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Modulating Sphingolipid Biosynthetic Pathway Rescues Photoreceptor Degeneration

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Science  14 Mar 2003:
Vol. 299, Issue 5613, pp. 1740-1743
DOI: 10.1126/science.1080549

Abstract

Mutations in proteins of the Drosophilaphototransduction 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. Here, modulating the sphingolipid biosynthetic pathway rescued retinal degeneration inDrosophila mutants. Targeted expression ofDrosophila neutral ceramidase rescued retinal degeneration in arrestin and phospholipase C mutants. Decreasing flux through the de novo sphingolipid biosynthetic pathway also suppressed degeneration in these mutants. Both genetic backgrounds modulated the endocytic machinery because they suppressed defects in a dynamin mutant. Suppression of degeneration in arrestin mutant flies expressing ceramidase correlated with a decrease in ceramide levels. Thus, enzymes of sphingolipid metabolism may be suitable targets in the therapeutic management of retinal degeneration.

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 (1, 2). Serine palmitoyl-CoA transferase (SPT) catalyzes the rate-limiting first step in the de novo biosynthesis of sphingolipids including ceramide (fig. S1). Ceramidases hydrolyze ceramide to sphingosine, and neutral or alka- line ceramidase is proposed to function in signaling (3). Mutant analyses in yeast have implicated enzymes of sphingolipid metabolism in endocytic membrane trafficking events (4). We modulated the sphingolipid biosynthetic pathway in vivo in Drosophila and examined its effects on mutants with endocytic defects in photoreceptors.

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 (5,6). Rhabdomere architecture is sensitive to perturbations in the phototransduction cascade and has been used to monitor photoreceptor degeneration (7).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 (8). Drosophila arrestin 2 also acts as a clathrin adaptor, mediating endocytosis of arrestin-rhodopsin complexes (9, 10). 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 (8, 11). Thus, arr23 photoreceptors provide a sensitive background for examining the in vivo effects of modulating the sphingolipid pathway in endocytosis.

We cloned the ceramidase gene into an upstream activating sequence (UAS) vector and expressed it in the Drosophila eye with the use of a glass multimer reporter (GMR)–Gal4 driver (12,13). Extracts from these fly heads showed increased neutral ceramidase activity, confirming that the protein was a bona fide neutral ceramidase (14) (Fig. 1A). Expression of GMR-Gal4; UAS-ceramidase in R1 to R6 did not affect photoreceptor integrity (Fig. 1B). Expression of ceramidase in arr23 rescued photoreceptor degeneration (Fig. 1D). In transmission electron micrographs (TEMs), rhabdomeres were intact, and multivacuolar bodies and degenerating photoreceptors, characteristic ofarr23 mutants (Fig. 1C), were completely absent in a 3-day arr23 fly expressing ceramidase (Fig. 1D). A near–wild-type level of rhodopsin was seen in rescued flies, reflecting photoreceptor integrity (Fig. 1E). Thus, expression of ceramidase in arr23 preserved rhabdomere structure and organization. Although defective, newly eclosedarr23 flies transduce light signals (8). 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, we carried out electroretinogram recordings (ERG) from 7-day-old flies exposed to light (Fig. 1, F to H). Because of extensive degeneration, ERGs ofarr23 flies had a very small amplitude, whereasarr23 flies expressing ceramidase showed a robust response (compare amplitudes of Fig. 1, G and H). However, the slow inactivation kinetics characteristic ofarr23 still persisted in the rescued flies. Rescued arrestin mutant flies transduced signals even on aging because the structural integrity of these photoreceptors was preserved.

Figure 1

Expression of ceramidase rescues degeneration in arr23 mutants. (A) Transgenic flies expressing ceramidase exhibit high neutral ceramidase (CDase) activity. Assays were initiated by the addition of membrane extracts from heads of control (GMR-Gal4) and GMR-Gal4; UAS-ceramidase flies to a buffer mixture containing 14C-labeled ceramide as described in (14), and specific activity was calculated (n = 5, where n is the number of experiments). (B) Expression of ceramidase does not affect photoreceptor integrity. Bars in (B) to (D) indicate 1 μm. (C) Rhabdomeres lacking arr23 degenerate. (D) Ceramidase expression inarr23 mutants rescued degeneration and results in ommatidia that are similar to those of the wild type. (Insets) Low-magnification TEMs of a section of a compound eye of the corresponding flies. (E) Ceramidase expression restored rhodopsin level in 3-day-old arr23 mutant flies. Immunoblot depicts rhodopsin 1 (Rh1) levels in wild type (lane 1), ceramidase expressor (lane 2), arr23 (lane 3), and arr23 expressing ceramidase (lane 4).arr23 mutants have greatly reduced rhodopsin levels, whereas CDase; arr23 show higher levels of Rh1. The blot was also probed with inositol polyphosphate 1-phosphatase (IPP) antibody as a loading control. (F) ERG of wild-type (w1118 ) flies. ERGs of 7-day-old control flies in bright light show normal inactivation kinetics and amplitude. (G) ERG of arr23 flies. Seven-day-old arr23 flies show slow deactivation and very small amplitudes of response. (H) ERG ofarr23 flies expressing ceramidase. Seven-day-oldarr23 flies expressing ceramidase show slow deactivation like the mutant flies but a large amplitude response due to suppression of degeneration. (I) Expression of ceramidase decreases ceramide levels in control and arrestin mutant flies. Individual ceramide species were estimated and total ceramide content was calculated in membrane extracts of heads from control flies (w1118), control flies expressing ceramidase (CDase), arrestin mutants (arr23 ), andarr23 -expressing ceramidase flies (CDase +arr23 ) as described in (14) (n = 3; ±SEM). Arrestin mutant flies show an increase in ceramide levels. Expression of ceramidase decreases the ceramide content by half in both CDase and CDase +arr23 flies. Cm:n (for example, C18:0) represents acyl amide with m (18) carbons and n (0) double bonds in ceramide species, and OH Cm:n represents 2-hydroxy Cm:n ceramide.

Because expression of ceramidase in arrestin mutant flies suppressed retinal degeneration and because ceramidase hydrolyzed ceramide, we reasoned that the rescue would be accompanied by a decrease in ceramide levels in these photoreceptors. We used electrospray ionization tandem mass spectrometry (ESI/MS/MS) to estimate ceramide levels in lipid extracts of membranes prepared from fly heads of control, ceramidase expressor, arrestin mutant, and arrestin mutant expressing ceramidase. We identified ceramide molecular species containing tetradecasphingenine and their 2-hydroxy counterparts by negative ion ESI/MS/MS with neutral loss of 200.2 and 271.2 mass units, respectively, as described in (15). As expected, expression of ceramidase reduces the ceramide levels in control animals (Fig. 1I). Lipid extracts from arrestin mutants showed an increase in ceramide levels, probably reflecting changes accompanying the severely degenerating photoreceptors (Fig. 1I). Expression of ceramidase in arrestin mutants decreased ceramide levels by 50% in all species measured (Fig. 1I). Thus, rescue of degeneration correlated with a decrease in ceramide levels in the mutant flies. Because sphingosine is a product of the ceramidase reaction, we evaluated whether increased sphingosine could suppress retinal degeneration in arrestin mutants. Viability of certain lace alleles (LCB2 subunit of SPT), which are deficient in de novo sphingosine biosynthesis, is increased when flies are raised in food supplemented with sphingosine (16). We raised arrestin mutant flies under similar conditions and examined their photoreceptors by electron microscopy. The rhabdomeres of R1 to R6 cells showed no suppression; instead, they showed enhanced degeneration of these photoreceptors (fig. S2). Thus, we believe 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.

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, we expressed ceramidase in a dynamin mutant background in the eye. Dynamin is a guanosine triphosphatase essential for clathrin-mediated endocytosis (17, 18). InDrosophila, a temperature-sensitive mutant of dynamin,shibire (shits1 ), has a general defect in endocytosis (19). These results were recapitulated in mammalian cells when a similar mutant dynamin was overexpressed (18). We used a temperature-sensitive dominant-negative mutant, UAS-shits1 , under the control of a GMR-Gal4 driver, to preferentially express the mutant protein in the eye (20). 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 (Fig. 2A). Ceramidase expression suppressed degeneration in UAS-shits1 photoreceptors. Rhabdomeres were largely intact, vacuolated cells were fewer, and trapezoidal arrangement of rhabdomeres was retained (Fig. 2B). As in other degenerating mutants, rhodopsin levels were low inshits1 mutants compared with those of the wild type but were restored upon ceramidase expression (Fig. 2C).

Figure 2

Ceramidase expression suppresses degeneration in UAS-shits1 mutant. (A) Expression of UAS-shits in R1 to R6 photoreceptors causes extensive degeneration in these cells due to defects in endocytosis. Bars in (A) and (B) indicate 1 μm. (B) Expression of ceramidase in photoreceptors expressing mutant dynamin partially suppresses degeneration. Rhabdomeres are intact, and the trapezoidal arrangement of ommatidial units is preserved. (Insets) Low-magnification TEMs of a section of a compound eye of the corresponding flies. (C) Rhodopsin levels are restored in dynamin mutants expressing ceramidase. Lane 1, Rh1 levels inw1118 control heads; lane 2, Rh1 levels in heads expressing mutant dynamin; lane 3, Rh1 levels in dynamin mutants expressing ceramidase. Experiments were done at 23°C, a temperature at which shits1 has been demonstrated to show endocytic defects in Drosophila photoreceptors (10). Suppression is also observed at higher temperatures; however, ultrastructural preservation is better appreciated at 23°C.

We then investigated whether SPT, the rate-limiting enzyme of the de novo sphingolipid biosynthetic pathway, could affect the degeneration observed in these mutants. InDrosophila, the Lace gene encodes the LCB2 subunit of SPT. The P-lacW-inserted lace allelel(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 (16). We crossedarr23 and UAS-shits1 mutants into the lace heterozygous background and examined photoreceptors by transmission electron microscopy. laceheterozygotes had intact photoreceptors (Fig. 3A). lace partially suppressed retinal degeneration in arr2 3 mutants (compareFig. 3, B and C) and in UAS-shits1 mutants (compare Fig. 3, D and E).

Figure 3

lace heterozygote suppresses degeneration in arr23 and dynamin mutants, and ceramidase and lace suppress norp A degeneration. Bars in (A) to (H) indicate 1 μm. (A) TEM of laceheterozygotes shows normal photoreceptor organization. (B) TEM of degenerating arr23 photoreceptors. (C) lace heterozygote partially suppresses arr23 degeneration. These flies show mostly intact rhabdomeres; however, some cellular degeneration characterized by vacuoles, dark multivesicular bodies, is still observed. (D) TEM of degenerating UAS-shits mutant photoreceptors. (E) lace heterozygotes show substantial suppression of dynamin mutant photoreceptor degeneration. (F) TEM of compound eye of norp Ap41 flies 3 days after eclosion. These photoreceptors show features of degeneration with disruption of rhabdomeres. (G) TEM of norp Ap41 flies expressing ceramidase have intact rhabdomeres. (H) TEM of norp Ap41 flies in a lace heterozygous background also show well-preserved rhabdomeres and subrhabdomeric cisternae.

Finally, we examined whether ceramidase and lace suppressed degeneration in a phospholipase C mutant, where endocytosis has been implicated in the degenerative process (11, 21).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 (22). Although norp A mutants degenerated slowly, these changes were obvious even in 3-day-old flies (Fig. 3F). Expression of ceramidase in a norp A mutant suppressed retinal degeneration (Fig. 3G). Lace heterozygotes also suppressednorp A degeneration (Fig. 3H). arr23 mutants undergo necrotic cell death, whereas norp A mutants accumulate rhodopsin-arrestin complexes and undergo apoptotic cell death (10, 11). Thus, regardless of the mode of cell death ceramidase expression and lace mutant rescued degeneration. Because they also suppressed degeneration in a dynamin mutant, we infer 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 (4). 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.

A large volume of work suggests that receptor desensitization, endocytosis, and recycling play a crucial role in GPCR signaling in higher organisms (23). In light of our 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 (24). Rescue of degeneration inDrosophila visual mutants provides a strong basis for exploring strategies that manipulate sphingolipid enzymes for therapeutic management of retinal degeneration in higher organisms.

Supporting Online Material

www.sciencemag.org/cgi/content/full/299/5613/1740/DC1

Materials and Methods

Figs. S1 and S2

  • * To whom correspondence should be addressed. E-mail: acharyaj{at}mail.ncifcrf.gov (J.K.A.), acharyau{at}mail.ncifcrf.gov (U.A.)

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