ERK1/2 activation is a therapeutic target in age-related macular degeneration

Abstract

Deficient expression of the RNase III DICER1, which leads to the accumulation of cytotoxic Alu RNA, has been implicated in degeneration of the retinal pigmented epithelium (RPE) in geographic atrophy (GA), a late stage of age-related macular degeneration that causes blindness in millions of people worldwide. Here we show increased extracellular-signal-regulated kinase (ERK) 1/2 phosphorylation in the RPE of human eyes with GA and that RPE degeneration in mouse eyes and in human cell culture induced by DICER1 depletion or Alu RNA exposure is mediated via ERK1/2 signaling. Alu RNA overexpression or DICER1 knockdown increases ERK1/2 phosphorylation in the RPE in mice and in human cell culture. Alu RNA-induced RPE degeneration in mice is rescued by intravitreous administration of PD98059, an inhibitor of the ERK1/2-activating kinase MEK1, but not by inhibitors of other MAP kinases such as p38 or JNK. These findings reveal a previously unrecognized function of ERK1/2 in the pathogenesis of GA and provide a mechanistic basis for evaluation of ERK1/2 inhibition in treatment of this disease.

Fig. 1.

DICER1 deficit in human GA RPE is accompanied by an increase in Alu RNA and ERK1/2 phosphorylation. (A) Fundus photographs and (B) flat mounts stained for ZO-1 (red) show cell degeneration and atrophy in RPE from a human GA eye compared with a normal eye. (Scale bar, 20 μm.) (C) ERK1/2 phosphorylation, assessed by Western blotting, is increased in human GA RPE compared with control RPE; however, p-p38 MAPK and p-JNK1/2 remained unchanged between the two groups. DICER1 quantification, assessed by quantitative PCR (D) and Western blotting (E), is lower in human GA RPE compared with control RPE (n = 5 independent experiments, *P = 0.012 by Student’s t test). (F) Alu RNA abundance is increased in human GA RPE compared with control RPE (n = 5 independent experiments, **P < 0.0001 by Student’s t test). Images are representative of three independent experiments (C and E).

Fig. 2.

DICER1 knockdown induces B1/B2 (Alu-like) RNA accumulation, RPE cell death, and ERK1/2 phosphorylation. (A) Fundus photographs and flat mounts stained for ZO-1 (in red) show RPE degeneration in Dicer1^f/f mice following subretinal injection of AAV1-BEST1-Cre but not AAV1-BEST1-GFP. (B) Subretinal administration of AAV1-BEST1-Cre in Dicer1^f/f mice induces ERK1/2 phosphorylation in the RPE compared with AAV1-BEST1-GFP. (C) Ad-Cre infection of RPE cells isolated from Dicer1^f/f mice promotes activation of ERK1/2 but not p38 MAPK or JNK1/2 compared with Ad-Null. DICER1 antisense (Dic-AS) increases ERK1/2 phosphorylation but not p-p38 MAPK or p-JNK1/2 in human RPE cells assessed by Western blotting (D) and immunofluorescence (E). Images are representative of three to four independent experiments (A–E).

Fig. 3.

Alu/B1/B2 overexpression or Alu RNA induces RPE degeneration and activates ERK1/2. (A and B) Fundus photographs and flat mounts stained for ZO-1 (in red) show RPE degeneration in wild-type mice following subretinal transfection of pAlu (A) and pB1 and pB2 (B). (C and D) Subretinal transfection of plasmids coding for Alu (C) or B1 or B2 RNA increases ERK1/2 phosphorylation in the RPE of wild-type mice (D). Overexpression of Alu or B1 or B2 promotes ERK1/2 activation in wild-type mouse RPE cells (E and H) and human RPE cells (F and G). (C–F and H) Western blotting. (G) Immunofluorescence. Images are representative of three to four independent experiments (A–H).

Fig. 4.

ERK1/2 inhibition rescues mouse RPE cell death induced by DICER1 knockdown. (A) The ERK1/2 inhibitor PD98059, but not p38 (SB202190) or JNK inhibitor (SP600125), protects the RPE of Dicer1^f/f mice from Dicer1 knockdown-induced degeneration after AAV1-BEST1-Cre administration. PD98059, but not SB202190 (p38 MAPK inhibitor) or SP600125 (JNK inhibitor), rescues viability of mouse (B) and human RPE cells (C) after DICER1 depletion. PD98059 inhibits Dicer1 knockdown-induced ERK1/2 activation in mouse and human RPE cells (D–F). Subretinal transfection of plasmid coding for full-length MAPKKK (MEK1) induces RPE degeneration in wild-type mouse (G). Values are mean ± SEM, n = 3 independent experiments, *P < 0.05 by ANOVA and post hoc Newman–Keuls test (B and C). Images are representative of three to four independent experiments (A and D–G).

Fig. 5.

ERK1/2 inhibition protects mouse and human RPE from Alu/B1/B2-induced cytotoxicity. PD98059, but not p38 (SB202190) or JNK inhibitor (SP600125), protects RPE cells from pAlu (A) or pB1/B2 cytotoxicity (B) in wild-type mice. PD98059 rescues human (C) and wild-type mouse RPE cell viability (D) after pAlu transfection. Values are mean ± SEM, n = 3, *P < 0.05 by ANOVA and post hoc Newman–Keuls test. Images are representative of three to four independent experiments (A–D).

Fig. 6.

Alu antisense inhibits ERK1/2 activation induced by DICER1 knockdown and rescue cell viability in human RPE cells. Alu antisense (Alu-AS) transfection inhibits ERK1/2 phosphorylation induced by DICER1 knockdown assessed by Western blotting (A) and immunofluorescence (B). Alu-AS protects human RPE cells from cytotoxicity induced by DICER1 knockdown (C). Values are mean ± SEM, n = 3, *P < 0.05 by ANOVA and post hoc Newman–Keuls test. Images are representative of three independent experiments (A–C).