Abstract
Purpose: :
In Retinitis Pigmentosa (RP) photoreceptor death occurs by apoptosis but the individual pathways of this process are unknown. We tested two hypotheses: 1) that the sphingolipid ceramide plays a pro-apoptotic role in the degeneration of photoreceptors in an animal model of RP; 2) that the in vivo administration of inhibitors of de novo ceramide biosynthesis decreases photoreceptor death.
Methods: :
rd10 mutant mice were used. These carry a missense mutation of the rod-specific phosphodiesterase leading to photoreceptor death from P14. rd10 and wt, control mice, aged P14, P22, P30 and P37, were anesthetized, their retinas isolated and frozen on dry ice. For ceramide quantification, the two retinas of each animal were washed, pooled, and endogenous ceramide content determined by the diacylglycerol kinase assay. Ceramide values of different animals were averaged and referred to total phospholipids.Intravitreal injections of non toxic amounts of commercially available inhibitors of serine palmitoyltransferase (SPT, the rate-limiting enzyme of ceramide biosynthesis), were performed in the right eyes of rd10 mice at P19. Left eyes were injected with vehicle. Treated and control eyes were isolated 48 hrs after injection and used for biochemical assays of ceramide or retinal histology. Photoreceptor degeneration rates were assessed by counting pyknotic (apoptotic) nuclei in the outer nuclear layer of whole mounted retinas after fluorescent nuclear staining and confocal microscopy.
Results: :
Retinal ceramide levels in rd10 mice double from P14 to P30, the time interval of maximum photoreceptor death. Endogenous ceramide content of wt, normalmice, is lower than that measured in rd10 retinas at the peak of apoptosis. Intraocular injections of SPT inhibitors have no toxic effects at local or general levels but reduce significantly retinal ceramide levels in rd10 compared to control retinas. Intraocular treatment with SPT inhibitors decrease the number of pycnotic photoreceptors in rd10 mutant mice by approximately 50%.
Conclusions: :
This study demonstrates for the first time in a mammalian model of RP that it is possible to decrease the rate of apoptotic death of photoreceptors by lowering retinal ceramide levels through inhibition of the de novo biosynthesis of this molecule. This approach can be regarded as a therapeutic strategy to maintain photoreceptors (otherwise doomed to death) viable in perspective of a subsequent RP treatment.
Keywords: apoptosis/cell death • photoreceptors • retinal degenerations: cell biology