Rho-associated Protein Kinase 2 Confers Epileptogenesis through the Activation of Astroglial Stat3 Pathway

Patients with temporal lobe epilepsy (TLE) are prone to tolerance to antiepileptic drugs. Based on the perspective of molecular targets for drug resistance, it is necessary to explore effective drug resistant genes and signaling pathways for the treatment of TLE. We performed gene expression proles in hippocampus of patients with drug-resistant TLE and identied ROCK2 as one of the 20 most signicantly increased genes in hippocampus. In vitro and in vivo experiments were performed to identify the potential role of ROCK2 in epileptogenesis. In addition, the activity of Stat3 pathway was tested in hippocampal tissues and primary cultured astrocytes. The expression levels of ROCK2 in the hippocampus of TLE patients were signicantly increased compared with the control group, which was due to the hypomethylation of ROCK2 promoter. Fasudil, a specic Rho-kinase inhibitor, alleviated epileptic seizures in the pilocarpine rat model of TLE. Furthermore, ROCK2 activated the Stat3 pathway in pilocarpine-treated epilepsy rats, and the spearman correlation method conrmed that ROCK2 is associated with Stat3 activation in TLE patients. In addition, ROCK2 was predominantly expressed in astrocytes during epileptogenesis, and induced epileptogenesis by activating astrocyte cell cycle progression via Stat3 pathway. The overexpressed ROCK2 plays an important role in the pathogenesis of drug-resistant epilepsy. ROCK2 accelerates astrocytes cell cycle progression via the activation of Stat3 pathway likely provides the key to explaining the process of epileptogenesis. 1% fungizone and 0.04% gentamicin]. The rst change ofmedium was performed after 24 h of culture. During the rst week, the medium change occurred once every 2 days and, from the second week, once every 4 days. From the third week onwards, the cells received medium supplemented with 20% FBS 23, 24 . Around the third to fourth weeks, cells reached conuence and were used for the experiments. The purity of the primary astrocyte cultures was assessed by immunocytochemistry for GFAP. 36 . The reactive astrocytosis may facilitate the development of epileptic seizures 37 . The cells of the high ROCK2 expression were found in pyramidal neurons of the hippocampus and cerebral cortex and Purkinje cells of the cerebellum 10 . By immuno-uorescence, ROCK2 was mainly localized in the neurons in our study. A previous study has shown that RhoA activation in the cortex and hippocampus 24 h after traumatic brain injury-related seizures 4 . In this study, we observed the expression of ROCK2 in hippocampal astrocytes in the Li-Pilo model. GFAP is the main intermediate lament protein in mature astrocytes. It has been demonstrated that GFAP-immunoreactive reactive astrocytes exhibited markedly increased RhoA expression in response to spinal cord injuries after kainic acid treatment 38 . We show reactive astrocyte co-localization with ROCK2 expression in the Li-Pilo-treated hippocampus,which is consistent with the Rho-ROCK pathway induces the generation of a reactive astrogliosis 39 . However, the expression of ROCK2-positive neurons is not induced by Li-Pilo treatment, indicating the increased expression of ROCK2 may be associated with pathological changes predominantly in astrocytes. components, and in ROCK2-dependent activation of 43, 44 . The transcription factor Stat3 is known to have important roles in regulating gene expression, specically increasing genes important to cell proliferation and cell cycle progression 31, 45 . In this study, we determined how ROCK2 induces epilepsy via Stat3 pathway, and found Stat3 target genes, Myc and Cyclin D1, were signicantly induced by ROCK2. Further, ROCK2 increased the occupancies of Stat3 on Myc and Cyclin D1 promoters, and accelerated astrocyte cell cycle progression. The CDK inhibitor protein p21 and p15 are important downstream targets of Myc and critical for cell cycle inhibition 46, 47 . We show here that p21 and p15 were further suppressed by ROCK2 activation of Myc via Stat3 pathway. Thus, we show ROCK2 induces astrocyte cell cycle progression in regulation of Stat3 pathway, and further contribute to the development of epileptic seizures. These experiments may explain, on one hand, the requirement of Stat3 for ROCK2-mediated activation of astrogliosis and, on the other, the ability of ROCK2 to provide a second input of cell cycle regulation for epileptogenesis in addition to the regulation of actin cytoskeleton dynamics and glutamatergic synaptic function as mentioned before 48, 49 . Rho-associated coiled-coil forming protein kinase; PTZ, Pentylenetetrazole; MES, Maximal electroconvulsive shock; IHC, Immunohistochemistry; CDK, cyclin-dependent kinase; AEDs, Anti-epileptic drugs; SD, Sprague Dawley; EEG, Electroencephalography; SE, Status epilepticus; FBS, Fetal bovine serum; NeuN, Neuronal nuclei; GFAP, Glial brillary acidic protein; HRP, horseradish peroxidase; qPCR, Quantitative PCR; ChIP, Chromatin immunoprecipitation; FLAIR-MRI, Fluid-attenuated inversion recovery magnetic resonance imaging; FDG-PET; Fluorodeoxyglucose-positron emission tomography; GTCS, Generalized tonic-clonic seizures.


Introduction
The Rho GTPases belong to the Ras superfamily of 20 to 30 kDa GTP-binding proteins that have been involved in the regulation of a broad spectrum of cellular functions 1 . Rho activation inhibits neurite outgrowth and promotes growth cone collapse and axon retraction 2 . RhoA is a member of the Rho GTPases and is abundantly expressed in cells of the CNS 3 . Recent research indicated that kainic acid-induced seizures result in bilateral RhoA activation in the cortex and hippocampus 4 .
ROCK, one of the most studied downstream effectors of RhoA, is a serine/threonine kinase of 160 kDa that exists in two isoforms in mammals: ROCK1 and ROCK2 5 . Both ROCK1 and ROCK2 are widely expressed in human, rat and mouse tissues [6][7][8] . ROCK1 is more prominent in liver, testes and kidney, whereas ROCK2 is more pronounced in brain 5 . Y-27632 and Fasudil are known as speci c Rho-kinase inhibitors and are clinically used to treat cerebral vasospasm following subarachnoid hemorrhage 9 .
ROCK2 immunoreactivity was observed in the pyramidal neurons of the cerebral cortex and hippocampus and in the Purkinje cells of the cerebellum 10 . ROCK activation is necessary for neurite retraction in the injured CNS 11 . Additionally, ROCK acts to reduce the degree of plasticity at hippocampal synapses during long-term potentiation 12 . Rho/ROCK pathway plays an important role in hippocampus-dependent long-term spatial memory 13 , and is even considered as a potential drug target in various neurological disorders 14 .
Recently, it has been reported that Rho/ROCK pathway plays a role in epilepsy induced by PTZ or MES 15 , and directly involved in seizure-induced cell death in vivo and in vitro 16 . In this study, we aimed to determine the expression and cellular distribution of ROCK2 in epilepsy and investigate the e cacy of Fasudil in lithium-pilocarpine induced rat model of epilepsy. Furthermore, we demonstrated that ROCK2 induces gliosis via activating Stat3 target gene Myc and Cyclin D1. Together, these observations showed that ROCK2 induces the development of epilepsy, potentially through the activation of astrocyte cell cycle progression via Stat3 pathway.

Human tissue specimens
All patients were diagnosed with TLE based on having recurrent epilepsy and being refractory to the maximal doses of at least 3 AEDs. All patients with mesial TLE with hippocampal sclerosis were treated surgically in Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University (Fig 2A.). All hippocampal specimens were randomly selected from an epilepsy brain bank. The criteria, informed consent, brain tissue processing, and how to match control groups were referenced in our previous publications [17][18][19] . The individual patient details are listed in Tables 1   and 2.  M  7  15  CPS  VPA, CBZ, TPM, LTG  LTHAR  g  3  2  WB, IHC,  MSP  27/M  7  22  CPS,SGS  VPA, LEV, LTG, TPM  RTHAR  FCD,g  6  1  WB, IHC,  MSP   26  32/F  3-4  13  CPS,SGS  VPA, LTG, LEV, OXC  RTHAR  FCD,g  8  3  WB, IHC,  MSP   27  26/M  4  19  CPS  VPA, CBZ, CZP, PHT  RTHAR  g,nl  4.5  2  WB, IHC,  MSP   28  23/F  1-3  22  SGS  LTG, OXC, LEV  LTHAR  g  5.5  1  WB, IHC,  MSP   29  27/M  7  16  CPS,SGS  VPA, LEV, LTG  RTHAR  g,nl  4  1  WB, IHC,  MSP   30  16/F  2-3  10  CPS  VPA,LTG,OXC,LEV,CZP  LTHAR  g  5  2  WB, IHC,  MSP   31  28/F  7-14  14  CPS,SGS  LTG, VPA, OXC, LEV  RTHAR  FCD,g  4   Preparation of the lithium-pilocarpine model of temporal lobe epilepsy and how animals were grouped were described in detail in our previously published articles 17, 18 . Human mRNA pro ling Blocks of hippocampus chosen randomly from the epileptic (n=8) and control (n=3) tissue groups were cut into small pieces and homogenized in Lambda. EEG electrodes were wired to a head mount that was also xed to the skull using dental cement (Fig. 4C). After 5-7 days of recovery from the surgery, EEG signals and synchronized video were recorded using the EEG-9200K (Nihon Kohden, Tokyo, Japan). The voltage differential between the pair of electrodes from each brain hemisphere were ampli ed with a high pass lter (1Hz) and recorded. EEG signals were sampled at 400 Hz and videos were recorded at 30 frames/sec. The detailed process is described in our previously published articles 21, 22 . Li-Pilo-induced SE and Behavioral grading of seizures supplemented with 20% FBS 23,24 . Around the third to fourth weeks, cells reached con uence and were used for the experiments. The purity of the primary astrocyte cultures was assessed by immunocytochemistry for GFAP.

Double immuno uorescence
Immuno uorescence was performed as described in our previously published articles 25,26 . The brain sections were incubated in a primary antibody solution containing rabbit anti-ROCK2 antibody (1:700; #PA5-78290, Invitrogen) and mouse anti-NeuN antibody (1:1000; #ab104224, Abcam) or mouse anti-GFAP antibody (1:1000; #ab10062, Abcam) that was dissolved in the rinse buffer for 12 h at 4 °C. Before secondary antibody incubation, the sections were washed 3 times for 30 min in PBS. The sections were then incubated in a secondary antibody solution containing FITC-conjugated goat anti-rabbit IgG (1:2000; #ab6717, Abcam) and Texas Red-conjugated goat anti-mouse IgG (1:2000; #ab6787, Abcam) in the rinse buffer for 4 h at 4 °C. After washing 3 times for 30 min in PBS, the sections were coverslipped with anti-fading medium.
Images of double immuno uorescence staining were obtained using a confocal laser-scanning microscope system (Nikon C2+, Tokyo, Japan).
Images were analyzed with ImageJ software (ver. 1 qPCR Extract the RNA and generate complementary DNA through GoScript Reverse Transcription System (Promega). The qPCR assay was performed as described previously 28 . Primers used for qPCR analysis of ROCK2, c-Myc, Cyclin D1, GFAP, P21, P15, and β-actin are listed on Table 3. Cell cycle assay was performed as described previously 29 .

ChIP
The ChIP analysis was performed using the ChIP Assay kit (Upstate Biotechnology, Charlottesville, VA). 10 7 cells were crosslinked with 1% formaldehyde for 10 min at 37 ℃ and then washed, lysed, and sonicated to generate 200-500 bp chromatin fragments. The samples were precleared with 60 μl of salmon sperm DNA-protein A-agarose and subsequently incubated at 4 ℃ overnight with 2 μg Stat3 antibody and rabbit IgG as control. Immunocomplexes were recovered, washed thoroughly, and eluted with the ChIP elution buffer. Following the reversal of crosslinks at 65 °C for 4 hours, samples were extracted with phenol/chloroform, precipitated with ethanol, and then used as templates for PCR ampli cation.
The sequences of PCR primers detecting c-Myc and Cyclin D1 promoter are available on request.

Methylation Assay
The methylation assay were performed as previously described 29 . Primers for the methylated promoter region were 5'-GATATACGATAGGAATTACGGGG-3' (sense) and 5'-TCTCTCTACCTTATCTAACCCG-3′ (antisense), and for the unmethylated region 5'-GATATATGATAGGAATTATGGGG-3' (sense) and 5'-CTCTACCTTATCTAACCCATTCCC-3' (antisense). The intensity of the methylated and the loading control products were analyzed by ImageJ software (NIH, Bethesda, MD) and compared with the base pairs of the amplicons and the cycles of individual PCR reactions. When the relative ratio of the methylated to the total DNA was more than 50 %, methylation was considered high, whereas it was considered low when the ratio was less than 50 %. Samples with no obvious methylation bands were considered as having no methylation.

IHC
Epileptic and control hippocampus tissues were collected at the tangdu Hospital of Fourth Military Medical University. The immunohistochemistry staining assay was performed and scored as previously described 17 . The primary antibodies for anti-ROCK2 (1:200, #PA5-78290, Invitrogen) and anti-p-Stat3 (1:150; #ab76315, Abcam) were applied. The protein levels were statistically analyzed by Student's t-test. Linear regression and Pearson's correlation signi cance were used to analyze ROCK2 and p-Stat3 correlation.

Statistical analysis
The data are expressed as the mean ±SEM. Statistical analyses were performed in GraphPad Prism 8.0 (GraphPad Software Inc, San Diego, CA).
One-way ANOVA analysis followed by Dunnett's multiple comparison test was used to determine the differences among multiple groups.
Correlations between the protein levels of ROCK2 and p-Stat3 were assessed using Pearson's rank correlation test. Statistical signi cance was based on a value of P < 0.05.

Results
mRNA Pro ling of human refractory epilepsy samples To gain mechanistic insight into system-level differences between normal and epileptic hippocampus tissues, we determined the gene expression pro les in 8 drug-resistant epilepsy samples and 3 control samples (Tables 1 and 2). Compared with the control samples, 1389 genes were downregulated and 1014 genes were up-regulated in refractory epilepsy samples (Fig. 1A, 1B). Further, we analyzed the top20 upregulated genes in epilepsy samples. Intriguingly, ROCK2 were found most dramatically highly expressed in epilepsy samples (Fig. 1C), suggesting the high levels of ROCK2 might be associated with epilepsy generation.
Elevated expression of ROCK2 in human epileptic hippocampus by the hypomethylation of ROCK2 promoter To evaluate the role of ROCK2 in epilepsy, we rstly analyzed the expression levels of ROCK2 in the normal (n=6) and epileptic (n=34) hippocampus tissues (Tables 1 and 2). Intriguingly, the densitometric analysis of western blots shows that ROCK2 was highly expressed in the hippocampus tissue of drug-resistant TLE patients (Fig.2B). The ROCK2 product OD in patients was signi cantly higher than that in controls (Fig.   2C). To determine the underlying mechanisms, we examined the potential changes in ROCK2 gene promoter region. Samples from TLE patients and control tissue samples were collected for methylation analysis by nested PCR. Methylation-speci c PCR (MSP) analysis shows that ROCK2 promoter region was highly methylated in control samples and only 1 of 6 TLE patients (Fig.2D). Further, a large scale of MSP analysis identi ed the hypermethylation of ROCK2 promoter in 9 of 12 normal tissues and 6 of 34 TLE patients (Fig.2E). Thus, epileptogenesis appears to be negatively associated with the hypomethylation of ROCK2 promoter and concomitant induced its expression.
Increased expression of ROCK2 in hippocampal and neocortical epilepsy Rats were divided randomly into eight subgroups of ten rats each and injected with lithium and 24 h later with saline (one subgroup) or the convulsant pilocarpine (seven subgroups) to establish a control and spontaneous epilepsy model groups. Neural ROCK2 expression was then determined at different times after seizure induction (1, 3, 5, 7, 15, 30, or 60 days). The lithium-pilocarpine model of epilepsy in rats were prepared and evaluated as previously described 17 . Western blot analysis of brain lysates prepared at these different times after pilocarpine treatment revealed increased ROCK2 expression compared with controls (Fig. 3). Compared with the control group, expression of ROCK2 in the rat temporal lobe increased gradually during the acute period (day 1-3) (Fig. 3A) before increasing obviously during the latent period (days 5-15) and the chronic phase (days 30-60). Expression of ROCK2 as expressed by product OD relative to that of β-actin was signi cantly different between control and every epilepsy subgroup (p<0.05 by Tukey's HSD test) except for the 1-day subgroup (p>0.05) (Fig. 3B). The expression of ROCK2 in epileptic hippocampus (Fig. 3C, D) followed a similar trend to that in the temporal cortex, with signi cant pair-wise differences in ROCK2 expression between the control and every epilepsy subgroup (p<0.05) except the 1-day subgroup. The expression of ROCK2 in the hippocampus increased even more obviously at 15 30 and 60 days after seizures(p<0.01) (Fig. 3D).

Fasudil alleviates rat epileptic seizures in vivo and protects against Li-Pilo-induced SE
Li-Pilo-induced epileptic rat model is one of the most widely used on epileptogenesis research and has been assess the seizures based on changes in the Racine stages. The rats were then divided into 3 groups (saline group PILO group and Fasudil treatment group). Fasudil, a speci c Rhokinase inhibitor, was used to treat the rats following Li-Pilo induced seizures for 2 weeks. Then seizures were induced by the low dose PILO (200 mg/kg, i.p.) again and assessed the effects of Fasudil on the epileptogenesis. In the present study, the video-EEG recording and racine stages were used to analyze seizure severity between Fasudil treatment group and PILO group. After administration of PILO, stage 3 behavioral scores earlier appeared in PILO group compared with Fasudil treatment group, stage 4 and 5 behavioral scores did not appear in Fasudil treated rats (Fig. 4A).

ROCK2 activates Stat3 pathway in Pilocarpine-Treated Epileptic Rats
To further investigate the role of ROCK2 in regulation of epileptogenesis, we determined the downstream targets in SE rat model. JAK/Stat3 pathway was previously shown constitutively activated during brain injuries like SE, and plays a key role in epilepsy development 30,31 . In the present study, we observed the phosphorylation and activation of Stat3 following SE. Intriguingly, Fassudil administration signi cantly decreased Stat3 phosphorylation, indicating ROCK2 potentially contributes to epileptogenesis through the regulation of Stat3 pathway (Fig. 5A, 4B). Further, we determined the downstream target gene expressions of Stat3. Myc and Cyclin D1, which play important roles in regulating cell proliferation and cell cycle progression, were induced following SE and signi cantly decreased after Fassudil treatment (Fig. 5C). Therefore, we suppose that ROCK2 induces cell proliferation via the activation of Stat3 pathway in brain tissue, and further contributes to the development and progression of epilepsy.

ROCK2 is predominantly expressed in astrocytes during epileptogenesis
Above data showed the elevated expression of ROCK2 in the brain tissues of TLE patients. To further address ROCK2 function in epileptogenesis, we detected the localization of ROCK2 in both neuron and astrocytes of rat brain tissue. The co-localization of ROCK2 with NeuN or GFAP was observed in normal brain tissue, indicating ROCK2 expression in certain level is necessary for the biological function of both neuron and astrocytes ( Fig. 6A, 6C). Whereas, from the third day after SE, ROCK2 expression in astrocytes was signi cantly increased in contrast with the constant level of ROCK2 in neuron tissue. Quantitative analysis showed nearly 2-fold increase of ROCK2 expression in astrocytes (Fig. 6B, 6D). Thus, we show that ROCK2 is predominantly expressed in astrocytes during epileptogenesis, indicating ROCK2 possibly induces epilepsy progression through the speci c regulation of astrocyte function.

ROCK2 induces epileptogenesis by activating astrocyte cell cycle progression via Stat3 pathway
Since accumulated evidence showed that astrogliosis plays important roles in epileptogenesis, it is reasonable to consider the involvement of ROCK2 in astrocyte proliferation and activation 32,33 . Based on this hypothesis, we set up the primary astrocytes from rat brain tissue, whose phenotype has been con rmed with GFAP in our previous study 17 . To investigate how ROCK2 confers astrocyte behavior, we overexpressed ROCK2 in astrocytes. Expectedly, the phosphorylation of Stat3 was dramatically induced by ROCK2 expression (Fig. 7A). Further, GFAP, the marker of astrocyte proliferation potential and Stat3 target gene, was induced by ROCK2 (Fig. 7B). During cell cycle analysis, we found ROCK2 accelerated astrocyte G1-S transition, indicating ROCK2 induces astrocyte proliferation which confers gliosis potentially through the regulation of cell cycle progression (Fig. 7C). Accordingly, Fasudil treatment blocked ROCK2 induction of pStat3 and GFAP expressions, and G1-S transition, which further con rmed our hypothesis (Fig. 7A-C).
To fully address the related mechanism, we then determined whether ROCK2 regulates cell cycle transition via Stat3 pathway. Previous data revealed Stat3 target genes, Myc and Cyclin D1, were ROCK2 dependently induced in SE rat brain tissue (Fig. 5C). Further study showed that Myc and Cyclin D1 were remarkably induced by ROCK2 in astrocytes (Fig. 7D). The CDK inhibitor p21 and p15, which are Myc direct targets, were decreased after ROCK2 overexpression. Additionally, ChIP experiments indicated that the binding of Stat3 to Myc and Cyclin D1 promoter was dramatically increased by ROCK2 overexpression (Fig. 7E). In the presence of Fasudil, ROCK2-induced expressions and Stat3 occupancies of Myc and cyclin D1 were signi cantly abolished in astrocytes (Fig. 7D, 7E). Taken together, these data suggest that ROCK2 induces the development of epilepsy, potentially through the activation of astrocyte cell cycle progression via Stat3 pathway.

ROCK2 is associated with Stat3 activation in TLE patients
To evaluate the clinical signi cance of ROCK2 induction of Stat3 pathway during epilepsy, we performed immunohistochemical analysis to assess the expressions and association of ROCK2 and pStat3 in TLE patients. The results revealed that both ROCK2 and pStat3 expressions were increased in patient samples (n=34) compared with control samples (n=12) (Fig. 8A). Indeed, in patient tissues versus control, ROCK2 levels were 7.82 versus 3.92 (Fig. 8B), respectively, and pStat3 levels were 6.88 versus 2.67 (Fig. 8C), respectively. An association study showed that ROCK2 expression positively correlated with pStat3 expression in the entire sample population (Fig. 8D) (p<0.001, ANOVA). Thus, the epilepsy-associated increase of ROCK2 is associated with the increased surface activation of Stat3 pathway.

Discussion
Active RhoA binds to its downstream targets to initiate numerous cellular responses. ROCK, a serine/threonine kinase, is one of the most studied RhoA effectors and is involved in regulation of the actin cytoskeleton, cell proliferation, focal adhesion /stress ber formation, and smooth muscle contraction 34 . The current study explored the role of ROCK2 in epilepsy, focusing on the possibility that ROCK2 inhibition is required for the suppression of seizures. Although ROCK2 has not previously been suspected to play a role in epilepsy, our data demonstrate the importance of ROCK2 in the Li-Pilo model and clinical epilepsy sample, by the inhibition of ROCK2 associated reduction in seizure activity. Thus, we identify ROCK2 as a key regulator of epilepsy, and a potentially novel therapeutic target for the prevention and treatment of epilepsy.
In this study, ROCK2 expression was rstly evaluated in normal and epileptic brain tissue. We show that increased ROCK2 expression in human epileptic brain tissue. The underlying mechanism study suggests that ROCK2 gene promoter region was hypomethylated in TLE patients, which potentially contributes to the elevated expression of ROCK2. To our knowledge this is the rst study providing direct evidence of increased ROCK2 expression in patients with drug-resistant TLE. Further, the highly isomorphic with human cases of TLE animal model was used, namely the model of TLE with pilocarpine-induced seizures 35 . In order to analyze the dynamics of epileptic seizure-induced alterations on the brain, samples taken from seizure experiencing animals 1, 3, 5, 7, 15, 30, and 60 days after proconvulsive agent administration were examined. Our study shows that the expression of ROCK2 protein increased dramatically in epilepsy rat cortex and hippocampus. Thus, we set up the association of ROCK2 with epileptogenesis.
Astrocytes are now known to be intimately involved in the pathogenesis of many neurological disorders, including epilepsy 32,36 . The reactive astrocytosis may facilitate the development of epileptic seizures 37 . The cells of the high ROCK2 expression were found in pyramidal neurons of the hippocampus and cerebral cortex and Purkinje cells of the cerebellum 10 . By immuno-uorescence, ROCK2 was mainly localized in the neurons in our study. A previous study has shown that RhoA activation in the cortex and hippocampus 24 h after traumatic brain injury-related seizures 4 . In this study, we observed the expression of ROCK2 in hippocampal astrocytes in the Li-Pilo model. GFAP is the main intermediate lament protein in mature astrocytes. It has been demonstrated that GFAP-immunoreactive reactive astrocytes exhibited markedly increased RhoA expression in response to spinal cord injuries after kainic acid treatment 38 . We show reactive astrocyte co-localization with ROCK2 expression in the Li-Pilotreated hippocampus,which is consistent with the Rho-ROCK pathway induces the generation of a reactive astrogliosis 39 . However, the expression of ROCK2-positive neurons is not induced by Li-Pilo treatment, indicating the increased expression of ROCK2 may be associated with pathological changes predominantly in astrocytes.
JAK/Stat3 pathway plays an important role in normal development and a number of disease processes, including epileptogenesis 30,40,41 . Recent evidence shows the aberrant activation of this pathway after a variety of brain insults that can lead to epilepsy, such as pilocarpine or kainateinduced SE 30,40 , and traumatic brain injury 42 . Whereas, the potential mechanism has not been fully addressed. Our study shows the highly expression ROCK2 can activate Stat3 activation in astrocytes, and further contribute to the development of epilepsy via the induction of astrogliosis. Thus, inhibiting ROCK2 expression or Stat3 phosphorylation may be a therapeutic approach to inhibit primary epileptogenesis.
Because ROCK is a serine/threonine kinase and cannot directly mediate tyrosine kinase phosphorylation of Stat3, it is likely that other intracellular signaling components, which may interface between ROCK and STAT3, are also engaged in this ROCK2-dependent activation of Stat3 43,44 .
The transcription factor Stat3 is known to have important roles in regulating gene expression, speci cally increasing genes important to cell proliferation and cell cycle progression 31,45 . In this study, we determined how ROCK2 induces epilepsy via Stat3 pathway, and found Stat3 target genes, Myc and Cyclin D1, were signi cantly induced by ROCK2. Further, ROCK2 increased the occupancies of Stat3 on Myc and Cyclin D1 promoters, and accelerated astrocyte cell cycle progression. The CDK inhibitor protein p21 and p15 are important downstream targets of Myc and critical for cell cycle inhibition 46,47 . We show here that p21 and p15 were further suppressed by ROCK2 activation of Myc via Stat3 pathway. Thus, we show ROCK2 induces astrocyte cell cycle progression in regulation of Stat3 pathway, and further contribute to the development of epileptic seizures. These experiments may explain, on one hand, the requirement of Stat3 for ROCK2-mediated activation of astrogliosis and, on the other, the ability of ROCK2 to provide a second input of cell cycle regulation for epileptogenesis in addition to the regulation of actin cytoskeleton dynamics and glutamatergic synaptic function as mentioned before 48,49 .
Since the continuous activation of astrocytosis confers epileptogenesis, our study provides the evidence of ROCK2 involvement in astrogliosis, which suggests ROCK2 could be considered as a potential target for the treatment of TLE patients. Further, we aimed to elucidate the role of ROCK2 activation of Stat3 pathway in the development and progression of epilepsy, speci cally whether brief inhibition of ROCK2 affects severity of SE. We thus introduced ROCK2 inhibitor Fasudil in the present study. Fasudil has been a commonly used rock signal inhibitor, which signi cantly reduced the duration of tonic hindlimb extensions and recovery latency for righting re ex in maximal electroconvulsive shock induced epilepsy mice, and prolonged the onset time of seizures in PTZ epilepsy mice 15  Furthermore, immunohistochemical staining of epileptic samples for ROCK2 and pStat3 also showed a correlation of protein accumulation between these two proteins, which is also positively correlated with the development of epilepsy. Thus ROCK2-mediated activation of Stat3 pathway likely serves as an important guardian for epileptogenesis. It should be noted that, as is often observed among clinical samples, no 1:1 correlation between ROCK2 and pStat3 expression in tissues was observed. Thus, in addition to ROCK2, other factors may also contribute to the activation of Stat3 pathway during epilepsy progression.
Taken together, our work provides the evidence that elevated expression of ROCK2 due to its promoter hypomethylation may contribute to the etiology of epilepsy. In terms of epileptogenesis, ROCK2 accelerates astrocytes cell cycle progression via the activation of Stat3 pathway, and further induces astrogliosis. As such, Targeting ROCK2 may lead to the inhibition of Stat3 phosphorylation in astrocytes, and that will be especially effective at restricting epilepsy progression. These data will extend the role of ROCK2 in the pathophysiology of drug-resistant TLE and de ne ROCK2 and its downstream signaling pathways as potential targets for therapeutic intervention for drug-resistant TLE.      promoter were determined respectively. Data are expressed as means ± SD (n = 3).