Dicer1 Protects Rpe Cells By Regulating Caspase-1 And Myd88 Activation

Abstract

Purpose: : DICER1 maintains RPE cell health by regulating the expression of toxic Alu RNA transcripts. Also, RPE can be rescued from Alu RNA toxicity by overexpression of DICER1. Until now, the mechanism of Alu RNA toxicity was unknown. Here, we reveal that DICER1 is an essential endogenous negative regulator of NLRP3 inflammasome activation, and that DICER1 deficiency leads to Alu RNA and Caspase-1-mediated, MyD88-dependent, microRNA-independent RPE degeneration.

Methods: : Caspase-1 activation was monitored by western blot analysis and by a fluorescent reporter. IRAK 1/4 phosphorylation was measured by western blot analysis. Subretinal injections of AAV1-BEST1-Cre vectors were performed in order to delete DICER1 from Dicer1f/f mice. DICER1 knockdown and Alu RNA blockade in vivo and in vitro was accomplished by antisense oligonucleotide transfection. RPE degeneration was monitored by fundus photography and retinal flat-mounts stained for ZO-1. Caspase-1 and MyD88 activation in vivo were suppressed by intravitreous injection of known inhibitors. miRNA levels were quantified by real-time qPCR.

Results: : Alu RNA-induced Caspase-1 activation in human RPE cells was inhibited by DICER1 overexpression. On the other hand, Caspase-1 cleavage induced by DICER1 knockdown in human RPE cells was inhibited by simultaneous antisense-mediated knockdown of Alu RNA. RPE degeneration and increased caspase-1 cleavage was observed in BEST1 Cre; Dicer1f/f mice, and also after subretinal delivery of AAV1-BEST1-Cre in Dicer1f/f mice. DICER1 deficit-induced pathology was blocked by intravitreous administration of the Caspase-1-inhibitory peptide but not a control peptide. Similarly, AAV1-BEST1-Cre-induced RPE degeneration in Dicer1f/f mice was also blocked by MyD88-inhibitory peptide but not a control peptide. Also, the MyD88-inhibitory peptide prevented cell death in human RPE cells treated with antisense oligonucleotides targeting DICER1. Furthermore, DICER1 knockdown in human RPE cells increased phosphorylation of IRAK1/4. The MyD88-inhibitory peptide also prevented cell death in Dicer1f/f mouse RPE cells treated with an adenoviral vector coding for Cre recombinase. Finally, MyD88 inhibition blocked RPE cell death without restoring the microRNA expression deficits induced by Dicer1 knockdown.

Conclusions: : Our data provide a novel anti-inflammatory role for DICER1: Dampening inflammasome activation in RPE cells. Until now, NLRP3 inflammasome activation in vivo has been largely restricted to immune cells, although our findings open the possibility that NLRP3 inflammasome activity may be more widespread than previously thought. Importantly, these results provide several new therapeutic strategies for patients with GA.

Keywords: age-related macular degeneration • inflammation • retinal pigment epithelium