Cell Biochem Biophys DOI 10.1007/s12013-014-9931-6
ORIGINAL PAPER
Fasudil Hydrochloride Protects Neurons in Rat Hippocampal CA1 Region through Inhibiting GluR6–MLK3–JNKs Signal Pathway Xiu-E Wei • Feng-Yu Zhang • Kai Wang Qing-Xiu Zhang • Liang-Qun Rong
•
Ó Springer Science+Business Media New York 2014
Abstract Fasudil hydrochloride (FH), a Rho kinase (ROCK) inhibitor, has been reported to prevent cerebral ischemia in vivo from increasing cerebral blood flow and inhibiting inflammatory responses. However, it is uncertain by what mechanism a ROCK inhibitor can directly protect neurons against ischemic damage. The present study was designed to evaluate whether FH decreased the increased phosphorylation of glutamate receptor 6 (GluR6) and its downstream in GluR6–MLK3–JNKs signal transduction pathway following global transient cerebral ischemia, as a result of protecting against neuronal apoptosis and death. Transient cerebral ischemia was induced by the Pulsinelli– Brierley four-vessel occlusion method. FH (15 mg/kg) was administered to rats by intraperitoneal injection 30 min before ischemia. The phosphorylation and protein expression of GluR6 at 6 h during reperfusion were detected using immunoprecipitation and immunoblotting analysis. The phosphorylation and protein expression of Mixed lineage kinase 3 (MLK3) at ischemia/reperfusion (I/R) 6 h and c-Jun N-terminal kinase (JNK) at I/R 3 d were detected using immunoblotting analysis, respectively. The same method was used to detect the expression of caspase-3 at I/R 6 h. Furthermore, we also use TUNEL staining and Cresyl violet staining to examine the survival neurons in rat hippocampal CA1 regions after 3 and 5 d reperfusion, respectively. Our study indicated that FH could inhibit the X.-E. Wei (&) K. Wang Q.-X. Zhang L.-Q. Rong Department of Neurology, The Second Affiliated Hospital of Xuzhou Medical College, Xuzhou 221000, People’s Republic of China e-mail: [email protected] F.-Y. Zhang Department of Neurology, Liao Cheng Hospital, Liaocheng 252000, People’s Republic of China
increased phosphorylation of GluR6 and MLK3 and the expression of caspase-3 at peaked 6 h of reperfusion and the phosphorylation of JNK (3 d) (p \ 0.5). The results of TUNEL staining and Cresyl violet showed that the number of surviving pyramidal neurons in rats hippocampal CA1 subfield increased markedly in FH-treated rats compared with ischemic groups after 3 or 5 d of reperfusion following ischemia (p \ 0.5). These results suggested that FH, as a ROCK inhibitor, may be partly responsible for its protective effects against such damage by taking part in GluR6-MLK3-JNKs signaling pathway which modulates ischemic damage. Taken together, this is the first study investigating Rho and ROCK as the upstream of GluR6 taking part in GluR6–MLK3–JNKs signal transduction pathway following cerebral ischemia. Keywords Fasudil hydrochloride GluR6 CA1 TUNEL staining Cerebral ischemia
Introduction Since cerebral infarction is one of the principal etiologies for neurological sequelae and/or death, it is very important to understand both its pathologic mechanisms and any effective treatments. After ROCK was found [9], it became clear that upregulated ROCK worked unfavorably to the host in many vascular diseases. Fasudil, initially characterized as an intracellular calcium antagonist, has played an important role in cerebral vasospasms occurring after subarachnoid hemorrhage by increasing regional CBF not only via an inhibition of smooth muscle contraction but also through an upregulation of endothelial nitric oxide synthase (eNOS) [13, 19] and a decrease in the inflammatory response [1, 16, 17, 19]. FH has an important role
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in cerebral ischemia, but by what molecular mechanism does it achieve the protective effect remains unknown. GluR6 receptor plays a prominent role and mediates serine phosphorylation in brain ischemia-mediated neuronal injury [3, 5]. FH inhibits the serine/threonine phosphorylation of ROCK. With this in mind, what is the relationship between ROCK and GluR6 or between FH and GluR6 serine phosphorylation in ischemic injury? Moreover, the RhoA/ROCK pathway provides promising pharmacological targets for treatment of a variety of cerebralvascular diseases. The present reports indicate that it is linked to glutamate receptor 6 (GluR6), the postsynaptic density protein 95 (PSD95), mixed lineage kinase 3 (MLK3), and JNK through phosphorylation triggering the cascade reaction in cerebral ischemic injury [4, 5, 7, 8]. What is the association between FH and MLK3, JNK3 serine phosphorylation in ischemic injury? Whether FH can inhibit GluR6 serine phosphorylation through inducing MLK3 and JNK3 serine phosphorylation or not? are the questions that remain unclear.
Materials and Methods Animals and Drugs Adult male Sprague–Dawley rats (SD rats, Shanghai Experimental Animal Center, Chinese Academy of Science) weighing 220–300 g were used. The experimental procedures were approved by the local legislation for ethics of experiments on animals, and efforts were carried out to minimize the pain or discomfort of rats. Four-vessel occlusion (4-VO) cerebral ischemia was induced as described before [18]. Male SD rats were randomly divided into four groups: given control, ischemia/reperfusion (I/R), I/R with FH treatments, and saline group. Briefly, under anesthesia with chloral hydrate (300 mg/kg, i.p.), vertebral arteries were electrocauterized, and common carotid arteries were exposed. Fasudil hydrochloride (15 mg/kg) was dissolved in saline at a concentration of 0.9 mg/ml, which was administered to rats by intraperitoneal injection 30 min before ischemia. Control rats were intraperitoneally given saline. Tissue Preparation Rats were decapitated immediately after 6 h or 3 d of reperfusion, and then the hippocampus was separated into CA1 and CA3/DG from hippocampal fissure and quickly frozen in –80 °C. Cytosol fraction was extracted as Ogita and Yoneda [10] described procedure. The homogenate
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was centrifuged at 12,0009g for 5 min at 4 °C. The supernatants were collected, and the protein concentration was determined by Lowry’s method. The protein was packed by the same concentration and boiled for 5 min before cooling. After dealing with that, samples stored at -80 °C were thawed until assays. Methods Immunoprecipitation and Immunoblotting Tissue homogenates (400 lg of protein) were processed following the methods of Li et al. [2]. Immunoprecipitation and immunoblotting-specific operation is as described by Li et al. [2]. The difference is Membranes were washed and then incubated with alkaline phosphatase-conjugated secondary antibodies in milk for 2 h and developed using NBT/BCIP color substrate (beyotime, China). The density of the bands on the membrane was scanned and analyzed with an image analyzer (LabWorks Software, UVP Upland, CA). TUNEL Staining TUNEL staining was performed using an ApopTagÒ Peroxidase In Situ Apoptosis Detection Kit according to the manufacturer’s protocol with minor modifications. Sections were treated with anti-digoxigenin conjugate for 30 min at room temperature and subsequently developed color in NBT/BCIP substrate. An initial dissector frame was positioned randomly in hippocampal sector and cells in every 10th section throughout the entire hippocampus. The cell numbers in hippocampus were assessed by means of previously published unbiased stereological techniques. In brief, cell counts were performed at 4009 magnification with the use of an Olympus BH-2 microscope connected to a Sony charge-coupled device video camera, amotorize stage system, and commercial stereology software. Cresyl Violet Staining The rats subjected to 5 d of reperfusion were perfusion fixed with 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under anesthesia. The paraffin-embedded brain sections (5 lm) were prepared and stained with Cresyl violet. Histological evaluations were performed with Cresyl violet staining for assessment of neuronal damage in the hippocampus. The Cresyl violet-positive cell numbers were counted by means of the unbiased stereological methods, which is mentioned above in TUNEL staining.
Cell Biochem Biophys
the lane-marked input was detected (Fig. 1). The results showed that GluR6 was phosphorylated at serine residues. When immunoprecipitated with nonspecific mouse IgG, no significant band corresponding to GluR6 was detected. Fig. 1 GluR6 is phosphorylated at the serine residue in hippocampus. Homogenates (400 lg of total protein) from sham-operated controls (Sham) and ischemic animals at 15 min of ischemia (I) were immuno precipitated (IP) with anti-phosphoserine antibody (anti-pSer) or nonspecific IgG (n.s. IgG), and the precipitates were analyzed by immunoblotting (Blot) with anti-GluR6. In the lane-marked input, 100 lg of protein without immunoprecipitation was loaded
Semiquantification of TUNEL and Cresyl Violet Staining Data Statistical Analysis Values expressed as mean ± SD were obtained independently from at least six rats. Statistical analysis of the results was carried out by one-way analysis of the variance (ANOVA) followed by Duncan’s new multiple range method or Newman–Keuls test. p \ 0.05 was considered significant.
FH Suppresses GluR6 Serine Phosphorylation Induced by I/R at 6 h Reperfusion in Rat Hippocampal CA1 Regions To further elucidate whether FH could inhibit GluR6 serine phosphorylation, we administered FH to detect serine phosphorylation of GluR6 at 6 h during reperfusion. Our result shows that FH suppressed GluR6 serine phosphorylation (Fig. 2). FH Inhibited the Phosphorylation of MLK3 Induced by I/R at 6 h Reperfusion in Rat Hippocampal CA1 Regions We selected 6 h after reperfusion to investigate MLK3 activation at the peak 6 h of MLK3 after reperfusion. We found that FH significantly diminished MLK3 activation at 6 h of reperfusion. It suggested that the increased ROCK activation might affect GluR6 downstream kinase MLK3 activation (Fig. 3).
Results Enhanced Serine Phosphorylation of GluR6 Induced by Brain I/R in Hippocampus Immunoprecipitation (IP) and immunoblot (IB) were used to examine serine phosphorylation of GluR6 after I/R in rat hippocampus. When Immunoprecipitated with anti-pSer followed by Blot with anti-GluR6, a band of about 115KDa was detected, and a band of the same molecular weight in
Fig. 2 Effects of fasudil hydrochloride (FH) on the GluR6 subunit serine phosphorylation at 6 h reperfusion in rat hippocampal CA1 regions. Rats were treated with FH, respectively, or saline operation before 15 min ischemia. Extracts from these groups were analyzed using immunoprecipitation and immunoblotting analysis. a Representative blots showing levels of the GluR6 and GluR6 serine
FH Inhibited the JNK3 Activation Induced by I/R at 3 d Reperfusion in Rat Hippocampal CA1 Regions We selected 3 d, the peak point of JNK3 activation after reperfusion, to investigate JNK3 activation to detect the effect of FH. We found that FH significantly diminished JNK3 activation at 3 d of reperfusion. It suggested that FH administered might affect GluR6 downstream kinase JNK3 activation (Fig. 4).
phosphorylation at 6 h reperfusion in rat hippocampal CA1 regions after 15 min ischemia. b Semiquantitative analysis of the GluR6 and GluR6 serine phosphorylation at 6 h reperfusion after 15 min ischemia. Data were mean ± SD. *p \ 0.05 versus sham, #p \ 0.05 versus I/R 6 h or saline
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Fig. 3 Effects of FH on the MLK3 activation in rat hippocampal CA1 regions. Rats were treated with FH, respectively, or saline operation before 15 min ischemia. Extracts from these groups were analyzed using immunoblotting analysis. a Representative blots showing levels of the phosphorylation and expression of MLK3 at
6 h reperfusion at different operated groups. b Semiquantitative analysis of the expression and activation of JNK3. Data were expressed as mean ± SD derived from six independent animals in each experiment group. *p \ 0.05 versus sham, #p \ 0.05 versus I/R 6 h or saline
Fig. 4 Effects of FH on the JNK3 activation in rat hippocampal CA1 regions. Rats were treated with FH, respectively, or saline operation before 15 min ischemia. Extracts from these groups were analyzed using immunoblotting analysis. a Representative blots showing levels of the phosphorylation and expression of JNK3 at 3 d reperfusion at
different operated groups. b Semiquantitative analysis of the expression and activation of JNK3. Data were expressed as mean ± SD derived from six independent animals in each experiment group. *p \ 0.05 versus sham, #p \ 0.05 versus I/R 3 d or saline
Fig. 5 Effects of FH on the caspase-3 activation in rat hippocampal CA1 regions. Rats were treated with FH, respectively, or saline operation before 15 min ischemia. Extracts from these groups were analyzed using immunoblotting analysis. a Representative blots showing levels of expression of caspase-3 at 6 h reperfusion at
different operated groups. b Semiquantitative analysis of the expression and activation of caspase-3. Data were expressed as mean ± SD derived from six independent animals in each experiment group. *p \ 0.05 versus sham, #p \ 0.05 versus I/R 6 h or saline
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Fig. 6 The neuroprotection of FH against brain ischemic injury in rat hippocampal CA1 regions. a, b (left) original magnification 940; (right) original magnification 9400; a Representative photograph of TUNEL-positive neurons of CA1 region from rats subjected to sham, ischemia-operated rats subjected to 15 min of ischemia followed by 3 d of reperfusion, vehicle-operated rats, FH-operated rats, respectively. b Example of Cresyl violet-strained sections of the hippocampi of
sham-operated rats and ischemia-operated rats subjected to 15 min of ischemia followed by 5 d of reperfusion, vehicle-operated rats, FHoperated rats also subjected to 15 min of ischemia followed by 5 d of reperfusion. Data were obtained from six independent animals in each experimental group, and the results of a typical experiment are presented. The areas shown with high magnification on the right panel and the number of vital neurons were counted
Table 1 Quantitative analysis of the numbers of TUNEL-positive neurons of CA1 region from different operated rats by FH
Table 2 Quantitative analysis of the protective effects of FH against transient ischemia followed by reperfusion
Group
Group
n
Numbers of neuronal profiles (mean ± SD) 193.3 ± 2.9
n
Positive numbers of neuronal profiles (mean ± SD)
Sham
6
8.60 ± 2.19
Sham
6
I/R 3 d
6
41.80 ± 3.03*
I/R 5 d
6
9.8 ± 2.1*
Saline 3 d
6
40.20 ± 2.77*
Saline 5 d
6
11.8 ± 1.6*
Fasudil 3 d
6
19.80 ± 2.86#
Fasudil 5 d
6
82.8 ± 3.2#
The numbers of positive neuronal profiles are expressed as numbers of apoptosis neurons per 1 mm length of CA1 pyramidal cells counted under light microscopy. Data are mean ± SD (n = 6)
The numbers of neuronal profiles are expressed as numbers of surviving neurons per 1 mm length of CA1 pyramidal cells counted under light microscopy. Data are mean ± SD (n = 6)
*p \ 0.05 versus sham, #p \ 0.05 versus I/R 3 d or saline 3 d
*p \ 0.05 versus sham, #p \ 0.05 versus I/R 5 d or saline 5 d
FH Inhibited the Activity of Caspase-3 Induced by I/R at 6 h at Reperfusion in Rat Hippocampal CA1 Regions
and 5 d reperfusion, respectively. The numbers of viable neurons per 1 mm length of CA1 pyramidal cells were quantitatively analyzed. As shown in Fig. 6a and Table 1, a significant number of TUNEL-positive cells were observed 3 d after reperfusion. FH also markedly decreased the neuronal injury. Transient brain ischemia followed 5 d of reperfusion induced sever cell death, while normal CA1 pyramidal cells in shamoperated groups showed round and pale-stained nuclei. However, FH obviously limited the neuronal loss (Fig. 6b; Table 2).
To investigate the effect of FH on caspase-3 activation, the study was carried out by Western immunoblotting. Caspase-3 had one activated peak at 6 h of reperfusion [7]. We confirmed that pretreatment with FH rescued the ischemia-induced increase in caspase-3 activation observed at 6 h of reperfusion (Fig. 5). FH Decreases Neural Injury of Rat Hippocampal CA1 Region Significantly after I/R
Discussion To examine whether FH could possess the potential protective effects against ischemic injury, TUNEL staining and Cresyl violet staining were used to examine the survival neurons after 3
Rho kinase is a serine/threonine kinase that is important in fundamental processes of cell migration, proliferation, and
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survival. However, the mechanism of ROCK-mediated ischemic injury is still not thoroughly understood. Fasudil is metabolized to hydroxyfasudil in human. Both fasudil and hydroxyfasudil are strong inhibitors of ROCK; furthermore, the major effect is considered to depend on FH rather than fasudil itself [14]. GluR6-PSD95-MLK3 signal pathway plays a crucial role in the pathogenesis of cerebral ischemic injury. Recent study has proved that GluR6 receptor plays a prominent role in brain I/R-mediated neuronal injury [3, 4]. Meanwhile, a previous study showed that the level of phosphorylation was time dependent by means of 4-VO occlusion models and also showed the peak of the GluR6 autophosphorylation occurring at 6 h [8, 20] after reperfusion. In this report, we found that brain ischemia could induce GluR6 serine phosphorylation, which was consistent with the previous study [5] (Fig. 1). Kainate receptor GluR6 subunit is largely expressed in the CA1 and CA3 of the brain hippocampus involved in learning and memory [11]. Kainate receptor GluR6 binds to PSD-95, which, in turn, anchors MLK3 via SH3 domain in rat brain tissue. MLK3 subsequently activates JNK via MAP kinase kinases (MKKs) [5]. MLK3, a member of mixed lineage kinase family, is a 93-kDa intracellular serine/threonine kinase, which triggers the activation of JNK3 [12]. Some reports have shown that autophosphorylation was required for MLK3 activation [6]. One member of the JNK family, JNK3, the only neural-specific isoform, is found predominantly in the brain within neurons [15] and is selectively activated during ischemia-induced apoptosis. Previous study also suggested that MLK3 and JNK3 played an important role in ischemia-induced neuronal apoptosis [1]. Either the nucleus or cytosol, JNK3 or MLK3 was activated and peaked at 30 min and 3 days during reperfusion [1]. MLK3 was activated and peaked at 30 min and 6 h during reperfusion [7]. Our present study indicated that FH inhibited autophosphorylation of GluR6 (6 h) (Fig. 2) and further reduced phosphorylation of MLK3 (6 h) (Fig. 3) and JNK3 (3 d) (Fig. 4). Thus, we speculated that there is a new pathway, namely Rho-GluR6-MLK3-JNKs signal pathway, in the I/R damage. Fasudil, a ROCK inhibitor, protects against ischemic damage in vitro and in vivo by acting directly on neurons. Our results indicate that GluR6 serine autophosphorylation is mediated following transient cerebral ischemia. So, we suppose that FH suppresses GluR6 serine phosphorylation. And our results certify this point. To further confirm the FH’s neuronal protection, we investigated the expression of caspase-3 following ischemia, a common molecule in cell apoptotic pathway. Our result shows that administration of FH can significantly suppress the activation of caspase-3 induced by I/R. Result
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from histology indicated that FH markedly decreased the number of TUNEL-positive cells at 3 d after ischemia. Meanwhile, it can obviously limit the neuronal death or injury. Based on the above data, autophosphorylation of GluR6, MLK3, and JNKs occurs in the serine site following transient cerebral ischemia. Furthermore, MLK3 and JNK3 autophosphorylation and the interaction occurred with the alteration of GluR6-PSD-95-MLK3-signaling module [1]. ROCK is one of the serine/threonine family. Our results confirm that FH, as a ROCK inhibitor, can decrease GluR6 autophosphorylation after I/R through inducing the downstream MLK3 and JNK3 autophosphorylation after reperfusion. Meanwhile, FH can also inhibit a common apoptotic pathway by inhibiting the expression of caspase3. Morphology results also show that FH can protect neurons in vivo. All in all, our results demonstrated that FH induced neuroprotection in vivo and may represent a useful therapeutic perspective by inhibiting GluR6-PSD-95MLK3-signaling pathway. From these results, we provide information for the first time that FH, a ROCK inhibitor, causes direct phosphorylation of GluR6 through the GluR6-PSD-95-MLK3-signaling pathway. But, it is unknown about the sites of serine phosphorylation of GluR6 which is mediated by FH. Therefore, it still needs further study to find out FH and GluR6, which would be our works to examine in the next step.
References 1. Hitomi, A., Satoh, S.-I., Ikegaki, I., Suzuki, Y., Shibuya, M., & Asano, T. (2000). Hemorheological abnormalities in experimental cerebral ischemia and effects of protein kinase inhibitor on blood fluidity. Life Science, 67, 1929–1939. 2. Li, C., Han, D., Zhang, F., Zhou, C., Yu, H. M., & Zhang, G. Y. (2007). Preconditioning ischemia attenuates increased neurexinneuroligin1–PSD-95 interaction after transient cerebral ischemia in rat hippocampus. Neuroscience Letters, 426, 192–197. 3. Mulle, C., Sailer, A., Perez-Otano, I., Dickinson-Anson, H., Castillo, P. E., Bureau, I., et al. (1998). Altered synaptic physiology and reduced susceptibility to kainate-induced seizures in GluR6deficient mice. Nature, 392, 601–605. 4. Tian, H., Zhang, G. Y., Li, H. C., & Zhang, Q. G. (2003). Antioxidant NAC and AMPA/KA receptor antagonist DNQX inhibited JNK3 activation following global ischemia in rat hippocampus. Neuroscience Research, 46, 191–197. 5. Tian, H., Zhang, Q. G., Zhu, G. X., Pei, D. S., Guan, Q. H., & Zhang, G. Y. (1061). Activation of c-Jun NH2-terminal kinase 3 is mediated by the GluR6PSD-95MLK3 signaling module following cerebral ischemia in rat hippocampus. Brain Research, 2005, 57–66. 6. Leung, I. W., & Lassam, N. (2001). The kinase activation loop is the key to mixed lineage kinase-3 activation via both back phosphorylation and hematopoietic progenitor kinase 1 phosphorylation. The Journal of Biological Chemistry, 276, 1961–1967.
Cell Biochem Biophys 7. Pan, J., Zhang, Q. G., & Zhang, G. Y. (2005). The neuroprotective effects of K252a through inhibiting MLK3/MKK7/JNK3 signaling pathway on ischemic brain injury in rat hippocampal region. Neuroscience, 131, 147–159. 8. Xu, J., Liu, Z. A., Pei, D. S., & Xu, T. J. (2010). Calcium/ calmodulin-dependent kinase II facilitated GluR6 subunit serine phosphorylation through GluR6–PSD95–CaMKII signaling module assembly in cerebral ischemia injury. Brain Research, 1366, 197–203. 9. Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., et al. (1996). Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-Kinase). Science, 273, 245–248. 10. Ogita, K., & Yoneda, Y. (1994). Selective potentiation of DNA binding activities of both activator protein 1 and cyclic AMP response element binding protein through in vivo activation of N-methyl-d-aspartate receptor complex in mouse brain. Journal Neurochemistry, 63, 525–534. 11. Tibbles, L. A., Ing, Y. L., Kiefer, F., Chan, J., Iscove, N., Woodgett, J. R., et al. (1996). MLK-3 activates the SAPK/JNK and p38/RK pathways via SEK1 and MKK3/6. EMBO Journal, 15, 7026–7035. 12. Darstein, M., Petralia, R. S., Swanson, G. T., Wenthold, R. J., & Heinemann, S. F. (2003). Distribution of kainate receptor subunits at hippocampal mossy fiber synapses. Journal of Neuroscience, 23, 8013–8019. 13. Shibuya, M., Suzuki, Y., Sugita, K., Saito, I., Sasaki, T., Takakura, K., et al. (1992). Effect of AT877 on cerebral vasospasm after aneurysmal subarachnoid hemorrhage. Results of a prospective placebo-controlled double-blind trial. Journal Neurosurgery, 76, 571–577.
14. Uehata, M., Ishizaki, T., Satoh, H., Ono, T., Kawahara, T., Morishita, T., et al. (1997). Calcium sensitization of smooth muscle mediated by Rho-associated protein kinase in hypertension. Nature, 389, 990–994. 15. Gupta, S., Barrett, T., Whitmarsh, A. J., Cavanagh, J., Sluss, H. K., Derijard, B., et al. (1996). Selective interaction of JNK protein kinase isoforms with transcription factors. EMBO Journal, 15, 2760–2770. 16. Satoh, S., Kobayashi, T., Hitomi, A., Ikegaki, I., Suzuki, Y., Shibuya, M., et al. (1999). Inhibition of neutrophil migration by a protein kinase inhibitor for the treatment of ischemic brain infarction. The Japanese Journal of Pharmacology, 30, 41–48. 17. Satoh, S., Utsunomiya, T., Tsurui, K., Kobayashi, K., Ikegaki, I., Sasaki, Y., et al. (2001). Pharmacological profile of hydroxyphasudil as a selective rho kinase inhibitor on ischemic brain damage. Life Science, 69, 1441–1453. 18. Pulsinelli, W. A., & Brierlsy, J. B. (1979). A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke, 3, 267–272. 19. Rikitake, Y., Kim, H., Huang, Z., Seto, M., Yano, K., Asano, T., et al. (2005). Inhibition of rho kinase(ROCK) leads to increased cerebral blood flow and stroke protection. Stroke, 36, 2251–2257. 20. Hao, Z. B., Pei, D. S., Guan, Q. H., & Zhang, G. Y. (2005). Calcium/calmodulin-dependent protein kinase II (CaMKII), through NMDA receptors and L-voltage-gated channels, modulates the serine phosphorylation of GluR6 during cerebral ischemia and early reperfusion period in rat hippocampus. Molecular Brain Research, 140, 55–62.
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ORIGINAL PAPER
Fasudil Hydrochloride Protects Neurons in Rat Hippocampal CA1 Region through Inhibiting GluR6–MLK3–JNKs Signal Pathway Xiu-E Wei • Feng-Yu Zhang • Kai Wang Qing-Xiu Zhang • Liang-Qun Rong
•
Ó Springer Science+Business Media New York 2014
Abstract Fasudil hydrochloride (FH), a Rho kinase (ROCK) inhibitor, has been reported to prevent cerebral ischemia in vivo from increasing cerebral blood flow and inhibiting inflammatory responses. However, it is uncertain by what mechanism a ROCK inhibitor can directly protect neurons against ischemic damage. The present study was designed to evaluate whether FH decreased the increased phosphorylation of glutamate receptor 6 (GluR6) and its downstream in GluR6–MLK3–JNKs signal transduction pathway following global transient cerebral ischemia, as a result of protecting against neuronal apoptosis and death. Transient cerebral ischemia was induced by the Pulsinelli– Brierley four-vessel occlusion method. FH (15 mg/kg) was administered to rats by intraperitoneal injection 30 min before ischemia. The phosphorylation and protein expression of GluR6 at 6 h during reperfusion were detected using immunoprecipitation and immunoblotting analysis. The phosphorylation and protein expression of Mixed lineage kinase 3 (MLK3) at ischemia/reperfusion (I/R) 6 h and c-Jun N-terminal kinase (JNK) at I/R 3 d were detected using immunoblotting analysis, respectively. The same method was used to detect the expression of caspase-3 at I/R 6 h. Furthermore, we also use TUNEL staining and Cresyl violet staining to examine the survival neurons in rat hippocampal CA1 regions after 3 and 5 d reperfusion, respectively. Our study indicated that FH could inhibit the X.-E. Wei (&) K. Wang Q.-X. Zhang L.-Q. Rong Department of Neurology, The Second Affiliated Hospital of Xuzhou Medical College, Xuzhou 221000, People’s Republic of China e-mail: [email protected] F.-Y. Zhang Department of Neurology, Liao Cheng Hospital, Liaocheng 252000, People’s Republic of China
increased phosphorylation of GluR6 and MLK3 and the expression of caspase-3 at peaked 6 h of reperfusion and the phosphorylation of JNK (3 d) (p \ 0.5). The results of TUNEL staining and Cresyl violet showed that the number of surviving pyramidal neurons in rats hippocampal CA1 subfield increased markedly in FH-treated rats compared with ischemic groups after 3 or 5 d of reperfusion following ischemia (p \ 0.5). These results suggested that FH, as a ROCK inhibitor, may be partly responsible for its protective effects against such damage by taking part in GluR6-MLK3-JNKs signaling pathway which modulates ischemic damage. Taken together, this is the first study investigating Rho and ROCK as the upstream of GluR6 taking part in GluR6–MLK3–JNKs signal transduction pathway following cerebral ischemia. Keywords Fasudil hydrochloride GluR6 CA1 TUNEL staining Cerebral ischemia
Introduction Since cerebral infarction is one of the principal etiologies for neurological sequelae and/or death, it is very important to understand both its pathologic mechanisms and any effective treatments. After ROCK was found [9], it became clear that upregulated ROCK worked unfavorably to the host in many vascular diseases. Fasudil, initially characterized as an intracellular calcium antagonist, has played an important role in cerebral vasospasms occurring after subarachnoid hemorrhage by increasing regional CBF not only via an inhibition of smooth muscle contraction but also through an upregulation of endothelial nitric oxide synthase (eNOS) [13, 19] and a decrease in the inflammatory response [1, 16, 17, 19]. FH has an important role
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in cerebral ischemia, but by what molecular mechanism does it achieve the protective effect remains unknown. GluR6 receptor plays a prominent role and mediates serine phosphorylation in brain ischemia-mediated neuronal injury [3, 5]. FH inhibits the serine/threonine phosphorylation of ROCK. With this in mind, what is the relationship between ROCK and GluR6 or between FH and GluR6 serine phosphorylation in ischemic injury? Moreover, the RhoA/ROCK pathway provides promising pharmacological targets for treatment of a variety of cerebralvascular diseases. The present reports indicate that it is linked to glutamate receptor 6 (GluR6), the postsynaptic density protein 95 (PSD95), mixed lineage kinase 3 (MLK3), and JNK through phosphorylation triggering the cascade reaction in cerebral ischemic injury [4, 5, 7, 8]. What is the association between FH and MLK3, JNK3 serine phosphorylation in ischemic injury? Whether FH can inhibit GluR6 serine phosphorylation through inducing MLK3 and JNK3 serine phosphorylation or not? are the questions that remain unclear.
Materials and Methods Animals and Drugs Adult male Sprague–Dawley rats (SD rats, Shanghai Experimental Animal Center, Chinese Academy of Science) weighing 220–300 g were used. The experimental procedures were approved by the local legislation for ethics of experiments on animals, and efforts were carried out to minimize the pain or discomfort of rats. Four-vessel occlusion (4-VO) cerebral ischemia was induced as described before [18]. Male SD rats were randomly divided into four groups: given control, ischemia/reperfusion (I/R), I/R with FH treatments, and saline group. Briefly, under anesthesia with chloral hydrate (300 mg/kg, i.p.), vertebral arteries were electrocauterized, and common carotid arteries were exposed. Fasudil hydrochloride (15 mg/kg) was dissolved in saline at a concentration of 0.9 mg/ml, which was administered to rats by intraperitoneal injection 30 min before ischemia. Control rats were intraperitoneally given saline. Tissue Preparation Rats were decapitated immediately after 6 h or 3 d of reperfusion, and then the hippocampus was separated into CA1 and CA3/DG from hippocampal fissure and quickly frozen in –80 °C. Cytosol fraction was extracted as Ogita and Yoneda [10] described procedure. The homogenate
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was centrifuged at 12,0009g for 5 min at 4 °C. The supernatants were collected, and the protein concentration was determined by Lowry’s method. The protein was packed by the same concentration and boiled for 5 min before cooling. After dealing with that, samples stored at -80 °C were thawed until assays. Methods Immunoprecipitation and Immunoblotting Tissue homogenates (400 lg of protein) were processed following the methods of Li et al. [2]. Immunoprecipitation and immunoblotting-specific operation is as described by Li et al. [2]. The difference is Membranes were washed and then incubated with alkaline phosphatase-conjugated secondary antibodies in milk for 2 h and developed using NBT/BCIP color substrate (beyotime, China). The density of the bands on the membrane was scanned and analyzed with an image analyzer (LabWorks Software, UVP Upland, CA). TUNEL Staining TUNEL staining was performed using an ApopTagÒ Peroxidase In Situ Apoptosis Detection Kit according to the manufacturer’s protocol with minor modifications. Sections were treated with anti-digoxigenin conjugate for 30 min at room temperature and subsequently developed color in NBT/BCIP substrate. An initial dissector frame was positioned randomly in hippocampal sector and cells in every 10th section throughout the entire hippocampus. The cell numbers in hippocampus were assessed by means of previously published unbiased stereological techniques. In brief, cell counts were performed at 4009 magnification with the use of an Olympus BH-2 microscope connected to a Sony charge-coupled device video camera, amotorize stage system, and commercial stereology software. Cresyl Violet Staining The rats subjected to 5 d of reperfusion were perfusion fixed with 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under anesthesia. The paraffin-embedded brain sections (5 lm) were prepared and stained with Cresyl violet. Histological evaluations were performed with Cresyl violet staining for assessment of neuronal damage in the hippocampus. The Cresyl violet-positive cell numbers were counted by means of the unbiased stereological methods, which is mentioned above in TUNEL staining.
Cell Biochem Biophys
the lane-marked input was detected (Fig. 1). The results showed that GluR6 was phosphorylated at serine residues. When immunoprecipitated with nonspecific mouse IgG, no significant band corresponding to GluR6 was detected. Fig. 1 GluR6 is phosphorylated at the serine residue in hippocampus. Homogenates (400 lg of total protein) from sham-operated controls (Sham) and ischemic animals at 15 min of ischemia (I) were immuno precipitated (IP) with anti-phosphoserine antibody (anti-pSer) or nonspecific IgG (n.s. IgG), and the precipitates were analyzed by immunoblotting (Blot) with anti-GluR6. In the lane-marked input, 100 lg of protein without immunoprecipitation was loaded
Semiquantification of TUNEL and Cresyl Violet Staining Data Statistical Analysis Values expressed as mean ± SD were obtained independently from at least six rats. Statistical analysis of the results was carried out by one-way analysis of the variance (ANOVA) followed by Duncan’s new multiple range method or Newman–Keuls test. p \ 0.05 was considered significant.
FH Suppresses GluR6 Serine Phosphorylation Induced by I/R at 6 h Reperfusion in Rat Hippocampal CA1 Regions To further elucidate whether FH could inhibit GluR6 serine phosphorylation, we administered FH to detect serine phosphorylation of GluR6 at 6 h during reperfusion. Our result shows that FH suppressed GluR6 serine phosphorylation (Fig. 2). FH Inhibited the Phosphorylation of MLK3 Induced by I/R at 6 h Reperfusion in Rat Hippocampal CA1 Regions We selected 6 h after reperfusion to investigate MLK3 activation at the peak 6 h of MLK3 after reperfusion. We found that FH significantly diminished MLK3 activation at 6 h of reperfusion. It suggested that the increased ROCK activation might affect GluR6 downstream kinase MLK3 activation (Fig. 3).
Results Enhanced Serine Phosphorylation of GluR6 Induced by Brain I/R in Hippocampus Immunoprecipitation (IP) and immunoblot (IB) were used to examine serine phosphorylation of GluR6 after I/R in rat hippocampus. When Immunoprecipitated with anti-pSer followed by Blot with anti-GluR6, a band of about 115KDa was detected, and a band of the same molecular weight in
Fig. 2 Effects of fasudil hydrochloride (FH) on the GluR6 subunit serine phosphorylation at 6 h reperfusion in rat hippocampal CA1 regions. Rats were treated with FH, respectively, or saline operation before 15 min ischemia. Extracts from these groups were analyzed using immunoprecipitation and immunoblotting analysis. a Representative blots showing levels of the GluR6 and GluR6 serine
FH Inhibited the JNK3 Activation Induced by I/R at 3 d Reperfusion in Rat Hippocampal CA1 Regions We selected 3 d, the peak point of JNK3 activation after reperfusion, to investigate JNK3 activation to detect the effect of FH. We found that FH significantly diminished JNK3 activation at 3 d of reperfusion. It suggested that FH administered might affect GluR6 downstream kinase JNK3 activation (Fig. 4).
phosphorylation at 6 h reperfusion in rat hippocampal CA1 regions after 15 min ischemia. b Semiquantitative analysis of the GluR6 and GluR6 serine phosphorylation at 6 h reperfusion after 15 min ischemia. Data were mean ± SD. *p \ 0.05 versus sham, #p \ 0.05 versus I/R 6 h or saline
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Fig. 3 Effects of FH on the MLK3 activation in rat hippocampal CA1 regions. Rats were treated with FH, respectively, or saline operation before 15 min ischemia. Extracts from these groups were analyzed using immunoblotting analysis. a Representative blots showing levels of the phosphorylation and expression of MLK3 at
6 h reperfusion at different operated groups. b Semiquantitative analysis of the expression and activation of JNK3. Data were expressed as mean ± SD derived from six independent animals in each experiment group. *p \ 0.05 versus sham, #p \ 0.05 versus I/R 6 h or saline
Fig. 4 Effects of FH on the JNK3 activation in rat hippocampal CA1 regions. Rats were treated with FH, respectively, or saline operation before 15 min ischemia. Extracts from these groups were analyzed using immunoblotting analysis. a Representative blots showing levels of the phosphorylation and expression of JNK3 at 3 d reperfusion at
different operated groups. b Semiquantitative analysis of the expression and activation of JNK3. Data were expressed as mean ± SD derived from six independent animals in each experiment group. *p \ 0.05 versus sham, #p \ 0.05 versus I/R 3 d or saline
Fig. 5 Effects of FH on the caspase-3 activation in rat hippocampal CA1 regions. Rats were treated with FH, respectively, or saline operation before 15 min ischemia. Extracts from these groups were analyzed using immunoblotting analysis. a Representative blots showing levels of expression of caspase-3 at 6 h reperfusion at
different operated groups. b Semiquantitative analysis of the expression and activation of caspase-3. Data were expressed as mean ± SD derived from six independent animals in each experiment group. *p \ 0.05 versus sham, #p \ 0.05 versus I/R 6 h or saline
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Fig. 6 The neuroprotection of FH against brain ischemic injury in rat hippocampal CA1 regions. a, b (left) original magnification 940; (right) original magnification 9400; a Representative photograph of TUNEL-positive neurons of CA1 region from rats subjected to sham, ischemia-operated rats subjected to 15 min of ischemia followed by 3 d of reperfusion, vehicle-operated rats, FH-operated rats, respectively. b Example of Cresyl violet-strained sections of the hippocampi of
sham-operated rats and ischemia-operated rats subjected to 15 min of ischemia followed by 5 d of reperfusion, vehicle-operated rats, FHoperated rats also subjected to 15 min of ischemia followed by 5 d of reperfusion. Data were obtained from six independent animals in each experimental group, and the results of a typical experiment are presented. The areas shown with high magnification on the right panel and the number of vital neurons were counted
Table 1 Quantitative analysis of the numbers of TUNEL-positive neurons of CA1 region from different operated rats by FH
Table 2 Quantitative analysis of the protective effects of FH against transient ischemia followed by reperfusion
Group
Group
n
Numbers of neuronal profiles (mean ± SD) 193.3 ± 2.9
n
Positive numbers of neuronal profiles (mean ± SD)
Sham
6
8.60 ± 2.19
Sham
6
I/R 3 d
6
41.80 ± 3.03*
I/R 5 d
6
9.8 ± 2.1*
Saline 3 d
6
40.20 ± 2.77*
Saline 5 d
6
11.8 ± 1.6*
Fasudil 3 d
6
19.80 ± 2.86#
Fasudil 5 d
6
82.8 ± 3.2#
The numbers of positive neuronal profiles are expressed as numbers of apoptosis neurons per 1 mm length of CA1 pyramidal cells counted under light microscopy. Data are mean ± SD (n = 6)
The numbers of neuronal profiles are expressed as numbers of surviving neurons per 1 mm length of CA1 pyramidal cells counted under light microscopy. Data are mean ± SD (n = 6)
*p \ 0.05 versus sham, #p \ 0.05 versus I/R 3 d or saline 3 d
*p \ 0.05 versus sham, #p \ 0.05 versus I/R 5 d or saline 5 d
FH Inhibited the Activity of Caspase-3 Induced by I/R at 6 h at Reperfusion in Rat Hippocampal CA1 Regions
and 5 d reperfusion, respectively. The numbers of viable neurons per 1 mm length of CA1 pyramidal cells were quantitatively analyzed. As shown in Fig. 6a and Table 1, a significant number of TUNEL-positive cells were observed 3 d after reperfusion. FH also markedly decreased the neuronal injury. Transient brain ischemia followed 5 d of reperfusion induced sever cell death, while normal CA1 pyramidal cells in shamoperated groups showed round and pale-stained nuclei. However, FH obviously limited the neuronal loss (Fig. 6b; Table 2).
To investigate the effect of FH on caspase-3 activation, the study was carried out by Western immunoblotting. Caspase-3 had one activated peak at 6 h of reperfusion [7]. We confirmed that pretreatment with FH rescued the ischemia-induced increase in caspase-3 activation observed at 6 h of reperfusion (Fig. 5). FH Decreases Neural Injury of Rat Hippocampal CA1 Region Significantly after I/R
Discussion To examine whether FH could possess the potential protective effects against ischemic injury, TUNEL staining and Cresyl violet staining were used to examine the survival neurons after 3
Rho kinase is a serine/threonine kinase that is important in fundamental processes of cell migration, proliferation, and
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survival. However, the mechanism of ROCK-mediated ischemic injury is still not thoroughly understood. Fasudil is metabolized to hydroxyfasudil in human. Both fasudil and hydroxyfasudil are strong inhibitors of ROCK; furthermore, the major effect is considered to depend on FH rather than fasudil itself [14]. GluR6-PSD95-MLK3 signal pathway plays a crucial role in the pathogenesis of cerebral ischemic injury. Recent study has proved that GluR6 receptor plays a prominent role in brain I/R-mediated neuronal injury [3, 4]. Meanwhile, a previous study showed that the level of phosphorylation was time dependent by means of 4-VO occlusion models and also showed the peak of the GluR6 autophosphorylation occurring at 6 h [8, 20] after reperfusion. In this report, we found that brain ischemia could induce GluR6 serine phosphorylation, which was consistent with the previous study [5] (Fig. 1). Kainate receptor GluR6 subunit is largely expressed in the CA1 and CA3 of the brain hippocampus involved in learning and memory [11]. Kainate receptor GluR6 binds to PSD-95, which, in turn, anchors MLK3 via SH3 domain in rat brain tissue. MLK3 subsequently activates JNK via MAP kinase kinases (MKKs) [5]. MLK3, a member of mixed lineage kinase family, is a 93-kDa intracellular serine/threonine kinase, which triggers the activation of JNK3 [12]. Some reports have shown that autophosphorylation was required for MLK3 activation [6]. One member of the JNK family, JNK3, the only neural-specific isoform, is found predominantly in the brain within neurons [15] and is selectively activated during ischemia-induced apoptosis. Previous study also suggested that MLK3 and JNK3 played an important role in ischemia-induced neuronal apoptosis [1]. Either the nucleus or cytosol, JNK3 or MLK3 was activated and peaked at 30 min and 3 days during reperfusion [1]. MLK3 was activated and peaked at 30 min and 6 h during reperfusion [7]. Our present study indicated that FH inhibited autophosphorylation of GluR6 (6 h) (Fig. 2) and further reduced phosphorylation of MLK3 (6 h) (Fig. 3) and JNK3 (3 d) (Fig. 4). Thus, we speculated that there is a new pathway, namely Rho-GluR6-MLK3-JNKs signal pathway, in the I/R damage. Fasudil, a ROCK inhibitor, protects against ischemic damage in vitro and in vivo by acting directly on neurons. Our results indicate that GluR6 serine autophosphorylation is mediated following transient cerebral ischemia. So, we suppose that FH suppresses GluR6 serine phosphorylation. And our results certify this point. To further confirm the FH’s neuronal protection, we investigated the expression of caspase-3 following ischemia, a common molecule in cell apoptotic pathway. Our result shows that administration of FH can significantly suppress the activation of caspase-3 induced by I/R. Result
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from histology indicated that FH markedly decreased the number of TUNEL-positive cells at 3 d after ischemia. Meanwhile, it can obviously limit the neuronal death or injury. Based on the above data, autophosphorylation of GluR6, MLK3, and JNKs occurs in the serine site following transient cerebral ischemia. Furthermore, MLK3 and JNK3 autophosphorylation and the interaction occurred with the alteration of GluR6-PSD-95-MLK3-signaling module [1]. ROCK is one of the serine/threonine family. Our results confirm that FH, as a ROCK inhibitor, can decrease GluR6 autophosphorylation after I/R through inducing the downstream MLK3 and JNK3 autophosphorylation after reperfusion. Meanwhile, FH can also inhibit a common apoptotic pathway by inhibiting the expression of caspase3. Morphology results also show that FH can protect neurons in vivo. All in all, our results demonstrated that FH induced neuroprotection in vivo and may represent a useful therapeutic perspective by inhibiting GluR6-PSD-95MLK3-signaling pathway. From these results, we provide information for the first time that FH, a ROCK inhibitor, causes direct phosphorylation of GluR6 through the GluR6-PSD-95-MLK3-signaling pathway. But, it is unknown about the sites of serine phosphorylation of GluR6 which is mediated by FH. Therefore, it still needs further study to find out FH and GluR6, which would be our works to examine in the next step.
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