Inhibition of ASK1-p38 pathway prevents neural cell death following optic nerve injury

Abstract

Optic nerve injury (ONI) induces retinal ganglion cell (RGC) death and optic nerve atrophy that lead to visual loss. 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 RGC apoptosis. In this study, we found that ONI-induced p38 activation and RGC loss were suppressed in ASK1-deficient mice. Sequential in vivo retinal imaging revealed that post-ONI treatment with a p38 inhibitor into the eyeball was effective for RGC protection. ONI-induced monocyte chemotactic protein-1 production in RGCs and microglial accumulation around RGCs were suppressed in ASK1-deficient mice. In addition, the productions of tumor necrosis factor and inducible nitric oxide synthase in microglia were decreased when the ASK1-p38 pathway was blocked. These results suggest that ASK1 activation in both neural and glial cells is involved in neural cell death, and that pharmacological interruption of ASK1-p38 pathways could be beneficial in the treatment of ONI.

Figure 1

Effect of ASK1 deficiency on RGC loss and IRL thickness following ONI. (a) H&E staining of retinal sections at 7 and 14 days after ONI in wild-type (WT) and ASK1 KO mice. Scale bar: 100 and 400 μm in the upper and immediately lower panels, respectively. ONL, outer nuclear layer. (b) Quantification of RGC number in WT and ASK1 KO mice. The number of neurons in the GCL was counted in retinal sections from one ora serrata through the optic nerve to the other ora serrata. (c) OCT cross-sectional images of retinas before (control) and 14 days after ONI. (d) Representative images of TUNEL staining in WT and ASK1 KO mouse retinas at 5 days after ONI. Scale bar: 100 μm. (e) Quantification of TUNEL-positive cells in WT and ASK1 KO mice. The data are presented as means±S.E.M. of six samples for each experiment. **P<0.01, *P<0.05

Figure 2

Effect of ASK1 on p38 activation following ONI. (a) Immunoblot analysis of phosphorylated p38, total p38, phosphorylated JNK and total JNK in whole retinas of wild-type (WT) and ASK1 KO mice at the indicated times after ONI. (b) Quantitative analysis of phosphorylated p38 in whole retinas of WT and ASK1 KO mice at the indicated times after ONI. The data are presented as means±S.E.M. of six samples for each experiment. *P<0.01. (c) Immunohistochemical analysis of phosphorylated p38 in retinal sections of WT and ASK1 KO mice 3 h after ONI. Scale bar: 100 and 200 μm in the upper and lower panels, respectively

Figure 3

Effect of p38 inhibitor on ONI-induced p38 activation in the retina. (a) Animal protocols. Phosphate-buffered saline (PBS) or SB203580 (20 μM), a p38 inhibitor, was intraocularly injected 1 h before ONI, and the animals were killed 4 h after ONI. (b) Effect of SB203580 on ONI-induced activation of the ASK1 signaling pathway assessed by immunoblot analyses of phosphorylated p38, total p38, phosphorylated JNK, total JNK, phosphorylated ASK1 and total ASK1 in whole retinas of wild-type (WT) mice. (c, d) Quantitative analyses of ONI-induced activation of p38 (c) and ASK1 (d) in whole retinas of WT mice. The data are presented as means±S.E.M. of six samples for each experiment. *P<0.01. (e) Immunohistochemical analysis of ONI-induced p38 phosphorylation in retinal sections. Scale bar: 100 and 200 μm in the upper and lower panels, respectively

Figure 4

Effect of p38 inhibitor on ONI-induced RGC loss. (a) Animal protocols. Phosphate-buffered saline (PBS) or SB203580 (20 μM) was intraocularly injected before or after ONI, and the animals were killed at 14 days after ONI. (b) H&E staining of retinal sections in WT and ASK1 KO mice. Pre- and post-treatment with SB203580 prevented ONI-induced RGC loss in WT mice. Scale bar: 100 and 400 μm in the upper and lower panels, respectively. (c, d) Quantification of RGC number and IRL thickness. (e) OCT cross-sectional images of retinas before (0 day) and after (7 and 14 days) ONI. (f) Longitudinal evaluation of IRL thickness. The data are presented as means±S.E.M. of six samples for each experiment. **P<0.01, *P<0.05

Figure 5

Effect of ASK1 deficiency on ONI-induced activation of RGCs. (a–c) Impaired chemokine productions in whole retinas of wild-type (WT) and ASK1 KO mice at 5 days after ONI. mRNA expression levels of MCP-1 (a), RANTES (b) and MIP-1α (c) were determined using quantitative real-time PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The data are presented as means±S.E.M. of six samples for each experiment. *P<0.01. (d) Immunohistochemical analysis of MCP-1 expression in the retinas of WT and ASK1 KO mice at 1 h after ONI. (e) Immunostaining of MCP-1-positive cells (green). Calretinin, TUJ1, GFAP and GLAST were used as cell type-specific markers (red). Overlapping immunoreactivities (yellow) of MCP-1 and calretinin or TUJ1 indicate the expression of MCP-1 in RGCs. Scale bar: 100 and 200 μm in the upper and lower panels, respectively

Figure 6

Effect of ASK1 deficiency on ONI-induced migration of microglia. (a) Double-labeling immunohistochemistry of iba1 (red) and MCP-1 (green) at 5 days after ONI. Overlapping immunoreactivities (yellow) indicate the expression of MCP-1 in microglial cells. (b) Quantification of iba1-stained areas in whole retinas. The data are presented as means±S.E.M. of six samples for each experiment. *P<0.01. (c) Double-labeling immunohistochemistry of iba1 (green) and phosphorylated p38 (p-p38; red) at 5 days after ONI. Overlapping immunoreactivities (yellow) indicate the activation of microglial cells. Scale bar: 100 μm

Figure 7

ASK1-p38 pathway is required for TNFα and iNOS productions in microglial cells. Cultured microglial cells were pre-treated with SB203580 (20 μM) or MSC2032964A (10 μM) for 1 h, and then stimulated with LPS (5 ng/ml) or TNFα (50 ng/ml) for 6 h. Cell lysates were subjected to quantitative real-time PCR to measure mRNA expression levels of TNFα (a) and iNOS (b, c). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The data are presented as means±S.E.M. of six samples for each experiment. *P<0.01

Figure 8

Effect of ASK1 deficiency on productions of TNFα and iNOS in whole retinas. mRNA expression levels of TNFα (a, c) and iNOS (b, d) were measured by quantitative real-time PCR after ONI (a, b) or intraocular injection of LPS (c, d). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The data are presented as means±S.E.M. of six samples for each experiment. *P<0.01