Abstract
Purpose: :
Rats treated with AY9944, an inhibitor of the final step of cholesterol biosynthesis, undergo progressive retinal degeneration and provide an animal model of Smith-Lemli-Opitz syndrome (SLOS). Prior proteomic and Western blot analysis (Tu et al., ARVO 2011) revealed increased cathepsin D (CathD) levels in AY9944-treated retinas, relative to controls; in contrast, cleaved caspase-3 (Casp3) was not detected, despite marked TUNEL-positive outer nuclear layer labeling. Here, we performed correlative immunohistochemical localization of CathD and cleaved Casp3 in the retinas of control and AY9944-treated rats.
Methods: :
Sprague-Dawley rats were treated with AY9944 as previously described (Fliesler et al., Pediatr. Res., 2007); untreated age-matched rats served as controls. Eyes were harvested at ca. 2 mo of age, fixed in buffered formalin, and processed for paraffin embedment; following antigen retrieval, tissue sections were incubated with primary antibody and subjected to secondary immunofluorescence detection. For CathD, summed z-stacks obtained by confocal microscopy were used to compare fluorescence signal intensity and distribution in control and experimental retinal sections of equivalent thickness. Etoposide-treated, paraffin-embedded Jurkat cells served as a cleaved Casp3 positive control.
Results: :
In control retinas, CathD immunolabeling was detected in the RPE, as expected; in addition, the perinuclear cytoplasm of cells in the inner nuclear and ganglion cell layers was labeled. Punctate CathD immunolabeling in the outer nuclear layer and outer limiting membrane, but not the plexiform layers, also was observed. A similar, but more robust, immunolabeling pattern was observed in retinas from AY9944-treated rats, particularly in ganglion cells; in addition, labeling of the plexiform layers and the interphotoreceptor space was observed. CathD immunolabeling did not colocalize with glutamine synthetase immunoreactivity, excluding Müller cells as a significant CathD source in retinas. Cleaved Casp3 immunoreactivity was not detected in either control or treated retinas, whereas etoposide-treated Jurkat cells were robustly immunopositive.
Conclusions: :
Retinal degeneration in the AY9944-induced SLOS rat model is Casp3-independent and involves CathD upregulation. The latter may be indicative of an autophagic response to oxidative stress, particularly in the inner retina.
Keywords: retinal degenerations: cell biology • apoptosis/cell death • proteolysis