38 34 26 31 42 33 18 39 32 27 23 40 36 38 29 37 18 25 26 23
10 6 5 13 19 8 17 9 7 12 13 8 5 15 19 6 11 19 20 21 16 8 5 7 8 10
CBZ,TPM,VPA VPA,LTG,CZP CBZ,TPM,CZP VPA,PHT,CBZ VPA,PB,CBZ,TPM VPA,CBZ,LTG VPA,PB.CBZ,LTG VPA,PHT,LTG,GBP CBZ,VPA,TPM,OXC PB,VPA,TPM, LTG VPA,PHT,LTG CBZ,VPA,TPM,LTG VPA,CBZ,TPM,PB VPA,PHT,CBZ VPA,CBZ,PHT VPA,PB,TPM VPA,CBZ,LTG VPA, TPM, CBZ VPA,PB,CBZ,PHT VPA,PB,CBZ,LTG CBZ, TPM, CZP VPA,CBZ,TPM,LTG VPA,CBZ,TPM,PB CBZ,VPA,TPM,PHT CBZ, VPA, TPM PB, CBZ, PHT, LTG
TNr TNr TNl TNl TNl TNr TNr TNr TNl TNr TNr TNl TNr TNl TNl TNr TNr TNr TNl TNr TNr TNl TNr TNl TNr TNl
nl nl nl, g nl nl, g nl nl nl, g nl nl, nl, g nl, g nl nl, nl, nl,g nl, nl, nl, nl,g nl nl,g nl nl,g nl, nl
F, female; M, male; y, year; AEDs, antiepileptic drugs; CBZ, carbamazepine; VPA, valproic acid; PB, phenobarbital; PHT, phenytoin; GBP, gabapentin; TPM, topamax; LTG, lamotrigine; OXC, oxcarbazepine; CZP, clonazepam; TN, temporal neocortex; l, left; r, right; nl, neuron loss; g, gliosis.
neurodegenerative disorders such as Alzheimer’s (Cancino et al., 2008, 2011), Parkinson’s (Imam et al., 2011; Ko et al., 2010) and Niemann Pick diseases (Alvarez et al., 2008), in which c-Abl activation has a central role in signal transduction pathways underlying pathogenesis of those diseases. Interestingly, epilepsy is one of the clinical findings of Niemann Pick disease. It has been shown that c-Abl modulates cyclindependent kinase-5 (Cdk5) activity. Cdk5 is a small serine/threonine kinase that plays a vital role in normal mammalian development (Zukerberg et al., 2000), it involves neurodegenerative pathologies in the brain of adult (Cheung et al., 2006) as well as young children (Chen et al., 2007). Recent evidence suggests that Cdk5 is involved in degeneration of hippocampal neurons induced by kainic acid (Putkonen et al., 2011). In our previous study, Cdk5 upregulation was observed in brain tissue of patients with drug-resistant epilepsy (Xi et al., 2009). These data suggest that c-Abl is likely to contribute to the neurodegeneration in epilepsy. However, to our knowledge, no study has addressed the potential role of c-Abl in epilepsy. In this study, we detected the expression of total and phosphorylated c-Abl in the temporal neocortex of patients with TLE. To extend the results gained through the analysis of human brain tissues, the expression of c-Abl was investigated in the hippocampus and adjacent cortex of a TLE rat model. MATERIALS AND METHODS Human brain tissue and clinical data Twenty-six patients who underwent resection surgery for medically intractable TLE and 12 cases of
nonepileptic control were included in this study. All brain samples were chosen randomly from our epilepsy brain tissue bank, which consisted of 223 epileptic tissues and 60 control tissues, obtained from the Departments of Neurosurgery of Beijing Tiantan Hospital, Xuanwu Hospital of the Capital University of Medical Sciences, Xinqiao Hospital of the Third Military Medical University and The First Affiliated Hospital of Chongqing Medical University. Informed consent was obtained from all the patients or their relatives. This study was approved by the ethics committees of Chongqing Medical University. All procedures were performed in compliance with the Declaration of Helsinki of the World Medical Association. All patients with TLE were refractory to maximal doses of three or more antiepileptic drugs (AEDs), including valproic acid, carbamazepine, phenobarbital, topiramate, phenytoin, lamotrigine, gabapentin, oxcarbazepine, and clonazepam. Presurgical assessment comprised detailed history and neurological examination, ictal and interictal electroencephalogram (EEG) recordings, neuropsychological testing, and neuroradiological studies, such as brain CT scan or magnetic resonance imaging (MRI). Intraoperative electrocorticography was used to localize the epileptic lesions for all patients with TLE and they had undergone surgical removal of temporal neocortex tissues. These patients had a mean age of 30.04 6 7.24 years (range: 18–42 years), and consisted of 16 men and 10 women. Histopathological findings included neuron loss (n 5 17) and neuron loss and gliosis (n 5 9). Clinical characteristics of TLE patients are summarized in Table I. Synapse
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L. CHEN ET AL. TABLE II. Clinical characteristics of control group
Gender Patients (M/F) 1 2 3 4 5 6 7 8 9 10 11 12
M M F F M F M M F M F M
Age (years)
Etiology diagnosis
19 32 40 37 27 22 28 34 32 29 21 33
Trauma Trauma Trauma Trauma Trauma Trauma Trauma Trauma Trauma Trauma Trauma Trauma
Resection tissue Pathology TNl TNr TNr TNl TNl TNr TNl TNr TNl TNl TNr TNr
n n n n n n n n n n n n
(Racine, 1972). Only those that reached Racine’s stages 4–5 were included in our later experiments. Control rats were intraperitoneally injected with saline rather than pilocarpine. All rats were videomonitored continuously until the day they were sacrificed. The experimental rats were sacrificed at 6, 72 h, 7, 14, 30, 60 days after onset of seizures, and both sides of temporal cortices and hippocampi were removed for study. All animal procedures were approved by the Commission of Chongqing Medical University for ethics of experiments on animals, conducted in accordance with international standards.
F, female; M, male; TN, temporal neocortex; l, left; r, right; n, normal.
Tissue processing For comparison, we obtained 12 histological normal temporal neocortex samples from individuals who underwent therapeutic surgical resection for increased intracranial pressure due to head trauma (7 males and 5 females with a mean age of 29.50 6 6.46 years; range: 19–40 years). All control patients had no history of epilepsy or exposure to antiepileptic drugs (AEDs). Brain samples from controls were taken soon after head trauma. A neuropathological examination revealed no signs of central nervous system disease. Table II shows the clinical features of the controls. There are no significant differences in age, sex, between TLE patients and controls (P > 0.05). Rat model of epilepsy A rat model of Lithium–pilocarpine-induced seizures was used in our study. Healthy 6–8-week-old male Sprague–Dawley rats (n 5 49), weighing 200– 250 g, were obtained from the Laboratory Animal Center of Chongqing Medical University. These animals were housed and maintained under a standard environmental condition with 12-h light/dark cycle, 22 6 1 C temperature, 55 6 5% humidity and given free access to food and water. All rats were randomly divided into the normal control group (n 5 7) or the experimental group (n 5 42). The experimental group was randomly divided into six subgroups (n 5 7 per subgroup) after onset of status epilepticus (SE): 6, 72 h, 7, 14, 30, and 60 days. The experimental rats were injected with lithium chloride (127 mg/kg, i.p., Sigma-Aldrich). 18 h later, atropine sulfate (1 mg/kg, i.p., Sigma) was administered to reduce the peripheral cholinomimetic effects of pilocarpine. Thirty minutes later, pilocarpine (50 mg/kg, i.p., SigmaAldrich) was injected to induce SE. Pilocarpine (10 mg/kg, i.p.) was given repeatedly every 30 min until the rats developed seizures. Diazepam (10 mg/kg, i.p., Sigma) was administered to terminate convulsions sixty minutes after the onset of the SE. The evoked seizures were classified according to Racine’s scale Synapse
For both human and animal tissue, one portion of these brains was frozen in liquid nitrogen, and stored at 280 C for Western blot analysis. Other portions of the resected brain tissues were fixed in 4% paraformaldehyde, frozen, and sectioned at 10 lm for double-immunofluorescence labeling analysis, or embedded in paraffin and sectioned at 5 lm for immunohistochemical analysis. One section from every embedded specimen was stained with hematoxylin–eosin (H&E). Double-immunofluorescence labeling Frozen tissue sections from humans and rats were air dried. Antigen recovery was performed by treating the sections with 10mmol/l sodium citrate buffer (pH 6.0) and heated with a microwave oven for 20 min at 92–98 C. Tissues were permeabilized with 0.5% Triton X-100 and then incubated in 5% goat serum (Zhongshan Golden Bridge, Beijing, China) for 1 h at room temperature. Tissue sections were then incubated with a mixture of polyclonal rabbit antitotal c-Abl antibody (1:100, catalogue number 2862; Cell signaling) and mouse antineuron specific enolase (NSE) antibody (1:50, Zhongshan Golden Bridge Inc, Beijing, China) or with a mixture of polyclonal rabbit antitotal c-Abl antibody and mouse antiglial fibrillary acidic protein (GFAP) antibody (1:50, Wuhan Boster Biological Technology, Wuhan, China) at 4 C overnight. For Abl-pY412 immunofluorescence, antitotal c-Abl antibody was replaced with a polyclonal rabbit anti-AblpY412 antibody (1:100, Bioss, Beijing, China). After washing in PBS, sections were incubated with fluorescein isothiocyanate-conjugated goat antirabbit IgG (1:50, Zhongshan Golden Bridge, Beijing, China) and tetramethylrhodamine isothiocyanate-conjugated goat antimouse IgG (1:50, Zhongshan Golden Bridge) in the dark for 1 h at room temperature. After rinsing, sections were coverslipped with 50% glycerol/PBS. Fluorescent-stained sections were evaluated by laser scanning confocal microscopy (Leica Microsystems Heidelberg GmbH, Wetzlar, Germany) on an Olympus IX 70 inverted microscope (Olympus, Tokyo, Japan) equipped with a Fluoview FVX confocal scan head.
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Immunohistochemistry analysis After deparaffinized in xylene and then rehydrated in graded ethanol, sections were incubated with 0.3% H2O2 for 10 min. Antigen recovery performed as described for immunofluorescence. Normal goat serum (Zhongshan Golden Bridge, Beijing, China) was used to block nonspecific binding for 30 min at room temperature. Sections were then incubated with the primary rabbit polyclonal antitotal c-Abl antibody (1:100, catalogue number 2862, Cell signaling) or polyclonal rabbit anti-Abl-pY412 antibody (1:100, Bioss, Beijing, China) at 4 C overnight. After washes with PBS, sections were incubated with biotinylated goat antirabbit secondary antibody (streptavidin–peroxidase kits, Zhongshan Golden Bridge, Beijing, China) for 30 min. Washed and incubated with an avidin–biotin peroxidase complex (Zhongshan Golden Bridge, Beijing, China) at 37 C for 30 min, The sections were then incubated with 3,3-diaminobenzidine (DAB, Zhongshan Golden Bridge, Beijing, China) for 5 min. Counterstaining was performed with Harris hematoxylin. For negative control, the primary antibody was replaced with PBS. Images were acquired by an OLYMPUS PM20 automatic microscope (Olympus, Osaka, Japan) and a TC-FY2050 pathology system (Yuancheng, Beijing, China). Ten visual fields were obtained randomly in each section. Image-Pro plus 5.0 software (Media, Cybemetrics) was used for the quantitative analysis of c-Abl expression. Mean optical density (OD) of each vision field was automatically measured by computer. Western blot analysis Total proteins were extracted from homogenated rat and human brain tissues using the whole protein extraction kit (Keygen Biotech, Nanjing, China). Protein concentrations were determined using the enhanced BCA protein assay kit (Beyotime, Haimen, China), according to the manufactures’ instructions. 50 mg protein was loaded into each gel lane and separated by 6% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), electrotransferred on to an Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore Corporation) using an electrophoretic transfer system (Bio-Rad Laboratories). The PVDF membrane was then incubated in 5% skim milk/Tween-20–Tris-buffered saline for 60 min at 37 C. The membranes were incubated overnight at 4 C with the following primary antibodies: rabbit antitotal c-Abl antibody (1:1000, #2862, Cell signaling), rabbit anti-Abl-pY412 antibody (1:300, Bioss, Beijing, China), rabbit anti-b-actin antibody (1:4000, Beijing 4A Biotech, Beijing, China) as internal control. Washed in Tween-20–Tris-buffered saline (TBST) three times, the membranes were then incubated with horseradish peroxidase-conjugated secondary
antibody (1:4000, goat antirabbit IgG-HRP, Zhongshan, Beijing, China) for 1 h at room temperature. An enhanced chemiluminescence kit (Pierce, Rockford, IL) and a charge-coupled device camera (BioRad Laboratories) were used to visualize the resulting protein bands, digitally scanned immune blots were quantified using Quantity One software (BioRad Laboratories). Band immune intensity ratio of cAbl and corresponding b-actin at the same time of electrophoresis were analyzed (Gassmann et al., 2009). The band intensity ratio of c-Abl relative to bactin (c-Abl/b-actin) was analyzed. Statistical analysis Data were expressed as mean 6 standard deviation (SD). Statistical analysis of the differences between the epileptic group and control group were conducted using Student’s t test (SPSS16.0). Differences among groups of experimental rats were assessed by oneway ANOVA analysis combined with Tukey’s HSD post hoc multiple comparison test (SPSS16.0), P values of less than 0.05 was considered statistically significant. RESULTS Neuronal localization of c-Abl To determine cell-type specific expression of c-Abl, we performed double-immunofluorescence staining. We found nonphosphorylated total c-Abl expressed exclusively in neurons of temporal neocortex samples from nonepileptic controls and patients with TLE, as shown by colocalization with the neuronal marker, NSE (Fig. 1A). Whereas GFAP1 astrocytes were not stained (Fig. 1B), indicating that total c-Abl was not expressed in astrocytes. Increased total c-Abl protein expression in the temporal neocortex of patients with TLE Immunohistochemistry images showed that total cAbl protein was expressed in the membrane and cytoplasm of neurons in the temporal neocortex of TLE patients and nonepileptic controls. Faint immunoreactivity for c-Abl was present in control cortices, whereas strong c-Abl staining was observed in TLE samples (Fig. 1C). No immunoreactivity was seen in negative controls in which the primary antibody had been omitted (data not shown). The mean OD value of c-Abl protein in the temporal neocortex of patients with TLE was significantly higher than that of the nonepileptic controls (P < 0.05) (Fig. 1C). To further verify the elevated c-Abl immunostaining observed in epileptic human brain sections, we performed western blot analysis. c-Abl protein immunoreactive bands were detected at about 135 kDa, and b-actin immunoreactive bands were seen at 42 kDa. Expression of c-Abl was evaluated in the temporal neocortex of all patients with TLE and Synapse
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Fig. 1. Double-immunofluorescent labeling, IHC, and Western blot analysis for total c-Abl in the temporal neocortex of patients with TLE and nonepileptic controls. A: Double-immunofluorescent labeling showed that c-Abl (green) and NSE (red) were coexpressed (merged) in the temporal neocortex of an epileptic patient. White arrows, c-Abl 1/ NSE1 cells. B: c-Abl (green) and GFAP (red) were not coexpressed (merged). White arrow, c-Abl1 cell; blue arrow, GFAP1 cell. C: IHC analysis for c-Abl in the temporal neocortex of humans indicated slight immunoreactive staining of c-Abl in the temporal neocortex of a control subject compared with strong immunoreactive staining of c-Abl in the
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temporal neocortex of a patient with TLE. Black arrows, c-Abl1 cells: the black scale bars indicate 75 mm. Comparison of the mean OD value (right) indicated significantly higher expression of c-Abl in the TLE group than that in the control group. *P < 0.05. D: Western blotting analysis for c-Abl in the temporal neocortex of humans. Proteins from individual brain homogenates from TLE and control subjects were separated with gradient SDS-PAGE. c-Abl was more strongly expressed in patients with TLE than that in controls. A comparison of the intensity ratio (right) indicated significantly higher expression of c-Abl in patients with TLE than that in controls. *P < 0.05.
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nonepileptic controls. Temporal neocortex specimens from TLE patients showed higher c-Abl immunoreactivity when compared with normal brain tissues from nonepileptic controls (Fig. 1D). The difference in the mean OD ratio between the TLE group and control group was statistically significant (P < 0.05) (Fig. 1D). Dynamic expression of total c-Abl in the hippocampus and adjacent cortex in the epileptic rats To rule out the possibility that altered c-Abl expression in patients with epilepsy may be caused by antiepileptic drugs, we performed an animal experiment using a rat model of epilepsy. c-Abl was expressed in neurons of the hippocampus and adjacent cortex from epileptic rats and controls, as shown by colocalization with the neuronal marker, NSE (Figs. 2A and 3A), cAbl positive cells were not colocalized with GFAP1 astrocytes (Fig. 2B), indicating that c-Abl was not expressed in astrocytes of rat hippocampus and adjacent cortex. c-Abl positive signals were detected in the membrane and cytoplasm of neurons of rat hippocampus and adjacent cortex (Figs. 2C and 3B), In the epileptic groups, stronger staining for c-Abl was observed in neurons of the dentate gyrus, CA1, and CA3 regions of hippocampus (Fig. 3B), and adjacent cortex (Fig. 2C,). In the cortex, IHC analysis showed that the mean OD value of each time point after pilocarpine induced seizure was higher than that of the control group (P < 0.01). A striking increase of c-Abl protein expression was found at 72 h (P < 0.01), at 7 days (P < 0.01), and at 14 days (P < 0.01) (Fig. 2C). In the hippocampus, the striking elevation of c-Abl protein was also observed at 72 h (P < 0.01), 7 days (P < 0.01) and 14 days (P < 0.01) (Fig. 3B), whereas the mean OD value returned to control level at 60 days. The western blot analysis result was in accordance with that of IHC: c-Abl upregulation in the cortex of epileptic rats appeared at 6 h after seizures, reached a peak level at 14 days, and was maintained at a relatively high level until 60 days (Fig. 2D). In the hippocampus, c-Abl expression elevated from 6 h to 30 days postseizures with the peak level at 72 h, the expression then returned to control level at 60 days (Fig. 3C). Semiquantitative densitometric analysis revealed that the difference in the c-Abl expression levels between control and each time point after seizures was significant (P < 0.01), except that between control and 60 days in the hippocampus (P > 0.05). Prominent expression of activated c-Abl in epileptic patients and rats Phosphorylation of c-Abl on tyrosine residue 412 has been shown to be important for the kinase activity (Reynolds et al., 2000). Therefore, we determined
the expression of activated c-Abl using phosphospecific antibody (Abl-pY412). We found that AblpY412 expressed in neurons but not in astrocytes of brain specimens from humans and rats, as shown by colocalization with the neuronal marker, NSE (Fig. 4A), but not colocalization with the astrocyte marker, GFAP (Fig. 5A). Abl-pY412 was detected in the neuronal cytoplasm and membrane in the temporal neocortex of controls and TLE patients (Fig. 4B) as well as in the hippocampus (Fig. 5B) and adjacent cortex (data not shown) of control and epileptic rats. Immunohistochemical study showed that neuronal AblpY412 immunoreactivity was stronger in patients with TLE than that in controls, which was significantly different (Fig. 4B) (P < 0.05). In the rat model, the Abl-pY412 immunoreactivity was also significantly elevated in the hippocampus (Fig. 5B) and adjacent cortex (data not shown) 60 days after seizures compared with controls. Western blotting study demonstrated that AblpY412 was significantly increased in the temporal neocortex of TLE patients than that in controls (Fig. 4C) (P < 0.05). In the rat model, Abl-pY412 levels were increased from 6 h to 60 days after seizures with the peak levels at 14 days (Fig. 5C), Semiquantitative densitometric analysis revealed that Abl-pY412 expression of each time point after seizures was significantly increased compared with the control group. (Fig 5C) (P < 0.05). DISCUSSION In the current study, we have examined the expression pattern of total and activated c-Abl in the temporal cortex of patients with TLE and in the hippocampus and adjacent cortex of a TLE rat model. We observed that both total and phosphorylated c-Abl were expressed in the neuronal membrane and cytoplasm in normal and epileptic brain tissue from humans and rats. Both total and phosphorylated cAbl were significantly upregulated in patients with TLE and epileptic rats compared to nonepileptic controls. In the rat model of epilepsy, the cortical expression of total and phosphorylated c-Abl increased from 6 h to 60 days after seizures, total c-Abl protein elevated from 6 h to 30 days and phosphorylated c-Abl was upregulated from 6 h to 60 days in the hippocampus. During epileptogenesis, neurodegeneration is one of the most common structural changes of brain (Pitk€ anen and Lukasiuk, 2011). Increasing evidence supports the participation of c-Abl in the signal cascade that regulates neuronal death in Alzheimer’s disease (AD) animal models (Alvarez et al., 2004b; Cancino et al., 2008; Wilson et al., 2004) and in Niemann Pick disease (Alvarez et al., 2008). Overexpression of c-Abl in adult mouse forebrain neurons resulted in neurodegeneration and neuronal loss, Synapse
Fig. 2. Double-immunofluorescent labeling, IHC, and Western blot analysis for total c-Abl in the temporal cortex of experimental rats. A: c-Abl (green) and NSE (red) were coexpressed (merged) in the temporal cortex of a TLE rat at 14 days after seizures. White arrow, c-Abl1/NSE1 cells. B: c-Abl (green) and GFAP (red) were not coexpressed (merged). White arrow, c-Abl1 cell; blue arrow, GFAP1 cell. C: Immunoreactive staining of c-Abl in the temporal cortex of rats. Slight immunoreactive staining of c-Abl in the cortex of control rats compared with strong staining in epileptic rats at 14 days after
seizures. Comparison of the mean OD value (right) of IHC staining between the control and epileptic rats at different time points after seizures. *P < 0.05 versus control. Black arrow, c-Abl1 cells. The black scale bars indicate 75 mm. D: Western blot analysis results from control and epileptic rats. Representative Western blot analysis images showed bands of c-Abl and b-actin (internal control) at different time points after seizures. Comparison of the mean intensity ratio (right) of immunoblotting between control and epileptic rats at each time point after seizures. *P < 0.05 versus control.
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Fig. 3. Double-immunofluorescent labeling, IHC, and Western blot analysis for total c-Abl in the hippocampus of experimental rats. A: c-Abl (green) and NSE (red) were coexpressed (merged) in the hippocampus of a TLE rat at 72 hours after seizures. White arrow, c-Abl1/NSE1 cells. B: Immunoreactive staining of c-Abl in the hippocampus of rats. Faint immunoreactive staining of c-Abl in the hippocampus (CA1 field) of control rats compared with strong staining in epileptic rats at 72 h after seizures. Comparison of the mean OD value (right) of IHC staining between the control and epi-
leptic rats at different time points after seizures. *P < 0.05 versus control. Black arrow, c-Abl1 cells. The black scale bars indicate 75 mm. C: Western blot analysis of c-Abl expression in the hippocampus of rats. Representative Western blot showed the dynamics of c-Abl expression at different time points after seizures in hippocampus, b-actin was used as an internal control. Comparison of the mean intensity ratio (right) of immunoblotting between control and epileptic rats at each time point after seizures. *P < 0.05 versus control.
particularly in the CA1 region of the hippocampus (Schlatterer et al., 2011). Moreover, c-Abl expression was increased in amyloid b-protein fibrils (Abf) injected hippocampus and the selective c-Abl inhibitor STI571 prevents the neurodegenerative changes induced by Abf (Cancino et al., 2008). It has been shown that c-Abl modulates Cdk5 kinases function during brain development (Zukerberg et al., 2000) and c-Abl activates Cdk5 in AD models (Cancino et al., 2011). The activation of c-Abl by Ab resulted in neuronal death in Drosophila through the deregulation of Cdk5 (Lin et al., 2007). c-Abl-Cdk5 signaling
activation also contributes to neuronal apoptosis induced by Enterovirus 71 (EV71) (Chen et al., 2007). Our previous study demonstrated Cdk5 upregulation in brain tissue of TLE patients (Xi et al., 2009), here, we found that total and activated c-Abl protein level elevated not only in brain tissue of patients with TLE but also in epileptic tissue of rats compared with the control groups. The Neuronal cytoplasm location of cAbl observed in our research is consistent with that found in AD brains (Jing et al., 2009). Insight into epileptogenic mechanisms has been revealed by investigating animal models of AD (Palop et al., Synapse
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Fig. 4. Double-immunofluorescent labeling, IHC, and Western blot analysis for phospho-c-Abl in the temporal neocortex of patients with TLE and controls. A: p-c-Abl (green) and NSE (red) were coexpressed (merged) in the temporal neocortex of an epileptic patient. White arrow, p-c-Abl1/NSE1 cells. B: IHC analysis for p-c-Abl in the temporal neocortex of humans. Black arrow, p-c-Abl1 cells. Slight immunoreactive staining of p-c-Abl is shown in the temporal neocortex of a control subject compared with strong staining in a TLE patient. Black scale bar 5 75 mm. Comparison of the mean OD
value (right) showed significantly higher expression of p-c-Abl in the TLE group than that in the control group. *P < 0.05. C: Western blot analysis for phospho-c-Abl in the temporal neocortex of humans. Immunoreactive stainings of p-c-Abl in patients with TLE are stronger than those in controls. Con: control. A comparison of the mean intensity ratio (right) indicated significantly higher expression of p-c-Abl in patients with TLE than that in controls. *P < 0.05.
2007), in which epilepsy can be a comorbidity. These data suggest a role of c-Abl in neurodegeneration in epilepsy. However, the mechanisms underlying this process remain unclear. For ethical consideration, we were not able to take normal human brain specimens, we used structurally normal brain samples obtained from temporal lobectomies performed for the treatment of traumatic brain injuries as control. The brain specimens of patients with TLE could only be obtained at the
drug-resistant stage of epilepsy. Thus we used lithium/pilocarpine rat model to further validate c-Abl expression found in patients with TLE. This model replicates many features of human epilepsy (Majores et al., 2007), in which an acute episode of seizures is frequently followed by a latency period with subsequent development of spontaneous recurrent seizures (Sharma et al., 2007). In our animal study, both total and activated c-Abl levels began to increase at 6 h after seizures (acute period), and maintained at a
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Fig. 5. Double-immunofluorescent labeling, IHC, and Western blot analysis for p-c-Abl in the hippocampus and adjacent cortex of experimental rats. A: p-c-Abl (green) and GFAP (red) were not coexpressed (merged) in the cortex of an epileptic rat at 14 days after seizures. White arrow, c-Abl1 cells; blue arrow, GFAP1 cells. B: Immunohistochemical staining of p-c-Abl in the hippocampus of rats. Faint immunoreactive staining of c-Abl in the hippocampus (CA1 field) of control rats compared with strong staining in epileptic rats at 14 days after seizures. Comparison of the mean OD
value (right) of IHC staining between the control and epileptic rats at different time points after seizures. Black arrow, p-c-Abl1 cells. black scale bar 5 75 mm. *P < 0.05 versus control. C: Western blot results from control and epileptic rats. Representative Western blot analysis images of p-c-Abl and b-actin (internal control) in the hippocampus at different time points after seizures. A comparison of the intensity ratio (right) of immunoblots between control and epileptic rats at each time point after seizures. *P < 0.05 versus control.
high level in latent period (7, 14 days), then continuously elevated in chronic period (30 days) in the hippocampus, which is supported by the results of Borges et al. (2003) who reported that extensive loss of neurons in the hippocampus occurred within 6 h post SE, and cell damage persisted for up to 31 days. In addition, our study showed that c-Abl immunoreactivity upregulation after seizures is predominantly limited to dentate gyrus, CA1 and CA3 subfields of hippocampus, neuronal necrosis was observed in these hippocampal regions in pilocarpine model (Clifford et al., 1987; Mello et al., 1993). These results
support the hypothesis that c-Abl plays a role in neuronal loss of epilepsy. In conclusion, our study reveals, for the first time to our knowledge, the increased expression of c-Abl protein in temporal lobe epileptic tissue of human and rat. These findings support the hypothesis that c-Abl plays a role in the pathogenesis of TLE. Because what we described was only a phenomenon, we could not conclude from current study whether c-Abl is a cause or a result of epilepsy. A better-designed experiment in the future is required to focus on the possible roles and specific mechanisms that c-Abl plays in epilepsy. Synapse
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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (No. 81071039). The authors sincerely thank the support of Beijing Tiantan Hospital, Xuanwu Hospital of the Capital University of Medical Sciences, Chongqing Xinqiao Hospital for supplying the brain tissues, the patients and their families for their participation in this study and the National Institutes of Health of China and the Ethics Committee on Human Research of the Chongqing Medical University. REFERENCES Alvarez AR, Godoy JA, Mullendorff K, Olivares GH, Bronfman M, Inestrosa NC. 2004a. Wnt-3a overcomes beta-amyloid toxicity in rat hippocampal neurons. Exp Cell Res 297:186–196. Alvarez AR, Klein A, Castro J, Cancino GI, Amigo J, Mosqueira M, Vargas LM, Ye’venes LF, Bronfman FC, Zanlungo S. 2008. Imatinib therapy blocks cerebellar apoptosis and improves neurological symptoms in a mouse model of Niemann Pick type C disease. FASEB J 22:3617–3627. Alvarez AR, Sandoval PC, Leal NR, Castro PU, Kosik KS. 2004b. Activation of the neuronal c-Abl tyrosine kinase by amyloid-betapeptide and reactive oxygen species. Neurobiol Dis 17:326–336. Brasher BB, Van Etten RA. 2000. c-Abl has high intrinsic tyrosine kinase activity that is stimulated by mutation of the Src homology 3 domain and by autophosphorylation at two distinct regulatory tyrosines. J Biol Chem 275:35631–35637. Borges K, Gearing M, McDermott DL, Smith AB, Almonte AG, Wainer BH, Dingledine R. 2003. Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model. Exp Neurol 182:21–34. Cancino GI, Perez de Arce K, Castro PU, Toledo EM, von Bernhardi R, Alvarez AR. 2011. c-Abl tyrosine kinase modulates tau pathology and Cdk5 phosphorylation in AD transgenic mice. Neurobiol Aging 32:1249–1261. Cancino GI, Toledo EM, Leal NR, Hernandez DE, Yevenes LF, Inestrosa NC, Alvarez AR. 2008. STI571 prevents apoptosis, tau phosphorylation and behavioural impairments induced by Alzheimer’s beta-amyloid deposits. Brain 131:2425–2442. Chen TC, Lai YK, Yu CK, Juang JL. 2007. Enterovirus 71 triggering of neuronal apoptosis through activation of Abl-Cdk5 signalling. Cell Microbiol 9:2676–2688. Cheung ZH, Fu AK, Ip NY. 2006. Synaptic roles of Cdk5: Implications in higher cognitive functions and neurodegenerative diseases. Neuron 50:13–18. Clifford DB, Olney JW, Maniotis A, Collins RC, Zorumski CF. 1987. The functional anatomy and pathology of lithium-pilocarpine and high-dose pilocarpine seizures. Neuroscience 23:953–968. Dorey K, Engen JR, Kretzschmar J, Wilm M, Neubauer G, Schindler T, Superti-Furga G. 2001. Phosphorylation and structure-based functional studies reveal a positive and a negative role for the activation loop of the c-Abl tyrosine kinase. Oncogene 20:8075–8084. Estrada LD, Zanlungo SM, Alvarez AR. 2011. C-Abl tyrosine kinase signaling: A new player in AD tau pathology. Curr Alzheimer Res 8:643–651. Gassmann M, Grenacher B, Rohde B, Vogel J. 2009. Quantifying western blots: Pitfalls of densitometry. Electrophoresis 30:1845– 1855. Imam SZ, Zhou Q, Yamamoto A, Valente AJ, Ali SF, Bains M, Roberts JL, Kahle PJ, Clark RA, Li S. 2011. Novel regulation of Parkin function through c-Abl-mediated tyrosine phosphorylation: Implications for Parkinson’s disease. J Neurosci 31:157–163. Jing Z, Caltagarone J, Bowser R. 2009. Altered subcellular distribution of c-Abl in Alzheimer’s disease. J Alzheimers Dis 17:409–422.
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Jones SB, Lu HY, Lu Q. 2004. Abl tyrosine kinase promotes dendrogenesis by inducing actin cytoskeletal rearrangements in cooperation with Rho family small GTPases in hippocampal neurons. J Neurosci 24:8510–8521. Ko HS, Lee Y, Shin JH, Karuppagounder SS, Gadad BS, Koleske AJ, Pletnikova O, Troncoso JC, Dawson VL, Dawson TM. 2010. Phosphorylation by the c-Abl protein tyrosine kinase inhibits Parkin’s ubiquitination and protective function. Proc Natl Acad Sci USA 107:16691–16696. Lin H, Lin TY, Juang JL. 2007. Abl deregulates Cdk5 kinase activity and subcellular localization in Drosophila neurodegeneration. Cell Death Differ 14:607–615. Majores M, Schoch S, Lie A, Becker AJ. 2007. Molecular neuropathology of temporal lobe epilepsy: Complementary approaches in animal models and human disease tissue. Epilepsia 48:4–12. McNamara JO, Huang YZ, Leonard AS. 2006. Molecular signaling mechanisms underlying epileptogenesis. Sci STKE 2006:re12. Mello LE, Cavalheiro EA, Tan AM, Kupfer WR, Pretorius JK, Babb TL, Finch DM. 1993. Circuit mechanisms of seizures in the pilocarpine model of chronic epilepsy: Cell loss and mossy fiber sprouting. Epilepsia 34:985–995. Moresco EM, Koleske AJ. 2003. Regulation of neuronal morphogenesis and synaptic function by Abl family kinases. Curr Opin Neurobiol 13:535–544. Ngugi AK, Bottomley C, Kleinschmidt I, Sander JW, Newton CR. 2010. Estimation of the burden of active and lifetime epilepsy: A meta-analytic approach. Epilepsia 51:883–890. Palop JJ, Chin J, Roberson ED, Wang J, Thwin MT, Bien-Ly N, Yoo J, Ho KO, Yu GQ, Kreitzer A, Finkbeiner S, Noebels JL, Mucke L. 2007. Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models of Alzheimer’s disease. Neuron 55:697–711. Pitk€ anen A, Lukasiuk K. 2009. Molecular and cellular basis of epileptogenesis in symptomatic epilepsy. Epilepsy Behav Suppl 1:16– 25. Pitk€ anen A, Lukasiuk K. 2011. Mechanisms of epileptogenesis and potential treatment targets. Lancet Neurol 10:173–186. Putkonen N, Kukkonen JP, Mudo G, Putula J, Belluardo N, Lindholm D, Korhonen L. 2011. Involvement of cyclin-dependent kinase-5 in the kainic acid-mediated degeneration of glutamatergic synapses in the rat hippocampus. Eur J Neurosci 34:1212– 1221. Racine RJ. 1972. Modification of seizure activity by electrical stimulation. II: Motor seizure. Electroencephalogr Clin Neurophysiol 32:281–294. Reynolds FH Jr., Oroszlan S, Stephenson JR. 1982. Abelson murine leukemia virus P120: Identification and characterization of tyrosine phosphorylation sites. J Virol 44:1097–1101. Rhee J, Mahfooz NS, Arregui C, Lilien J, Balsamo J, VanBerkum MF. 2002. Activation of the repulsive receptor Roundabout inhibits Ncadherin-mediated cell adhesion. Nat Cell Biol 4:798–805. Schlatterer SD, Tremblay MA, Acker CM, Davies P. 2011. Neuronal c-Abl overexpression leads to neuronal loss and neuroinflammation in the mouse forebrain. J Alzheimers Dis 25:119–133. Wang JY, Ledley F, Goff S, Lee R, Groner Y, Baltimore D. 1984. The mouse c-abl locus: Molecular cloning and characterization. Cell 36:349–356. Wilson C, Henry S, Smith MA, Bowser R. 2004. The p53 homologue p73 accumulates in the nucleus and localizes to neurites and neurofibrillary tangles in Alzheimer disease brain. Neuropathol Appl Neurobiol 30:19–29. Xiao L, Chen D, Hu P, Wu J, Liu W, Zhao Y, Cao M, Fang Y, Bi W, Zheng Z, Ren J, Ji G, Wang Y, Yuan Z. 2011. The c-Abl-MST1 Signaling pathway mediates oxidative stress-induced neuronal cell death. J Neurosci 31:9611–9619. Xi ZQ, Xiao F, Yuan J, Wang XF, Wang L, Quan FY, Liu GW. 2009. Gene expression analysis on anterior temporal neocortex of patients with intractable epilepsy. Synapse 63:1017–1028. Zukerberg LR, Patrick GN, Nikolic M, Humbert S, Wu CL, Lanier LM, Gertler FB, Vidal M, Van Etten RA, Tsai LH. 2000. Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth. Neuron 26: 633–646.
Altered Expression of c-Abl in Patients with Epilepsy and in a Rat Model LING CHEN,* ZHIHUA WANG, BO TANG, MIN FANG, JIE LI, GUOJUN CHEN, AND XUEFENG WANG* Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Neurology, Chongqing 400016, China
KEY WORDS
temporal lobe epilepsy; c-Abl; neurodegeneration
ABSTRACT c-Abl is an ubiquitous nonreceptor tyrosine kinase involved in signal transduction pathways that promote cytoskeleton remodeling and apoptosis. In brain, c-Abl plays important roles in neuronal development, neurogenesis, neuronal migration, neurite outgrowth, and synaptic plasticity. Neuronal death, gliosis and synaptic remodeling are thought to be involved in the development of epilepsy. Here we investigated the expression pattern and distribution of total and phosphorylated c-Abl in patients with temporal lobe epilepsy (TLE) and a rat model of epilepsy to explore the probable relationship between c-Abl expression and TLE. Double immunolabeling, Immunohistochemistry, and immunoblotting results showed that both total and phosphorylated c-Abl were upregulated in the temporal neocortex of 26 patients with TLE compared to nonepileptic controls. In the temporal neocortex of pilocarpine-treated rats, upregulation of total and phosphorylated c-Abl began 6 hours after seizures, with relatively high expression for 60 days. In the hippocampus of experimental rats, total unphosphorylated c-Abl elevated from 6 hours to 30 days after seizures, the expression then returned to normal levels at 60 days, while phosphorylated c-Abl increased along with the time and maintained at significant high levels for up to 60 days. These results indicate that c-Abl may play an important role in the development of TLE. Synapse 68:306–316, 2014. VC 2014 Wiley Periodicals, Inc. INTRODUCTION Epilepsy is a common, chronic neurological disorder characterized by recurrent spontaneous seizures that affects about 70 million people worldwide (Ngugi et al., 2010). Onethird of the patients treated with antiepileptic drugs continue to experience seizures. Furthermore, most current therapies for epilepsy are merely symptomatic treatments, which suppress seizures without affecting the underlying mechanisms of epileptogenesis (the process by which a normal brain becomes epileptic) and seizure-related brain injury (Pitk€ anen and Lukasiuk, 2011). Thus, understanding the molecular events underlying epileptogenesis is critical for devising novel therapeutic strategies. Temporal lobe epilepsy (TLE), the most common form of epilepsy, is often resistant to antiepileptic drugs. Neurodegeneration, gliosis, neurogenesis, axonal sprouting, dentritic plasticity are thought to be involved in the process of TLE development (McNamara et al., 2006; Pitk€ anen and Lukasiuk, 2009). Yet, to date, our understanding of molecular mechanisms underlying epileptogenesis is still incomplete. c-Abl is a nonreceptor tyrosine kinase belongs to Abl family and involved in the signal transduction pathways that promote apoptotic signaling and cytosÓ 2014 WILEY PERIODICALS, INC.
keletal rearrangement (Estrada et al., 2011). c-Abl is a homolog of the transforming element of the Abelson murine leukemia virus, and its genetic rearrangement causes chronic myelogenous leukemia (Wang et al.,1984). In brain, c-Abl kinase plays roles in different neuronal processes including neurogenesis, neuronal migration, axon path finding, and synaptic plasticity (Jones et al., 2004; Moresco and Koleske, 2003; Rhee et al., 2002; Zukerberg et al., 2000). Phosphorylation of tyrosine residue in the c-Abl catalytic domain (Y412) is necessary for activity of c-Abl (Brasher and Van Etten, 2000; Dorey et al., 2001). Growing evidence has suggested a role for c-Abl in neurodegeneration in culture neurons and the brains exposed to oxidative stress or fibrillar Ab (Alvarez et al., 2004a; Schlattere et al., 2011; Xiao et al., 2011). In addition, c-Abl has been implicated in several *Correspondence to: Ling Chen; Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, 1 You Yi Road, Chongqing 400016, China. E-mail: [email protected] or Xuefeng Wang; Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, 1 You Yi Road, Chongqing 400016, China. E-mail: [email protected] Received 12 November 2013; Revised 18 February 2014; Accepted 8 March 2014 DOI: 10.1002/syn.21741 Published online 13 (wileyonlinelibrary.com).
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ALTERED EXPRESSION OF C-ABL TABLE I. Clinical characteristics of TLE patients Patients 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
Gender (M/F)
Age (years)
Course (years)
AEDs before surgery
Resectiontissue
Pathology
M F M M F M F M M F M F M F M F M M F M M F M F M M
26 19 28 39 32 22 38 34 26 31 42 33 18 39 32 27 23 40 36 38 29 37 18 25 26 23
10 6 5 13 19 8 17 9 7 12 13 8 5 15 19 6 11 19 20 21 16 8 5 7 8 10
CBZ,TPM,VPA VPA,LTG,CZP CBZ,TPM,CZP VPA,PHT,CBZ VPA,PB,CBZ,TPM VPA,CBZ,LTG VPA,PB.CBZ,LTG VPA,PHT,LTG,GBP CBZ,VPA,TPM,OXC PB,VPA,TPM, LTG VPA,PHT,LTG CBZ,VPA,TPM,LTG VPA,CBZ,TPM,PB VPA,PHT,CBZ VPA,CBZ,PHT VPA,PB,TPM VPA,CBZ,LTG VPA, TPM, CBZ VPA,PB,CBZ,PHT VPA,PB,CBZ,LTG CBZ, TPM, CZP VPA,CBZ,TPM,LTG VPA,CBZ,TPM,PB CBZ,VPA,TPM,PHT CBZ, VPA, TPM PB, CBZ, PHT, LTG
TNr TNr TNl TNl TNl TNr TNr TNr TNl TNr TNr TNl TNr TNl TNl TNr TNr TNr TNl TNr TNr TNl TNr TNl TNr TNl
nl nl nl, g nl nl, g nl nl nl, g nl nl, nl, g nl, g nl nl, nl, nl,g nl, nl, nl, nl,g nl nl,g nl nl,g nl, nl
F, female; M, male; y, year; AEDs, antiepileptic drugs; CBZ, carbamazepine; VPA, valproic acid; PB, phenobarbital; PHT, phenytoin; GBP, gabapentin; TPM, topamax; LTG, lamotrigine; OXC, oxcarbazepine; CZP, clonazepam; TN, temporal neocortex; l, left; r, right; nl, neuron loss; g, gliosis.
neurodegenerative disorders such as Alzheimer’s (Cancino et al., 2008, 2011), Parkinson’s (Imam et al., 2011; Ko et al., 2010) and Niemann Pick diseases (Alvarez et al., 2008), in which c-Abl activation has a central role in signal transduction pathways underlying pathogenesis of those diseases. Interestingly, epilepsy is one of the clinical findings of Niemann Pick disease. It has been shown that c-Abl modulates cyclindependent kinase-5 (Cdk5) activity. Cdk5 is a small serine/threonine kinase that plays a vital role in normal mammalian development (Zukerberg et al., 2000), it involves neurodegenerative pathologies in the brain of adult (Cheung et al., 2006) as well as young children (Chen et al., 2007). Recent evidence suggests that Cdk5 is involved in degeneration of hippocampal neurons induced by kainic acid (Putkonen et al., 2011). In our previous study, Cdk5 upregulation was observed in brain tissue of patients with drug-resistant epilepsy (Xi et al., 2009). These data suggest that c-Abl is likely to contribute to the neurodegeneration in epilepsy. However, to our knowledge, no study has addressed the potential role of c-Abl in epilepsy. In this study, we detected the expression of total and phosphorylated c-Abl in the temporal neocortex of patients with TLE. To extend the results gained through the analysis of human brain tissues, the expression of c-Abl was investigated in the hippocampus and adjacent cortex of a TLE rat model. MATERIALS AND METHODS Human brain tissue and clinical data Twenty-six patients who underwent resection surgery for medically intractable TLE and 12 cases of
nonepileptic control were included in this study. All brain samples were chosen randomly from our epilepsy brain tissue bank, which consisted of 223 epileptic tissues and 60 control tissues, obtained from the Departments of Neurosurgery of Beijing Tiantan Hospital, Xuanwu Hospital of the Capital University of Medical Sciences, Xinqiao Hospital of the Third Military Medical University and The First Affiliated Hospital of Chongqing Medical University. Informed consent was obtained from all the patients or their relatives. This study was approved by the ethics committees of Chongqing Medical University. All procedures were performed in compliance with the Declaration of Helsinki of the World Medical Association. All patients with TLE were refractory to maximal doses of three or more antiepileptic drugs (AEDs), including valproic acid, carbamazepine, phenobarbital, topiramate, phenytoin, lamotrigine, gabapentin, oxcarbazepine, and clonazepam. Presurgical assessment comprised detailed history and neurological examination, ictal and interictal electroencephalogram (EEG) recordings, neuropsychological testing, and neuroradiological studies, such as brain CT scan or magnetic resonance imaging (MRI). Intraoperative electrocorticography was used to localize the epileptic lesions for all patients with TLE and they had undergone surgical removal of temporal neocortex tissues. These patients had a mean age of 30.04 6 7.24 years (range: 18–42 years), and consisted of 16 men and 10 women. Histopathological findings included neuron loss (n 5 17) and neuron loss and gliosis (n 5 9). Clinical characteristics of TLE patients are summarized in Table I. Synapse
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L. CHEN ET AL. TABLE II. Clinical characteristics of control group
Gender Patients (M/F) 1 2 3 4 5 6 7 8 9 10 11 12
M M F F M F M M F M F M
Age (years)
Etiology diagnosis
19 32 40 37 27 22 28 34 32 29 21 33
Trauma Trauma Trauma Trauma Trauma Trauma Trauma Trauma Trauma Trauma Trauma Trauma
Resection tissue Pathology TNl TNr TNr TNl TNl TNr TNl TNr TNl TNl TNr TNr
n n n n n n n n n n n n
(Racine, 1972). Only those that reached Racine’s stages 4–5 were included in our later experiments. Control rats were intraperitoneally injected with saline rather than pilocarpine. All rats were videomonitored continuously until the day they were sacrificed. The experimental rats were sacrificed at 6, 72 h, 7, 14, 30, 60 days after onset of seizures, and both sides of temporal cortices and hippocampi were removed for study. All animal procedures were approved by the Commission of Chongqing Medical University for ethics of experiments on animals, conducted in accordance with international standards.
F, female; M, male; TN, temporal neocortex; l, left; r, right; n, normal.
Tissue processing For comparison, we obtained 12 histological normal temporal neocortex samples from individuals who underwent therapeutic surgical resection for increased intracranial pressure due to head trauma (7 males and 5 females with a mean age of 29.50 6 6.46 years; range: 19–40 years). All control patients had no history of epilepsy or exposure to antiepileptic drugs (AEDs). Brain samples from controls were taken soon after head trauma. A neuropathological examination revealed no signs of central nervous system disease. Table II shows the clinical features of the controls. There are no significant differences in age, sex, between TLE patients and controls (P > 0.05). Rat model of epilepsy A rat model of Lithium–pilocarpine-induced seizures was used in our study. Healthy 6–8-week-old male Sprague–Dawley rats (n 5 49), weighing 200– 250 g, were obtained from the Laboratory Animal Center of Chongqing Medical University. These animals were housed and maintained under a standard environmental condition with 12-h light/dark cycle, 22 6 1 C temperature, 55 6 5% humidity and given free access to food and water. All rats were randomly divided into the normal control group (n 5 7) or the experimental group (n 5 42). The experimental group was randomly divided into six subgroups (n 5 7 per subgroup) after onset of status epilepticus (SE): 6, 72 h, 7, 14, 30, and 60 days. The experimental rats were injected with lithium chloride (127 mg/kg, i.p., Sigma-Aldrich). 18 h later, atropine sulfate (1 mg/kg, i.p., Sigma) was administered to reduce the peripheral cholinomimetic effects of pilocarpine. Thirty minutes later, pilocarpine (50 mg/kg, i.p., SigmaAldrich) was injected to induce SE. Pilocarpine (10 mg/kg, i.p.) was given repeatedly every 30 min until the rats developed seizures. Diazepam (10 mg/kg, i.p., Sigma) was administered to terminate convulsions sixty minutes after the onset of the SE. The evoked seizures were classified according to Racine’s scale Synapse
For both human and animal tissue, one portion of these brains was frozen in liquid nitrogen, and stored at 280 C for Western blot analysis. Other portions of the resected brain tissues were fixed in 4% paraformaldehyde, frozen, and sectioned at 10 lm for double-immunofluorescence labeling analysis, or embedded in paraffin and sectioned at 5 lm for immunohistochemical analysis. One section from every embedded specimen was stained with hematoxylin–eosin (H&E). Double-immunofluorescence labeling Frozen tissue sections from humans and rats were air dried. Antigen recovery was performed by treating the sections with 10mmol/l sodium citrate buffer (pH 6.0) and heated with a microwave oven for 20 min at 92–98 C. Tissues were permeabilized with 0.5% Triton X-100 and then incubated in 5% goat serum (Zhongshan Golden Bridge, Beijing, China) for 1 h at room temperature. Tissue sections were then incubated with a mixture of polyclonal rabbit antitotal c-Abl antibody (1:100, catalogue number 2862; Cell signaling) and mouse antineuron specific enolase (NSE) antibody (1:50, Zhongshan Golden Bridge Inc, Beijing, China) or with a mixture of polyclonal rabbit antitotal c-Abl antibody and mouse antiglial fibrillary acidic protein (GFAP) antibody (1:50, Wuhan Boster Biological Technology, Wuhan, China) at 4 C overnight. For Abl-pY412 immunofluorescence, antitotal c-Abl antibody was replaced with a polyclonal rabbit anti-AblpY412 antibody (1:100, Bioss, Beijing, China). After washing in PBS, sections were incubated with fluorescein isothiocyanate-conjugated goat antirabbit IgG (1:50, Zhongshan Golden Bridge, Beijing, China) and tetramethylrhodamine isothiocyanate-conjugated goat antimouse IgG (1:50, Zhongshan Golden Bridge) in the dark for 1 h at room temperature. After rinsing, sections were coverslipped with 50% glycerol/PBS. Fluorescent-stained sections were evaluated by laser scanning confocal microscopy (Leica Microsystems Heidelberg GmbH, Wetzlar, Germany) on an Olympus IX 70 inverted microscope (Olympus, Tokyo, Japan) equipped with a Fluoview FVX confocal scan head.
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Immunohistochemistry analysis After deparaffinized in xylene and then rehydrated in graded ethanol, sections were incubated with 0.3% H2O2 for 10 min. Antigen recovery performed as described for immunofluorescence. Normal goat serum (Zhongshan Golden Bridge, Beijing, China) was used to block nonspecific binding for 30 min at room temperature. Sections were then incubated with the primary rabbit polyclonal antitotal c-Abl antibody (1:100, catalogue number 2862, Cell signaling) or polyclonal rabbit anti-Abl-pY412 antibody (1:100, Bioss, Beijing, China) at 4 C overnight. After washes with PBS, sections were incubated with biotinylated goat antirabbit secondary antibody (streptavidin–peroxidase kits, Zhongshan Golden Bridge, Beijing, China) for 30 min. Washed and incubated with an avidin–biotin peroxidase complex (Zhongshan Golden Bridge, Beijing, China) at 37 C for 30 min, The sections were then incubated with 3,3-diaminobenzidine (DAB, Zhongshan Golden Bridge, Beijing, China) for 5 min. Counterstaining was performed with Harris hematoxylin. For negative control, the primary antibody was replaced with PBS. Images were acquired by an OLYMPUS PM20 automatic microscope (Olympus, Osaka, Japan) and a TC-FY2050 pathology system (Yuancheng, Beijing, China). Ten visual fields were obtained randomly in each section. Image-Pro plus 5.0 software (Media, Cybemetrics) was used for the quantitative analysis of c-Abl expression. Mean optical density (OD) of each vision field was automatically measured by computer. Western blot analysis Total proteins were extracted from homogenated rat and human brain tissues using the whole protein extraction kit (Keygen Biotech, Nanjing, China). Protein concentrations were determined using the enhanced BCA protein assay kit (Beyotime, Haimen, China), according to the manufactures’ instructions. 50 mg protein was loaded into each gel lane and separated by 6% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), electrotransferred on to an Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore Corporation) using an electrophoretic transfer system (Bio-Rad Laboratories). The PVDF membrane was then incubated in 5% skim milk/Tween-20–Tris-buffered saline for 60 min at 37 C. The membranes were incubated overnight at 4 C with the following primary antibodies: rabbit antitotal c-Abl antibody (1:1000, #2862, Cell signaling), rabbit anti-Abl-pY412 antibody (1:300, Bioss, Beijing, China), rabbit anti-b-actin antibody (1:4000, Beijing 4A Biotech, Beijing, China) as internal control. Washed in Tween-20–Tris-buffered saline (TBST) three times, the membranes were then incubated with horseradish peroxidase-conjugated secondary
antibody (1:4000, goat antirabbit IgG-HRP, Zhongshan, Beijing, China) for 1 h at room temperature. An enhanced chemiluminescence kit (Pierce, Rockford, IL) and a charge-coupled device camera (BioRad Laboratories) were used to visualize the resulting protein bands, digitally scanned immune blots were quantified using Quantity One software (BioRad Laboratories). Band immune intensity ratio of cAbl and corresponding b-actin at the same time of electrophoresis were analyzed (Gassmann et al., 2009). The band intensity ratio of c-Abl relative to bactin (c-Abl/b-actin) was analyzed. Statistical analysis Data were expressed as mean 6 standard deviation (SD). Statistical analysis of the differences between the epileptic group and control group were conducted using Student’s t test (SPSS16.0). Differences among groups of experimental rats were assessed by oneway ANOVA analysis combined with Tukey’s HSD post hoc multiple comparison test (SPSS16.0), P values of less than 0.05 was considered statistically significant. RESULTS Neuronal localization of c-Abl To determine cell-type specific expression of c-Abl, we performed double-immunofluorescence staining. We found nonphosphorylated total c-Abl expressed exclusively in neurons of temporal neocortex samples from nonepileptic controls and patients with TLE, as shown by colocalization with the neuronal marker, NSE (Fig. 1A). Whereas GFAP1 astrocytes were not stained (Fig. 1B), indicating that total c-Abl was not expressed in astrocytes. Increased total c-Abl protein expression in the temporal neocortex of patients with TLE Immunohistochemistry images showed that total cAbl protein was expressed in the membrane and cytoplasm of neurons in the temporal neocortex of TLE patients and nonepileptic controls. Faint immunoreactivity for c-Abl was present in control cortices, whereas strong c-Abl staining was observed in TLE samples (Fig. 1C). No immunoreactivity was seen in negative controls in which the primary antibody had been omitted (data not shown). The mean OD value of c-Abl protein in the temporal neocortex of patients with TLE was significantly higher than that of the nonepileptic controls (P < 0.05) (Fig. 1C). To further verify the elevated c-Abl immunostaining observed in epileptic human brain sections, we performed western blot analysis. c-Abl protein immunoreactive bands were detected at about 135 kDa, and b-actin immunoreactive bands were seen at 42 kDa. Expression of c-Abl was evaluated in the temporal neocortex of all patients with TLE and Synapse
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Fig. 1. Double-immunofluorescent labeling, IHC, and Western blot analysis for total c-Abl in the temporal neocortex of patients with TLE and nonepileptic controls. A: Double-immunofluorescent labeling showed that c-Abl (green) and NSE (red) were coexpressed (merged) in the temporal neocortex of an epileptic patient. White arrows, c-Abl 1/ NSE1 cells. B: c-Abl (green) and GFAP (red) were not coexpressed (merged). White arrow, c-Abl1 cell; blue arrow, GFAP1 cell. C: IHC analysis for c-Abl in the temporal neocortex of humans indicated slight immunoreactive staining of c-Abl in the temporal neocortex of a control subject compared with strong immunoreactive staining of c-Abl in the
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temporal neocortex of a patient with TLE. Black arrows, c-Abl1 cells: the black scale bars indicate 75 mm. Comparison of the mean OD value (right) indicated significantly higher expression of c-Abl in the TLE group than that in the control group. *P < 0.05. D: Western blotting analysis for c-Abl in the temporal neocortex of humans. Proteins from individual brain homogenates from TLE and control subjects were separated with gradient SDS-PAGE. c-Abl was more strongly expressed in patients with TLE than that in controls. A comparison of the intensity ratio (right) indicated significantly higher expression of c-Abl in patients with TLE than that in controls. *P < 0.05.
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nonepileptic controls. Temporal neocortex specimens from TLE patients showed higher c-Abl immunoreactivity when compared with normal brain tissues from nonepileptic controls (Fig. 1D). The difference in the mean OD ratio between the TLE group and control group was statistically significant (P < 0.05) (Fig. 1D). Dynamic expression of total c-Abl in the hippocampus and adjacent cortex in the epileptic rats To rule out the possibility that altered c-Abl expression in patients with epilepsy may be caused by antiepileptic drugs, we performed an animal experiment using a rat model of epilepsy. c-Abl was expressed in neurons of the hippocampus and adjacent cortex from epileptic rats and controls, as shown by colocalization with the neuronal marker, NSE (Figs. 2A and 3A), cAbl positive cells were not colocalized with GFAP1 astrocytes (Fig. 2B), indicating that c-Abl was not expressed in astrocytes of rat hippocampus and adjacent cortex. c-Abl positive signals were detected in the membrane and cytoplasm of neurons of rat hippocampus and adjacent cortex (Figs. 2C and 3B), In the epileptic groups, stronger staining for c-Abl was observed in neurons of the dentate gyrus, CA1, and CA3 regions of hippocampus (Fig. 3B), and adjacent cortex (Fig. 2C,). In the cortex, IHC analysis showed that the mean OD value of each time point after pilocarpine induced seizure was higher than that of the control group (P < 0.01). A striking increase of c-Abl protein expression was found at 72 h (P < 0.01), at 7 days (P < 0.01), and at 14 days (P < 0.01) (Fig. 2C). In the hippocampus, the striking elevation of c-Abl protein was also observed at 72 h (P < 0.01), 7 days (P < 0.01) and 14 days (P < 0.01) (Fig. 3B), whereas the mean OD value returned to control level at 60 days. The western blot analysis result was in accordance with that of IHC: c-Abl upregulation in the cortex of epileptic rats appeared at 6 h after seizures, reached a peak level at 14 days, and was maintained at a relatively high level until 60 days (Fig. 2D). In the hippocampus, c-Abl expression elevated from 6 h to 30 days postseizures with the peak level at 72 h, the expression then returned to control level at 60 days (Fig. 3C). Semiquantitative densitometric analysis revealed that the difference in the c-Abl expression levels between control and each time point after seizures was significant (P < 0.01), except that between control and 60 days in the hippocampus (P > 0.05). Prominent expression of activated c-Abl in epileptic patients and rats Phosphorylation of c-Abl on tyrosine residue 412 has been shown to be important for the kinase activity (Reynolds et al., 2000). Therefore, we determined
the expression of activated c-Abl using phosphospecific antibody (Abl-pY412). We found that AblpY412 expressed in neurons but not in astrocytes of brain specimens from humans and rats, as shown by colocalization with the neuronal marker, NSE (Fig. 4A), but not colocalization with the astrocyte marker, GFAP (Fig. 5A). Abl-pY412 was detected in the neuronal cytoplasm and membrane in the temporal neocortex of controls and TLE patients (Fig. 4B) as well as in the hippocampus (Fig. 5B) and adjacent cortex (data not shown) of control and epileptic rats. Immunohistochemical study showed that neuronal AblpY412 immunoreactivity was stronger in patients with TLE than that in controls, which was significantly different (Fig. 4B) (P < 0.05). In the rat model, the Abl-pY412 immunoreactivity was also significantly elevated in the hippocampus (Fig. 5B) and adjacent cortex (data not shown) 60 days after seizures compared with controls. Western blotting study demonstrated that AblpY412 was significantly increased in the temporal neocortex of TLE patients than that in controls (Fig. 4C) (P < 0.05). In the rat model, Abl-pY412 levels were increased from 6 h to 60 days after seizures with the peak levels at 14 days (Fig. 5C), Semiquantitative densitometric analysis revealed that Abl-pY412 expression of each time point after seizures was significantly increased compared with the control group. (Fig 5C) (P < 0.05). DISCUSSION In the current study, we have examined the expression pattern of total and activated c-Abl in the temporal cortex of patients with TLE and in the hippocampus and adjacent cortex of a TLE rat model. We observed that both total and phosphorylated c-Abl were expressed in the neuronal membrane and cytoplasm in normal and epileptic brain tissue from humans and rats. Both total and phosphorylated cAbl were significantly upregulated in patients with TLE and epileptic rats compared to nonepileptic controls. In the rat model of epilepsy, the cortical expression of total and phosphorylated c-Abl increased from 6 h to 60 days after seizures, total c-Abl protein elevated from 6 h to 30 days and phosphorylated c-Abl was upregulated from 6 h to 60 days in the hippocampus. During epileptogenesis, neurodegeneration is one of the most common structural changes of brain (Pitk€ anen and Lukasiuk, 2011). Increasing evidence supports the participation of c-Abl in the signal cascade that regulates neuronal death in Alzheimer’s disease (AD) animal models (Alvarez et al., 2004b; Cancino et al., 2008; Wilson et al., 2004) and in Niemann Pick disease (Alvarez et al., 2008). Overexpression of c-Abl in adult mouse forebrain neurons resulted in neurodegeneration and neuronal loss, Synapse
Fig. 2. Double-immunofluorescent labeling, IHC, and Western blot analysis for total c-Abl in the temporal cortex of experimental rats. A: c-Abl (green) and NSE (red) were coexpressed (merged) in the temporal cortex of a TLE rat at 14 days after seizures. White arrow, c-Abl1/NSE1 cells. B: c-Abl (green) and GFAP (red) were not coexpressed (merged). White arrow, c-Abl1 cell; blue arrow, GFAP1 cell. C: Immunoreactive staining of c-Abl in the temporal cortex of rats. Slight immunoreactive staining of c-Abl in the cortex of control rats compared with strong staining in epileptic rats at 14 days after
seizures. Comparison of the mean OD value (right) of IHC staining between the control and epileptic rats at different time points after seizures. *P < 0.05 versus control. Black arrow, c-Abl1 cells. The black scale bars indicate 75 mm. D: Western blot analysis results from control and epileptic rats. Representative Western blot analysis images showed bands of c-Abl and b-actin (internal control) at different time points after seizures. Comparison of the mean intensity ratio (right) of immunoblotting between control and epileptic rats at each time point after seizures. *P < 0.05 versus control.
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Fig. 3. Double-immunofluorescent labeling, IHC, and Western blot analysis for total c-Abl in the hippocampus of experimental rats. A: c-Abl (green) and NSE (red) were coexpressed (merged) in the hippocampus of a TLE rat at 72 hours after seizures. White arrow, c-Abl1/NSE1 cells. B: Immunoreactive staining of c-Abl in the hippocampus of rats. Faint immunoreactive staining of c-Abl in the hippocampus (CA1 field) of control rats compared with strong staining in epileptic rats at 72 h after seizures. Comparison of the mean OD value (right) of IHC staining between the control and epi-
leptic rats at different time points after seizures. *P < 0.05 versus control. Black arrow, c-Abl1 cells. The black scale bars indicate 75 mm. C: Western blot analysis of c-Abl expression in the hippocampus of rats. Representative Western blot showed the dynamics of c-Abl expression at different time points after seizures in hippocampus, b-actin was used as an internal control. Comparison of the mean intensity ratio (right) of immunoblotting between control and epileptic rats at each time point after seizures. *P < 0.05 versus control.
particularly in the CA1 region of the hippocampus (Schlatterer et al., 2011). Moreover, c-Abl expression was increased in amyloid b-protein fibrils (Abf) injected hippocampus and the selective c-Abl inhibitor STI571 prevents the neurodegenerative changes induced by Abf (Cancino et al., 2008). It has been shown that c-Abl modulates Cdk5 kinases function during brain development (Zukerberg et al., 2000) and c-Abl activates Cdk5 in AD models (Cancino et al., 2011). The activation of c-Abl by Ab resulted in neuronal death in Drosophila through the deregulation of Cdk5 (Lin et al., 2007). c-Abl-Cdk5 signaling
activation also contributes to neuronal apoptosis induced by Enterovirus 71 (EV71) (Chen et al., 2007). Our previous study demonstrated Cdk5 upregulation in brain tissue of TLE patients (Xi et al., 2009), here, we found that total and activated c-Abl protein level elevated not only in brain tissue of patients with TLE but also in epileptic tissue of rats compared with the control groups. The Neuronal cytoplasm location of cAbl observed in our research is consistent with that found in AD brains (Jing et al., 2009). Insight into epileptogenic mechanisms has been revealed by investigating animal models of AD (Palop et al., Synapse
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Fig. 4. Double-immunofluorescent labeling, IHC, and Western blot analysis for phospho-c-Abl in the temporal neocortex of patients with TLE and controls. A: p-c-Abl (green) and NSE (red) were coexpressed (merged) in the temporal neocortex of an epileptic patient. White arrow, p-c-Abl1/NSE1 cells. B: IHC analysis for p-c-Abl in the temporal neocortex of humans. Black arrow, p-c-Abl1 cells. Slight immunoreactive staining of p-c-Abl is shown in the temporal neocortex of a control subject compared with strong staining in a TLE patient. Black scale bar 5 75 mm. Comparison of the mean OD
value (right) showed significantly higher expression of p-c-Abl in the TLE group than that in the control group. *P < 0.05. C: Western blot analysis for phospho-c-Abl in the temporal neocortex of humans. Immunoreactive stainings of p-c-Abl in patients with TLE are stronger than those in controls. Con: control. A comparison of the mean intensity ratio (right) indicated significantly higher expression of p-c-Abl in patients with TLE than that in controls. *P < 0.05.
2007), in which epilepsy can be a comorbidity. These data suggest a role of c-Abl in neurodegeneration in epilepsy. However, the mechanisms underlying this process remain unclear. For ethical consideration, we were not able to take normal human brain specimens, we used structurally normal brain samples obtained from temporal lobectomies performed for the treatment of traumatic brain injuries as control. The brain specimens of patients with TLE could only be obtained at the
drug-resistant stage of epilepsy. Thus we used lithium/pilocarpine rat model to further validate c-Abl expression found in patients with TLE. This model replicates many features of human epilepsy (Majores et al., 2007), in which an acute episode of seizures is frequently followed by a latency period with subsequent development of spontaneous recurrent seizures (Sharma et al., 2007). In our animal study, both total and activated c-Abl levels began to increase at 6 h after seizures (acute period), and maintained at a
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Fig. 5. Double-immunofluorescent labeling, IHC, and Western blot analysis for p-c-Abl in the hippocampus and adjacent cortex of experimental rats. A: p-c-Abl (green) and GFAP (red) were not coexpressed (merged) in the cortex of an epileptic rat at 14 days after seizures. White arrow, c-Abl1 cells; blue arrow, GFAP1 cells. B: Immunohistochemical staining of p-c-Abl in the hippocampus of rats. Faint immunoreactive staining of c-Abl in the hippocampus (CA1 field) of control rats compared with strong staining in epileptic rats at 14 days after seizures. Comparison of the mean OD
value (right) of IHC staining between the control and epileptic rats at different time points after seizures. Black arrow, p-c-Abl1 cells. black scale bar 5 75 mm. *P < 0.05 versus control. C: Western blot results from control and epileptic rats. Representative Western blot analysis images of p-c-Abl and b-actin (internal control) in the hippocampus at different time points after seizures. A comparison of the intensity ratio (right) of immunoblots between control and epileptic rats at each time point after seizures. *P < 0.05 versus control.
high level in latent period (7, 14 days), then continuously elevated in chronic period (30 days) in the hippocampus, which is supported by the results of Borges et al. (2003) who reported that extensive loss of neurons in the hippocampus occurred within 6 h post SE, and cell damage persisted for up to 31 days. In addition, our study showed that c-Abl immunoreactivity upregulation after seizures is predominantly limited to dentate gyrus, CA1 and CA3 subfields of hippocampus, neuronal necrosis was observed in these hippocampal regions in pilocarpine model (Clifford et al., 1987; Mello et al., 1993). These results
support the hypothesis that c-Abl plays a role in neuronal loss of epilepsy. In conclusion, our study reveals, for the first time to our knowledge, the increased expression of c-Abl protein in temporal lobe epileptic tissue of human and rat. These findings support the hypothesis that c-Abl plays a role in the pathogenesis of TLE. Because what we described was only a phenomenon, we could not conclude from current study whether c-Abl is a cause or a result of epilepsy. A better-designed experiment in the future is required to focus on the possible roles and specific mechanisms that c-Abl plays in epilepsy. Synapse
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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (No. 81071039). The authors sincerely thank the support of Beijing Tiantan Hospital, Xuanwu Hospital of the Capital University of Medical Sciences, Chongqing Xinqiao Hospital for supplying the brain tissues, the patients and their families for their participation in this study and the National Institutes of Health of China and the Ethics Committee on Human Research of the Chongqing Medical University. REFERENCES Alvarez AR, Godoy JA, Mullendorff K, Olivares GH, Bronfman M, Inestrosa NC. 2004a. Wnt-3a overcomes beta-amyloid toxicity in rat hippocampal neurons. Exp Cell Res 297:186–196. Alvarez AR, Klein A, Castro J, Cancino GI, Amigo J, Mosqueira M, Vargas LM, Ye’venes LF, Bronfman FC, Zanlungo S. 2008. Imatinib therapy blocks cerebellar apoptosis and improves neurological symptoms in a mouse model of Niemann Pick type C disease. FASEB J 22:3617–3627. Alvarez AR, Sandoval PC, Leal NR, Castro PU, Kosik KS. 2004b. Activation of the neuronal c-Abl tyrosine kinase by amyloid-betapeptide and reactive oxygen species. Neurobiol Dis 17:326–336. Brasher BB, Van Etten RA. 2000. c-Abl has high intrinsic tyrosine kinase activity that is stimulated by mutation of the Src homology 3 domain and by autophosphorylation at two distinct regulatory tyrosines. J Biol Chem 275:35631–35637. Borges K, Gearing M, McDermott DL, Smith AB, Almonte AG, Wainer BH, Dingledine R. 2003. Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model. Exp Neurol 182:21–34. Cancino GI, Perez de Arce K, Castro PU, Toledo EM, von Bernhardi R, Alvarez AR. 2011. c-Abl tyrosine kinase modulates tau pathology and Cdk5 phosphorylation in AD transgenic mice. Neurobiol Aging 32:1249–1261. Cancino GI, Toledo EM, Leal NR, Hernandez DE, Yevenes LF, Inestrosa NC, Alvarez AR. 2008. STI571 prevents apoptosis, tau phosphorylation and behavioural impairments induced by Alzheimer’s beta-amyloid deposits. Brain 131:2425–2442. Chen TC, Lai YK, Yu CK, Juang JL. 2007. Enterovirus 71 triggering of neuronal apoptosis through activation of Abl-Cdk5 signalling. Cell Microbiol 9:2676–2688. Cheung ZH, Fu AK, Ip NY. 2006. Synaptic roles of Cdk5: Implications in higher cognitive functions and neurodegenerative diseases. Neuron 50:13–18. Clifford DB, Olney JW, Maniotis A, Collins RC, Zorumski CF. 1987. The functional anatomy and pathology of lithium-pilocarpine and high-dose pilocarpine seizures. Neuroscience 23:953–968. Dorey K, Engen JR, Kretzschmar J, Wilm M, Neubauer G, Schindler T, Superti-Furga G. 2001. Phosphorylation and structure-based functional studies reveal a positive and a negative role for the activation loop of the c-Abl tyrosine kinase. Oncogene 20:8075–8084. Estrada LD, Zanlungo SM, Alvarez AR. 2011. C-Abl tyrosine kinase signaling: A new player in AD tau pathology. Curr Alzheimer Res 8:643–651. Gassmann M, Grenacher B, Rohde B, Vogel J. 2009. Quantifying western blots: Pitfalls of densitometry. Electrophoresis 30:1845– 1855. Imam SZ, Zhou Q, Yamamoto A, Valente AJ, Ali SF, Bains M, Roberts JL, Kahle PJ, Clark RA, Li S. 2011. Novel regulation of Parkin function through c-Abl-mediated tyrosine phosphorylation: Implications for Parkinson’s disease. J Neurosci 31:157–163. Jing Z, Caltagarone J, Bowser R. 2009. Altered subcellular distribution of c-Abl in Alzheimer’s disease. J Alzheimers Dis 17:409–422.
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