Cell-nonautonomous function of ceramidase in photoreceptor homeostasis.
ABSTRACT Neutral ceramidase, a key enzyme of sphingolipid metabolism, hydrolyzes ceramide to sphingosine. These sphingolipids are critical structural components of cell membranes and act as second messengers in diverse signal transduction cascades. Here, we have isolated and characterized functional null mutants of Drosophila ceramidase. We show that secreted ceramidase functions in a cell-nonautonomous manner to maintain photoreceptor homeostasis. In the absence of ceramidase, photoreceptors degenerate in a light-dependent manner, are defective in normal endocytic turnover of rhodopsin, and do not respond to light stimulus. Consistent with a cell-nonautonomous function, overexpression of ceramidase in tissues distant from photoreceptors suppresses photoreceptor degeneration in an arrestin mutant and facilitates membrane turnover in a rhodopsin null mutant. Furthermore, our results show that secreted ceramidase is internalized and localizes to endosomes. Our findings establish a role for a secreted sphingolipid enzyme in the regulation of photoreceptor structure and function.
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ABSTRACT: The effects of ceramide analogs on the uptake of markers for fluid-phase (horseradish peroxidase, HRP) and receptor-mediated (low density lipoprotein, LDL) endocytosis were studied in Chinese hamster fibroblasts. N-Hexanoyl-D-erythro-sphingosine (C6-Cer) decreased the uptake of HRP in a dose-dependent manner. Internalization was inhibited > 40% with 25 microM C6-Cer, relative to controls, and was apparent within 5 min. Internalization of HRP was also inhibited by other Cer analogs and by treatment with agents that raise levels of endogenous Cer (sphingomyelinase or the glycosphingolipid synthesis inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP)), but not by N-hexanoyl-D-erythrosphinganine (C6-dihydro-Cer) or sphingosine. Internalization of LDL was also inhibited by C6-Cer in a concentration-dependent manner, but was less pronounced than the effect on HRP internalization (10% versus 40% inhibition with 25 microM C6-Cer), suggesting that ceramide might affect fluid-phase and receptor-mediated endocytosis to different extents. C6-Cer also slowed HRP and LDL transport from endosomes to lysosomes as studied by analysis of endocytic vesicles on Percoll density gradients and induced a redistribution of endocytic organelles as determined by fluorescence microscopy of intact cells using appropriate markers. This resulted in decreased degradation of 125I-labeled LDL in the presence of C6-Cer. These results suggest that endogenous ceramide may modulate endocytosis.Journal of Biological Chemistry 07/1995; 270(22):13291-7. · 4.77 Impact Factor
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ABSTRACT: A variety of molecular changes occur during the process of apoptosis. Much of the recent work has focused on changes in critical cellular proteins, proteins necessary for the initiation and continuation of the apoptotic process. Given the fact that numerous membrane changes occur throughout the apoptotic process, we initiated an investigation aimed at determining the major lipid changes that occurred during programmed cell death. When ionizing radiation was used to initiate the apoptotic process in Jurkat cells, one of the major changes that occurred within 24 h was an increase in a species with a m/z of 572 as determined by negative ion electrospray mass spectrometry. This particular mass ion displayed high performance liquid chromatography characteristics of a neutral lipid species. Further analysis by collision-induced-dissociation tandem mass spectrometry indicated only one daughter species indicative of a Cl adduct and therefore a parental mass of 537. Comparison to a commercial C16 ceramide yielded identical spectra by mass spectrometry (MS) and MS/MS analysis in the negative ion mode. Increases in C16 ceramide levels occurred 2 h after initiation of apoptosis by ionizing radiation, and its accumulation paralleled apoptosis as determined by cellular morphology. Interestingly, radiation-sensitive Jurkat cells displayed increased levels of long term C16 ceramide accumulation, whereas radiation-resistant K562 cells did not. These findings were supported by increases in caspase-3 activity in Jurkat cells, whereas caspase-3 activity in K562 cells remained unchanged. C16 ceramide accumulation and sensitivity to ionizing radiation was investigated further in a melanoma cell line. Only those cells that were radiation sensitive (approximately 70-75%) displayed increases in long term ceramide accumulation. Taken together, these results indicated a correlation between increases in C16 ceramide accumulation and radiation sensitivity. Increases in long term C16 ceramide accumulation were also seen in Fas-induced apoptosis, which occurred at time points greater than 2 h. Analysis of mitochondrial modifications using the mitochondrial probe nonyl acridine orange (NAO) indicated that initial increases in C16 ceramide levels closely paralleled the decrease in mitochondrial mass during Fas or radiation-induced apoptosis. Taken together, these results support a role for C16 ceramide in the effector (mitochondrial) phase of apoptosis.Journal of Biological Chemistry 11/1999; 274(43):30580-8. · 4.77 Impact Factor
Article: Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration.[show abstract] [hide abstract]
ABSTRACT: Retinitis pigmentosa (RP) is a group of hereditary human diseases that cause retinal degeneration and lead to eventual blindness. More than 25% of all RP cases in humans appear to be caused by dominant mutations in the gene encoding the visual pigment rhodopsin. The mechanism by which the mutant rhodopsin proteins cause dominant retinal degeneration is still unclear. Interestingly, the great majority of these mutants appear to produce misfolded rhodopsin. We now report the isolation and characterization of 13 rhodopsin mutations that act dominantly to cause retinal degeneration in Drosophila; four of these correspond to identical substitutions in human autosomal dominant RP patients. We demonstrate that retinal degeneration results from interference in the maturation of wild-type rhodopsin by the mutant proteins.Proceedings of the National Academy of Sciences 04/1995; 92(7):3070-4. · 9.68 Impact Factor