Inhibition of RhoA/Rho kinase signaling pathway by fasudil protects against kainic acid‐induced neurite injury

Abstract Aim RhoA/Rho kinase pathway is essential for regulating cytoskeletal structure. Although its effect on normal neurite outgrowth has been demonstrated, the role of this pathway in seizure‐induced neurite injury has not been revealed. The research examined the phosphorylation level of RhoA/Rho kinase signaling pathway and to clarify the effect of fasudil on RhoA/Rho kinase signaling pathway and neurite outgrowth in kainic acid (KA)‐treated Neuro‐2A cells and hippocampal neurons. Method Western blotting analysis was used to investigate the expression of key proteins of RhoA/Rho kinase signaling pathway and the depolymerization of actin. After incubated without serum to induce neurite outgrowth, Neuro‐2A cells were fixed, and immunofluorescent assay of rhodamine‐phalloidin was applied to detect the cellular morphology and neurite length. The influence of KA on neurons was detected in primary hippocampal neurons. Whole‐cell patch clamp was conducted in cultured neurons or hippocampal slices to record action potentials. Result KA at the dose of 100–200 μmol/L induced the increase in phosphorylation of Rho‐associated coiled‐coil‐containing protein kinase and decrease in phosphorylation of Lin11, Isl‐1 and Mec‐3 kinase and cofilin. The effect of 200 μmol/L KA was peaked at 1–2 hours, and then gradually returned to baseline after 8 hours. Pretreatment with Rho kinase inhibitor fasudil reversed KA‐induced activation of RhoA/Rho kinase pathway and increase in phosphorylation of slingshot and 14‐3‐3, which consequently reduced the ratio of G/F‐actin. KA treatment induced inhibition of neurite outgrowth and decrease in spines both in Neuro‐2a cells and in cultured hippocampal neurons, and pretreatment with fasudil alleviated KA‐induced neurite outgrowth inhibition and spine loss. Conclusion These data indicate that inhibiting RhoA/Rho kinase pathway might be a potential treatment for seizure‐induced injury.


INTRODUCTION
Epilepsy is a group of neurological disorders characterized by transient disturbance caused by abnormal excessive or synchronous neuronal discharges (Fisher et al., 2005). Recurrent epileptic seizures lead to a series of neurobiological, cognitive, psychological and social consequences. Cognitive impairment is one of the commonest comorbidities of epilepsy. Though recent treatments are effective in controlling seizures, their impacts on cognitive function remain unsatisfactory (Dias et al., 2017). Apart from the effects of chronic epilepsy on brain anatomy and physiology, the underlying brain dysfunction responsible for epilepsy also contributes significantly to cognitive morbidity in a dependent way. An 8-to 9-year follow-up of adolescents with diagnosed epilepsy in childhood showed that up to 39% of them also suffered from neurodevelopmental disorders (Baca et al., 2011). Cognitive impairments shown in epileptic children include difficulties in learning, memory, problem solving, as well as concept formation. Anxiety, depression and attention deficit disorders are the most common psychiatric comorbidities (Gulati et al., 2014). Thus, cognitive impairment and psychiatric comorbidities have pronounced impacts on long-term life quality of people with childhood-onset epilepsy.
It has been widely considered that plasticity of dendritic spines is one of the key mechanisms and substrates of cognitive function (McCann & David, 2017;Park et al., 2017 ). It has been reported that kainic acid (KA)-induced seizures caused irreversible dendritic beading morphological change and loss of spines (Zeng et al., 2007). Furthermore, KA-induced seizures also resulted in activation of actindepolymerizing factors and a corresponding decrease in filamentous actin (Zeng et al., 2007). The actin cytoskeleton is crucial for neurite growth cone, spine development, and formation of synapses in neurons (Eiseler et al., 2010;Yamada et al., 2013 ). Cofilin is one of the key proteins of the RhoA/Rho kinase pathway which is essential for regulating cytoskeletal structure (Patel et al., 2017). At present, pharmacological inhibitors of Rho-associated protein kinase 2 (ROCK2) have been extensively investigated. Among them, fasudil is the best characterized and most frequently used (Feng et al., 2016). Previous studies have revealed that fasudil has antiepileptic effects and significantly reduces the duration of epileptic seizure as well as recovery latency for righting reflex, and also prolongs the onset of seizures in acute seizure models (Inan & Büyükafsar, 2008;Xu et al., 2014 ). These results indicated that RhoA/Rho kinase pathway might play an important role in regulating dendritic structure after seizures and that inhibitors of RhoA/Rho kinase pathway have potential clinical application for preventing or reversing seizure-induced cognitive impairment and psychiatric comorbidities.
Neuro-2a is a fast-growing mouse neuroblastoma cell line, with the potential to differentiate into neuron-like cells and easy to culture. In the present study, the effect of RhoA/Rho kinase signaling pathway on neurite morphology in KA-treated Neuro-2a cells was investigated.
Moreover, the impact of RhoA/Rho kinase signaling pathway on neurite morphology in the KA-treated primary cultured hippocampal neurons was also examined. The study aimed at illustrating the effects of the specific intracellular signaling and mechanistic elements on seizure-induced dendrite injury and laying the foundation for a potential treatment of seizure-induced cognitive impairment. They were both initially dissolved in PBS, stored at −4 • C, and diluted by medium immediately before treatment, as described previously (Zhu et al., 2011).

Primary culture of hippocampal neurons
The cultured hippocampal neurons were obtained from the 18-day embryonic (E18) Sprague-Dawley rats. Glass coverslips (20 mm in diameter) were placed in dishes and coated with 0.5 mg/mL poly-L- Cytarabine (Ara-C, Pfizer, USA) was added to the culture medium at a final concentration of 2.5 μg/mL, and all of the culture medium was replaced after 2 days. Then medium was renewed every 4 days in the following 2 weeks.

Active RhoA detection
The detection of active RhoA was conducted according to the protocol of Active Rho Detection Kit purchased from Cell Signaling Technology.
Briefly, the hippocampus tissue was isolated and lysated with 500 μL 1× Lysis Buffer(add 1 mM PMSF before using

Western blot analysis
The Analysis of the ratio of F-actin to G-actin was also made by western blot according to a previously published method (Zeng et al., 2007). After serum deprivation for 1 hour and then drug treatment (refer to the previous paragraph for drug delivery), cells were collected and homogenized in G-actin lysis buffer and then centrifuged at 15,000 rpm for 30 minutes. The supernatant was used to determine the amount of G-actin. The pellets were resuspended in G-actin lysis buffer plus an equal volume of F-actin, and gently mixed every 5 minutes for 1 hour to depolymerize F-actin. The samples were centrifuged at 15,000 rpm for 30 minutes, and the supernatant was also used for measurement of F-actin. G-actin and F-actin samples were both determined and analyzed according to western blot analysis.

Measuring of the neurite outgrowth in Neuro-2a cells under different interface contrast microscopy
The Neuro-2a cells were seeded onto glass coverslips and exposed to

Calculation of neurite length and spine number
Images either taken by optical or confocal microscope for each group were analyzed by Image J. Images with clear background and uniform cell density were selected with a precondition that the neurites were not connected with other cells. The length of the longest neurites of each cell was calculated using NeuronJ function of Image J. One hundred cells were observed and measured in each group. The numbers of the spines in each cell neurites were also counted. NaCl, 20 D-glucose, 1 sodium pyruvate, 1.25 NaH 2 PO 4 , 2 CaCl 2 , and 1

Statistics
Results were presented as mean ± standard deviation (SD

KA significantly activated the RhoA/Rho kinase pathway in Neuro-2a cells
Our previous research demonstrated that KA seizures induced a rapid activation of cofilin and corresponding depolymerization of actin filaments in dendrites in mice (Zeng et al., 2007). To deter-  (Figures 2A, B). Simultaneously, the phosphorylation levels of LIMK1 and cofilin proteins in Neuro-2a cells started to decrease at 0.5 hour and peaked at 1-2 hours, and then turned to baseline gradually. These data further indicated that KA significantly changed the phosphorylation levels of RhoA/Rho kinase pathway (Figures 2A-D).

Pretreatment of fasudil effectively reversed KA-induced change of the RhoA/Rho kinase pathway and alleviated KA-induced actin depolymerization
The effect of fasudil on RhoA/Rho kinase pathway was investigated.
As shown in Figure  It has been showed by several groups that protein slingshot phosphatase isoform 1 (P-SSH1) could dephosphorylate P-cofilin and reactivate cofilin (Dhruba & Peng, 2016;Govek, 2005 ). Thus, we investigated whether KA treatment had an effect on the expression of phosphorylated SSH1 and its upstream protein P-14-3-3. It was found that the KA treatment significantly increased the expression of P-SSH1 and P-14-3-3 at both 1 and 4 hours ( Figures 4A, B). By contrast, pretreatment with fasudil significantly reversed the KA-induced increase of phosphorylated P-SSH1 and P-14-3-3 in Neuro-2a cells ( Figures 4A, B).
In addition, KA caused a marked decrease of filamentous actin at 1 hour after treatment, and fasudil blocked the actin depolymerization.
The actin depolymerization recovered partially 4 hours after KA treatment but the effect of fasudil still persisted ( Figure 4C).  The rhodamine-phalloidin immunofluorescence result showed the consistent results ( Figures 5B, D). At 1 hour after KA treatment, the length of cell neurites was significantly decreased, compared with control group, and fasudil reversed KA-induced inhibition ( Figure 5D). The lengths of cell neurites in the KA-treated cells did not differ significantly from that of the control cells at 4 hours after treatment, and neurite extensions were longer in the presence of fasudil when compared with the KA-treated group and control group at 4 hours ( Figure 5D).

Fasudil pretreatment reversed KA-induced neurite outgrowth inhibition in Neuro-2a cells
In addition, we also found that the number of spines was significantly   (Figures 6E-H).

Fasudil pretreatment reversed KA-induced neurite outgrowth inhibition in primary hippocampal cells
These results further demonstrated that inhibition of RhoA/Rho kinase signaling pathway protected against KA-induced neurite injury.
To explore whether KA induced seizure-like electrical activity in the cultured hippocampal cells, we performed whole-cell patch-clamp recording from the cultured hippoampal cells. Indeed, it was found seizure-like electrical activity from patched neurons after adding 200 μM KA ( Figure 6I). Next, we explored whether fasudil had antiseizure like activity in more physiological condition using hippocampal slice.
We applied fasudil before KA and found that KA induced less action potentials when in the presence of fasudil compared to the control group ( Figures 6J, H).

DISCUSSION
In the present study, we found that (1)  Previous studies suggested that KA-induced high-stage seizures caused activation of the actin-depolymerizing factor cofilin and the following actin depolymerization, which was associated with morphologic changes in dendrites (Zeng et al., 2007). Cofilin modulation was a probable mechanism for mediating this seizure-induced actin depolymerization and dendritic injury. In addition, several studies revealed the antiepileptic effect of Rho kinase inhibitors (Defert & Boland, 2017;Dhruba & Peng, 2016 ). The RhoA/Rho kinase pathway is one of the classic cellular pathways regulating cofilin (Liu et al., 2018). Both dendritic spines and synapses are regulated by the actin cytoskeleton mediated by the Rho GTPases and LIMK1/cofilin (Bernard, 2007;Carcak et al., 2018;Govek, 2005;Rust, 2015). Thus, it is reasonable to assume that RhoA/Rho kinase pathway plays crucial roles in seizure-induced neurite injury (Jeon et al., 2013 The role of the RhoA/Rho kinase pathway in neurite outgrowth has been addressed in numerous studies, but the specific effects on this pathway after seizures remain little known (Lars, 2011). The RhoA/Rho kinase pathway consists of a cohort of kinases and phos-phatases maintaining the balance of F-actin dynamics (Gopalakrishnan et al., 2008;Meng et al., 2002). Cofilin is inactivated by P-LIMKmediated phosphorylation at serine 3 (Ser3) and dephosphorylated by SSH1, which reactivates cofilin-1 (Bernard, 2007;Mitsuharu et al., 2003;Soosairajah et al., 2005). LIMK and SSH1 show the highest substrate specificity compared to other kinases and phosphatases that affect cofilin-1 activity (Mitsuharu et al., 2003;Soosairajah et al., 2005;Zafar et al., 2017). Moreover, SSH1 is also regulated by 14-3-3 protein (Mitsuharu et al., 2003). ROCK2 (brain type), an upstream kinase, can regulate LIMK and then inactivate cofilin by phosphorylation (Koch et al., 2014;Shi et al., 2013 This subsequently caused the activation of cofilin and further depolymerization of F-actin, which was associated with dramatic neurite outgrowth inhibition. It was speculated that RhoA/Rho kinase pathway may be one of the key pathways meditating neurodegeneration in epilepsy. The inhibitors of ROCK2, such as fasudil and Y-27632, have been reported with an antiepileptic effect (Xu et al., 2014). However, the specific intracellular signaling and mechanistic elements involved are still unclear. In this research, we applied fasudil to Neuro-2a cells before Most of all, this study provides exciting preclinical data that support the initiation of clinical trials using fasudil for treating comorbidity of epilepsy after additional animal and clinical studies since fasudil is a FDA-approved drug. Nevertheless, a potential limitation of these experiments was the use of Neuro-2a cells and primary hippocampal cells in vitro. Future studies will examine these mechanisms further in in vivo epilepsy models.

CONFLICT OF INTEREST
All authors claim that there are no conflicts of interest.