Selective degeneration of photoreceptors in retinal tissue in vitro: a new experimental model for retinitis pigmentosa

SELECTIVE DEGENERATION OF PHOTORECEPTORS IN RETINAL TISSUE IN VITRO: A NEW EXPERIMENTAL MODEL FOR RETINITIS PIGMENTOSA^*

RAFAEL LINDEN, FABIOLA G. DE FREITAS, MONA L. LEAL-FERREIRA AND LUCIANA B. CHIARINI

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil

Retinitis pigmentosa (RP) is a degenerative retinal disease, that starts with the death of rod photoreceptors. Various forms of RP are mapped to at least 23 chromosomal loci, including more than 100 mutations. Many of these affect components of the visual transduction cascade, but the mechanisms of induction of cell death are unknown. Apart from attempts at selective gene therapy for specific mutations, photoreceptor protective measures may help the design of novel treatments for RP. We established a model of selective photoreceptor apoptosis within retinal tissue in vitro. Retinal explants from 5-6 day-old rats were maintained in vitro for 24 hours in the presence of thapsigargin, an inhibitor of Calcium-ATPase of the endoplasmic reticulum; okadaic acid, a phosphatase inhibitor; anisomycin, an inhibitor of protein synthesis; or forskolin, an activator of adenylyl cyclase. Sections of the explants were examined for apoptosis by either Neutral-Red staining or TUNEL. Photoreceptors were identified by rhodopsin immunohistochemistry. Protein synthesis-dependent apoptosis of photoreceptors was selectively induced by either thapsigargin or okadaic acid, and was blocked by an increase of intracellular cAMP. Thus, apoptosis may be selectively induced in photoreceptors within the histotypical environment of retinal tissue in controlled in vitro conditions. It is not clear why photoreceptors are selectively vulnerable to these two metabolic inhibitors, which affect many intracellular processes in all retinal cells. The redox factor/AP endonuclease Ref-1 (APE, APEX, HAP1) affects both DNA repair and the activity of various transcription factors, and controls sensitivity to genotoxic insults. Ref-1 protein was detected immunohistochemically both in retinae of various ages and in explants in vitro. Ref-1 content increased progressively within the nucleus of differentiating retinal cells, but remained less in rod photoreceptors than in other cells of the mature retina. During cell death, Ref-1 invariably disappeared from the nucleus of apoptotic cells. Inhibition of protein synthesis both prevented the loss of Ref-1 and rescued the neurons. The data suggest that Ref-1 is an anti-apoptotic protein associated with cell differentiation in the retina. This study indicates that a low content of Ref-1 may be associated with the highest sensitivity of photoreceptors to degeneration, and warrants attempts at controlling photoreceptor cell death by targeted overexpression of Ref-1. — ( June 27, 2000 ).

Publication Dates

• Publication in this collection
  05 Oct 2000
• Date of issue
  Sept 2000