MDCK cells were treated with the indicated concentrations of -carrageenan

yer; inner nuclear layer; inner plexiform layer and ganglion cell layer. Morphometric analyses of the retinas from ASMase KO and WT mice. Data are expressed as mean SE; n !3, !4 mice. Indicates significant difference between responses in WT and ASMase KO mice. doi:10.1371/journal.pone.0133032.g003 No significant difference of c-wave amplitudes was detected between 1 and 2 months old WT mice. In the eye, the RPE is responsible for the daily phagocytosis of photoreceptor outer segments, a process coupled to their daily renewal. Phagocytosed outer segment material enters the RPE lysosomal compartment where it is degraded. Undigested material accumulates in the form of lipofuscin granules. Excessive accumulation of lipofuscin is associated with retinal degeneration. We therefore examined lipofuscin accumulation in the RPE of WT, ASMase heterozygous knockout and ASMase homozygous knockout mice at 6 months-of- age. RPE cells from 6-month-old WT and ASMase+/- mice show significant accumulation of lipofuscin granules. There is extensive accumulation of lipofuscin granules in the RPE cells from 6-month-old ASMase KO mice to the extent of filling up the cytoplasm. The fluorescence emission MedChemExpress Elesclomol spectra of individual granules from WT, ASMase+/-, and ASMase KO 7 / 14 ASMase and Autophagic Stress-Related Retinal Degeneration Fig 4. Effect of deletion of ASMase on RPE. Representative c-waves in 2-month-old WT and KO mice. Data analyses of c-wave amplitudes in WT and KO mice at 1 month and 2 months-of-age. Data are presented as mean SE, n = 8. Indicates significant difference between responses in WT PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19747411 and ASMase KO mice. doi:10.1371/journal.pone.0133032.g004 Fig 5. RPE lipofuscin of ASMase KO mice. True color fluorescence micrographs of flat-mounted RPEs from 6-month-old ASMase+/+, ASMase+/-, ASMase-/-. Scale bar is 10m; Emission spectra of the granule autofluorescence from ASMase+/+, ASMase+/ and ASMase-/- granules. Error bars represent S.E. doi:10.1371/journal.pone.0133032.g005 8 / 14 ASMase and Autophagic Stress-Related Retinal Degeneration Fig 6. Effect of deletion ASMase on SPL profiles. Sphingomyelin profile and dihydrosphingosine and sphingosine profiles and ceramide profile in WT and ASMase KO retinas from 1-, 2-, 4-, 6- and 8-month-old mice. Data are presented as mean SD, n = 3. doi:10.1371/journal.pone.0133032.g006 were broadly similar, with a peak at ~600 nm. The emission spectrum is characteristic of the presence of by-products of vitamin A metabolism, such as bis-retinoids. Retinal sphingolipid levels in ASMase KO and WT mice As shown in Fig 6, the deletion of ASMase significantly increased PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19748727 SM levels in the retina at various ages. In addition, several very long chain SMs displayed large age-dependent increases. Interestingly, sphingosine and dihydrosphingosine levels were also elevated in the ASMase KO mice in an age-dependent manner. However, no significant changes in the ceramide level were observed between ASMase KO and WT retinas. Retinal S1P levels were below the level of detection. Autophagy dysfunction in ASMase KO eyecups Autophagy is an intracellular lysosomal clearance process associated with retinal degeneration and sphingolipid disorders. As shown in Fig 7A, deletion of ASMase resulted in a 58 12.8% increase in LC3-II in the eyecups, suggesting the accumulation of autophagosomes in ASMase KO eyecups. However, LC3-I and beclin-1, the upstream regulators of autophagy signaling and crucial to the initiation of the formation of autophago