ASK1 Deficiency Attenuates Neural Cell Death in GLAST-Deficient Mice, a Model of Normal Tension Glaucoma

Cell Death and Differentiation (2010) 17, 1751–1759 & 2010 Macmillan Publishers Limited All rights reserved 1350-9047/10 $32.00 www.nature.com/cdd ASK1 deﬁciency attenuates neural cell death in GLAST-deﬁcient mice, a model of normal tension glaucoma

C Harada1, K Namekata1, X Guo1, H Yoshida2, Y Mitamura3, Y Matsumoto4, K Tanaka5, H Ichijo6 and T Harada*,1,2

Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase and has an important role in stress-induced retinal ganglion cell (RGC) apoptosis. In the mammalian retina, glutamate/ aspartate transporter (GLAST) is a major glutamate transporter, and the loss of GLAST leads to optic nerve degeneration similar to normal tension glaucoma (NTG). In GLASTÀ/À mice, the glutathione level in the retina is decreased, suggesting the involvement of oxidative stress in NTG pathogenesis. To test this hypothesis, we examined the histology and visual function of GLAST þ /À:ASK1À/À and GLASTÀ/À:ASK1À/À mice by multifocal electroretinograms. ASK1 deficiency protected RGCs and decreased the number of degenerating axons in the optic nerve. Consistent with this finding, visual function was significantly improved in GLAST þ /À:ASK1À/À and GLASTÀ/À:ASK1À/À mice compared with GLAST þ /À and GLASTÀ/À mice, respectively. The loss of ASK1 had no effects on the production of glutathione or malondialdehyde in the retina or on the intraocular pressure. Tumor necrosis factor (TNF)-induced activation of p38 MAPK and the production of inducible nitric oxide synthase were suppressed in ASK1-deficient Mu¨ller glial cells. In addition, TNF-induced cell death was suppressed in ASK1-deficient RGCs. These results suggest that ASK1 activation is involved in NTG-like pathology in both neural and glial cells and that interrupting ASK1-dependent pathways could be beneficial in the treatment of glaucoma, including NTG. Cell Death and Differentiation (2010) 17, 1751–1759; doi:10.1038/cdd.2010.62; published online 21 May 2010

It is estimated that glaucoma affects nearly 70 million disease progression, and elucidating these factors is neces- individuals worldwide, including at least 6.8 million who are sary to better understand the pathogenesis of glaucoma, bilaterally blind.1 The disease is characterized by the slow especially in the context of NTG. For this purpose, an animal progressive degeneration of the retinal ganglion cells (RGCs) model representing disease characteristics of NTG would be and their axons, which are usually associated with elevated extremely useful. To date, some animal models have been intraocular pressure (IOP). Recent studies have shown that introduced, for example, the optic nerve ligation model shows glaucoma is affected by multiple genes and environmental RGC loss with normal IOP,8 but this is more suitable as a factors,2,3 and there are several inherited and experimentally model of ischemia or optic nerve injury. In addition, prepara- induced animal models of high IOP glaucoma, including tion of these artificial models requires a high level of technical DBA/2J mice and laser-induced chronic ocular hypertension skills, but unfortunately, long-term reproducibility seems to be model.4–6 There is a subtype of glaucoma termed normal somewhat limited. Thus, there has been a great demand tension glaucoma (NTG), however, that presents with to create suitable animal models of NTG. statistically normal IOP. The number of NTG patients has In addition to more extensively studied factors such been thought to be small relative to the total number of as reduced ocular blood flow and systemic blood pressure glaucoma patients, but recent studies have revealed an changes, excessive stimulation of the glutamatergic unexpectedly high prevalence of NTG.7 These findings system has been proposed to contribute to the death of suggest that non-IOP-dependent factors may contribute to RGCs in glaucoma. Excessive extracellular concentrations

1Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo Metropolitan Organization for Medical Research, Tokyo, Japan; 2Department of Neuro-ophthalmology, Tokyo Metropolitan Neurological Hospital, Tokyo, Japan; 3Department of Ophthalmology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan; 4Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Tokyo Metropolitan Organization for Medical Research, Tokyo, Japan; 5Laboratory of Molecular Neuroscience, School of Biomedical Science and Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan and 6Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, Strategic Approach to Drug Discovery and Development in Pharmaceutical Sciences, Global Center of Excellence program, and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, The University of Tokyo, Tokyo, Japan *Correspondence: T Harada, Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo Metropolitan Organization for Medical Research, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan. Tel: þ 81 42 325 3881; Fax: þ 81 42 321 8678; E-mail: [email protected] Keywords: glaucoma; ASK1; glutamate transporter; glia; neural cell death Abbreviations: ASK1, apoptosis signal-regulating kinase 1; GLAST, glutamate/aspartate transporter; NTG, normal tension glaucoma; Ab, amyloid beta; GDNF, glial cell line-derived neurotrophic factor; GLT-1, glutamate transporter 1; iNOS, inducible nitric oxide synthase; IL-1, interleukin-1; IOP, intraocular pressure; MAPK, mitogen- activated protein kinase; mfERGs, multifocal electroretinograms; NO, nitric oxide; NTN, neurturin; RGC, retinal ganglion cell; ROS, reactive oxygen species; TLR4, Toll- like receptor 4; TNF, tumor necrosis factor Received 28.12.09; revised 22.3.10; accepted 15.4.10; Edited by N Bazan; published online 21.5.10 ASK1 deﬁciency prevents normal tension glaucoma C Harada et al 1752

of glutamate induce uncontrolled elevation of intracellular not only to keep the extracellular glutamate concentration calcium, which enters through chronically activated glutamate below a neurotoxic level but also to maintain glutathione levels receptors. Glutamate uptake by the glial cells is a well-known by transporting glutamate, which is a substrate for glutathione mechanism to maintain low extracellular levels of glutamate synthesis, into Mu¨ller cells. As retinal concentration of and promote efficient interneuronal signaling in the central glutathione, a major cellular antioxidant in the retina, was nervous system (CNS). Furthermore, the same process is decreased in GLAST-deficient mice, both glutamate neuro- considered to be neuroprotective during neurodegeneration. toxicity and oxidative stress may be involved in NTG-like Clearance of glutamate from the extracellular space is pathology.13 Together with the evidence that downregulation accomplished primarily by the action of glutamate transpor- of GLAST (human EAAT1) in the retina and of glutathione ters.9 In the CNS, the glutamate/aspartate transporter level in the plasma are found in human glaucoma (GLAST) and glutamate transporter 1 (GLT-1) are patients,14,15 it is appropriate to consider GLAST-deficient Na þ -dependent glutamate transporters found in astrocytes. mice as a valid and adequate model that offer a powerful Genetic deletion of GLAST and/or GLT-1 causes abnormal system to determine the mechanisms of and evaluate new brain development and neurological symptoms such as motor treatments for NTG. deficits.9–11 We have previously reported that GLAST, located Apoptosis signal-regulating kinase 1 (ASK1) has key in Mu¨ller glial cells, is the only glial-type glutamate transporter roles in human diseases closely related to the dysfunction of in the retina, whereas GLT-1 is expressed in neurons, cellular responses to oxidative stress and endoplasmic reti- including bipolar cells and photoreceptors.12 Not surprisingly, culum stressors, including neurodegenerative diseases.16,17 GLAST is more active than GLT-1 in preventing glutamate We have previously reported that ASK1 is primarily expressed neurotoxicity after ischemia.12 In addition, we recently found in RGCs, and ASK1À/À mice are less susceptible to that GLAST-deficient (GLASTÀ/À and GLAST þ /À) mice show ischemic injury.18 The role of ASK1 in glaucoma, however, spontaneous RGC death and optic nerve degeneration is unknown. In an attempt to identify the apoptotic signals without elevated IOP.13 Interestingly, GLAST is essential regulating RGC death in GLAST-deficient mice, we generated

Figure 1 Effect of ASK1 on visual response in GLAST þ /À and GLASTÀ/À mice. (a) Summed responses of the second-order kernel examined using multifocal electroretinograms. (b) Averaged responses of the second-order kernel are demonstrated using three-dimensional plots. The degree of retinal function is presented in the color bar. A higher score (red) indicates highly sensitive visual function and a lower score (green) indicates retinal dysfunction. (c) Quantitative analysis of the visual response amplitude. Note the improved visual function of GLAST þ /À:ASK1À/À and GLASTÀ/À:ASK1À/À mice compared with GLAST þ /À and GLASTÀ/À mice, respectively. Values are given in nV per square degree (nV/deg2). **Po0.01, *Po0.05

Cell Death and Differentiation ASK1 deﬁciency prevents normal tension glaucoma C Harada et al 1753

GLAST þ /À:ASK1À/À and GLASTÀ/À:ASK1À/À mice and retina. Consistent with the results of the mfERGs, the retina determined the effect of ASK1 deficiency on the NTG-like of ASK1À/À mice showed normal organization at 3 weeks phenotype. (3 W), 3 M and 6 M (Figure 2). Cell number in the ganglion cell layer (GCL) was significantly decreased after 3 M in GLAST þ /À mice and after 3 W in GLASTÀ/À mice (Figures 2 Results and 3a). In addition, the thickness of the inner retinal layer (IRL) was decreased after 3 M in both strains (Figure 3b). In ASK1 deficiency protects visual function in GLAST- GLAST þ /À:ASK1À/À mice, however, GCL cell number was deficient mice. To determine whether ASK1 deficiency significantly increased at 3 M and 6 M compared with is capable of preventing the NTG-like phenotype in þ /À 13 þ /À þ /À GLAST mice (Figure 3a). IRL thickness was increased GLAST-deficient mice, GLAST :ASK1 mice were ± ± þ /À À/À to a normal level (105 11% at 3 M and 95 11% at 6 M; interbred and genotyped at weaning. GLAST :ASK1 þ /À À/À À/À À/À À/À n ¼ 6) in GLAST :ASK1 mice (Figure 3b). In GLAST : and GLAST :ASK1 mice were born in accordance ASK1À/À mice, IRL thickness was significantly increased at with Mendelian inheritance ratios, survived into adulthood 3 M and 6 M compared with GLASTÀ/À mice (Figure 3b). In and were fertile. We first examined the visual function addition, GCL cell number was increased at 3 W and 3 M, but of these mice at 3 months of age (3 M) using multifocal not at 6 M (Figure 3a). These results suggest that ASK1 electroretinograms (mfERGs), an established noninvasive 13 deficiency prevents the loss of RGCs and secondary retinal method. Figure 1a and b show the averaged responses degeneration in GLAST-deficient mice. of the second-order kernel in each group. The visual function À/À of WT and ASK1 mice was indistinguishable (Figure 1c). ASK1 deficiency prevents optic nerve degeneration in As we have previously reported, visual function in the þ /À À/À GLAST-deficient mice. As nearly half of the cells in the GLAST and GLAST mice was impaired in all rodent GCL are displaced amacrine cells, we needed to visual fields, but was clearly improved by ASK1 deficiency distinguish RGCs from displaced amacrine cells by (Figure 1a–c). In particular, the amplitude of the secondary retrograde labeling.18 As ASK1 deficiency was most effec- þ /À À/À ± 2 kernel in GLAST :ASK1 mice (4.7 0.4 nV/deg ; n ¼ 8) tive in 3 M GLAST þ /À mice, we examined RGC number in was not significantly different compared with WT mice WT, ASK1À/À, GLAST þ /À and GLAST þ /À:ASK1À/À mice at ± 2 (5.0 0.3 nV/deg ; n ¼ 9) (P ¼ 0.67, Figure 1c). These 3 M (Figure 4a–h). RGC number per square millimeter in results suggest that ASK1 deficiency has no harmful effects ASK1À/À mice (4200±238; n ¼ 3) was normal compared during development and prevents visual disturbances in with WT mice (4050±170; n ¼ 3) (P ¼ 0.64, Figure 4m). In GLAST-deficient mice. GLAST þ /À mice, RGC number (3358±180; n ¼ 3) was significantly reduced compared with WT mice (Po0.05). ASK1 deficiency protects retinal neurons in GLAST- However, RGC number in GLAST þ /À:ASK1À/À mice deficient mice. We next analyzed the histopathology of the (4067±121; n ¼ 3) was clearly increased compared with

Figure 2 Effect of ASK1 on the progress of glaucoma. H&E staining of retinal sections at 3 weeks, 3 months and 8 months. WT, GLAST þ /À and GLASTÀ/À mice were littermates. GLAST þ /À:ASK1À/À and GLASTÀ/À:ASK1À/À mice were littermates. Scale bar: 100 and 400 mm in the upper and lower rows, respectively. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer

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To determine the effect of ASK1 on IOP, we examined the IOP of ASK1À/À, GLAST þ /À:ASK1À/À and GLASTÀ/À: ASK1À/À mice. IOP measurements were carried out at around 2100 hours, when IOP is highest in mouse eyes.19 The IOP values of ASK1À/À, GLAST þ /À:ASK1À/À and GLASTÀ/À:ASK1À/À mice were not signiﬁcantly decreased compared with WT and GLASTÀ/À mice (Figure 5). These results suggest that the recovery of NTG-like pathology in GLAST þ /À:ASK1À/À and GLASTÀ/À:ASK1À/À mice is IOP independent.

Role of oxidative stress and glutamate neurotoxicity in GLAST/ASK1 double-deficient mice. Oxidative stress has been proposed to contribute to RGC death in glaucoma, and a reduction in glutathione levels was reported in the plasma of human glaucoma patients.15 Consistent with these findings, we have previously reported a decreased glutathione concentration in the retina of GLASTÀ/À mice.13 To determine the effect of ASK1 on glutathione synthesis, we examined the glutathione concentration in the retina of 6 M ASK1À/À and GLASTÀ/À:ASK1À/À mice and found that it was not significantly increased compared with WT and GLASTÀ/À mice, respectively (Figure 6a). In addition, the malondialdehyde concentration in the retina of ASK1À/À and GLASTÀ/À:ASK1À/À mice was indistinguishable from that of WT and GLASTÀ/À mice, respectively (Figure 6b). We have previously reported that intravitreal glutamate concentration is normal, but memantine, N-methyl-D-aspartate receptor antagonist, partially protected RGCs in GLASTÀ/À mice.13 In addition, we showed that GLAST has a major role in glutamate uptake into Mu¨ller glial cells.20 To explore the possibility that ASK1 is involved in glutamate transport, Figure 3 Quantification of the severity of glaucoma. (a) RGC number in we examined glutamate uptake activity in Mu¨ller glial cells WT, ASK1À/À, GLAST þ /À, GLAST þ /À:ASK1À/À, GLASTÀ/À and GLASTÀ/À: À/À þ /À À/À À/À prepared from ASK1 and GLAST :ASK1 mice, and ASK1 mice. The number of neurons in the GCL was counted in retinal sections found that it was not significantly increased compared with WT from one ora serrata through the optic nerve to the other ora serrata. (b) Thickness þ /À of the inner retinal layer in WT, ASK1À/À, GLAST þ /À, GLAST þ /À:ASK1À/À, and GLAST mice, respectively (Figure 7). These findings GLASTÀ/À and GLASTÀ/À:ASK1À/À mice. **Po0.01, *Po0.05 suggest that ASK1 deficiency attenuates NTG-like degeneration without affecting the conditions of oxidative stress and glutamate neurotoxicity in GLAST-deficient mice. GLAST þ /À mice (Po0.05) and in normal range compared with WT mice (P ¼ 0.94, Figure 4m). Similarly, RGC number Effect of ASK1-p38 mitogen-activated protein kinase in GLASTÀ/À:ASK1À/À mice (3392±102; n ¼ 3) was increa- (MAPK) signaling in Mu¨ ller glial cells and RGCs. ASK1 is sed compared with GLASTÀ/À mice (2592±269; n ¼ 3) activated in response to cytotoxic stresses, including reactive (Po0.05,Figure 4m). oxygen species (ROS) and tumor necrosis factor (TNF), Degeneration of the optic nerve is one of the hallmarks of and relays these signals to p38 MAPK.16,17 To determine glaucoma. To analyze morphological changes in the optic whether this pathway is active in Mu¨ller glial cells, we nerve, semi-thin transverse sections were cut and stained first examined the effects of TNF on cultured Mu¨ller cells with toluidine blue (Figure 4i–l). Consistent with severe RGC from WT and ASK1À/À mice. Western blot analysis demon- loss, the degenerating axons in 3 M GLAST þ /À mice had strated that stimulation of WT Mu¨ller cells with TNF leads abnormally dark axonal profiles (arrowheads in Figure 4k). to strong phosphorylation of p38 in a dose-dependent Such degenerating axons, however, were almost absent manner (Figure 8a). The activation of p38, however, was in GLAST þ /À:ASK1À/À mice (Figure 4l). Taken together, significantly suppressed in ASK1-deficient Mu¨ller cells these results demonstrate that ASK1 deficiency protects (Figure 8a). Nitric oxide (NO) generated by inducible nitric against RGC loss and optic nerve degeneration in GLAST- oxide synthase (iNOS) is involved in retinal neuronal deficient mice, which leads to improved visual function as cell death,21,22 and a previous study has reported that detected by mfERG (Figure 1). TNF-induced iNOS expression and NO release are suppressed by a specific inhibitor of p38 in mouse IOP measurement in GLAST/ASK1 double-deficient astrocytes.23 These results suggest that the ASK1-p38 mice. We have previously reported that GLAST-deficient pathway regulates TNF-induced iNOS expression in Mu¨ller mice show normal IOP compared with WT mice.13 cells. To evaluate this possibility, we next examined iNOS

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Figure 4 Effect of ASK1 on RGC and optic nerve degeneration. (a–h) Retrogradely labeled RGCs in WT, ASK1À/À, GLAST þ /À and GLAST þ /À:ASK1À/À mice. (e–h) Magnified images of (a–d), respectively. (i–l) Staining of semi-thin sections with toluidine blue revealed the presence of abnormally dark axonal profiles (arrowheads) and reduced axons in GLAST þ /À mice (k), which was ameliorated in GLAST þ /À:ASK1À/À mice (l). (m) Quantification of labeled RGC number in WT, ASK1À/À, GLAST þ /À, GLAST þ /À:ASK1À/À, GLASTÀ/À and GLASTÀ/À:ASK1À/À mice. Scale bar: 1 mm (a–d); 200 mm(e–h); 20 mm(i–l). *Po0.05 protein levels in cultured Mu¨ller cells. In untreated Mu¨ller decreased (41±9%; n ¼ 6) compared with that from WT cells, iNOS protein was almost absent, but TNF clearly mice (Po0.05, Figure 8c). Taken together, loss of ASK1 increased iNOS expression levels (Figure 8b). Similar prevents TNF-induced RGC death through both the direct iNOS induction was detected in GLAST-deficient Mu¨ller pathway and the indirect pathway through Mu¨ller cells that cells (Figure 8b). However, TNF-induced iNOS expression is independent of GLAST. was completely suppressed in ASK1-deficient Mu¨ller cells (Figure 8b). These results suggest that the ASK1-p38 Discussion pathway is required in Mu¨ller cells for the TNF-induced iNOS production, which may lead to the death of retinal In this study, we show that ASK1 is associated with neurons including RGCs. We further examined the direct progressive RGC loss, glaucomatous optic nerve degenera- effect of TNF on cultured RGCs.18 TNF-induced cell death tion and visual disturbances in GLAST-deficient mice. We in cultured RGCs from ASK1-deficient mice was significantly previously suggested the possibility that dysfunction of

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Figure 5 Effect of ASK1 on intraocular pressure in GLAST þ /À and GLASTÀ/À mice. Intraocular pressure in GLAST þ /À:ASK1À/À and GLASTÀ/À:ASK1À/À mice was not signiﬁcantly decreased compared with GLASTÀ/À mice

GLAST (human EAAT1) has a role in RGC death in human NTG.13 It has been reported that EAAT1 is downregulated in the retinas of human patients with glaucoma14 and in fibroblasts from patients with Alzheimer’s disease.24 Con- sidering the high frequency of glaucoma in Alzheimer’s disease patients,25 common mechanisms such as GLAST dysfunction might contribute to both diseases. Interestingly, ROS-mediated ASK1 activation is a key mechanism for amyloid beta (Ab)-induced neurotoxicity, which has a central role in Alzheimer’s disease.26 The accumulation of Ab is also observed in apoptotic RGCs in a rat model of glaucoma due to high IOP.27 In this model, inhibiting amyloidogenic pathways by agents affecting multiple stages of the Ab pathway reduces RGC apoptosis in vivo.27 This suggests a new hypothesis Figure 6 Effect of ASK1 on oxidative stress in GLAST þ /À and GLASTÀ/À À/À for RGC death in glaucoma involving ASK1-dependent mice. (a) Mean glutathione concentration in whole retinas from WT, ASK1 , GLAST þ /À, GLAST þ /À:ASK1À/À, GLASTÀ/À and GLASTÀ/À:ASK1À/À mice. Ab neurotoxicity, mimicking Alzheimer’s disease.28 In addi- (b) Malondialdehyde concentration in whole retinas from WT, ASK1À/À, GLAST þ /À, tion, multiple single-nucleotide polymorphisms in the Toll-like GLAST þ /À:ASK1À/À, GLASTÀ/À and GLASTÀ/À:ASK1À/À mice. *Po0.05 receptor 4 (TLR4) gene have been associated with the risk of NTG.29 As ASK1 is required for innate immune responses dependent on TLR4,30 TLR4-ASK1 signaling may be involved increases glutamate uptake by Mu¨ller cells, primarily through in the development of NTG. Taken together, these findings the activation of GLAST, and protects RGCs from glutamate suggest that ASK1 may have roles in various neuro- neurotoxicity.20,31 IL-1 is a mediator of brain injury induced degenerative disorders, including glaucoma.16–18 by ischemia or trauma and has been implicated in chronic Our present results demonstrate reduced RGC death, brain diseases, such as Alzheimer’s disease, Parkinson’s decreased axonal loss and mild visual disturbance in disease and multiple sclerosis.32 Therefore, we need to GLAST þ /À:ASK1À/À and GLASTÀ/À:ASK1À/À mice, high- examine the beneficial and detrimental roles of IL-1 during lighting ASK1 as a potential therapeutic target for NTG. Loss glaucoma in vivo. Furthermore, we are undertaking experi- of ASK1 had no effect on IOP (Figure 5), the production of ments to determine the neuroprotective effect of GLAST malondialdehyde (Figure 6) or glutamate uptake activity by against neurotoxicity, axotomy and neuroinflammation in Mu¨ller cells (Figure 7). Whereas TNF-induced iNOS produc- mice overexpressing GLAST. tion was suppressed in ASK1-deficient Mu¨ller cells, and A recent study has shown that upregulation of GLAST in ASK1-deficient RGCs were more resistant to TNF-induced Mu¨ller cells by glial cell line-derived neurotrophic factor death compared with WT RGCs (Figure 8). Thus, in combi- (GDNF) and neurturin (NTN) is required to rescue RGCs nation with conventional treatments to lower IOP, inhibition following optic nerve transection.33 As the receptors for GDNF of ASK1 signaling may be useful in the treatment of glaucoma. and NTN are increased in Mu¨ller cells after RGC axotomy, the In addition, we recently reported that interleukin-1 (IL-1) neuroprotective effects of GDNF and NTN may be indirect, at

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least partly, through the enhancement of glutamate uptake in Mu¨ller cells. Similar upregulation of the receptors for GDNF and NTN has been observed in a rat model of photoreceptor degeneration.34,35 In this animal model, trophic factors such as nerve growth factor, brain-derived neurotrophic factor and basic ﬁbroblast growth factor increase the production of multiple trophic factors in Mu¨ller cells, which indirectly leads to photoreceptor survival.35–37 In addition, nerve growth factor eye drops may prevent the progress of human glaucoma.38 In this study, we found that the loss of ASK1 prevented the activity of p38 and TNF-induced iNOS production in Mu¨ller cells (Figure 8). Recent studies have shown that TNF and NO can induce RGC death and participate in the pathophysiology of glaucoma.39–41 Our results suggest that the ASK1-p38 pathway is involved in the process of TNF-induced RGC degeneration in neighboring glial cells, as well as in the RGC itself (Figure 8).18 Taken together, these ﬁndings suggest that such a glial–neuronal network may be functional in various forms of neurodegenerative diseases and that ASK1, NO, GLAST and trophic factors in Mu¨ller cells have important roles in this network during glaucoma.42,43 Thus, further efforts to discover new compounds that can enhance glutamate uptake and inhibit ASK1 signaling for a prolonged period may lead to Figure 7 Glutamate uptake activity in Mu¨ller cells derived from WT, ASK1À/À, the development of novel strategies for the management of GLAST þ /À and GLAST þ /À:ASK1À/À mice. The data are presented as means± glaucoma, including NTG. S.E.M. of eight samples for each group. *Po0.05

Materials and Methods Mice. Experiments were carried out using ASK1À/À,18 GLAST þ /À and GLASTÀ/À mice12,13 in accordance with the Tokyo Metropolitan Institute for Neuroscience Guidelines for the Care and Use of Animals. After mating ASK1À/À and GLASTÀ/À mice, GLAST þ /À:ASK1 þ /À mice were interbred to obtain GLAST þ /À:ASK1À/À and GLASTÀ/À:ASK1À/À mice.

Histological and morphometric studies. Paraffin retinal sections of 7 mm thickness were cut through the optic nerve and stained with hematoxylin and eosin. RGC number and the extent of retinal degeneration were quantified in three ways.18 First, the thickness of the IRL (between the internal limiting membrane and the interface of the outer plexiform layer and the outer nuclear layer) was analyzed. Second, in the same sections, the number of neurons in GCL was counted from one ora serrata through the optic nerve to the other ora serrata. Third, RGCs were retrogradely labeled from the superior colliculus with Fluoro-Gold (FG; Fluorochrome, Englewood, CO, USA). At 7 days after FG application, the eyes were enucleated, and the retinas were detached and prepared as flattened whole mounts in 4% PFA in 0.1 M PBS solution. The GCL was examined in whole- mounted retinas with fluorescence microscopy to determine RGC density. Four standard areas (0.04 mm2) of each retina at a point 0.1 mm from the optic disc were randomly chosen. Labeled cells were counted by observers blinded to the identity of the mice, and the average number of RGCs/mm2 was calculated. The changes in RGC number were expressed as a percentage of the WT control eyes. For detailed morphological analysis, optic nerves were fixed in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer overnight at 41C. After dissection, the pieces of tissue were placed in 1% osmium tetroxide, and after dehydration, the pieces were embedded in Epon (Nisshin EM, Tokyo, Japan). Transverse semi-thin (1 mm) sections were stained with 0.2% toluidine blue in 1.0% 13,44 Figure 8 ASK1 is required for TNF-induced p38 activation and iNOS production sodium borate. in Mu¨ller glial cells. (a) Effect of ASK1 on TNF-induced p38 activation. Mu¨ller cells derived from WT or ASK1À/À mice were stimulated with the indicated concentration IOP measurement. IOP was measured by a previously validated commercial of TNF for 20 min, followed by immunoblot analysis of total and phosphorylated p38 rebound tonometer (TonoLab; Colonial Medical Supply, Franconia, NH, USA) in in cell lysates. (b) Effect of GLAST and ASK1 on TNF-induced iNOS production. anesthetized mice as reported previously.45 To minimize variation, the data were Mu¨ller cells derived from WT, GLASTÀ/À or ASK1À/À mice were stimulated for 16 h collected during a time window 4–6 min after injection of the anesthetic during which with TNF (50 ng/ml) or left unstimulated. (c) Effect of TNF on RGC death. RGCs IOP plateaus. The animals were 6 months of age, and their body weights ranged derived from WT or ASK1À/À mice were treated for 2 days with TNF (400 ng/ml), from 22–36 g at the time of IOP measurement. As the 24-h IOP pattern in mouse and the number of PI-positive cells was counted. The data are presented as eyes is biphasic, with IOP being highest around 2100 hours,19 we examined mean ± S.E.M. of six samples for each group IOP between 2000 and 2300 hours.

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mfERG. Mice (3 months old) were anesthetized by intraperitoneal injection of a 7. Iwase A, Suzuki Y, Araie M, Yamamoto T, Abe H, Shirato S et al. The prevalence of mixture of xylazine (10 mg/kg) and ketamine (25 mg/kg). The pupils were dilated primary open-angle glaucoma in Japanese: the Tajimi Study. Ophthalmology 2004; 111: with 0.5% phenylephrine hydrochloride and 0.5% tropicamide. mfERGs were 1641–1648. recorded using a VERIS 6.0 system (Electro-Diagnostic Imaging, Redwood City, 8. Zhang X, Cheng M, Chintala SK. Optic nerve ligation leads to astrocyte-associated CA, USA). The visual stimulus consisted of seven hexagonal areas scaled with matrix metalloproteinase-9 induction in the mouse retina. Neurosci Lett 2004; 356: 140–144. eccentricity. The stimulus array was displayed on a high-resolution black and white 9. Tanaka K, Watase K, Manabe T, Yamada K, Watanabe M, Takahashi K et al. Epilepsy and monitor driven at a frame rate of 100 Hz. The second-order kernel, which is impaired exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 1997; 13,44 in patients with glaucoma, was analyzed. 276: 1699–1702. 10. Watase K, Hashimoto K, Kano M, Yamada K, Watanabe M, Inoue Y et al. Motor Malondialdehyde and glutathione assay. The concentrations of discoordination and increased susceptibility to cerebellar injury in GLAST mutant mice. malondialdehyde and glutathione were measured using the Bioxytech LPO-586 Eur J Neurosci 1998; 10: 976–988. (Oxis Research, Beverly Hills, CA, USA) and Glutathione Assay Kit (Cayman 11. Matsugami TR, Tanemura K, Mieda M, Nakatomi R, Yamada K, Kondo T et al. Indispensability of the glutamate transporters GLAST and GLT1 to brain development. Chemical, Ann Arbor, MI, USA), according to the manufacturers’ protocols. Proc Natl Acad Sci USA 2006; 103: 12161–12166. 12. Harada T, Harada C, Watanabe M, Inoue Y, Sakagawa T, Nakayama N et al. Functions of Glutamate uptake assay. 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