Mechanisms of cell recovery: Anastasis in retinal photoreceptors

Abstract

Retinal neurodegeneration, characterized by apoptotic photoreceptor cell death, is associated with several ocular pathologies which remain the chief causes of irreversible blindness. Given the lack of reversible treatment options, neuroprotection has been an important focus of investigation. Recent studies have shown that apoptosis can be halted and reversed in a process known as “anastasis”. This study aims to determine if anastasis can occur in photoreceptors and if so, what may control this mechanistically. 661W mouse photoreceptor cells were treated with staurosporine (STS) for 15 hours to induce apoptosis. The drug was subsequently removed and replaced with fresh media, and cells were allowed to recover for 24 or 48 hours. MTT and Crystal violet assays were performed to assess cell viability and proliferation/morphology, respectively. PARP/cleaved- PARP expression in cells was measured using Western blotting. RT-PCR was conducted to evaluate relative mRNA expression of three early-response genes associated with anastasis (IER5, EGR1, FOS). After 48 hours of recovery in fresh media, photoreceptors that exhibited hallmarks of apoptosis, regained healthy cell morphology. PARP-1 (mean Fold change=0.968, pandlt;0.05) and Cleaved-PARP-1 (mean Fold change=0.685, pandlt;0.05) protein expression significantly decreased in recovery cells, matching that of untreated controls. Compared to treatment-induced apoptotic cells, recovered cells demonstrated increased cell proliferation and enrichment of IER5, EGR1, and FOS, early-response genes associated with anastasis (Mean relative mRNA expression: IER5: 5.860, pandlt;0.05; EGR1: 0.899, pandlt;0.05; FOS: 3.000, pandlt;0.05). Our data suggests that photoreceptors can recover from apoptosis, and that they show the hallmark signs of anastasis.