Abstract
Purpose: :
Leber's congenital amaurosis (LCA), an autosomal recessive form of RP resulting in blindness, may be caused by mutations in the Rpe65 gene. As several other mutations in genes expressed in retinal pigment epithelium (RPE), those affecting RPE65 are linked to the degeneration of photoreceptor cells. However, the relationship between genetic mutation, retinal defect and final loss of photoreceptors remains unresolved. Photoreceptor cell death can be caused by any changes that alter the composition of the signal transduction cascade or disturb the structural and functional interactions between the RPE and the underlying photoreceptors. Although apoptosis is a common pathway for photoreceptor degeneration in animal models of RP, the cellular and molecular events leading to cell death in RPE65–/– mouse remain largely unknown.
Methods: :
Expression of apoptosis–related genes was evaluated in Rpe65–/– mouse model of LCA before and at the onset of photoreceptor degeneration. Retina RNA was isolated from wild–type (wt) and Rpe65–/– mice at 2, 4, 6 and 12 months. Modulated gene expression level was assessed in microarry and and real–time PCR experiments.
Results: :
Modulated expression of genes of the Bcl–2 protein family is observed in Rpe65–deficient retinas. Regulation of the anti–apoptotic Bcl–2 gene is altered, as reflected by the inhibition of age–dependent increased mRNA in 2, 4, and 6 month–old Rpe65–/– as compared to wt retinas. A parallel increased expression of pro–apoptotic Bmf and Bax genes is also observed in mutant retinas, whereas Bad and JNK1 are upregulated later on during disease progression, namely in 12 month–old diseased retinas. Protein tyrosine kinase 9, involved in actin filament assembly, as well as trombospondin 1, a regulatory protein of the extracellular matrix (ECM), are significantly upregulated in Rpe65–/– retinas.
Conclusions: :
These results suggest that RPE65 defect may trigger signaling pathways such as those related to cell death promoted by disruption of ECM contact and/or loss of cytoskeletal architecture that in turn affect the levels of Bcl–2 protein family. This may impact on the life or death of photoreceptor cells and favor the balance towards apoptosis as reflected by upregulation of pro–apoptotic and inhibition of anti–apoptotic proteins in Rpe65–deficient retinas.
Keywords: retinal degenerations: cell biology • apoptosis/cell death • cell-cell communication