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Abstract

This work aims to investigate the effect of compromised lysosomal enzyme activity on the accumulation of photoreceptor-derived debris in the retinal pigment epithelial (RPE) cells and to examine if this accelerated debris accumulation can induce retinal abnormalities similar to those observed in aged individuals. A mutated, enzymatically inactive form of cathepsin D (CatD), generated by site-directed mutagenesis was used to produce stable cell lines and transgenic mice. There was a strong increase in enzymatically inactive CatD protein production in the mutated CatD DNA transfected D407 cells (D407MCD). The presence of the inactive CatD has been linked to an impairment in bovine rod outer segment (BROS) digestion and was confirmed by a statistically significant increase of undigested residual BROS in the medium of D407MCD when compared to the control vector-transfected D407 cells (t-test, P < or = 0.016, P < or = 0.003) or untransfected D407 cells (t-test, P < or = 0.008, P < or = 0.003). The impairment was also confirmed in vivo by demonstration of BROS-derived debris accumulation in the RPE cell layer of transgenic mice. These results demonstrated that the mutated and inactive CatD form could lead to impairment of photoreceptor outer segments (POS) proteolysis. It is proposed that this initial impairment of POS proteolysis may result in the accumulation of CatD-opsin-like complexes in the pigment epithelium, which further compromises RPE cell functions and thus causes the changes observed in aging humans.

Chemoproteomic profiling reveals that cathepsin D off-target activity drives ocular toxicity of β-secretase inhibitors

“…Our results suggest that BACE1 inhibitors that induce AFG increases have >80% in vivo target engagement of CatD. On the basis of the precedent in transgenic mice heterozygous for catalytically inactive CatD 51 , 80% inhibition of CatD alone would be more than sufficient to cause accumulation of autofluorescent material in the RPE. This level of CatD inhibition would likely have severe long-term consequences as sheep 18 and humans 19 with CatD loss-of-function mutations have not just visual issues, but also accumulated neuronal autofluorescent material and other symptoms of neuronal ceroid lipofuscinosis including loss of speech, impaired motor functions, and other neurodegenerative symptoms.…”

Phagosome maturation during endosome interaction revealed by partial rhodopsin processing in retinal pigment epithelium

“…In RPE cells, the lysosomal enzyme cathepsin D is very highly expressed (Rakoczy et al, 1999; Yamada et al, 1990; Zhang et al, 2002; Zimmerman et al, 1983), and RPE cells expressing a mutant inactive form of cathepsin D accumulate undigested POS products (Rakoczy et al, 2002), suggesting an important role for this enzyme in POS degradation. We therefore co-stained ultrathin cryosections for cathepsin D and rhodopsin, using 1D4 or RET-P1 (Fig.…”

Animal models of age related macular degeneration

“…The accumulation of inactive forms of procathepsin D have been linked to RPE dysfunction (Rakoczy et al, 1996). To study the effects of inactive cathepsin accumulation on photoreceptors, transgenic mice with a mutant form of cathepsin ( mcd ) have been created (Zhang et al, 2002). Mice homozygous for the transgene ( mcd/mcd ) demonstrate areas of RPE atrophy starting at 9 months of age (Rakoczy et al, 2002).…”