InteractiveFly: GeneBrief

Ceramidase: Biological Overview | References

Gene name - Ceramidase

Synonyms -

Cytological map position - 99F5-99F6

Function - enzyme

Keywords - sphingolipid metabolism, photoreceptor homeostasis, endocytosis, photoreceptor signal transduction

Symbol - CDase

FlyBase ID: FBgn0039774

Genetic map position - 3R:26,285,512..26,291,458 [+]

Classification - Neutral/alkaline non-lysosomal ceramidase

Cellular location - secreted and internalized into endosomes

NCBI link: EntrezGene

CDase orthologs: Biolitmine

Neutral ceramidase, a key enzyme of sphingolipid metabolism (see Long-chain base synthesis resulting in ceramide formation from Shayman, 2000), hydrolyzes ceramide to sphingosine. These sphingolipids are critical structural components of cell membranes and act as second messengers in diverse signal transduction cascades. This study isolated and characterized functional null mutants of Drosophila ceramidase. 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, the results show that secreted ceramidase is internalized and localizes to endosomes. These findings establish a role for a secreted sphingolipid enzyme in the regulation of photoreceptor structure and function (Acharya, 2008).

Sphingolipids are essential structural components of membranes and regulate membrane architecture (Holthuis, 2001). Many sphingolipids, such as ceramide, sphingosine, and sphingosine 1-phosphate, are also bioactive molecules that regulate diverse cellular processes, including growth, differentiation, apoptosis, and angiogenesis, among others (Dickson, 1998; Futerman, 2004; Hla, 2004; Spiegel, 2003). Generally, ceramide and sphingosine promote apoptosis and inhibit proliferation, while sphingosine 1-phosphate promotes growth and inhibits apoptosis (Hannun, 2002; Le Stunff, 2004). Ceramide is a precursor for sphingosine, sphingosine 1-phosphate, sphingomyelin, and complex sphingolipids. Enzymes involved in the generation and conversion of these sphingolipids are conserved across species (Futerman, 2005). Ceramidases (CDases) hydrolyze ceramide to sphingosine, which is subsequently phosphorylated to sphingosine 1-phosphate. CDases are thus key enzymes that attenuate ceramide-mediated effects and regulate ceramide/sphingosine/sphingosine 1-phosphate levels in cells. CDases are classified as acid, neutral, or alkaline based on their pH optimum. A deficiency of acid CDase causes Farber's disease in humans, in which ceramide accumulates in the lysosomes. Drosophila has one neutral CDase and no known acid CDase homolog (Acharya, 2005) (Acharya, 2008).

Drosophila phototransduction, a prototypic G protein-coupled receptor (GPCR) signaling cascade, is initiated when the visual pigment rhodopsin absorbs light. Rhodopsin activates a heterotrimeric G protein, Gq, and the effector for Gq is phospholipase C (NorpA). Activation of NorpA leads to the opening of light-sensitive cation channels (Trp and Trpl), by a mechanism that is not completely understood. Termination of the cascade is achieved by the binding of arrestins (Arr1 and Arr2) to rhodopsin, leading to its inactivation (Acharya, 2008).

The current study has shown that CDase acts in a cell-nonautonomous manner to maintain photoreceptor homeostasis. CDase null mutations, obtained by chemical mutagenesis, are embryonic lethal. Clones of cdase null mutant photoreceptors generated in a CDase heterozygous background do not degenerate. However, a genetic background has been identified from which cdase null adult flies can be recovered. The photoreceptors of these CDase null flies undergo light-dependent degeneration, signifying that cell-nonautonomous function of CDase suppresses degeneration in mosaic mutant clones (Acharya, 2008).

Earlier studies have shown that targeted overexpression of ceramidase in photoreceptors suppresses retinal degeneration in certain Drosophila phototransduction mutants (arrestin and norpA) and facilitates the dissolution of incompletely formed rhabdomeric membranes in a rhodopsin mutant (Acharya, 2003; Acharya, 2004). Consistent with a cell-nonautonomous function, targeted expression of CDase in a tissue distant from photoreceptors (such as the fat body or mushroom body) is capable of suppressing retinal degeneration in an arrestin mutant and accelerating the turnover of involuting rhabdomeric membranes in a rhodopsin mutant. This study also shows that secreted CDase is internalized from the cell surface and localizes to the endosomes. The data indicate that CDase participates in the normal endocytic turnover of the arrestin-rhodopsin complex in photoreceptors (Acharya, 2008).

The findings that CDase acts in a cell-nonautonomous manner to promote the survival and function of photoreceptors may have important clinical applications for suppressing retinal degeneration in humans. In addition, these results provide direct evidence that sphingolipid metabolism plays important physiological roles both extracellularly, at the plasma membrane, and within the endocytic compartments (Acharya, 2008).

To isolate cdase mutants, a western blot-based ethylmethanesulfonate (EMS) mutagenesis screen was used (Acharya, 2006). The screening strategy was based on the loss of CDase antigen in immunoblots from EMS-mutagenized flies that were viable over a deficiency that uncovers the CDase gene region. Lethal lines that were generated in the screen were carried over a balancer and subjected to transgenic rescue with a genomic copy of CDase. 2392 lines were established, of which 21 lines were lethal over the deficiency. Transgenic rescue experiments with these lines led to the isolation of the cdase1 mutation, which harbors a G to A transition at amino acid 641, converting a tryptophan residue into a stop codon. The mutated gene encodes an extremely unstable protein, given that no endogenous protein corresponding to the mutant version is visible in western blots of cdase1 null flies. Because EMS causes random mutations throughout the chromosomes, all other incidental mutations within the cdase1 mutant were removed by three successive outcrossings to control w1118 chromosomes (Acharya, 2008).

The cdase1 mutation was a homozygous lethal; mutant animals died at late embryonic stages. Adult flies could be recovered when carrying a transgene with a genomic copy of CDase. To analyze the cdase1 lethal mutants, mosaics were generated in the eye using the ey-FLP/FRT system in combination with a cell-lethal mutation on the wild-type chromosome. In these mosaics, almost the entire eye is homozygous for the mutation while the rest of the animal is heterozygous for CDase (Acharya, 2008).

Based on earlier results showing that targeted overexpression of CDase rescues photoreceptor degeneration in certain phototransduction mutants, it was predicted that CDase would play a significant role in photoreceptor homeostasis (Acharya, 2003). Therefore, cdase1 mosaic photoreceptors were examined for ultrastructural abnormalities by transmission electron microscopy (TEM). Each of the 800 ommatidia of a Drosophila compound eye consists of eight photoreceptor cells (R1 to R8). Each cell has a rhabdomere, a specialized microvillar structure derived from the plasma membrane that houses the phototransduction machinery. Rhabdomere architecture is sensitive to perturbations in the phototransduction cascade and has been used to monitor photoreceptor degeneration. cdase1 mosaics aged to 15 days posteclosion showed a normal rhabdomere architecture (Acharya, 2008).

Because the cdase1 mosaic photoreceptors were morphologically normal, immunocytochemistry was performed on mosaic eye discs with antibodies to CDase to ascertain the lack of CDase protein in the developing eye. Surprisingly, the cdase1 mosaic tissue still showed staining for CDase, despite being genetically null for this protein. Similarly, full-length CDase (not the truncated product encoded by the mutant message) was detected in western blots of retinal extracts from the mosaic eyes. As a control for this method, similar photoreceptor null mutations were generated in a crumbs mutant, whose gene product, crumbs, is required for photoreceptor morphogenesis and maintenance. Retinal extracts from crumbs mosaic tissue do not show any crumbs protein, unlike cdase mosaics that show CDase protein) (Acharya, 2008).

It was reasoned that these findings were due to CDase being a secreted protein (Tani, 2003; Yoshimura, 2002). Most likely, CDase synthesized elsewhere in the animal was transported to the mutant eye tissue. This interpretation could account for CDase staining observed in the homozygous null eyes and perhaps their lack of structural defects. In case of a nonsecreted protein like crumbs, genetically null clones are truly devoid of protein because crumbs will not be transported to the mutant cells from heterozygous tissues. The results of these mosaic analysis experiments also indicated that this approach was not a feasible way to analyze CDase function in vivo. Further study would require the development of a functional cdase null mutant that gave rise to viable adult flies (Acharya, 2008).

During the course of these studies, it was observed that the original cdase1 chromosome, though a homozygous lethal, generated some viable progeny (less than 5%) when crossed to a deficiency that uncovers the CDase region, because of the presence of a closely linked suppressor mutation on the same chromosome. The frequency of viable flies was rescued to normal Mendelian ratios with the introduction of a genomic copy of CDase, which indicated that the CDase mutation contributed to the lethality in this genotype and that the suppressor mutation rescued some of the lethality. The identity of this viable suppressor mutation is currently unknown (Acharya, 2008).

Further examination of the viable adults by western analysis of lysates with several polyclonal and monoclonal antibodies against CDase showed that these flies were devoid of CDase protein. Furthermore, a measurement of neutral CDase activity showed no detectable activity. Immunostaining of eye discs from mutant third-instar larvae of this line did not show any CDase immunoreactivity either. Therefore, it is concluded that these flies were functional null mutants for CDase. The identification of this viable CDase null mutant has permitted continuation of in vivo studies (Acharya, 2008).

Given that photoreceptor-targeted overexpression of CDase rescues degenerating photoreceptors in certain phototransduction mutants (Acharya, 2003), photoreceptor structure-function studies were conducted with the cdase1 functional null mutants (hereafter called cdase1 null). The adult mutant flies were raised in regular light and dark cycles and maintained for varying numbers of days after eclosion; their photoreceptors were then examined by TEM. Five-day-old flies showed severe photoreceptor degeneration. Many photoreceptor cells were missing their rhabdomeres and had vacuolated cell bodies. This degenerative phenotype was completely rescued by introducing a genomic copy of CDase, indicating that the loss of CDase was responsible for the observed photoreceptor degeneration. While the suppressor mutation rescues some lethality, it is unable to compensate for lack of CDase in photoreceptors. One likely explanation is that the high turnover of membranes and signaling molecules in active photoreceptors may create a greater requirement of CDase in this cell type compared to others (Acharya, 2008).

To find out whether the retinal degeneration of cdase1 null mutants depended on light activation of the phototransduction cascade, mutant flies were raised in constant darkness for varying periods, and their photoreceptors were observed by TEM. As expected for a light-dependent effect, photoreceptors of the dark-raised, 5-day-old mutant flies did not show morphological signs of degeneration. This finding also ruled out the possibility that the photoreceptor degeneration was the result of a developmental defect. Furthermore, because CDase localizes to the rhabdomeres and the subrhabdomeric areas of wild-type photoreceptors, it seems that CDase plays a local role in the survival and function of the photoreceptors (Acharya, 2008).

The observation from mosaic experiments that CDase null photoreceptors in a heterozygous background did not degenerate had led to a belief that CDase, which is secreted, acts in a cell-nonautonomous manner. To determine whether CDase expressed in a different tissue could indeed reach the photoreceptors, the enzyme was expressed using UAS-CDase transgenic lines in the fat bodies of wild-type flies using a fat-body-specific GAL4 line. (The fat body of Drosophila is the functional equivalent of mammalian liver and adipose tissue.) As expected, western analysis of extracts from dissected fat bodies of these flies show overexpression of CDase. Retina were isolated from these fat-body-driven flies for western analysis. The blots showed that expression of CDase in the fat body resulted in significant accumulation of CDase in retinal lysastes. In contrast, a nonsecreted protein, crumbs, when overexpressed under the control of the same fat body driver, did not accumulate in the retina (Acharya, 2008).

The fat-body-driven CDase that accumulates in the retinal tissue was visualized using the cdase1 null background. Eye discs were isolated from larvae expressing fat-body-driven CDase in the cdase1 null and these discs were stained for the protein. Mutant eye discs did not show CDase staining, while eye discs from mutants expressing CDase in the fat body displayed robust staining for CDase. Thus, protein made in the fat body reached the eye discs of the cdase1 null animals. To test whether CDase transported to photoreceptors is active, enzyme activity was measured after dissecting retina from cdase1 null flies and cdase1 null flies expressing fat-body-driven CDase. While the mutant shows no activity, retinal extracts from mutants expressing fat-body-driven CDase show high enzyme activity. These results show that CDase that reaches the target site (i.e., photoreceptors) from a distant tissue is active in hydrolyzing ceramide (Acharya, 2008).

Next whether CDase indeed functioned cell-nonautonomously, as was suspected, was examined. To do so, it was asked whether overexpression of CDase by the fat body was sufficient to rescue the photoreceptor degeneration of an arrestin (arr23) mutant and promote membrane turnover in a rhodopsin null (ninaEI17) mutant. Fat body expression of CDase in arr23 indeed suppressed photoreceptor degeneration, and its expression in ninaEI17 enhanced the turnover of involuting rhabdomeric elements. These results are similar to those obtained by expressing CDase specifically in the photoreceptors of these mutants (Acharya, 2003; Acharya, 2004), and they demonstrate that extracellular CDase is indeed capable of modulating the phenotypes caused by these mutations. To further validate that CDase can function cell-nonautonomously, similar experiments were performed after ectopically overexpressing CDase in the mushroom body neurons. Mushroom bodies are centers of olfactory memory in Drosophila. Here too, CDase reaches the retina and has similar effects in arr23 and ninaEI17 mutants (Acharya, 2008).

Staining experiments indicate that extracellular CDase can be transported to the photoreceptors. Because CDase catalyzes the hydrolysis of ceramide to sphingosine and a fatty acid, experiments were carried out to test whether a decrease in substrate (ceramide) or formation of products (sphingosine or fatty acid) could be responsible for CDase function in this study. Electrospray tandem mass spectrometry was used to estimate the ceramide levels in lipid extracts prepared from control, cdase1 null mutant, and fat-body-driven CDase in cdase1 null flies, and molecular species of ceramide were identified containing tetradecasphingenine by negative ion ESI/MS/MS. Lipid extracts from the mutant flies showed an increase in total ceramide levels compared with those from wild-type controls, showing that one of the consequences of a CDase mutation is increased ceramide levels. Overexpression of CDase in the fat body in CDase mutant background results in significant decrease in ceramide level. CDase action not only decreases ceramide levels, it also leads to formation of sphingosine. To evaluate whether formation of sphingosine was responsible for CDase's action, whether increased sphingosine could suppress the degeneration in cdase1 null mutant was examined. Lace encodes for one of the subunits of Drosophila serine palmitoyltransferase, the first and rate-limiting enzyme of sphingolipid biosynthesis, and lace mutants are lethal (Adachi-Yamada, 1999). It has been shown earlier that viability of lace alleles deficient in de novo biosynthesis of sphingosine is promoted when they are raised in food supplemented with sphingosine. Using rescue of lace lethality as evidence for sphingosine function in vivo, cdase1 null mutant flies were raised in food supplemented with sphingosine, and their photoreceptors were examined by electron microscopy. Wild-type photoreceptors are not affected by feeding sphingosine. Sphingosine feeding does not suppress degeneration observed in cdase1 null photoreceptors. This experiment suggests that sphingosine on its own is not a likely candidate for the observed effects of CDase in the present study. N-linked saturated fatty acid is the second product of CDase action. While a role for a fatty acid in this process cannot be ruled out, observations with ceramide kinase mutant and its overexpression in CDase mutants support the notion that CDase's function correlates with ceramide levels rather than sphingosine or fatty acid. The observations that CDase reaches photoreceptors from other tissues combined with the fact that ceramides are very hydrophobic and found only in membranous structures lead to a proposal that local action of CDase is responsible for its effect on photoreceptors (Acharya, 2008).

CDase might first function at the cell surface and then affect intracellular events, such as membrane turnover or endocytosis and/or function, after being internalized from the cell surface. To investigate CDase's mechanism of action, whether extracellular CDase could bind ceramide on the cell surface was examined in vitro. A V5 epitope-tagged CDase was expressed in Schneider cells. Protein expression in this cell line was induced, and the culture medium containing the tagged, extracellular CDase was collected and partially purified. The tagged CDase had ceramide-hydrolyzing activity, as demonstrated by the conversion of NBD-ceramide to NBD-fatty acid (Acharya, 2008).

To determine whether extracellular CDase in fact hydrolyzed ceramide at the cell surface, endogenous CDase expression was knocked down in S2 cells by RNAi, and the cells were incubated with fluorescent ceramide (C12 NBD-ceramide) to incorporate this analog into the plasma membrane. The RNAi-treated cells were pelleted, washed, and incubated with either V5-tagged CDase or buffer alone. The lipids were then extracted from the cells and separated by thin-layer chromatography. Increased hydrolysis (about 40%) was detected of the C12 NBD-ceramide in the cells that were incubated with the tagged CDase (NBD-Cer + CDase), compared with the control cells (NBD-Cer no CDase). Therefore, extracellularly added CDase metabolized the C12 NBD-ceramide at the cell surface (Acharya, 2008).

In a fat body CDase expression experiment, punctate intracellular staining of the CDase protein was detected in eye discs, which suggested that CDase might be internalized. Similar intracellular staining was also seen for CDase in normal eye imaginal discs. Therefore, S2 cells to were used investigate further whether extracellular CDase is internalized. S2 cells were incubaed with the V5-tagged enzyme as the source of extracellular CDase, and the fate of the tagged enzyme was monitored by immunofluorescence analysis. The analysis showed the appearance of punctate fluorescent dots within the cytoplasm, which indicated that the extracellular CDase had been internalized. To determine this, S2 cells were stained with markers of intracellular compartments, and their localization was examined relative to that of the V5-tagged CDase. The endocytic markers, Rab 11 dextran and Drosophila Rab5, colocalized with the internalized CDase, while a Golgi marker, Lava lamp, did not show colocalization. Thus, extracellular CDase is internalized and sorted to the endosomes (Acharya, 2008).

Previous work on CDase has suggested that it functions in membrane-associated intracellular trafficking events. It facilitates membrane turnover in a rhodopsin null mutant (Acharya, 2004). Phenotypic studies on cdase deletion mutants generated by P element excision showed that CDase is important for synaptic function at larval neuromuscular junctions (Rohrbough, 2004). Because its mode of action is unknown, this study set out to elucidate how CDase functions in photoreceptors (Acharya, 2008).

During photoreceptor development, rhodopsin is transported to rhabdomeres through the secretory pathway. Several elegant studies have shown that proper transport of rhodopsin is crucial for photoreceptor stability. As in vertebrates, Drosophila rhodopsin 1 (Rh1) is synthesized in the endoplasmic reticulum (ER) and glycosylated. It is then transported through different compartments of the Golgi complex where it undergoes further modification into mature Rh1 and is finally delivered to the rhabdomeres. The 34 kDa mature Rh1 is rhabdomeric; immature forms have a higher molecular weight (40 kDa) and are predominantly localized to the ER. Furthermore, several mutants where immature Rh1 accumulates, which is indicative of defective transport, show retinal degeneration (Acharya, 2008).

To test whether the CDase mutants were defective in the forward transport of rhodopsin to the rhabdomeres, the accumulation of immature Rh1 was examined for in CDase mutants. Light-raised cdase1 mutant flies showed reduced Rh1 levels by day 3 and its almost complete loss by day 10. This reduction is not surprising, since many degenerative mutations cause a precipitous drop in Rh1 levels. However, despite the reduction in Rh1 levels in cdase1 mutants, only the mature, 34 kDa form of Rh1 was detected in the retinal lysates and not the immature 40 kDa form. Rh1 levels in were examined dark-raised flies, which do not show the morphological degeneration observed in light-raised flies. The Rh1 levels in the dark-raised cdase1 mutants were similar to the levels in control flies, and the Rh1 detected was the mature form. Thus, the cdase1 mutants are not defective in the forward transport of Rh1 from the ER to rhabdomeres (Acharya, 2008).

Invertebrate Rh1 is normally endocytosed upon light activation, and the two visual arrestins have been implicated in its endocytosis in Drosophila photoreceptors, a function similar to that of nonvisual mammalian arrestins, which are involved in the desensitization and endocytosis of activated GPCRs. To test whether cdase1 mutants are defective in Rh1 endocytosis, a biochemical assay was used that monitors the formation of a protein complex between Rh1 and Arr2 in photoreceptors. Blue light converts rhodopsin to active metarhodopsin, accompanied by the binding of arrestin 2. This binding is followed by endocytosis of the Rh1-Arr2 complex. Orange light converts Rh1 to an inactive form accompanied by the release of Arr2. Wild-type and cdase1 dark-raised flies were exposed to blue or blue followed by orange light, extracts were prepared and fractionated into supernatant and pellet fractions. In this assay, Arr2 bound to Rh1 pellets upon centrifugation, while released Arr2 is found in the supernatant. In the dark, Arr2 was found in the pellet and supernatant in both control and cdase1 null mutant photoreceptors. When exposed to blue light, Arr2 bound Rh1 in both the control and the mutant flies. However, upon photoconversion of Rh1 by orange light, Arr2 was efficiently released into the supernatant in the control whereas, in cdase1 null flies, Arr2 release was defective. The identical experiment performed with cdase1 mutants carrying a copy of genomic CDase (rescued flies) showed efficient release of Arr2 into the supernatant (Acharya, 2008).

The binding of Arr1 to Rh1 was tested in the wild-type and mutant backgrounds using the same assay. In wild-type adult flies, unlike Arr2, Arr1 does not partition differently upon exposure to blue and orange light. Rather, almost all the Arr1 was found in the pellet fraction upon either blue or orange light exposure. However, for the cdase1 mutants, more Arr1 was found in the supernatant than for wild-type flies. This defect was rescued by introducing a genomic copy of CDase. Thus, in cdase1 null mutants, Arr1 associates less tightly with Rh1 than in wild-type flies. Thus, the dynamics of interaction between rhodopsin and arrestins are altered in cdase1 null mutants. These alterations could be indicative of defective endocytosis in cdase1 null photoreceptors (Acharya, 2008).

To test whether photoreceptor function in the cdase1 mutants is also defective, electroretinogram recordings (ERGs) were performed from control, mutant, and rescued flies. Because dark-raised cdase1 mutant flies did not show morphological degeneration, the recordings were performed on dark-raised flies. Interestingly, despite their intact photoreceptor morphology, the mutant flies did not respond to even high-intensity light stimulus. However, the rescued cdase1 mutants showed normal ERGs. The block of phototransduction in the cdase1 mutants shows that CDase is critical for normal photoreceptor signal transduction. The lack of response to light in the absence of morphological changes suggests that CDase has additional roles in photoreceptor function beyond its effects on endocytosis (Acharya, 2008).

Kinetic evidence and the pharmacological manipulation of ceramide levels in mammalian cells have demonstrated a close link between ceramide levels and the induction of apoptosis (Hannun, 2000; Hannun, 2002). To detect apoptosis in the eye imaginal discs of third-instar cdase1 larvae, an antibody against activated caspases was used. The anti-activated-caspase staining was stronger in the cdase1 mutant imaginal discs than in wild-type discs. This increased apoptosis was largely in the undifferentiated cells, anterior to the morphogenetic furrow. Whether damage-induced apoptosis was enhanced in the cdase1 mutants was checked, since several studies in mammalian tissue culture have shown that stress stimuli, including ionizing radiation, lead to ceramide accumulation by interfering with enzymes involved in its metabolism (Hannun, 2002; Kolesnick, 2003]). The imaginal discs of irradiated late third-instar larvae from cdase1 mutants showed higher levels of caspase staining than those from irradiated wild-type controls. Therefore, the cdase1 null mutation results in an increased propensity for apoptosis compared with wild-type flies (Acharya, 2008).

Because cdase1 mutants are prone to apoptosis, it was reasoned that the degeneration of mutant photoreceptors might be caused by apoptosis. To test this, the caspase inhibitor p35, using an eye-specific promoter, was expressed in cdase1 mutants. A TEM analysis showed that the expression of p35 in the mutant photoreceptors suppressed their degeneration. Thus, activation of apoptosis mediates the degeneration observed in cdase1 mutant photoreceptors (Acharya, 2008).

In summary, this study used a functional null mutant of Drosophila neutral CDase to show that this enzyme plays a critical role in regulating photoreceptor structure and function, cell-nonautonomously. Extracellular CDase is internalized from the cell surface, localizes to the endocytic compartment, and participates in the endocytic turnover of rhodopsin-arrestin complexes to maintain photoreceptor homeostasis (Acharya, 2008).

In mutants such as arrestin, rhodopsin remains chronically active, leading to cytotoxicity and photoreceptor cell death. Retinal degeneration also occurs in a subset of visual system mutants (norpA, rdgc, rdgb) through the accumulation of an abnormally stable Rh1-Arr2 complex in the photoreceptor cells. In this situation, the activated Rh1-Arr2 complexes undergo massive internalization and disrupt the endocytic pathway, but the exact mechanism by which these complexes impair endocytic function is not known. Nonetheless, the persistence of these complexes induces retinal degeneration, eventually leading to photoreceptor apoptosis. Although vertebrate rhodopsin is not normally endocytosed, recent studies show that certain mutations in rhodopsin associated with retinitis pigementosa (RP) cause the formation of stable rhodopsin-arrestin complexes that alter the morphology of the endosomal compartments and impair receptor-mediated endocytic functions (Chuang, 2004). Given CDase's ability to rescue similar mutations in flies, it may be possible to exploit this function clinically to suppress retinal degeneration in humans via the targeted overexpression of CDase (Acharya, 2008).

Recent studies show that sphingolipid-metabolizing enzymes function both intracellularly and extracellularly. An isoform of sphingosine kinase 1 that acts on S1-P is secreted and contributes to establishing the vascular S1-P gradient that regulates angiogenesis (Ancellin, 2002). Similarly, these findings support both extracellular and intracellular roles for CDase. Furthermore, studies in mammalian tissue culture systems and in yeast have noted the modulation of endocytosis by ceramide or sphingoid base. This is consistent with the idea that intracellular endocytic processes cannot proceed without cooperative changes in the outer leaflet of the plasma membrane bilayer; however, the nature of these changes has been largely unexplored. The current findings highlight the role of CDase in bringing about such cooperative changes. CDase-mediated alterations of the microenvironment of the lipid bilayer could potentially affect membrane dynamics, not only at the cell surface, where intracellular events would be initiated, but also in internalized compartments along the endocytic route. Recently, it has been demonstrated that ceramide intrinsically moves between the two leaflets of the plasma membrane efficiently (Mitsutake, 2007). Thus, changes in ceramide levels on the extracellular leaflet of membranes will affect ceramide levels in the cytoplasmic leaflet. This phenomenon could influence arrestin interactions in the cytoplasmic leaflet when CDase hydrolyzes ceramide on the extracellular leaflet (Acharya, 2008).

While acid and neutral CDases have been identified in mammals, Drosophila encodes only one neutral Cdase, and no acid CDase homolog has been identified. While a null mutation of neutral CDase is lethal in Drosophila, mice whose corresponding homolog is disrupted do not show obvious abnormalities (Kono, 2006). Rather, they have a normal lifespan with no major alterations in ceramide levels in their tissues, but they are deficient in the intestinal degradation of ceramide. Knockout mice for acid CDase, however, die as embryos, and the heterozygotes show progressive lipid storage disease (Li, 2002). It is tempting to speculate that neutral CDase may also play the role of acid CDase in Drosophila (Acharya, 2008).

In conclusion, this study found that CDase functions in a cell-nonautonomous manner to maintain photoreceptor homeostasis. Furthermore, these results show the functional coupling of the sphingolipid biosynthetic pathway with Drosophila photoreceptor survival and function (Acharya, 2008).

Modulating sphingolipid biosynthetic pathway rescues photoreceptor degeneration

Mutations in proteins of the Drosophila phototransduction cascade, a prototypic guanine nucleotide-binding protein-coupled receptor signaling system, lead to retinal degeneration and have been used as models to understand human degenerative disorders. In this study, modulating the sphingolipid biosynthetic pathway rescued retinal degeneration in Drosophila mutants. Targeted expression of Drosophila neutral ceramidase rescues retinal degeneration in arrestin and phospholipase C mutants. Decreasing flux through the de novo sphingolipid biosynthetic pathway also suppresses degeneration in these mutants. Both genetic backgrounds modulate the endocytic machinery because they suppressed defects in a dynamin mutant. Suppression of degeneration in arrestin mutant flies expressing ceramidase correlates with a decrease in ceramide levels. Thus, enzymes of sphingolipid metabolism may be suitable targets in the therapeutic management of retinal degeneration (Acharya, 2003).

Sphingolipids are integral components of eukaryotic cell membranes and also a rich source of second messengers for several signal transduction cascades. Sphingolipid metabolism generates and interconverts various metabolites including ceramide, sphingosine, and sphingosine 1-phosphate, which are second messengers in diverse signaling pathways that affect cell cycle, apoptosis, and angiogenesis, among others. Serine palmitoyl-CoA transferase (SPT) catalyzes the rate-limiting first step in the de novo biosynthesis of sphingolipids including ceramide. Ceramidases hydrolyze ceramide to sphingosine, and neutral or alkaline ceramidase is proposed to function in signaling. Mutant analyses in yeast have implicated enzymes of sphingolipid metabolism in endocytic membrane trafficking events. This study modulated the sphingolipid biosynthetic pathway in vivo in Drosophila and examined its effects on mutants with endocytic defects in photoreceptors (Acharya, 2003).

Each of the 800 ommatidia of a Drosophila compound eye consists of eight photoreceptor cells (R1 to R8). Each cell has a rhabdomere, a specialized microvillar structure derived from the plasma membrane that houses the phototransduction machinery. Rhabdomere architecture is sensitive to perturbations in the phototransduction cascade and has been used to monitor photoreceptor degeneration. Drosophila phototransduction is a prototypic GTP-binding protein-coupled receptor (GPCR) cascade that is initiated by light activation of rhodopsin. Association of arrestin 2 with phosphorylated rhodopsin leads to deactivation of rhodopsin. Drosophila arrestin 2 also acts as a clathrin adaptor, mediating endocytosis of arrestin-rhodopsin complexes. arr23 mutants (Val52 to Asp) make less than 1% of the protein, are defective in endocytosis, accumulate abnormal multivesicular bodies, show extensive retinal degeneration, and undergo necrotic cell death. These changes also result in a precipitous drop in rhodopsin levels in these photoreceptors. Thus, arr23 photoreceptors provide a sensitive background for examining the in vivo effects of modulating the sphingolipid pathway in endocytosis (Acharya, 2003).

The ceramidase gene was cloned into an UAS vector, and it was expressed in the Drosophila eye with the use of a glass multimer reporter (GMR)-Gal4 driver. Extracts from these fly heads showed increased neutral ceramidase activity, confirming that the protein was a bona fide neutral ceramidase. Expression of GMR-Gal4; UAS-ceramidase in R1 to R6 did not affect photoreceptor integrity. Expression of ceramidase in arr23 rescued photoreceptor degeneration. In transmission electron micrographs (TEMs), rhabdomeres were intact, and multivacuolar bodies and degenerating photoreceptors, characteristic of arr23 mutants, were completely absent in a 3-day arr23 fly expressing ceramidase. A near-wild-type level of rhodopsin was seen in rescued flies, reflecting photoreceptor integrity. Thus, expression of ceramidase in arr23 preserved rhabdomere structure and organization. Although defective, newly eclosed arr23 flies transduce light signals. As they age, they undergo progressive degeneration and lose their ability to transmit signals. To test whether rescued flies retain their functional ability to signal, electroretinogram recordings (ERG) were carried out from 7-day-old flies exposed to light. Because of extensive degeneration, ERGs of arr23 flies had a very small amplitude, whereas arr23 flies expressing ceramidase showed a robust response. However, the slow inactivation kinetics characteristic of arr23 still persisted in the rescued flies. Rescued arrestin mutant flies transduced signals even on aging because the structural integrity of these photoreceptors was preserved (Acharya, 2003).

Because expression of ceramidase in arrestin mutant flies suppressed retinal degeneration and because ceramidase hydrolyzed ceramide, it was reasoned that the rescue would be accompanied by a decrease in ceramide levels in these photoreceptors. Electrospray ionization tandem mass spectrometry (ESI/MS/MS) was used to estimate ceramide levels in lipid extracts of membranes prepared from fly heads of control, ceramidase expressor, arrestin mutant, and arrestin mutant expressing ceramidase. Ceramide molecular species containing tetradecasphingenine and their 2-hydroxy counterparts were identified by negative ion ESI/MS/MS with neutral loss of 200.2 and 271.2 mass units, respectively. As expected, expression of ceramidase reduces the ceramide levels in control animals. Lipid extracts from arrestin mutants showed an increase in ceramide levels, probably reflecting changes accompanying the severely degenerating photoreceptors. Expression of ceramidase in arrestin mutants decreased ceramide levels by 50% in all species measured. Thus, rescue of degeneration correlated with a decrease in ceramide levels in the mutant flies. Because sphingosine is a product of the ceramidase reaction, whether increased sphingosine could suppress retinal degeneration in arrestin mutants was evaluated. Viability of certain lace alleles (LCB2 subunit of SPT, a.k.a. Serine palmitoyltransferase subunit II), which are deficient in de novo sphingosine biosynthesis, is increased when flies are raised in food supplemented with sphingosine. Arrestin mutant flies were raised under similar conditions and their photoreceptors were examined by electron microscopy. The rhabdomeres of R1 to R6 cells showed no suppression; instead, they showed enhanced degeneration of these photoreceptors. Thus, it is believed that sphingosine on its own is not a likely candidate for suppression of degeneration in the present study; instead, suppression correlated with decreased ceramide levels in rescued mutant flies (Acharya, 2003).

Ceramidase suppressed degeneration in arr23 mutants with chronically active rhodopsin and defects in clathrin-dependent endocytosis. It is possible that ceramidase suppressed degeneration by altering the balance of the endocytic pathway, thereby alleviating cytotoxicity arising from defective endocytosis. To test this, ceramidase was expressed in a dynamin mutant background in the eye. Dynamin is a guanosine triphosphatase essential for clathrin-mediated endocytosis. In Drosophila, a temperature-sensitive mutant of dynamin, shibire (shits1), has a general defect in endocytosis. These results were recapitulated in mammalian cells when a similar mutant dynamin was overexpressed. A temperature-sensitive dominant-negative mutant, UAS-shits1, under the control of a GMR-Gal4 driver, was used to preferentially express the mutant protein in the eye. These photoreceptors showed profound retinal degeneration characterized by loss of rhabdomere and accumulation of multivesicular bodies and vacuoles in R1 to R6, whereas R7 was largely unaffected. Ceramidase expression suppressed degeneration in UAS-shits1 photoreceptors. Rhabdomeres were largely intact, vacuolated cells were fewer, and trapezoidal arrangement of rhabdomeres was retained. As in other degenerating mutants, rhodopsin levels were low in shits1 mutants compared with those of the wild type but were restored upon ceramidase expression (Acharya, 2003).

Whether SPT, the rate-limiting enzyme of the de novo sphingolipid biosynthetic pathway, could affect the degeneration observed in these mutants was tested. In Drosophila, the Lace gene encodes the LCB2 subunit of SPT. The P-lacW-inserted lace allele l(2)k05305 is an insertion of a P-element 8 to 9 base pairs upstream of the transcription start site of lace and is homozygous lethal. arr23 and UAS-shits1 mutants were crossed into the lace heterozygous background and photoreceptors were examined by transmission electron microscopy. lace heterozygotes had intact photoreceptors. lace partially suppressed retinal degeneration in arr23 mutants and in UAS-shits1 mutants (Acharya, 2003).

Finally, whether ceramidase and lace suppressed degeneration in a phospholipase C mutant, where endocytosis has been implicated in the degenerative process, was examined. Norp A encodes an eye-specific phospholipase C that activates GPCR signaling by generating inositol trisphosphate and diacylglycerol. norp A mutant flies do not show light-induced receptor potential and are blind. Although norp A mutants degenerated slowly, these changes were obvious even in 3-day-old flies. Expression of ceramidase in a norp A mutant suppressed retinal degeneration. Lace heterozygotes also suppressed norp A degeneration. arr23 mutants undergo necrotic cell death, whereas norp A mutants accumulate rhodopsin-arrestin complexes and undergo apoptotic cell death. Thus, regardless of the mode of cell death ceramidase expression and lace mutant rescued degeneration. Because they also suppressed degeneration in a dynamin mutant, it is inferred that the sphingolipid pathway exerts its beneficial effect by altering the dynamics of the endocytic process. This is supported by observations that a sphingoid base is required for yeast endocytosis and that in mammalian cells ceramide analogs modulate fluid-phase and receptor-mediated endocytosis (Acharya, 2003).

The molecular details of suppression of retinal degeneration by ceramidase overexpression and lace mutant remain to be elucidated. A common denominator in both situations is the likely decrease in concentrations of ceramide, which could be responsible for activating a cascade that suppresses degeneration (Acharya, 2003).

A large volume of work suggests that receptor desensitization, endocytosis, and recycling play a crucial role in GPCR signaling in higher organisms. In light of the current finding, it will be interesting to study sphingolipid metabolism in GPCR-mediated processes. Several inherited forms of human retinal degenerations result from mutations in rhodopsin, arrestin, and phosphodiesterase, among others. Individuals with Oguchi disease have mutations in visual arrestin and a form of degenerative night blindness. Rescue of degeneration in Drosophila visual mutants provides a strong basis for exploring strategies that manipulate sphingolipid enzymes for therapeutic management of retinal degeneration in higher organisms (Acharya, 2003).

Ceramidase expression facilitates membrane turnover and endocytosis of rhodopsin in photoreceptors

Transgenic expression of ceramidase suppresses retinal degeneration in Drosophila arrestin and phospholipase C mutants. This study shows that expression of ceramidase facilitates the dissolution of incompletely formed and inappropriately located elements of rhabdomeric membranes in ninaEI17 mutants lacking the G protein receptor Rh1 in R1-R6 photoreceptor cells. Ceramidase expression facilitates the endocytic turnover of Rh1. Although ceramidase expression aids the removal of internalized rhodopsin, it does not affect the turnover of Rh1 in photoreceptors maintained in dark, where Rh1 is not activated and thus has a slower turnover and a long half-life. Therefore, the phenotypic consequence of ceramidase expression in photoreceptors is caused by facilitation of endocytosis. This study provides mechanistic insight into the sphingolipid biosynthetic pathway-mediated modulation of endocytosis and suppression of retinal degeneration I (Acharya, 2004).

Rh1, a major component of rhabdomeres, is not only the receptor for transducing light signal in R1-R6, but is also required for organization of the rhabdomere terminal web (RTW), an actin-based cytoskeletal scaffold, believed to orchestrate the biogenesis of this organelle. In Rh1 null mutant ninaEI17, the RTW is not organized during the late pupal stage, and rhabdomere biogenesis is defective. Consequently, photoreceptors examined by transmission electron microscopy 3 days after eclosion show the ill formed rhabdomeres, and membranes of rhabdomeres are seen involuting into the photoreceptors. These involuting curtains of rhabdomere elements are then slowly cleared over two weeks (Acharya, 2004).

Transgenic expression of ceramidase does not affect either the development or function of photoreceptors. However, ceramidase expression suppresses degeneration in arrestin and norpA mutants and in photoreceptors expressing a dominant-negative form of dynamin. This led to the proposal that ceramidase mediates its effect by modulating the endocytic pathway. It was reasoned that the phenotypic consequence of ceramidase expression in Rh1 mutant ninaEI17 would be different from that observed in arrestin and dynamin mutants. If ceramidase affects endocytosis, then in a ninaEI17 mutant it should influence the process of involution and clearance of the incompletely formed and inappropriately localized rhabdomeric membranes (Acharya, 2004).

Indeed, expression of ceramidase facilitates the removal of these inappropriately positioned rhabdomeric elements. Three-day-old ninaEI17 flies have ill-formed rhabdomeres, and remnant rhabdomeres are seen as long contiguous elements of plasma membrane involuting into the cell body in R1-R6 photoreceptors. In contrast, 3-day-old ninaEI17 flies expressing ceramidase have very little rhabdomeric elements at the apical surface of the photoreceptors. Most of the rhabdomeric membranes have been internalized and are in the process of being cleared. Although R1-R6 cells of almost all ommatidial sections of a ninaEI17 compound eye have a significant density of remnant rhabdomeric membranes in the apical portion of the cells, more than 95% of the R1-R6 photoreceptors expressing ceramidase have no significant rhabdomeric membranes in the apical region. Instead, the apical regions have plasma membranes that are contiguous with the rest of the photoreceptor cells. The cell- cell adherens junction is intact, indicating the structural integrity of these photoreceptor cells (Acharya, 2004).

Because the development of rhabdomeres is initiated in the pupae and because GMR-Gal4 initiates ceramidase expression during its formation, it can be argued that ceramidase expression could affect the biogenesis of rhabdomeric elements in ninaEI17, thus resulting in the observed phenotype. To resolve this issue, ceramidase was expressed, after eclosion, in an adult ninaEI17 mutant. In these experiments, UAS-ceramidase expression was driven by a heat shock Gal4 driver. Newly eclosed ninaEI17 flies and ninaEI17 flies with ceramidase transgene were incubated at 37°C for 1 h/day for 3 days. Control ninaEI17 flies heat shocked for 3 days showed features similar to non-heat-shocked mutant flies. Rhabdomeres were incompletely formed but slightly compact, and membranes were seen involuting into the R1-R6 photoreceptors. In contrast, flies expressing ceramidase cleared most of the rhabdomere from the apical region. Thus, expression of ceramidase specifically accelerates the intracellular dissolution of rhabdomeric membranes in ninaEI17 mutant photoreceptor cells. The internalized tubular and vesicular elements generated by expression of ceramidase in ninaEI17 was examined for an antigen specific to rhabdomere. Chaoptin is a photoreceptor specific, leucine-repeat containing, cell adhesion plasma membrane protein that localizes to the outer leaflet and is enriched in rhabdomeres of photoreceptors. ninaEI17 flies expressing ceramidase were examined by immunoelectron microscopy for chaoptin. Chaoptin immunoprecipitates were localized on internalized membranous tubular and vesicular structures, providing additional evidence of their rhabdomeric origin (Acharya, 2004).

Like other G protein-coupled receptors, rhodopsins undergo a ligand-dependent endocytic turnover. Given the previous data on the consequence of ceramidase expression in endocytic mutants and the current observations with ninaEI17 mutant, itwas decided to evaluate the effects of ceramidase expression on ligand (light)-induced rhodopsin turnover in wild-type photoreceptors (Acharya, 2004).

The endocytic turnover of Rh1 can be followed using an inducible and uniquely tagged Rh1. Rh1- 1D4 transgenic flies express a heat-shock inducible Drosophila Rh1 carrying a specific tag, 1D4, derived from the C terminus of bovine Rh1. Rh1- 1D4 transgene is functional, because it rescues the phenotypic defects in ninaEI17. Rh1-1D4 synthesis was initiated in wild-type and ceramidase transgenic flies by a single heat shock at 37°C, and was followed by western analysis for 1D4 tag over a period while being maintained in a normal 12-h light/12-h dark cycle at 25°C. Under these conditions, heat shock induction initiated the synthesis of Rh1, which peaked after 24-48 h, decreased over the next several days in wild-type photoreceptors and was still visible around day 8. Under similar conditions in flies expressing ceramidase, Rh1 levels peaked similar to wild-type flies; however, Rh1 disappeared rapidly thereafter, and none was visible after 4 days. These experiments indicate that ceramidase expression enhances the light-dependent turnover of Rh1 (Acharya, 2004).

Drosophila phototransduction is a prototypic G protein-coupled receptor-signaling cascade. Like other G protein signaling cascades, invertebrate rhodopsin undergoes light-dependent turnover. In the blowfly, it has been demonstrated that rhodopsin has an extended half-life in flies maintained in the dark, whereas in flies maintained in light it has a short half-life. Although rhodopsins undergo photochemical interconversion, light-activated rhodopsins are eventually endocytosed and degraded. In Drosophila, visual arrestins act as clathrin adaptors and have been demonstrated to bind and internalize light-activated Rh1. It was hypothesized that if ceramidase facilitates a downstream process of endocytosis, although it can facilitate the turnover of light-activated rhodopsin it should have minimal effect on the half-life of an unactivated rhodopsin in flies maintained in dark. Therefore the effect was examined of ceramidase on rhodopsin turnover in flies maintained in the dark. A pulse of rhodopsin in flies reared in the dark results in longer half-life, and a greater fraction of the pulsed rhodopsin is still visible 8 days after heat shock. Similar levels of rhodopsin were seen in ceramidase expressors maintained in the dark, thus suggesting that ceramidase does not accelerate the process of rhodopsin turnover in flies maintained in the dark. It is therefore concluded that ceramidase enhances turnover of light-activated rhodopsin by facilitating endocytosis. Because ceramidase does not affect rhodopsin that is not light-activated, it is believed that it facilitates an existing normal mechanism for rhodopsin turnover. Although the molecular details of rhabdomere and rhodopsin turnover are yet to be elucidated, it has been known for several years that in flies the photoreceptor membrane is shed into the photoreceptor cell and cleared by endocytosis. Ceramidase expression results in the clearing of ill-formed rhabdomeric elements in ninaEI17 mutants, whereas its expression in endocytic mutants such as arrestin and dynamin suppresses degeneration. Therefore, the consequence of ceramidase expression is determined by the underlying pathology of the phototransduction mutant (Acharya, 2004).

This study has used a sensitized background to reveal the effects of ceramidase on membrane turnover. By following the phenotypic changes from the long involuting rhabdomeric membranes seen in ninaEI17 mutant photoreceptor cells to cells almost devoid of rhabdomeres in ninaEI17 expressing ceramidase, it was shown that ceramidase expression facilitates membrane turnover in these cells. The use of an inducible, tagged Rh1 (hs-Rh1-1D4) has allowed the turnover of rhodopsin to be followed in wild-type photoreceptors. Using the inducible rhodopsin, this study has demonstrated that ceramidase specifically facilitates the turnover of light-activated receptor. Light is the ligand for rhodopsin, and these receptors are not engaged when photoreceptors are maintained in the dark. Ceramidase expression had no effect on the half-life of rhodopsin when maintained in the dark, and it was therefore concluded that ceramidase-facilitated ligand induced endocytic turnover of rhodopsin. The study of rhodopsin turnover in wild-type photoreceptors permitted examination of receptor endocytosis without complications of an underlying mutant phenotype. Ceramidase expression suppresses degeneration in endocytic mutants such as arr23 and mutant photoreceptors expressing a dominant-negative form of dynamin. Indeed, it would be very interesting to evaluate the turnover of rhodopsin in these mutants. Attempts were made to follow rhodopsin turnover in an inducible dominant-negative dynamin mutant background. However, the mutant photoreceptors degenerated, and the function of these photoreceptors was severely compromised. Because of the degeneration, heat-shock induction of 1D4-tagged Rh1 in these mutants did not result in synthesis of appreciable amounts of protein, and hence turnover could not be followed. A similar degenerative pathology complicates analysis in arrestin mutant background, and the lack of these critical controls makes it difficult to evaluate effects of ceramidase on Rh1 turnover in these mutant backgrounds. Another approach is to investigate the interaction of ceramidase expressors with components of pathways that have been implicated in membrane transport and photoreceptor degeneration. A recent study has addressed the probable dual role of phosphoinositides in activation and adaptation of phototransduction cascade. Arrestin 2 specifically bound phoshphoinositide/inositol phosphates. In this study, flies expressing engineered mutants of arrestin that were defective in phosphoinositide binding but not Rh1 binding were generated. The photoreceptors from these flies show delayed and decreased translocation of arrestin to the rhabdomere upon light activation, and those that were translocated were not internalized efficiently after binding Rh1. In a reverse approach, the this study also showed that light-dependent translocation of arrestin was defective in mutants that disrupt phosphoinositide metabolism. Earlier work on CDP-DAG synthase, an enzyme required for phosphoinositide biosynthesis, and RDGB, a phosphoinositide transfer protein, have implicated phosphoinositides in membrane turnover and signaling in Drosophila photoreceptors. Because phosphoinositides have been implicated in transport of phototransduction components to and from rhabdomeric membranes and sphingolipids are integral membrane components, specific interactions could influence phototransduction at multiple steps. It would thus be worthwhile to examine whether enzymes of the sphingolipid biosynthetic pathway, such as serine palmitoyltransferase and ceramidase, do interact with the phosphoinositide signaling pathway. It is also important to discern whether these interactions, if any, are mediated by specific protein- protein interactions or are caused by effects of changes in metabolite concentrations, such as ceramide and sphingosine, or rather are the result of a combined effect. Use of mass spectrometry and feeding experiments suggested that a decrease in steady-state levels of ceramide contribute to the beneficial effect of ceramidase in suppressing degenerations. It is now believed that facilitation of endocytosis observed in ceramidase expressors is also consequent to its action on ceramide levels in photoreceptors. If so, then mutants such as lace that are defective in ceramide synthesis and upstream of neutral ceramidase in the de novo biosynthetic pathway should have a similar effect as ceramidase expression in ninaEI17mutant. Formation and breakdown of ceramide can affect the structure of membranes because of its topology, membrane sidedness, and limited flip-flop across membranes. It is believed that these results lend credence to suggestions that many of the actions of ceramide are caused by its role in membrane domain formation, membrane vesiculation, fusion and fission reactions, and trafficking. Sphingolipids are being increasingly implicated in yeast as important regulators of cell growth, heat stress response, and membrane trafficking. In conclusion, whereas earlier studies showed that expression of ceramidase suppresses degeneration in arr23, norpA, and dynamin mutant backgrounds, the current study has led to the proposal that it does so by facilitating endocytosis and a decrease in ceramide contributes to these processes (Acharya, 2004).

This approach of genetically modulating sphingolipid biosynthetic pathway in Drosophila phototransduction mutants, a prototypic G protein-coupled receptor signaling cascade, will help in integrating signaling, lipid metabolism, membrane turnover, and degenerative pathways (Acharya, 2004).

Ceramidase regulates synaptic vesicle exocytosis and trafficking

A screen for Drosophila synaptic dysfunction mutants identified slug-a-bed (slab). The slab gene encodes ceramidase, a central enzyme in sphingolipid metabolism and regulation. Sphingolipids are major constituents of lipid rafts, membrane domains with roles in vesicle trafficking, and signaling pathways. Null slab mutants arrest as fully developed embryos with severely reduced movement. The SLAB protein is widely expressed in different tissues but enriched in neurons at all stages of development. Targeted neuronal expression of slab rescues mutant lethality, demonstrating the essential neuronal function of the protein. C5-ceramide applied to living preparations is rapidly accumulated at neuromuscular junction (NMJ) synapses dependent on the SLAB expression level, indicating that synaptic sphingolipid trafficking and distribution is regulated by SLAB function. Evoked synaptic currents at slab mutant NMJs are reduced by 50%-70%, whereas postsynaptic glutamate-gated currents are normal, demonstrating a specific presynaptic impairment. Hypertonic saline-evoked synaptic vesicle fusion is similarly impaired by 50%-70%, demonstrating a loss of readily releasable vesicles. In addition, FM1-43 dye uptake is reduced in slab mutant presynaptic terminals, indicating a smaller cycling vesicle pool. Ultrastructural analyses of mutants reveal a normal vesicle distribution clustered and docked at active zones, but fewer vesicles in reserve regions, and a twofold to threefold increased incidence of vesicles linked together and tethered at the plasma membrane. These results indicate that SLAB ceramidase function controls presynaptic terminal sphingolipid composition to regulate vesicle fusion and trafficking, and thus the strength and reliability of synaptic transmission (Rohrbough, 2004).

The slug-a-bed gene was identified in a forward screen for synaptic dysfunction mutants and encodes a long-chain Cdase that regulates the ceramide level. At slab mutant synapses, postsynaptic receptor function is unaltered, whereas nerve-evoked transmission and HO saline-evoked SV fusion is decreased by 50%-70%. These results indicate a specific presynaptic impairment consistent with a loss of the readily releasable pool. However, slab synapses exhibit normal numbers of clustered and docked SVs at the AZ, ruling out defects in forming a localized releasable pool (RRP). It is therefore concluded that the primary transmission impairment is a reduced ability of SVs to complete priming/fusion steps after docking (Rohrbough, 2004).

Neurotransmitter release is most directly mediated by the readily releasable pool, a small cycling SV pool that includes a subpopulation of docked, fusion-competent vesicles at the active zone (AZ). Provided that the essential vesicle fusion machinery is functional, release efficacy is predictably correlated to the size of this docked pool, which may be functionally assayed by hypertonic saline-evoked fusion. This correlation is genetically supported by C. elegans mutations in the Sec-1-related protein UNC-18, which causes a parallel loss of docked SVs and hypertonic saline-evoked fusion. Similarly, mutants in the mammalian UNC-18 homolog reduce docking and exocytosis of dense core secretory vesicles. Disruptions to downstream priming and fusion steps, including mutations in the SNARE (SNAP receptor) core complex proteins syntaxin-1, N-synaptobrevin, and SNAP-25 as well as in UNC-13 (uncoordinated-13) and RIM (Rab3-interacting molecule), which regulate SV priming, also severely reduce or eliminate Ca2+- and hypertonic saline-evoked release. In contrast, however, these priming/fusion mutants typically exhibit increased SV docking and a larger overall vesicle pool because of selective block of the exocytosis step(s) (Rohrbough, 2004 and references therein).

The slab mutant phenotypes place the functional transmission requirement downstream of docking at the level of vesicle priming/fusion, with the insignificant increase (~20%) in docked SVs consistent with a pronounced but incomplete inhibition of fusion. SNARE complex proteins have been shown to localize to sphingolipid- and sterol-enriched membrane raft environments in secretory cells. Other exocytic proteins, such as nSEC-1 and αSNAP, are reported to localize to distinct sterol-rich raft-like domains. An accumulation and spatial misregulation of ceramide, the predominant sphingolipid in flies, is expected to disrupt the topology, lipid environment, and protein content of membrane exocytic domains. Ceramide has established roles regulating membrane domain fluidity and curvature, cholesterol aggregation, vesicle formation, and fusion (Li, 1999; Venkataraman, 2000; van Blitterswijk, 2003). Increased membrane ceramide levels may therefore interfere with SV fusion competency by inhibiting lipid restructuring required for exocytosis, by inhibiting functional interactions between release machinery proteins, or by a combination of both lipid- and protein-mediated processes (Rohrbough, 2004).

The results indicate that sphingolipids are also involved in regulating SV number and distribution. In slab mutant terminals, the endo/exo cycling pool labeled by FM1-43 is reduced by ~30%. Although overall mutant SV density is not changed, the number of SVs not localized to AZs is reduced by a similar percentage. Most interestingly, slab terminals exhibit a striking increase in the percentage of SVs linked together or tethered to the plasma membrane (PM). Filamentous tethers often clearly appear to connect SVs and to connect SVs to the PM, singly and in multi-vesicle arrays. SVs sometimes appear both linked and tethered, suggesting that these forms of linkage are similar structurally and serve to sequester SVs from the AZ. Although previously overlooked, normal synapses exhibit a small number of linked/tethered SVs, indicating they represent normal intermediate trafficking steps that are abnormally accumulated in the absence of SLAB (Rohrbough, 2004).

C. elegans and Drosophila endophilin (endo) and synaptojanin (synj) mutants also exhibit prominent arrays or 'strings' of linked SVs and SVs tethered to the PM by cytoskeletal filaments. Both endo and synj SV trafficking phenotypes are associated with severe primary defects in endocytosis, resulting in a substantial depletion (50%-70%) of SVs from the entire terminal, including AZ regions, and an accumulation of intermediate endocytic structures at or near the PM. The qualitative similarities between the slab, endo, and synj phenotypes reinforce the conclusion that presynaptic lipid environments are important for regulating SV pool size, trafficking, and availability for fusion. However, the slab ultrastructural phenotype differs from these endocytic mutants in two significant respects. First, the reduction in vesicles is far less pronounced and restricted to non-AZ domains, whereas the clustered and docked populations appear unaffected. Second, the slab phenotype is more specific to an accumulation of later SV trafficking states (Rohrbough, 2004).

How might SLAB Cdase and ceramide regulate SV trafficking? Ceramide resides both in plasma and vesicle membranes and is concentrated in raft domains, interacts with raft-associated proteins, and modulates general membrane endocytosis and trafficking (Brown, 2000; van Blitterswijk, 2003; Acharya, 2004) depending on its production and topological location in the membrane. Asymetrical generation of ceramide, as in rafts, promotes negative membrane curvature, vesicle budding, and vesicle aggregation (van Blitterswijk, 2003). Conversely, certain ceramide analogs inhibit membrane internalization and trafficking. Ceramide may therefore have similar roles in synaptic PMs and SVs and is likely trafficked between the inner PM leaflet and the SV surface by endocytosis. SV recycling is thought to occur in specialized, spatially defined regions in which lipid and protein constituents are preassembled before vesicle budding. This process is potentially modulated by changes in PM ceramide levels and raft distribution. The slab ultrastructural and FM1-43 loading phenotypes do not indicate a severe endocytosis defect at the NMJ. However, given the pronounced SV fusion impairment in the absence of SLAB, a reduced level or spatial specificity of endocytosis may be sufficient to maintain a pool of SVs trafficked to release sites. A recent analysis of RRP organization at the frog NMJ suggests that, contrary to general assumption, the RRP may be widely dispersed throughout the overall vesicle population and not necessarily recruited from regions nearest the AZ. If this is the case in Drosophila NMJ terminals, the increased SV sequestration observed in slab boutons could more directly underlie reduced RRP availability and weakened transmission, particularly during maintained activity. Raft domains have functions consistent with a role in SV tethering, including serving as sites for actin nucleation and polymerization and regulating actin cytoskeleton stability. In the absence of SLAB, altered raft environments may inhibit F-actin-mediated vesicle trafficking or interactions between SV and the cytoskeletal scaffold. For example, disruption of membrane sphingolipid composition may interfere with SV binding to the tethering protein synapsin, which mediates the activity-dependent sequestration and mobilization of SVs (Rohrbough, 2004).

Drosophila detergent-insoluble embryonic membranes are enriched in sphingolipids, sterols, and proteins, supporting the existence of functional raft domains in flies analogous to those in vertebrates. Drosophila sphingolipids consist predominantly of saturated long-chain ceramides, glycoceramides, and phosphoethanolamine ceramide (PECer), a sphingomyelin analog present in insects. Ceramide may therefore be produced in the membrane by PECer hydrolysis. Sterols constitute ~18% of Drosophila membranes and over 30% of raft lipid fractions, relative to phospholipids. Cholesterol, an essential vertebrate membrane component, contributes only a fraction of sterols but may, nevertheless, have an important role in membrane and raft structure and in the regulation of proteins such as signaling protein Hedgehog, known to undergo cholesterol modification. Notably, Hedgehog and numerous glycosylphosphatidylinositol (GPI)-linked proteins are localized to Drosophila rafts, indicating that GPI linkage is a conserved mechanism for targeting proteins to rafts by (Rohrbough, 2004).

SLAB Cdase contains a secretory signal sequence and is secreted in an N-glycosylated form when expressed in Drosophila S2 cells. SLAB overexpression in the Drosophila eye suppresses retinal degeneration by reducing ceramide levels and facilitating membrane recycling (Acharya, 2003; Acharya, 2004), consistent with the current findings. Protein localization and mutant loss-of-function studies were used to examine expression and functional requirement of the endogenous Cdase. SLAB is expressed in numerous tissues and cells and prominently enriched in central neurons. Mutant lethality is rescued by neuronally targeted gene expression, showing that SLAB neuronal expression is essential. C5-ceramide, an analog known to be trafficked and metabolized in cells, is rapidly accumulated at synaptic boutons, and its synaptic concentration is dependent on the slab expression level. These results suggest that endogenous sphingolipids are enriched and dynamically trafficked at Drosophila synapses and regulated by SLAB activity. SLAB may regulate synaptic sphinolipid environments by several plausible pathways. In neurons, SLAB is clearly cytoplasmically localized, consistent with either an intracellular or secreted function. SLAB may be present at low levels and function in the synaptic terminal; alternatively, it may be primarily secreted and act on the neuronal PM. If the essential protein function is secreted, however, these rescue results support the conclusion that neuronal rather than general secretion is required. Finally, SLAB may regulate sphingolipid production and content in membrane trafficked to and incorporated at the synapse (Rohrbough, 2004).

These results provide further evidence for the involvement of sphingolipid raft domains in specialized SV trafficking and exocytosis. A role for ceramide-rich membrane domains in SV priming or fusion processes represents the most direct potential involvement in neurotransmitter release. Likewise, sphinoglipid-dependent interactions between SV and tethering proteins potentially regulate recycling and trafficking steps. Future studies will investigate mechanistic links between SLAB, lipid raft domains, and the established SV fusion and trafficking machinery (Rohrbough, 2004).

Molecular cloning and characterization of a secretory neutral ceramidase of Drosophila melanogaster

This study reports the molecular cloning and characterization of the Drosophila neutral ceramidase (CDase). Using the BLAST program, a neutral CDase homologue was found in the Drosophila GenBank and cloned from a cDNA library of Drosophila imaginal discs. The open reading frame of 2,112 nucleotides encoded a polypeptide of 704 amino acids having five putative N-glycosylation sites and a putative signal sequence composed of 23 residues. When a His-tagged CDase was overexpressed in D. melanogaster Schneider's line 2 (S2) cells, the enzyme was continuously secreted into the medium through a vesicular transport system. Treatment of the secretory 86.3-kDa CDase with glycopeptidase F resulted in the generation of a 79.3-kDa protein, indicating that the enzyme is actually glycosylated with N-glycans. The enzyme hydrolyzed various N-acylsphingosines but not galactosylceramide, GM1a or sphingomyelin, and exhibited a peak of activity at pH 6.5-7.5, and thus was classified as a neutral CDase. RNAi for the enzyme remarkably decreased the CDase activity in a cell lysate as well as a culture supernatant of S2 cells mostly at neutral pH, indicating that both activities were derived from the same gene product (Yoshimura, 2002).


Search PubMed for articles about Drosophila Ceramidase

Acharya, J. K., Dasgupta, U., Rawat, S. S., Yuan, C., Sanxaridis, P. D., Yonamine, I., Karim, P., Nagashima, K., Brodsky, M. H., Tsunoda, S. and Acharya, U. (2008). Cell-nonautonomous function of ceramidase in photoreceptor homeostasis. Neuron 57(1): 69-79. PubMed ID: 18184565

Acharya, U., et al. (2003). Modulating sphingolipid biosynthetic pathway rescues photoreceptor degeneration. Science 299: 1740-1743. PubMed ID: 12637747

Acharya, U., et al. (2004). Ceramidase expression facilitates membrane turnover and endocytosis of rhodopsin in photoreceptors. Proc. Natl. Acad. Sci. 101: 1922-1926. PubMed ID: 14769922

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Biological Overview

date revised: 15 March 2008

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