Furthermore, the roles of HIF-1 and HIF-2 in cancer seem context dependent

somes, showed no measureable difference between WT and ASMase KO eyecups. To determine if the autophagy disorder is a consequence caused by the ASMase deletion, we evaluated the levels of LC-3 isoforms in cultured cells, including a control fibroblast cell line and a Niemann-Pick type A dermal fibroblast cell line. As shown in Fig 7B, significant accumulation of LC-3II was observed in the NPD fibroblast cells, while no changes in LC3-I were observed. The accumulation of LC3-II could be caused by either the activation of autophagocytosis or by defects in the fusion process of autophagosomes with lysosomes. To discriminate between these two possibilities, the cells were treated with two lysosomal inhibitors, pepstatin A and E64D. The inhibitors PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1974940 increased the levels of LC3-II in the control cells, but not in the NPD cells. This result demonstrated that the fusion of autophagosome and lysosome was impaired in NPD cells. 9 / 14 ASMase and Autophagic Stress-Related Retinal Degeneration Fig 7. Effect of the deletion of ASMase on LC3 levels. purchase GSK126 expression levels of LC3-I, LC3-II and beclin-1 in WT and ASMase KO eyecups from 1-, 2-, 4-, 6- and 8-month-old mice. Beta-actin was used for loading controls. Expression of LC3-II levels in control fibroblast cell line and Niemann-Pick type A dermal fibroblast cell line with and without the lysosomal inhibitors, pepstatin A and E-64D. doi:10.1371/journal.pone.0133032.g007 Discussion ASMase is an essential enzyme for sphingolipid metabolism. The absence of ASMase expression in NPD individuals has been shown to lead to neurodegeneration in the brain; however, studies in the retina are limited and have produced conflicting results. Examination of postmortem eyes from a NPD type A patient revealed abnormal storage inclusion bodies in various retinal cells. In NPD type B patients, two independent studies showed phenotypes of vision loss in some patients, while another study failed to identify any evidence of retinal degeneration in these individuals. Our results demonstrated that age-dependent neural retinal degeneration occurs in the ASMase KO animals. However, the observation of normal retinal morphology and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19747545 limited functional deficits at 1 month supports the idea that ASMase has a limited role, if any, in neural retinal development. However, additional studies are needed to definitively answer questions about the role of ASMase in retinal development and vascular function. In young ASMase KO mice, the significant decrease of a-wave amplitude and eventual loss of photoreceptor outer segments and cell bodies is consistent with the idea that photoreceptor degeneration is a central feature of these animals. To investigate if additional outer retinal changes are expressed in these mice, ERG c-waves and RPE lipofuscin were evaluated. As shown in Fig 4, 1-month-old ASMase KO mice also exhibited significant reductions in c-wave amplitudes. Comparing the accumulation of lipofuscin in the RPE of to WT and ASMase+/mice, there was a dramatic elevation, with lipofuscin granules virtually filling the cytoplasm. The fluorescence emission spectra of the RPE granules are broadly similar to those reported previously. The slightly broader emission spectrum of the ASMase KO granules may however indicate the presence of some differences in lipofuscin composition or environment in that case. The reduction in ERG c-wave amplitude demonstrated that in general RPE function is reduced, while the large accumulation of autofluorescent