Increased Expression of Rac1 in Epilepsy Patients and Animal Models
Jie Li1 • Hongxia Xing1 • Guohui Jiang2 • Zhou Su1 • Yuqing Wu1 • Yi Zhang1 • Shuangxi Guo1
Abstract
The mechanisms of epilepsy remain incompletely understood. Rac1 (ras-related C3 botulinum toxin substrate 1) belongs to the Rho family of small GTPases. Rac1 play important roles in cytoskeleton rearrangement and neuronal synaptic plasticity, which had also been implicated in epilepsy. However, little is known regarding the expression of Rac1 in the epileptic brain or whether Rac1-targeted interventions affect the progression of epilepsy. The aim of this study was to investigate the expression profile of Rac1 in brain tissues from patients suffering from temporal lobe epilepsy (TLE) and experimental epileptic rats and determine the possible role of Rac1 in epilepsy. We demonstrated that the expression of Rac1 is significantly increased in TLE patients and in lithium-pilocarpine epilepsy model animals compared to the corresponding controls. Rac1 inhibitor NSC23766 reduced the severity of status epilepticus during the acute stage in a lithium-pilocarpine animal model. Consistent with these results, the latent period of a PTZ kindling animal model also increased. Our results demonstrated that the increased expression of Rac1 may contribute to pathophysiology of epilepsy.
Keywords Epilepsy Rac1 Temporal lobe epilepsy
Introduction
Epilepsy is a common neurological disease characterized by the recurrent appearance of spontaneous seizures. Although there are more than 20 types of antiepileptic drugs (AEDs), approximately 30 % of newly diagnosed patients will develop intractable epilepsy [1, 2]. However, to date, the mechanisms of epilepsy are still not fully understood. Many studies showed neurogenesis, gliosis, axonal sprouting and dendritic plasticity participate in epileptogenesis [3]. Abnormal synapses connect between neurons provide the anatomic basis for epilepsy [4]. Molecules involved in the regulation of cytoskeletal proteins play important roles in these dynamic changes during epileptogenesis.
Rac1 (ras-related C3 botulinum toxin substrate 1) is a small guanosine triphosphatase (GTPase) belonging to the Rho family of small GTPases and plays an important role in neuronal synaptic plasticity, cytoskeletal rearrangement, intercellular adhesion and gene expression [5–8]. Rho family proteins act as molecular switches that cycle between the inactive GDP-bound form and an active GTPbound form. The activated GTP-bound Rac1 (Rac1-GTP) interacts with downstream effector proteins and elicits a vast range of cellular effects [9].
It is known that Rac1 regulates dendritic spine density and shape in rat and mouse pyramidal neurons [10]. In neurons, genetic approaches suggest Rac1 is required for axon outgrowth, guidance, and branching [11, 12]. Rac1 stimulates the kinase activity of Lim kinase-1 (Limk1), which leads to filopodia formation and axonal extension [5, 13]. Conditional deletion of Rac1 in ventricular zone progenitors has indicated a role for Rac1 in axon guidance while axonal outgrowth is not affected [14]. Double knockouts for Rac1 and Rac3 GTPases correlated with a large decrease of the hilar mossy cells, an important class of excitatory neurons targeting dentate granule cells [15]. Extensive loss of hilar mossy cells has been reported in patients with temporal lobe epilepsy [16]. Indeed, Rac1 has also been associated with long-term potentiation in hippocampal slices and plays a role in synaptic plasticity by modulating NMDA receptors [17]. Loss of Rac1 resulted in severe impairment of long-term potentiation (LTP) in both CA1 and dentate gyrus neurons. Selective elimination of Rac1 in excitatory neurons in the forebrain in vivo not only affects spine structure, but also impairs synaptic plasticity in the hippocampus [18].
These studies suggested that Rac1 may play an important role in the formation of abnormal neuronal networks and synaptic plasticity via regulation of cytoskeletal reorganization. Here, we investigated the expression profile of Rac1 in epilepsy and demonstrated that Rac1 is involved in the process of epilepsy. Rac1 inhibitor NSC23766 was also used to determine the effect of Rac1 on behavioral changes.
Materials and Methods
Patients and Clinical Data
Twenty-four brain tissue samples of intractable temporal lobe epilepsy were randomly selected from our epilepsy brain bank, which had been reported in our previous studies [19, 20]. The seizure types were classified according to the International League Against Epilepsy (ILAE) classification. All the patients were refractory to maximal tolerable doses of at least two AEDs, including carbamazepine, valproic acid, phenobarbital, phenytoin, oxcarbazepine, topiramate, clonazepam, gabapentin or lamotrigine. The clinical data of each patient is presented in Table 1 (supplementary material). Intraoperative electrocorticogram was used during the surgical procedure to identify the epileptogenic focus for subsequent resection. Pathological findings of the epileptic group from the resected tissue included neuronal loss and gliosis.
For comparison, 12 histologically normal tissue samples selected randomly from our brain bank were used as controls. Histological examination of all specimens showed normal. All the patients of control group were suffered from head trauma (Table 2, supplementary material). These subjects reported no history of epilepsy and were not exposed to any AEDs. All experimental protocols in this study were approved by the National Institutes of Health and the Committee on Human Research at Chongqing Medical University. Informed consent was obtained from each patient for the use of brain tissue and for access to medical records for research purposes. Lithium-Pilocarpine Epilepsy Model
Adult male Sprague–Dawley rats weighing approximately 190–230 g were used for animal experiments. We obtained the animals from the Experimental Animal Center of Chongqing Medical University, China. All animals were raised in a temperature-controlled room with a 12-h light– dark cycle and were given free access to standard water and food. The animals were randomly separated into the control group (n = 5) or the experimental group (n = 35). The experimental group was randomly separated into 7 subgroups that differed according to the time point after onset of status epilepticus: 6, 24, 72 h, 1, 2 weeks, 1 and 2 months (chronic epilepsy state with spontaneous recurrent seizures, most of the first spontaneous recurrent seizures occurred in 3–4 weeks). Pilocarpine-induced seizures were classified according to Racine’s standard criteria [21]. The experimental animals were injected intraperitoneally (i.p.) with lithium chloride (127 mg/kg, Sigma-Aldrich, USA) 20 h before the first pilocarpine administration (40 mg/kg, i.p., Sigma-Aldrich, USA). Pilocarpine (10 mg/ kg, i.p.) was administered repeatedly every 30 min until the rats developed seizures. Only rats that exhibited stages 4 or 5 according to Racine’s scale were used for this experiment. Diazepam (10 mg/kg, i.p., Sigma-Aldrich, USA) was administered 1 h after the onset of status epilepticus. The animals in the control group were i.p. injected with the same volume of saline.
Pentylenetetrazole (PTZ) Chronic Kindling Model
Animals were intraperitoneally injected with subconvulsive doses of PTZ (35 mg/kg, Sigma-Aldrich, USA) diluted in saline between 8:00 and 10:00 a.m. everyday as described by Maciejak et al. [22]. After each PTZ injection, convulsive behavior was monitored for at least 30 min and scored according to Racine’s criteria [21]. The animals were considered to be kindled after experiencingfour consecutive Class 4 or 5 seizures.
Surgical Procedures and Drug Injection
The animals were anesthetized via i.p. injection of chloral hydrate (0.35 g/kg, Sigma-Aldrich, USA). A midline skin incision was performed under a stereotaxic apparatus (Stoelting, Wood Dale, USA) to expose the dorsal surface of the skull. Then a 1 mm hole was drilled in the skull at the following stereotaxic coordinates: 1.0 mm posterior to Bregma, 1.5 mm left to the midline, according to Paxinos and Watson [23]. A guide cannula (O.D: 0.64 mm/I.D: 0.45 mm, RWD Life Science Co., Ltd; Shenzhen, China) was implanted into the right lateral ventricle (3.5 mm below the skull surface). A matched cannula core was screwed to the guide cannula, which could be removed at the time of injection. All animals were maintained at 36.5–38.5 C until fully awake and allowed to recover from surgery for 7 days prior to the experimental manipulations. NSC23766 (15 ll/ kgdilutedto 50 lMindistilleddeionized water, Tocris,UK) was intracerebroventricularly injected 1 h before pilocarpine or PTZ administration using a 5 ll Hamilton syringe (Hamilton, Bonaduz, Switzerland). NSC23766 was injected slowly (0.5 ll/min) into the lateral ventricle, and the needle was kept in the brain for 1 min after the drug injection to allow for diffusion of the drug from the needle tip. Animal Behavior Investigations
In the lithium-pilocarpine epilepsy model, during the acute stage, we recorded the seizure class and the latent period as described previously [24]; the latent period was defined as the time from the injection of pilocarpine to the first onset of the class 4 or 5 seizure. During the chronic period of the pilocarpine epilepsy model, the behavior of the animals was recorded using a closed-circuit video system 24 h per day to detect Class 4 and 5 seizures through the 6th week. The animals were provided free access to water and food. The video system used an infrared camera for video recording of animal activity during the dark periods. For every animal, class 4 or 5 seizure times were accumulated for 1 week. The latent period of the PTZ kindling model was defined as the period from the day of PTZ injection to the day in which four consecutive Class 4 or 5 seizures occurred.
Tissue Preparation
The animals were sacrificed using 3.5 % chloral hydrate (350 mg/kg, i.p.) at 6, 24, 72 h, 1, 2 weeks, 1 or 2 months after the onset of status epilepticus. And the hippocampus was removed quickly using RNase-free instruments. Both the human and animal tissues were immediately placed in liquid nitrogen and later used for protein extraction.
Western Blot
Western blot analysis of Rac1expression was performed as described previously [25]. Total protein was extracted according to the manufacturer’s instructions (Keygen Biotech, Nanjing, China). BCA Protein Assay Kits (Beyotime Institute of Biotechnology, Shanghai, China) were used to determine the protein concentrations. Then, 50 lg of total protein was loaded and separated via SDS-PAGE (5 % spacer gel; 10 % separating gel). Electrophoresis was performed for 50 min at 80 V. The protein was then electrotransferred to a polyvinylidene fluoride membrane (PVDF; pore size 0.22 lm, Millipore) at 250 mA for 120 min. Equivalent protein loading and transfer were confirmed via Ponceau S staining of the membranes. The PVDF membrane was then incubated at 37 C for 60 min in 5 % skim milk to block nonspecific binding. The primary antibodies used were mouse anti-Rac1 (1:1000, Millipore Corp., Temecula, CA, USA) and rabbit antiGAPDH (1:4000, Beijing 4A Biotech Co., Ltd., Beijing, China), which were incubated at 4 C overnight. After washing with Tris-buffered saline-20 % Tween-20 (TTBS) three times (15 min each), the membranes were incubated in a horseradish peroxidase-conjugated secondary antibody (1:4000, Zhongshan, Beijing, China) for 60 min at 37 C and washed with TTBS three times (15 min each). The resulting protein bands were visualized using an enhanced chemiluminescence substrate kit (Beyotime Institute of Biotechnology, China) followed by digital scanning (BioRad Laboratories). The resulting pixel density was quantified using Quantity One software (Bio-Rad Laboratories Inc., CA, USA) [26].
Measurements of Rac1 Activation
Activated Rac1 (Rac1-GTP) was measured using a Rac1 activation Assay Kit (Upstate, Millipore, Billerica, MA) according to the manufacturer’s protocol. We performed the pulldown assay using PBD-GST (p21-binding domain from PAK), which specifically binds to GTP-bound forms of Rac1. Bound small GTPases were separated by 15 % SDS-PAGE and detected by immunoblotting with antibodies against Rac1 [17].
Statistical Analysis
All dataare presented as the mean ± SEM. Statistical analysis was performed using SPSS 16.0 statistical software. Student’s t test was used for statistical analysis of two different groups. One-way analysis of variance (ANOVA) analysis followed by a post hoc Bonferroni’s test was used to determine the differences between multiple groups. Values of p\0.05 were considered to be statistically significant.
Results
Comparison of Clinical Characteristics
There were no significant differences in age (p[0.05) or gender (p[0.05) between the epilepsy patients and the control subjects. In this study, the mean age of the epilepsy patients was 27.38 ± 1.63 years, with 14 males and 10 females in the experimental group. The control group had a mean age of 26.92 ± 2.30 years and consisted of seven males and five females. All of the patients had taken three or more AEDsand had reported at least a 3-year history of seizure recurrence.
Increased Expression of Rac1/Rac1-GTP in the Lithium-Pilocarpine Induced Epilepsy Model and Human TLE
We examined the expression of Rac1and Rac1-GTP via Western blot in TLE patients and the epilepsy model rat brain tissue (Fig. 1a). Based on Western blot analysis, the expression of Rac1 was significantly increased in the brain tissue from the TLE patients compared to those from the control subjects (Fig. 1b). In the pilocarpine-induced epilepsy animals, Rac1 expression was increased from 6 h to 2 months after pilocarpine-induced status epilepticus (chronic stage with spontaneous recurrent seizure) compared to the control animals (p\0.05; Fig. 1c, d). The activated form of Rac1 (Rac1-GTP) was also examined using Western blot. We found the expression of Rac1-GTP in TLE group was significantly higher compared with control group. One-way ANOVA analysis of revealed a statistically significant difference in the expression of Rac1-GTP between the control and epileptic groups in the pilocarpine animal model (p\0.05; Fig. 2a–d).
Behavioral Changes After Administration of Rac1 Inhibitor NSC23766
Spontaneous seizures typically occur frequently in pilocarpine epilepsy model animals but are seldom detected in the kindling rodent epilepsy model [27, 28]. Therefore, these two epilepsy models were used to explore the effect of Rac1 expression on epileptogenesis. Intracerebroventricular injection of NSC23766 was performed to inhibit the expression of activated Rac1-GTP in vivo. Western blot analysis revealed that the expression of Rac1-GTP was significantly decreased6 h after NSC23766 administration compared to control rats (p\0.05; Fig. 2e–f).
For the pilocarpine-induced epilepsy model, as shown in Fig. 3, the NSC23766-treated rats exhibited significantly attenuated seizure classes and prolonged latencyafter pilocarpine injection (Fig. 3a, b). We also found that the frequency of spontaneous seizures was significantly decreased in the NSC23766-treatedrats compared to the control group during the chronic stage of the pilocarpine epilepsy model (6th week after pilocarpine administration) (p\0.5; Fig. 3c). To confirm the effect of NSC23766 on seizures, additional 2 groups of pilocarpine-induced spontaneous epilepsy model were used. NSC23766 was intracerebroventricularly injected at the 6th week after pilocarpine administration. We also found that SRSs times in NSC23766-treated group significantly decreased compared with the control group during the chronic stage (p\0.5; Fig. 3d). For the PTZ chronic kindling model, the seizure class of the NSC23766-treated group was also significantly different compared to the control groups at the time points of 5, 6, 8–10, 12–15 and 17 days (p\0.05; Fig. 3e). The number of days required to be fully kindled was markedly increased in the NSC23766-treated group. The NSC23766-treated rats exhibited markedly prolonged kindled duration in response to repeated PTZ injection (Fig. 3f). These data suggest that down-regulation of activated Rac1 decreased the susceptibility of rats to PTZkindled seizure.
Discussion
In the present study, we detected increased Rac1 and Rac1GTP expression in brain tissue from TLE patients and epilepsy model rats compared to controls. We also found that administration of NSC23766 alleviated the severity of status epilepticus during the acute stage and decreased the SRS frequency during the chronic stage of the lithiumpilocarpine model. Consistent with this result, the latent period of the PTZ kindled seizure model was also increased due to administration of NSC23766.
We used the lithium-pilocarpine model to investigate the expression levels and pattern of Rac1 because this model is the classical model for exploring the mechanism of epilepsy [29]. A recent study by Dang et al. [30] has reported that Rac1 was significantly up-regulated in the PTZ group and played a role in mossy fibers sprouting. Another study also showed Rac1 mRNA levels was significantly high on Days 3, 14 and 28 in a kainic acidinduced rat epilepsy model [31]. As epilepsy is a complicated disease, two distinct epilepsy models are used in this experiment. We demonstrated that down regulation of Rac1 exerts prolonged seizure-suppressive effects. The NSC23766-treated rats exhibited markedly prolonged latent periods in response to repeated PTZ injection. Additionally, the frequency of spontaneous seizures was significantly decreased in the NSC23766-treated rats compared to the control groups during the chronic stage of the pilocarpine-induced epilepsy model. Previous studies have demonstrated that Rho-kinase inhibitor Y-27632 significantly suppressed the percentage of the tonic convulsion index in the maximal electroconvulsive shock model and prolonged the onset of PTZ seizures in the acute PTZ seizure test group. They also found that repeated administration of Y-27632 prevented the development of PTZ kindling by reducing the mean seizure stage [32]. However, while we observed this phenomenon, the exact mechanism is still not understood.
TLE is characterized by recurrent spontaneous seizures which originate in brain structures such as the hippocampus. The abnormal structure of hippocampal disturbed the balance of excitability and inhibitory and caused refractory spontaneous epileptic activity [33, 34]. Rac1 drives actin polymerization and is an important integrator of signals from integrins and growth factor receptors [35]. Actin remodeling may be particularly important for the establishment and structural modification of dendritic spines on which the great majority of excitatory synapses are formed in the mammalian CNS [36, 37]. Given the importance of Rac1 in the actin cytoskeletal organization, we hypothesize that up-regulation of Rac1/Rac1-GTP might participate in the regulation of cytoskeletal reorganization and be involved in epileptogenesis. This abnormal hippocampal structure disturbs the balance of excitation and inhibition, causing refractory spontaneous epileptic activity [38, 39].
Our study showed that up-regulation of Rac1/Rac1-GTP may be a consequence rather than a cause of the epileptic seizures for the following reasons. First, the expression of Rac1/Rac1-GTP was higher compared to control groups after status epilepticus in the acute stage. It was the higher expression of Rac1/Rac1-GTP that caused spontaneous recurrent seizure in the chronic stage. Second, the expression did not further increase after spontaneous epilepsy. Third, when Rac1 inhibitor NSC23766 was administered, the animals also exhibited behavioral changes indicating attenuated epilepsy.
In conclusion, the expression of Rac1 is up-regulated in a pilocarpine animal model and human TLE. Selective inhibition of Rac1 decreased spontaneous seizure times in the chronic stage of epilepsy. These reports suggested that Rac1/Rac1-GTP plays an important role in the process of epilepsy. Future studies should focus on animal experiments to determine the mechanisms by which Rac1 is involved in epilepsy.
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