Valproic acid and ASK1 deficiency ameliorate optic neuritis and neurodegeneration in an animal model of multiple sclerosis

Neuroscience Letters 639 (2017) 82–87

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Research article

Valproic acid and ASK1 deficiency ameliorate optic neuritis and neurodegeneration in an animal model of multiple sclerosis Yuriko Azuchi a,b , Atsuko Kimura a , Xiaoli Guo a , Goichi Akiyama a , Takahiko Noro a , Chikako Harada a , Atsuko Nishigaki b , Kazuhiko Namekata a,b,∗ , Takayuki Harada a a b

Visual Research Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan Department of Environmental Science, Graduate School of Science, Toho University, Chiba, Japan

h i g h l i g h t s • • • •

We examine the effects of VPA on optic neuritis in EAE mice. VPA suppresses demyelination in the optic nerve and protects retinal neurons. VPA shows enhanced effects on retinal protection in ASK1-deficient EAE mice. VPA and ASK1 inhibition may be useful for treatment of optic neuritis and MS.

a r t i c l e

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Article history: Received 9 November 2016 Received in revised form 21 December 2016 Accepted 23 December 2016 Available online 28 December 2016 Keywords: Valproic acid ASK1 Optic neuritis EAE Neuroprotection

a b s t r a c t Optic neuritis, which is an acute inflammatory demyelinating syndrome of the central nervous system, is one of the major complications in multiple sclerosis (MS). Herein, we investigated the therapeutic potential of valproic acid (VPA) on optic neuritis in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. EAE was induced in C57BL/6 mice by immunization with MOG35-55 and VPA (300 mg/kg) was administered via intraperitoneal injection once daily from day 3 postimmunization until the end of the experimental period (day 28). VPA treatment suppressed neuroinflammation and decreased the clinical score of EAE at an early phase (from day 12–14 after immunization). We also examined the effects of apoptosis signal-regulating kinase 1 (ASK1), an evolutionarily conserved signaling intermediate for innate immunity, in EAE mice. ASK1 deficiency strongly suppressed microglial activation and decreased the clinical score of EAE at a late phase (day 25, 27 and 28 after immunization). When VPA was administered to ASK1-deficient EAE mice, the clinical score was suppressed in both early and late phases (from day 12–28 after immunization) and showed synergistic effects on protection of retinal neurons. Our findings raise intriguing possibilities that the widely prescribed drug VPA and ASK1 inhibition may be useful for neuroinflammatory disorders including optic neuritis and MS. © 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Optic neuritis is inflammation of the optic nerve and is the most common type of optic neuropathy. Patients usually present with an acute reduction of visual acuity, orbital pain exacerbated by eye movements, dyschromatopsia, and an afferent papillary defect, with or without swelling of the optic nerve head. Optic neuritis is the initial presentation in approximately 20% of multiple sclerosis (MS) cases and 30–70% of MS patients develop optic neuritis dur-

∗ Corresponding author at: Visual Research Project, Tokyo Metropolitan Institute of Medical Science 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan. E-mail address: [email protected] (K. Namekata). http://dx.doi.org/10.1016/j.neulet.2016.12.057 0304-3940/© 2016 Elsevier Ireland Ltd. All rights reserved.

ing the course of their disease [5,22]. Since optic neuritis can cause severe visual loss, especially in the optic-spinal form of MS or neuromyelitis optica [13,20], and this loss is irreversible currently, it draws much attention to finding a treatment that will restore the visual function. Valproic acid (VPA) is a short-chain fatty acid and is used worldwide clinically for treatment of epilepsy, mood disorders, migraines and neuropathic pain [4,11,17,27]. The pharmacological action of VPA involves multiple mechanisms including those that affect intracellular signal transduction pathways. For example, VPA may modulate enzymatic activities such as extracellular-signalregulated kinases (ERK), phosphatidylinositol 3-kinase/Akt-1, and glycogen synthase kinase 3␤, as well as histone deacetylase (HDAC) [6,9,23,25]. Recently, the concept that VPA exerts neuro-

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protective effects has emerged [2,3,15,16,29] and in addition, VPA may ameliorate inflammation of the spinal cord in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by suppressing the activation of T cells [18]. Another factor that is involved in the severity of EAE is apoptosis signal-regulating kinase 1 (ASK1), which is one of a growing number of mitogen-activated protein kinase (MAPK) kinase kinases identified in the upstream of the c-Jun N-terminal kinase and p38 MAPK pathways [12]. We previously reported that Toll-like receptor (TLR)-ASK1 signaling is required for chemokine productions in astrocytes and for recruitment of activated microglia into the lesion site during EAE [7]. In addition, the same signaling pathways in microglia seem to modulate progression of demyelination by altering the release of proinflammatory components. These results suggest that VPA and ASK1 may regulate the activity of different cell types that play important roles during EAE. To determine this possibility, in this study, we investigated the therapeutic potential of VPA against optic neuritis in EAE mice and examined whether ASK1 deficiency has synergistic effects with VPA. 2. Materials and methods 2.1. Animals Female C57BL/6J and ASK1−/− (ASK1 KO) mice [10] were 6–8 weeks of age at the time of immunization. Animal treatments were performed in accordance with the Tokyo Metropolitan Institute of Medical Science Guidelines for the Care and Use of Animals. 2.2. EAE induction, VPA administration, and clinical scoring EAE was induced with MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) as previously reported [7,21]. Briefly, mice were subcutaneously injected with 100 ␮g of MOG35–55 mixed with 500 ␮g of heat-killed Mycobacterium tuberculosis H37RA (Difco, Detroit, MI, USA) emulsified in complete Freund’s adjuvant. Each mouse also received intraperitoneal injections of 500 ng pertussis toxin (Merck Millipore, Billerica, MA, USA) immediately and 48 h after the immunization. To evaluate the effect of VPA, mice were treated with either VPA (300 mg/kg) or vehicle (PBS) once daily by intraperitoneal administration from day 3 postimmunization until the end of the experimental period (day 28). Clinical signs were scored daily as follows: 0, no clinical signs; 1, loss of tail tonicity; 2, flaccid tail; 3, impairment of righting reflex; 4, partial hind limb paralysis; 5, complete hind limb paralysis; 6, partial body paralysis; 7, partial forelimb paralysis; 8, complete forelimb paralysis or moribund; 9, death. 2.3. Histopathology and immunohistochemistry At the end of the experimental period, mice were perfused with Zamboni’s Fixative (2% paraformaldehyde and 15% picric acid in 0.1 M phosphate buffer). Eyes were enucleated and post-fixed in 3% glutaraldehyde solution (3% glutaraldehyde, 9% formaldehyde, 37.5% ethanol and 12.5% acetic acid in distilled water) for 2 h. Paraffin embedded retinal sections of 7 ␮m thickness were cut through the optic nerve and stained with hematoxylin and eosin (HE). The extent of retinal degeneration was quantified by counting the number of neurons in the ganglion cell layer (GCL) from one ora serrata through the optic nerve to the other ora serrata [10]. Optic nerves and spinal cords were post-fixed in Zamboni’s Fixative for 2 h and cut into 10 ␮m thick sections. Immunohistochemistry was performed using the following primary antibodies: rabbit antiGFAP (1:2; Abcam, Cambridge, MA, USA), goat anti-Iba1 (1:400; Abcam), and mouse anti-CD3 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). To quantify the stained region, the spinal cord

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Fig. 1. Effects of VPA on clinical evaluation of EAE in WT and ASK1 KO mice during a period of 28 days after MOG immunization. Mice were treated with either VPA (300 mg/kg) or vehicle (PBS) for 25 days (once daily, i.p.) from 3 days after MOG immunization. Data are presented as mean ± S.E.M. WT; n = 6, WT + VPA; n = 9, ASK1 KO; n = 7, ASK1 KO + VPA; n = 8. **p < 0.01; *p < 0.05.

sections were divided into four quadrants and the immunointensity in the area of one quadrant of the dorsal lateral region was calculated using ImageJ 1.50c4 (NIH, Bethesda, MD, USA). To analyze the optic nerve, the area 2 mm from the optic nerve head (0.04 mm2 ), was evaluated. GFAP and Iba1 immunointensity are expressed as fold changes relative to the WT non-EAE mice. The number of T cells is expressed as percentage of the WT EAE mice. To evaluate the extent of demyelination, optic nerve sections were stained with Luxol fast blue (LFB) followed by HE. Demyelination is expressed as percentage decrease in myelinated (stained) area relative to WT non-EAE mice. 2.4. Statistics Data are presented as means ± S.E.M. When statistical analyses were performed, the one-way ANOVA with Tukey-Kramer post hoc test was used to estimate the significance of the results. P < 0.05 was regarded as statistically significant. 3. Results 3.1. Effects of VPA and ASK1 deficiency on disease severity during EAE Mice were observed for a period of 28 days after MOG immunization. Wild-type (WT) EAE mice started to show disease signs about 10 days after disease induction (Fig. 1), and reached an incidence of 100%. Daily treatment of VPA significantly suppressed the severity of clinical symptoms in WT EAE mice from day 12–14. As we previously reported [7], the clinical score in ASK1 KO EAE mice was decreased at day 25, 27 and 28 compared with WT EAE mice. When ASK1 KO mice were treated with VPA, the average clinical score was decreased from day 12 through to the end of the experimental period compared with WT EAE mice. We then examined the spinal cords histopathologically at day 28 (Fig. 2A). The immunointensity of glial fibrillary acidic protein (GFAP)-positive astrocytes (Fig. 2B) and Iba1-positive microglia (Fig. 2C) was increased in WT EAE mice, but this increase was milder in VPA-treated WT EAE mice. The immunointensity of Iba1positive microglia in ASK1 KO EAE mice was significantly lower than those in VPA-treated WT EAE mice (Fig. 2C). Since T cells play critical roles in EAE, we also examined the infiltration of CD3-

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Fig. 2. Histological analyses of spinal cords in WT and ASK1 KO EAE mice. (A) Lumbar spinal cords were stained with an anti-GFAP, anti-Iba1 or anti-CD3 antibody. Scale bar: 300 ␮m for the upper and middle panels and 50 ␮m for the lower panel. (B,C) GFAP and Iba1 immunopositive areas are expressed as fold changes relative to the WT non-EAE mice. (D) The number of T cells is expressed as percentage of the WT EAE mice. Data are presented as mean ± SEM. n = 3 per group. ***p < 0.001; **p < 0.01; *p < 0.05.

positive T cells in the spinal cord. As previously reported [18], the number of T cells was significantly decreased with VPA treatment in WT EAE mice (Fig. 2D). The immunointensity for astrocytes and microglia in VPA-treated ASK1 KO EAE mice was significantly suppressed compared with VPA-treated WT mice, but additional effects were not detected compared with PBS-treated ASK1 KO EAE mice.

3.2. Effects of VPA and ASK1 deficiency on the optic nerve and retina We then examined the optic nerves histopathologically at day 28 (Fig. 3A). We found that the effects of VPA and ASK1 deficiency on glial cell activation in the EAE optic nerve were similar to those on the spinal cord (Fig. 3B and C). On the other hand, there was no statistical difference in the number of T cells between WT EAE

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Fig. 3. Histological analyses of optic nerves in WT and ASK1 KO EAE mice. (A) Optic nerves were stained with an anti-GFAP, anti-Iba1, anti-CD3 antibody or LFB and HE. Scale bar: 65 ␮m for Iba1 and 50 ␮m for other panels. (B,C) GFAP and Iba1 immunopositive areas are expressed as fold changes relative to the WT non-EAE mice. (D) The number of T cells is expressed as percentage of the WT EAE mice. (E) Demyelination is expressed as percentage decrease in myelinated (stained) area relative to WT non-EAE mice. Data are presented as mean ± SEM. n = 4 per group. ***p < 0.001; **p < 0.01; *p < 0.05.

mice and ASK1 KO EAE mice, but it was significantly decreased with VPA treatment in these mice (Fig. 3D). EAE-induced demyelination in the optic nerves was milder with VPA treatment in WT mice (Fig. 3E). In addition, demyelination in VPA-treated ASK1 KO EAE mice was significantly suppressed compared with VPA-treated WT

mice, but additional effects were not detected compared with PBStreated ASK1 KO EAE mice. We next examined the effects of VPA and ASK1 deficiency on EAE-induced retinal degeneration that leads to permanent visual impairment [8]. The histopathological examination of the retina demonstrated that EAE induced significant loss of cells in the

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Fig. 4. Effects of VPA on retinal degeneration in WT and ASK1 KO EAE mice. (A) Representative histology of the retina in EAE mice. Scale bar: 80 ␮m for the upper panel and 40 ␮m for the lower panel. (B) Quantitative analyses of (A). Data are presented as mean ± SEM. n = 6 per group. ***p < 0.001; **p < 0.01; *p < 0.05.

GCL in WT mice, and the extent of retinal degeneration was not reduced in VPA-treated WT EAE and PBS-treated ASK1 KO EAE mice (Fig. 4). However, the number of surviving neurons was significantly increased in VPA-treated ASK1 KO EAE mice, suggesting the synergistic effect of VPA and ASK1 deficiency on retinal neuroprotection during EAE.

4. Discussion Herein, we demonstrate that both VPA and ASK1 deficiency attenuate EAE-induced neuroinflammation in spinal cords and optic nerves and they exert synergistic effects on protection of retinal neurons. Previous studies have shown that VPA induces apoptosis in activated T cells and maintains the immune homeostasis [18]. In addition, VPA treatment in EAE suppressed the polarization of Th1 and Th17 cells but induced the Th2 and Treg cells, and inhibited lymphocyte proliferation and macrophage activation in vitro [30]. These findings, together with decreased T cell infiltration into optic nerves in our study (Fig. 3D), indicate that VPA targets T cells during EAE to reduce neuroinflammation. Interestingly, VPA also suppressed the activation of glial cells in both spinal cords and optic nerves, which may be a secondary effect after T cell suppression. In contrast to VPA, ASK1 has no effect on T cell proliferation capability in vitro [7]. ASK1 signaling in astrocytes regulates chemokine production and recruitment of activated microglia after the infiltration of T cells into the lesion site. In addition, the same signaling pathway in microglial cells seems to modulate the progress of demyelination by altering the release of proinflammatory components [7]. Thus, the beneficial effects of ASK1 inhibition during EAE may mainly

come from suppression of glial cell activation in the late phase of EAE, although T cell activation was suppressed in the spinal cord of ASK1 KO EAE mice (Fig. 2D). In fact, VPA and ASK1 deficiency decreased the clinical score in the early and late phase, respectively (Fig. 1). These findings suggest a possibility that VPA and ASK1 inhibition are effective in different cell types and at different timepoints during EAE. Further studies are required to examine the pathology of spinal cords and optic nerves at various time points during EAE. In this study, we demonstrate that VPA and ASK1 deficiency show clear synergistic effects on retinal neuroprotection during EAE (Fig. 4). There is increasing evidence that MS is not only an inflammatory disease, but also a neurodegenerative disease, and that neuroprotective agents may be effective for MS therapy [19]. VPA has been reported to exert neuroprotective effects by stimulating the ERK pathway in cortical neurons [9] and in the retina following optic nerve injury [2,31]. Also, VPA is an effective HDAC inhibitor [6,25] and increased histone acetylation is associated with VPA-mediated neuroprotection [1,16,26]. In addition, VPA reduces glutamatergic excitatory neurotransmission [28], suggesting that excitotoxic damage in EAE mice may be decreased by VPA treatment. Furthermore, VPA may accelerate the recovery phase of EAE and increase the number of remyelinated axons in the lesion area by recruiting neural stem cells and oligodendrocyte progenitors [24]. Therefore, VPA could effectively ameliorate EAE by exerting neuroprotective effects through multiple mechanisms, in addition to its suppressive effects on T cells. On the other hand, ASK1 deficiency stimulates neuroprotection through different pathways, probably suppression of p38 MAPK etc. [7,10], during EAE as well as after optic nerve injury [14]. Therefore, robust synergistic neuroprotective effects may be achieved by combination of the two.

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In conclusion, we report that the widely prescribed drug VPA exerts therapeutic effects on optic nerve demyelination and retinal degeneration in a mouse model of MS. Our findings raise an interesting possibility that combination therapy of VPA and ASK1 inhibition may be useful for treatment of autoimmune demyelinating disorders including optic neuritis and MS. We are planning to utilize an ASK1 inhibitor [7] in combination with VPA, especially after the onset of EAE, in our future studies. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements We would like to thank Prof. H. Ichijo for providing ASK1deficient mice, and M. Kunitomo, K. Okabe and S. Ihara for their technical assistance. This work was supported in part by JSPS KAKENHI Grants-in-Aid for Scientific Research (JP26861479 to A.K.; JP16K07076 to X.G.; 16K20341 to G.A.; JP16K11308 to C.H.; JP16K08635 to K.N.; JP15H04999 to T.H.); and the Takeda Science Foundation (T.H.). References [1] O. Alsarraf, J. Fan, M. Dahrouj, C.J. Chou, P.W. Yates, C.E. Crosson, Acetylation preserves retinal ganglion cell structure and function in a chronic model of ocular hypertension, Invest. Ophthalmol. Visual Sci 55 (2014) 7486–7493. [2] J. Biermann, P. Grieshaber, U. Goebel, G. Martin, S. Thanos, S.D. Giovanni, W.A. Lagrèze, Valproic acid-mediated neuroprotection and regeneration in injured retinal ganglion cells, Invest. Ophthalmol. Visual Sci. 51 (2010) 526–534. [3] S. Chen, H. Wu, D. Klebe, Y. Hong, J. Zhang, Valproic Acid: a new candidate of therapeutic application for the acute central nervous system injuries, Neurochem. Res. 39 (2014) 1621–1633. [4] C.T. Chiu, Z. Wang, J.G. Hunsberger, D.M. Chuang, Therapeutic potential of mood stabilizers lithium and valproic acid: beyond bipolar disorder, Pharmacol. Rev. 65 (2013) 105–142. [5] E.M. Frohman, J.G. Fujimoto, T.C. Frohman, P.A. Calabresi, G. Cutter, L.J. Balcer, Optical coherence tomography: a window into the mechanisms of multiple sclerosis, Nat. Clin. Pract. Neurol. 4 (2008) 664–675. [6] M. Göttlicher, S. Minucci, P. Zhu, O.H. Krämer, A. Schimpf, S. Giavara, J.P. Sleeman, F.L. Coco, C. Nervi, P.G. Pelicci, T. Heinzel, Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells, EMBO J. 20 (2001) 6969–6978. [7] X. Guo, C. Harada, K. Namekata, A. Matsuzawa, M. Camps, H. Ji, D. Swinnen, C. Jorand-Lebrun, M. Muzerelle, P.A. Vitte, T. Rückle, A. Kimura, K. Kohyama, Y. Matsumoto, H. Ichijo, T. Harada, Regulation of the severity of neuroinflammation and demyelination by TLR-ASK1-p38 pathway, EMBO Mol. Med. 2 (2010) 504–515. [8] X. Guo, K. Namekata, A. Kimura, T. Noro, Y. Azuchi, K. Semba, C. Harada, H. Yoshida, Y. Mitamura, T. Harada, Brimonidine suppresses loss of retinal neurons and visual function in a murine model of optic neuritis, Neurosci. Lett. 592 (2015) 27–31. [9] Y. Hao, T. Creson, L. Zhang, P. Li, F. Du, P. Yuan, T.D. Gould, H.K. Manji, G. Chen, Mood stabilizer valproate promotes ERK pathway-dependent cortical neuronal growth and neurogenesis, J. Neurosci. 24 (2004) 6590–6599. [10] C. Harada, K. Nakamura, K. Namekata, A. Okumura, Y. Mitamura, Y. Iizuka, K. Kashiwagi, K. Yoshida, S. Ohno, A. Matsuzawa, K. Tanaka, H. Ichijo, T. Harada, Role of apoptosis signal-regulating kinase 1 in stress-induced neural cell apoptosis in vivo, Am. J. Pathol. 168 (2006) 261–269.

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