InteractiveFly: GeneBrief

Sterol regulatory element binding protein: Biological Overview | References

BIOLOGICAL OVERVIEW

Animal cells exert exquisite control over the physical and chemical properties of their membranes, but the mechanisms are obscure. Phosphatidylethanolamine, the major phospholipid in Drosophila, controls the release of sterol regulatory element-binding protein (SREBP) from Drosophila cell membranes, exerting feedback control on the synthesis of fatty acids and phospholipids. The finding that SREBP processing is controlled by different lipids in mammals and flies (sterols and phosphatidylethanolamine, respectively) suggests that an essential function of SREBP is to monitor cell membrane composition and to adjust lipid synthesis accordingly (Dobrosotskaya, 2002).

The lipid composition of membranes in animal cells is maintained within strict limits, primarily by feedback regulation of lipid biosynthesis. The mechanism for this homeostasis is beginning to be understood. Recent insights have emerged from the study of membrane-bound transcription factors called SREBPs that activate genes encoding enzymes of lipid biosynthesis in insect cells (Seegmiller, 2002) as well as in mammalian cells (Brown, 1997; Goldstein, 2002). The activities of SREBPs are inhibited in a feedback fashion by membrane lipids, but these regulatory lipids differ in mammalian and Drosophila cells. In mammalian cells, SREBP activity is inhibited by sterols and polyunsaturated fatty acids (Brown, 1997, Wang, 1994; Osborne, 2000). In Drosophila cells, SREBP activity is blocked when palmitate but not sterols or other fatty acids is added to the culture medium (Seegmiller, 2002). Although the regulatory agents differ, the mechanism is conserved. In Drosophila and mammalian cells, control is attained through regulated proteolytic release of the active fragments of SREBPs from cell membranes (Dobrosotskaya, 2002).

The SREBPs are synthesized as intrinsic proteins of the endoplasmic reticulum (ER) membrane. To activate transcription, SREBPs must be transported to the Golgi complex, where they are cleaved by two proteases that liberate the basic helix-loop-helix-leucine-zipper domains so they can enter the nucleus (Goldstein, 2002). This transport requires an escort protein, SCAP (SREBP cleavage-activating protein) (Nohturfft, 2000). In mammalian cells, SCAP serves as a sterol sensor; it loses the ability to move to the Golgi complex when sterol concentrations are high. Drosophila cells express genes that encode a single SREBP (dSREBP) and orthologs of mammalian SCAP and the two SREBP proteases (Seegmiller, 2002). Experiments with RNA interference (RNAi) indicate that SCAP is required for dSREBP processing in Drosophila cells, as it is in animal cells (Dobrosotskaya, 2002).

A major difference between mammalian and Drosophila cells relates to the genes activated by SREBPs. In mammalian cells, SREBPs activate genes that encode enzymes of cholesterol and unsaturated fatty acid biosynthesis (Brown, 1997; Osborne, 2000; Edwards, 2000). Drosophila cells, like those of other insects, do not produce sterols. The major SREBP targets in Drosophila S2 cells are enzymes required for saturated fatty acid biosynthesis (Dobrosotskaya, 2002).

An important question is whether palmitate regulates SREBP processing in Drosophila cells or whether it must be incorporated into another product, such as a phospholipid, in order to act. The inhibitory effect of palmitate [16 carbons, 0 double bonds (16:0)] on dSREBP processing in Drosophila cells is highly specific (Seegmiller, 2002). Other saturated fatty acids such as stearate (18:0) are much less effective, as is the monounsaturated fatty acid oleate (18:1). Polyunsaturated fatty acids had no activity. This finding suggested that palmitate might act by incorporation into another lipid through the action of a highly specific enzyme. Enzyme inhibitors and RNAi were used in this study to block incorporation of palmitate into various end products in Drosophila S2 cells. The results indicate that palmitate must be converted to phosphatidylethanolamine (PE) to inhibit SREBP cleavage and that this conversion occurs through the sphingolipid pathway (Dobrosotskaya, 2002).

PE synthesis in eukaryotic cells has been well characterized. Activated palmitate (palmitoyl-CoA) can be converted to PE by condensing with serine through the action of serine palmitoyltransferase (SPT), which forms an intermediate that is converted to sphinganine. Addition of another fatty acid and introduction of a double bond converts sphinganine to ceramide, which is converted to sphingosine through loss of the additional fatty acid. Phosphorylation by either of two sphingosine kinases (SK1 or SK2) produces sphingosine-1-phosphate, which is broken down by sphingosine-1-phosphate lyase (SPL) to produce the key intermediate phosphoethanolamine plus trans-2-hexadecenal (Dobrosotskaya, 2002).

The net result of this pathway is to convert palmitate plus serine plus phosphate [from adenoside triphosphate (ATP)] into trans-2-hexadecenal plus phosphoethanolamine. The trans-2-hexadecenal can be converted back to palmitate via hexadecanal by reducing the double bond and oxidizing the carbonyl with fatty aldehyde dehydrogenase (FALDH). Phosphoethanolamine is attached to cytidine 5'-diphosphate (CDP) by phosphoethanolamine cytidylyltransferase (PECT). CDP-ethanolamine donates phosphoethanolamine to diacylglycerol to produce PE. When ethanolamine is available, it can be converted directly to phosphoethanolamine by ethanolamine kinase (EK), bypassing the sphingolipid intermediates. PE can also be created from phosphatidylserine through decarboxylation (via phosphatidylserine decarboxylase) or by base exchange when free ethanolamine is available (via phosphatidylserine synthase) (Dobrosotskaya, 2002).

To study conversion of palmitate into phospholipids, Drosophila S2 cells were incubated in a chemically defined medium (IPL-41) supplemented with delipidated, dialyzed fetal calf serum (FCS). This medium is devoid of fatty acids and contains all 20 amino acids, including 1.9 mM serine. The medium contains 140 µM choline but no ethanolamine. To this defined medium, palmitate, ceramide, or sphingosine were added for 4 hours before harvest. Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted with an antibody to dSREBP. In the absence of any additions, the membrane-bound precursor and the cleaved nuclear forms of dSREBP were detected, that migrated with apparent molecular masses of ~125 and 72 kD, respectively. Addition of palmitate, ceramide, or sphingosine selectively reduced the nuclear form, which indicates inhibition of proteolytic processing. To show the requirement for converting palmitate to a sphingolipid, ISP-1, a specific inhibitor of SPT, which catalyzes the first step in this conversion, was used. In the absence of ISP-1, palmitate and ceramide both inhibited dSREBP processing. In the presence of ISP-1, palmitate no longer inhibited dSREBP processing, but ceramide remained effective. To confirm the specificity of ISP-1, the block was reproduced by treating the cells with double-stranded RNAs (dsRNAs) directed at the mRNAs for the two subunits of SPT. Such treatment abolishes the corresponding endogenous mRNAs selectively through RNAi. As a control, the cells were treated with dsRNA directed against an irrelevant messenger RNA (mRNA) (rat CYP7A1). RNAi against either of the two subunits of SPT blocked the ability of palmitate to inhibit dSREBP cleavage, whereas the control CYP7A1 dsRNA had no effect (Dobrosotskaya, 2002).

To determine whether inhibition of dSREBP cleavage by palmitate or sphingosine requires converting sphingolipids to PE, some of the necessary enzymes were eliminated by RNAi. Elimination of SPL blocked inhibition by palmitate, ceramide, and sphingosine, indicating that the inhibitory effect of all three of these compounds requires conversion to either phosphoethanolamine or trans-2-hexadecenal. Elimination of PECT also blocked the actions of palmitate, ceramide, and sphingosine, indicating that the required metabolite is phosphoethanolamine and that the phosphoethanolamine must be converted to CDP-ethanolamine, the final precursor of PE. To test the effect of the other product of the SPL reaction, trans-2-hexadecenal, hexadecanal was added to cells. Hexadecanal is produced in cells from trans-2-hexadecenal by reducing the double bond. Although hexadecanal inhibited dSREBP processing, RNAi directed against FALDH abolished its effect, indicating that hexadecanal acts by being converted to palmitate, which initiates the whole sequence of reactions. FALDH RNAi did not block inhibition by palmitate, and it had only a partial effect on the action of ceramide (Dobrosotskaya, 2002).

The preceding data indicate that palmitate inhibits dSREBP processing at least in part by supplying phosphoethanolamine through the sphingolipid pathway. Palmitate may also supply the fatty acid component necessary for PE biosynthesis. To sort out these separate effects, cells were incubated with palmitate alone, ethanolamine alone, or the two together. As before, palmitate had a major effect in reducing nuclear dSREBP. Ethanolamine alone had no effect. Palmitate plus ethanolamine was similar to palmitate alone. As observed here, the palmitate effect was abolished when the sphingolipid pathway was blocked by RNAi directed against SPT-I or SPL. It was also abolished by RNAi against SK1 plus SK2. Addition of ethanolamine restored complete inhibition by palmitate in the presence of these RNAi treatments. These data strongly suggest that the sphingolipid pathway is required only to produce phosphoethanolamine and that the alternative supply of phosphoethanolamine through the direct pathway obviates the need for sphingolipid intermediates in the palmitate-mediated inhibition of dSREBP processing (Dobrosotskaya, 2002).

Inasmuch as ethanolamine is never sufficient to inhibit processing in the absence of palmitate, the data indicate that palmitate is required for one or more other reactions in addition to production of phosphoethanolamine. One of these reactions may be the glycerol 3-phosphate acyltransferase (GPAT)-catalyzed addition of fatty acids to glycerol 3-phosphate to form diacylglycerol, a precursor of all phospholipids, including PE. If this is true, then other fatty acids should inhibit dSREBP processing in the presence of ethanolamine, because the fatty acid substrate specificity of GPAT is broader than that of SPT. This idea was tested by treating S2 cells with various fatty acids in the absence and presence of exogenous ethanolamine. In the absence of ethanolamine, only palmitate completely blocked processing of dSREBP. Palmitoleate (16:1) had no effect, whereas oleate (18:1) caused partial inhibition. In the presence of ethanolamine, palmitoleate inhibited partially, and oleate inhibited strongly. These data suggest that the palmitate specificity stems from its requirement for phosphoethanolamine synthesis through the sphingolipid pathway. Once this requirement is satisfied, oleate can replace palmitate for the nonspecific function (Dobrosotskaya, 2002).

To demonstrate the role of dSREBP in phospholipid synthesis, S2 cells were treated with RNAi against dSREBP or the control CYP7A1 and then incubated the cells with [32P]orthophosphate. RNAi against dSREBP reduced 32P incorporation into PE by 60% compared with the control RNAi. Addition of ceramide enhanced PE synthesis in the control cells and restored PE synthesis to baseline levels in the dSREBP-deficient cells. RNAi against dSREBP also reduced PC synthesis by 45%, perhaps because of the reduction in the mRNA encoding phosphocholine cytidylyltransferase. In contrast to PE synthesis, PC synthesis was not restored by ceramide (Dobrosotskaya, 2002).

Taken together, the data indicate that dSREBP controls membrane lipid production in Drosophila cells by regulating the synthesis of fatty acids and their incorporation into PE and PC. The activity of dSREBP is inhibited in a feedback fashion by the end product PE. The phosphoethanolamine component of PE can be derived from palmitate through the sphingolipid pathway, or it can be derived from external ethanolamine through EK. In contrast, in mammalian cells SREBP processing is controlled by cholesterol, and this regulates cholesterol synthesis (Dobrosotskaya, 2002).

Given the similarities in the pathway for SCAP-dependent proteolytic processing of SREBPs in Drosophila and mammalian cells, it is surprising that this pathway would be controlled by different end products. How can two different lipids such as cholesterol and PE regulate the same biological process? One clue emerges from a comparison of their physical properties. PE is a hexagonal (H_II)-phase lipid. Unlike PC and PS, which form flat bilayers spontaneously on hydration, PE forms extended monolayer tubes in which the polar headgroups face inward. The exposed hydrophobic tails interact with those of other tubes to form stacked arrays that appear hexagonal in cross section. These structures have not been observed in vivo because the bilayer is stabilized by the presence of bilayer-forming lipids. However, some have speculated that the presence of H_II-phase lipids, such as PE in cell membranes, alters membrane structure and properties. This may be true in Drosophila, where PE is the predominant phospholipid in membranes (~55% of total for PE versus ~20% for PC). In mammalian cells, on the other hand, the major membrane phospholipid is PC (~50% of total for PC versus ~20% for PE). Although cholesterol does not form H_II structures in isolation, it is remarkable for its ability to induce H_II-phase formation in bilayer systems, especially those containing PE. These observations suggest that PE and cholesterol may perturb membranes through their tendency to form hexagonal structures. Inhibition of SREBP processing may result from such perturbations in the local environment surrounding SCAP (Dobrosotskaya, 2002).

The SREBP Pathway in Drosophila: Regulation by Palmitate, Not Sterols

In mammals, synthesis of cholesterol and unsaturated fatty acids is controlled by SREBPs, a family of membrane-bound transcription factors. This study shows that the Drosophila genome encodes all components of the SREBP pathway, including a single SREBP (dSREBP), SREBP cleavage-activating protein (dSCAP), and the site 1 and site 2 proteases (S1P and S2P), which release the bHLH-zip domain from the membrane (Brown, 1999). In cultured Drosophila S2 cells, dSREBP is processed at sites 1 and 2, and the liberated fragment increases mRNAs encoding enzymes of fatty acid biosynthesis, but not sterol or isoprenoid biosynthesis. Processing requires dSCAP, but is not inhibited by sterols as in mammals. Instead, dSREBP processing is blocked by palmitic acid. These findings suggest that the ancestral SREBP pathway functions to maintain membrane integrity rather than to control cholesterol homeostasis (Seegmiller, 2002).

The current data establish the following points regarding the SREBP pathway in Drosophila: (1) the Drosophila genome contains one SREBP gene whose product resembles mammalian SREBP-1a and SREBP-2; (2) the Drosophila genome encodes orthologs of mammalian SCAP, S1P, and S2P; (3) dSREBP is processed at site 1 and site 2 in a process that requires dSCAP, thereby generating an NH₂-terminal nuclear fragment; (4) nuclear dSREBP enhances transcription of genes encoding enzymes of fatty acid biosynthesis, but not cholesterol or isoprenoid biosynthesis; and (5) processing of dSREBP at site 1 is decreased in the presence of palmitate, but not other fatty acids or sterols. It is not yet clear whether palmitate itself inhibits processing of dSREBP or whether palmitate must be incorporated into a more complex lipid, such as a sphingolipid or a glyceryl phospholipid, to exert its regulatory effect (Seegmiller, 2002).

The studies in Drosophila cells provide interesting similarities and contrasts with the SREBP pathway in mammalian cells. In mammalian cells, SCAP transports SREBPs from the ER to the Golgi apparatus where S1P and S2P reside. This transport is inhibited by sterols, which thereby block SREBP processing (DeBose-Boyd, 1999; Nohturfft, 2000). Sterol inhibition is mediated through the membrane domain of SCAP, which contains a polytopic membrane-spanning segment of ~150 amino acids, termed the sterol-sensing domain. Drosophila SCAP contains a sequence that shares 47% identity with this sterol-sensing domain. In mammalian SCAP, sensitivity to sterols is disrupted by one of two mutations; that is, when Tyr-298 is mutated to Cys or when Asp-443 is mutated to Asn (Nohturfft, 1998). In Drosophila SCAP, the amino acid corresponding to Tyr-298 is Tyr-382, as determined by sequence alignment. In contrast, the Drosophila equivalent of mammalian Asp-443, rather than being an Asp, is Asn-525, which corresponds to one of the two mutant versions of mammalian SCAP that renders it sterol resistant. Whether this single amino acid difference is the basis for the sterol resistance of dSCAP remains to be determined (Seegmiller, 2002).

Although the processing of dSREBP is not inhibited by sterols, it is effectively blocked by palmitate, which does not block processing in mammalian cells. If palmitate acts through the sterol-sensing domain in dSCAP, this finding would suggest that this domain can respond to other lipids beside sterols. Perhaps this domain senses some physical property of the ER membrane, such as membrane thickness or fluidity, which in mammalian cells is altered by sterols and in Drosophila cells by palmitate itself or, more likely, a lipid derived from palmitate (Seegmiller, 2002).

Although palmitate and other saturated fatty acids do not suppress SREBP cleavage in mammalian cells, evidence has been provided that polyunsaturated fatty acids, such as arachidonate, are capable of achieving such suppression. Under certain conditions, these lipids act synergistically with sterols. No additive effect was found when palmitate or other fatty acids were added to Drosophila cells in combination with sterols. Whether arachidonate in mammalian cells acts by the same mechanism as palmitate in Drosophila cells remains to be determined (Seegmiller, 2002).

In an evolutionary sense, the current data suggest that the original function of the SREBP pathway may have been to maintain the integrity of the cell membrane by adjusting fatty acid synthesis in response to an excess or deficiency of palmitic acid or a lipid derived from palmitic acid. During evolution, the single ancestral SREBP gene became duplicated in the lineage leading to vertebrates, and the resulting mammalian SREBP proteins acquired additional roles in regulating cholesterol homeostasis and in mediating increased lipid synthesis in liver in response to insulin (Matsuda, 2001). Further evolutionary insights will require the identification of the palmitate-derived lipid that regulates the processing of dSREBP (Seegmiller, 2002).

Fatty acid auxotrophy in Drosophila larvae lacking SREBP

SREBPs are membrane bound transcription factors that are crucial for normal lipid synthesis in animal cells. This study shows that Drosophila lacking dSREBP die before the third larval instar. Mutant larvae exhibit pronounced growth defects prior to lethality, along with substantial deficits in the transcription of genes required for fatty acid synthesis. Compared to wild-type larvae, mutants contain markedly less fatty acid, although its composition is unaltered. Dietary supplementation with fatty acids rescues mutants to adulthood. The most effective fatty acid, oleate, rescues 80% of homozygotes. Rescue by dSREBP requires expression only in fat body and gut. Larvae expressing dSREBP prior to pupariation complete development and are viable as adults even when dSREBP expression is subsequently extinguished. The role, if any, of dSREBP in adults is not yet apparent. These data indicate that dSREBP deficiency renders Drosophila larvae auxotrophic for fatty acids (Kunte, 2006).

This study reports that loss of dSREBP activity is lethal during larval development in Drosophila. Almost all homozygous mutants die by the end of the second instar. This lethality occurs solely as a result of the loss of dSREBP function. dSREBP¹⁸⁹ homozygotes are rescued by expressing a cDNA, a genomic fragment, or by dietary supplementation. Such treatments also rescue dSREBP⁵²/dSREBP¹⁸⁹ transheterozygotes; loss of exon 1 of Gyc76C does not contribute to the observed lethality. Dispensability of 'exon 1' accords with the observation that a a viable line, l(3)L0909-a, harbors a P element insertion in exon 1 of Gyc76C. When the P{Switch} system was used to express active dSREBP only during larval life, this rescued lethality. Once mutants reached adulthood, dSREBP is not strictly required for viability (Kunte, 2006).

Multiple lines of evidence demonstrate that dSREBP's essential role is the transcription of genes needed for fatty acid synthesis and uptake: (1) In larvae, dSREBP activity is readily observed in tissues involved in lipid uptake (midgut) and synthesis (fat body and oenocytes). (2) Homozygous mutant larvae are rescued by expressing dSREBP in fat body and gut. (3) Mutant larvae show reduced levels of transcripts for fatty acid synthetic genes (e.g. ACC, ACS, and FAS). This deficit is not reversed by lipid supplementation, indicating that it results from lack of nuclear dSREBP and not as a secondary consequence of end product deficiency. (4) Mutant larvae contain markedly less total fatty acid than heterozygotes or wild-type larvae. (5) In a classic end product-mediated feedback mechanism, dSREBP cleavage in growing larvae is strongly suppressed by dietary lipids. (6) Feeding extra fatty acids rescues lethality in dSREBP mutants. Homozygous mutants rescued by supplementation are indistinguishable from their heterozygous siblings in mass and morphology (Kunte, 2006).

Wild-type flies can develop on defined medium lacking all lipids save cholesterol and can therefore synthesize all fatty acids required for growth. dSREBP mutant larvae, however, are unable to grow even on regular cornmeal-molasses-agar unless supplemented with fatty acids. Therefore, flies lacking dSREBP are fatty acid auxotrophs and an important role of dSREBP in Drosophila physiology is the maintenance of fatty acid prototrophy (Kunte, 2006).

Furthermore, the data indicate that regulation of dSREBP activity enables the organism to adjust the level of de novo lipid synthesis in response to the supply of lipids in the diet. Thus, the growing larva can allocate resources efficiently between the syntheses of various macromolecules in response to its environment. SREBPs have been shown to similarly regulate cholesterol synthesis in the liver in mice and hamsters (Brown, 1997). The benefits of balancing endogenous synthesis with dietary input and lipid demand likely provide the selective pressure for conservation of the SREBP pathway in evolution (Kunte, 2006).

This study has identified multiple genes involved in de novo fatty acid synthesis as dSREBP targets. No changes were detected in transcript abundance for genes involved in the elongation or desaturation of fatty acids. This differs from mouse liver, where manipulation of the SREBP pathway causes transcriptional changes leading to altered fatty acid composition (Shimomura, 1998). In dSREBP mutants, a global deficit in the fatty acid content was detected but no change was found in the relative abundance of the various species. In addition, dietary supplementation with any of the major fatty acids of flies served to compensate for lack of dSREBP, albeit with varying efficiency. These data indicate that the mechanisms necessary for interconversion among various species of fatty acids continue to function in the absence of dSREBP-mediated transcription (Kunte, 2006).

Activation of the SREBP pathway in mammals results in the preferential production of oleate (C18:1). This may reflect the need for a substrate for the esterification and storage of the other major product of the SREBP pathway, cholesterol (Repa, 2000; Shimomura, 1998). In Drosophila, the SREBP pathway is not involved in cholesterol synthesis (Seegmiller, 2002) and this distinction may underlie the observed differences in fatty acid production between mammals and Drosophila (Kunte, 2006).

The reduced fatty acid content of dSREBP mutant larvae is unlikely to result from a selective deficit in a particular class of lipids. An inference may be drawn by comparing the abundance of myristate (C14:0) and oleate (C18:1). Myristate is relatively enriched in di- and tri-glycerides, while oleate is enriched in phospholipids. The lack of change in the relative abundance of C14:0 and C18:1 suggests a coordinate decrease in the production of both classes of lipid (Kunte, 2006).

Lethality of dSREBP mutant larvae occurs following growth arrest at the end of the second larval instar, a time when wild-type larvae begin to increase enormously in mass. Rapid growth undoubtedly places a great demand on lipid metabolism. Growing larvae must make additional cell membrane to accommodate increasing cell size. Drosophila, like other insects, must achieve a critical mass in order to complete development, termed the threshold size for metamorphosis (~0.45 mg during second instar). Inability to carry out this task may determine the timing of growth arrest and lethality. How flies monitor mass is unknown, but recent work demonstrates a key role for the prothoracic gland (Kunte, 2006 and references therein).

Larvae reared on non-nutritive agar fail to grow and die within 2-3 days after hatching. Loss of dSREBP mimics features of starvation. For example, homozygotes fail to grow to normal size under standard culture conditions, remaining about the size of first instar larvae. When wild-type larvae are starved for nutrients that must be acquired exogenously, such as certain amino acids, choline and cholesterol, pyrimidines, or vitamins, growth is arrested but the larvae can survive for an extended period. Transfer to complete medium within 6-8 days permits these starved animals to finish development. While flies have mechanisms such as arrested growth and delayed development for coping with deficits in exogenous nutrients, these mechanisms apparently do not respond to a deficit in nutrients that are typically supplied endogenously in wild-type animals (Kunte, 2006).

Death of dSREBP homozygotes prior to second to third instar transition may in part reflect a failure to achieve a critical mass of neutral lipid stores in fat body. Regulation of larval growth by fat body has been demonstrated previously (Kunte, 2006).

A third possible reason for the observed lethality in dSREBP mutants is that dSREBP may be directly required for the synthesis of a specific signaling molecule that controls growth in an endocrine fashion. The present data do not permit distinguishing conclusively between these mechanisms. An endocrine mechanism is considered the least likely, however, owing to the variety of different fatty acids that can rescue dSREBP mutants (Kunte, 2006).

The lack of specificity in the fatty acid requirement contrasts with previous observations in Schneider S2 cells. There, a specific requirement was observed for palmitate (C16:0) in the regulation of dSREBP cleavage. This specificity reflected the need for palmitate as a precursor for the head group of phosphatidylethanolamine (Dobrosotskaya, 2002). In the present study, additional lipids were added to regular cornmeal-molasses-agar medium that already contained lipids from yeast and corn. This may explain the relaxed specificity of the fatty acids required. Indeed, in cultured S2 cells, addition of exogenous ethanolamine to the medium relaxed the specific requirement for palmitate (Kunte, 2006).

In addition to tissues involved in de novo lipid synthesis (fat body and oenocytes), dSREBP activity is also required in a tissue that is predominantly associated with nutrient digestion and absorption (midgut). Free fatty acids rescue dSREBP mutants at a much lower concentration than needed when fatty acids are supplied as phospholipids or triglycerides. dSREBP activity may be needed for the animal to generate absorbable free fatty acids from phospholipids and/or triglycerides. Interestingly, a major, previously unrecognized, transcriptional target of dSREBP, CG6295, is highly similar in predicted amino acid sequence to mammalian pancreatic lipases and contains a conserved catalytic triad (Kunte, 2006).

Alternative processing of sterol regulatory element binding protein during larval development in Drosophila

Sterol regulatory element binding protein (SREBP) is a major transcriptional regulator of lipid metabolism. Nuclear Drosophila SREBP (dSREBP) is essential for larval development in Drosophila melanogaster but dispensable in adults. dSREBP- larvae die at second instar owing to loss of dSREBP-mediated transcription but survive to adulthood when fed fatty acids. Activation of SREBP requires two separate cleavages. Site-1 protease (S1P) cleaves in the luminal loop of the membrane-bound SREBP precursor, cutting it in two. The NH2- and COOH-terminal domains remain membrane bound owing to their single membrane-spanning helices. The NH2-terminal cleavage product is the substrate for site-2 protease (S2P), which cleaves within its membrane-spanning helix to release the transcription factor. In mice, loss of S1P is lethal but the consequences of loss of S2P in animals remain undefined. All known functions of SREBP require its cleavage by S2P. Drosophila mutants that eliminate all dS2P function (dS2P^-) were isolated. Unexpectedly, larvae lacking dS2P are viable. They are deficient in transcription of some dSREBP target genes but less so than larvae lacking dSREBP. Despite loss of dS2P, dSREBP is processed in mutant larvae. Therefore, larvae have an alternative cleavage mechanism for producing transcriptionally active dSREBP, and this permits survival of dS2P mutants (Matthews, 2009).

Mutant Drosophila harboring a deficiency that removes the entire dS2P transcription unit were isolated. No dS2P mRNA is detectable in these animals and no dSREBP processing is observed in mutant adults under conditions where it is readily observed in wild-type flies. Instead, the substrate for dS2P cleavage, the intermediate form of dSREBP, accumulates in membranes. Therefore, the dS2P¹ deletion is a null allele of dS2P (Matthews, 2009).

Phenotypes of the P-element insertion allele, dS2P², are indistinguishable from dS2P¹ and are no more severe in trans to the deletion allele. Transcripts from dS2P² cannot yield catalytically active dS2P. Thus, dS2P² is a null allele by genetic and molecular criteria. Surprisingly, animals harboring either allele are viable and can be readily maintained as homozygous stocks. Reciprocally, dSREBP¹⁸⁹ flies can be rescued by expressing a dSREBP cDNA harboring an N₄₆₂P > FL mutation that renders dSREBP refractory to cleavage by dS2P. Thus, the site-2 protease is not essential for the development and growth of Drosophila (Matthews, 2009).

The dS2P¹ allele must also be null for the predicted gene CG34229 that encodes a putative component of the higher eukaryotic NADH complex. The predicted sequence of the encoded polypeptide is highly conserved, supporting the case for this gene (Matthews, 2009).

Are there consequences of the loss of CG34229 that influence the phenotypes that are reported here? The possibility cannot be absolutely excluded that some phenotypes could result, in part, from haplo-insufficiency for CG34229 in dS2P trans-heterozygotes. However, CG34229 cannot be an essential gene; dS2P¹ homozygotes are viable. Most of the experiments presented in this study were performed with mutants trans-heterozygous for dS2P¹ and dS2P². In parallel experiments, indistinguishable results were found with flies homozygous for either dS2P¹ or dS2P², which indicates that the phenotypes that were observed are not the result of the loss of CG34229. Further, the reduced survival of dS2P mutants is rescued by feeding fatty acids, a treatment that also rescues lethality in animals lacking dSREBP. This indicates that reduced survival is a consequence of reduced dSREBP activity (Matthews, 2009).

The phenotype informative for the most important finding described in this study is cleavage of dSREBP in the absence of dS2P. Whether or not insufficiency for CG34229 (or any gene yet to be identified in this region) contributes in some way to reduced viability, smaller-average-size, or delayed development in dS2P homozygotes, dS2P is absent and dSREBP does reach the nucleus without cleavage by dS2P (Matthews, 2009).

In mammals, S2P is needed to process other membrane-bound transcription factors, ATF-6α and -β, that play a crucial role in the endoplasmic reticulum (ER)-stress response [also known as the unfolded protein response or UPR. The Drosophila genome encodes a protein highly similar to mammalian ATF-6, CG3136. In mammals, ATF6 is required to transcribe XBP1 mRNA, and mutant cells lacking S2P are deficient in the induction of the spliced form of XBP1 mRNA. When dS2P^– larvae are challenged with dithiothreitol or tunicamycin, treatments that elicit the UPR, no difference is seen in XBP1 splicing compared to wild-type larvae. If the Drosophila UPR is closely similar to the mammalian UPR, these data suggest that ATF6 processing is relatively unimpaired in dS2P^– larvae. It might be that the Drosophila homolog of ATF6 is not required for the fly UPR or that its activity does not require cleavage by dS2P. If dS2P is required to activate this homolog in flies, the observed developmental delay of dS2P^– larvae may result from defects in ATF6 activation. Nevertheless, while these putative additional functions of dS2P may be important, the crucial function of dS2P in flies is to process dSREBP (Matthews, 2009).

In striking contrast to dS2P^– adults, which lack nuclear dSREBP under conditions where it is readily detected in wild type, dSREBP can reach the nucleus and activate transcription of target genes in dS2P mutant larvae. Thus, Drosophila larvae lacking dS2P have an alternative means of releasing the nuclear transcription factor domain of dSREBP from the membrane-bound precursor. This explains the greater abundance of dSREBP target transcripts in dS2P¹/dS2P² mutants compared with dSREBP¹⁸⁹ mutants (Matthews, 2009).

What is the role of this alternative mechanism for producing nuclear dSREBP? The current data show only that it occurs in the absence of dS2P. It is not yet known if it is a normal, physiologically relevant mechanism or whether it happens fortuitously in the absence of normal dSREBP processing. It is, however, sufficient to afford the survival, over many generations, of flies completely lacking dS2P (Matthews, 2009).

How is the transcription factor domain of dSREBP produced in dS2P mutants? A possible mechanism is production of alternative transcripts that encode only the dSREBP transcription factor domain without the membrane-spanning helices. These might arise from different promoter usage or from differential splicing. Arguing against these possibilities is the fact that only a single transcript is detected for dSREBP in flies from embryogenesis through adulthood and in various tissues examined (Theopold, 1996). A single band was likewise observed on Northern blots for dSREBP. Any putative alternative transcripts or splice forms would have to be present at levels too low to be detected in these assays, while the activity of nuclear dSREBP in dS2P¹/dS2P² larvae is readily detected. Moreover, a cDNA construct harboring the N₄₆₂P > FL mutation and under control of a single, heterologous promoter rescues dSREBP mutants. This construct has no exons; it is not subject to alternative splicing nor is it cleaved by dS2P (Seegmiller, 2002; Matthews, 2009).

The hypothesis if favored that in larvae lacking dS2P, dSREBP is released from the membrane by some other protease(s). This posited protease is unlikely to cleave within the first membrane-spanning helix of dSREBP: flies have no other S2P homologs, and other intramembrane-cleaving proteases display different substrate preferences. The signal peptide peptidase (SPP) is an intramembrane protease of the ER. SPPs are unlikely candidates for cleavage of SREBPs, however. Like S2P, the SPPs require prior cleavage of the substrate by a separate protease. Chinese hamster ovary (CHO) cells express active SPP, but multiple, independently isolated lines of CHO cells lacking S2P show no processing of SREBPs. If SPPs could cleave SREBPs, one would expect some evidence of SREBP processing in S2P^– cells. Cleavage of dSREBP following its first membrane-spanning helix cannot release the NH₂-terminal domain. It is most probable that the alternative cleavage occurs in the cytoplasm, between the transcription factor domain and the first membrane-spanning helix of dSREBP. This portion of dSREBP was termed the "stalk" (Matthews, 2009 and references therein).

Cleavage of SREBPs within the stalk has been reported previously. Wang, showed that caspases 3 and 7 could each cleave mammalian SREBPs (Wang, 1995; Pai, 1996) and that this cleavage was detectable during apoptosis (Wang, 1996). The physiological significance of this cleavage is presently unclear. The caspase cleavage sites identified by Wang (1995) are highly conserved among vertebrate SREBP isoforms, however, and all metazoan SREBPs (except those from Nematoda) contain potential caspase cleavage sites within their stalk regions. Using reporter constructs, Higgins showed that SREBP cleaved during apoptosis by caspases can be transcriptionally active (Higgins, 2001). There is precedent for caspase cleavage of SREBPs releasing the functional transcription factor (Matthews, 2009).

Current data do not suggest that the production of nuclear dSREBP in dS2P mutants has any involvement with apoptosis. However, nonapoptotic roles of caspases have been found in Drosophila and other systems. Cleavage of dSREBP in the absence of dS2P may be an example of a nonapoptotic caspase function. The hypothesis that dSREBP is cleaved by caspases to produce transcriptionally active dSREBP in dS2P^- larvae is currently being tested (Matthews, 2009).

Activation of sterol regulatory element-binding protein by the caspase Drice in Drosophila larvae

During larval development in Drosophila, transcriptional activation of target genes by sterol regulatory element-binding protein (dSREBP) is essential for survival. In all cases studied to date, activation of SREBPs requires sequential proteolysis of the membrane-bound precursor by site-1 protease (S1P) and site-2 protease (S2P). Cleavage by S2P, within the first membrane-spanning helix of SREBP, releases the transcription factor. In contrast to flies lacking dSREBP, flies lacking dS2P are viable. The Drosophila effector caspase Drice cleaves dSREBP, and cleavage requires an Asp residue at position 386, in the cytoplasmic juxtamembrane stalk. The initiator caspase Dronc does not cleave dSREBP, but animals lacking dS2P require both drice and dronc to complete development. They do not require Dcp1, although this effector caspase also can cleave dSREBP in vitro. Cleavage of dSREBP by Drice releases the amino-terminal transcription factor domain of dSREBP to travel to the nucleus where it mediates the increased transcription of target genes needed for lipid synthesis and uptake. Drice-dependent activation of dSREBP explains why flies lacking dS2P are viable, and flies lacking dSREBP itself are not (Amarneh, 2009).

Cleavage of SREBP by caspases was first reported in 1995 (Pai, 1995; Wang, 1995; Wang, 1996), but the significance of those observations has remained unclear. Both in transfected S2 cells and in vitro, with purified enzyme, Drice cleaves dSREBP, and this cleavage requires an Asp residue at position 386 in the juxtamembrane stalk. Other Asp residues in this region are not required for cleavage. As caspases typically cleave following an Asp residue, the parsimonious interpretation of these data is that Drice cleaves dSREBP following Asp-386 (Amarneh, 2009).

Cleavage of dSREBP by Drice releases the amino terminus. The present data demonstrate that dS2P mutants require drice for survival. Animals doubly mutant for dS2P and drice phenocopy dSREBP null mutants; they survive extremely poorly on unsupplemented medium and show greatly improved survival on medium supplementedwith fatty acids. Conversely, larvae whose only source for dSREBP is a dSREBP transgene, P{dSREBPg(D386A)}, which harbors a substitution of Asp-386 for Ala at the putative cleavage site, are viable in the presence of wild type dS2P. If these transgenic animals lack dS2P, the mutant transgenic larvae die at the end of second instar. Just as observed for the double mutant dS2P, drice larvae, the P{dSREBPg(D386A)} transgenic larvae are substantially rescued by dietary supplementation with fatty acids. The extent of rescue is similar to the rescue of dSREBP¹⁸⁹ homozygotes in the same experiment, confirming that lethality results from deficits in fatty acid metabolism (Amarneh, 2009).

The results with the dS2P¹/dS2P²; dronc⁵¹/dronc⁵¹ double mutants are very similar to the results of experiments with drice^δ1, even though purified Dronc cannot cleave dSREBP directly. This likely reflects the requirement for the initiator caspase Dronc to cleave the effector caspase Drice, such that in the absence of Dronc, Drice is not activated and cannot cleave dSREBP. Cleavage of Drice by Dronc, for example, is required for activation of Drice during apoptosis. These data suggest that Drice, activated by Dronc, cleaves dSREBP in larvae lacking dS2P (Amarneh, 2009).

Little apoptosis is observed in Drosophila between embryogenesis and pupariation. Cleavage of dSREBP by Drice during larval growth therefore does not appear to be related to apoptosis. Even in the absence of substantial apoptosis, however, mRNAs for Drice and Dronc are detected at low levels in larvae. Genetic data indicate that at least some of that message is translated to yield enzyme that is active during larval life when apoptosis is not observed. Constitutive activation of effector caspases by the apoptosome in the absence of apoptosis is seen elsewhere during Drosophila growth and development. For example, in embryos, there is a basal level of effector caspase activity several hours before the onset of programmed cell death. Caspase activity is also detected in hemocytes from 3rd instar larvae that are likewise not undergoing apoptosis (Amarneh, 2009).

Does caspase cleavage of dSREBP play a role in normal larval physiology or is this phenomenon seen only when dS2P is absent? In larvae lacking dS2P, activation of dSREBP is readily detected in the fat body. The cleavage of dSREBP by Drice in the larval fat body could be a fortuitous consequence of the presence of a caspase site in the juxtamembrane stalk and the coincidental expression of Drice in the larval fat body. Alternatively, caspase cleavage may represent an important and pervasive physiologically relevant means of activating SREBP independently of the previously described machinery (Scap, S1P, and S2P). The present data do not allow firm distinction between these possibilities but an inference may be drawn from sequence data (Amarneh, 2009).

The site of caspase cleavage in SREBP-1 is conserved among its vertebrate homologues, and the site of caspase cleavage in SREBP-2, which is not homologous to the caspase site in SREBP-1, is likewise highly conserved among vertebrate SREBP-2s. Similarly, the caspase site Asp in dSREBP is well conserved among Drosophila SREBPs as is the preceding Thr residue. In the case of mammalian SREBP-1 and -2, and now for dSREBP, the seputative sites have been validated experimentally. The differing positions of these confirmed caspase sites indicate that the absolute position of caspase cleavage within the juxtamembrane stalk is not crucial, whereas the presence of a caspase cleavage site is. Cleavage almost anywhere within this region may offer suitable means of releasing active SREBPand thus bypassing the normal processing machinery (Amarneh, 2009).

Cleavage of dSREBP is usually tightly controlled by end product feedback regulation. If SREBP in the ER membrane is the substrate for caspase cleavage, this would bypass feedback regulation, which relies of the control of ER-to-Golgi transport of SREBP (Amarneh, 2009).

Under what circumstances might a cell or organism need to bypass end product-mediated feedback suppression of the transcription of the genes of lipid synthesis? It may be desirable during periods of rapid membrane synthesis, such as fetal development in mammals. Rapid deposition of large stores of lipid may be another such a case. The mass of the Drosophila larvae increases roughly 200-fold between the time it emerges from the egg and the onset of pupariation about 5 days later. The majority of this increase in mass results from the storage of lipid in the fat body, which is needed to fuel metamorphosis. End product-mediated suppression of the transcription of the genes of lipid synthesis may be incompatible with the need for continued high levels of lipid accumulation and synthesis in the presence of large amounts of lipid already stored (Amarneh, 2009).

In dS2P mutant larvae, transcript abundance of dSREBP target genes is much greater than in larvae lacking dSREBP itself, and activation of dSREBP is readily detected in the fat body. This activation permits the survival of the mutant animals. However, in the complete absence of dS2P, the mutant offspring of mutant mothers survive only half as well as their heterozygous siblings. Their reduced survival results from a deficit in lipid metabolism; they survive at nearly the expected rate on medium supplemented with fatty acids. Therefore, cleavage of dSREBP by Drice is not fully redundant with the usual processing mechanism. Instead, it may serve to augment dSREBP activation in specific tissues to support the rapid deposition of lipid stores during larval life (Amarneh, 2009).

Activation of SREBP in the Absence of Scap in Drosophila melanogaster

The escort factor Scap is essential in mammalian cells for the regulated activation of sterol regulatory element binding proteins (SREBPs), which are membrane-bound transcription factors. Cells lacking Scap cannot activate SREBP and are therefore deficient in the transcription of numerous genes involved in lipid synthesis and uptake; they cannot survive in the absence of exogenous lipid. This study reports that, conversely, Drosophila completely lacking dscap are viable. Flies lacking dscap emerge at about 70% of the expected rate and readily survive as homozygous stocks. These animals continue to cleave dSREBP in some tissues. Transcription of dSREBP target genes in dscap mutant larvae is reduced compared to wild type but is greater than in mutants lacking dSREBP and remains responsive to dietary lipids in dscap mutants. In contrast to flies lacking ds2p, a gene encoding a protease that releases the transcription factor domain of dSREBP from the membrane, dscap mutants do not require the caspase Drice to activate dSREBP. Larvae doubly mutant for dscap and ds2p exhibit phenotypes similar to those of ds2p single mutants. Thus, dScap and dS2P, essential components of the SREBP activation machinery in mammalian cells, are dispensable in Drosophila owing to different compensatory mechanisms (Matthews, 2010).

Mammalian cells lacking Scap or S2P (or S1P) are auxotrophic for cholesterol and unsaturated fatty acids owing to their failure to activate SREBP (Goldstein, 2002). Although mammalian models lacking all forms of SREBP have not been reported, they would presumably also evince lethality. The Drosophila genome harbors a single form of SREBP and null mutants in this gene die at the end of the second instar owing to an insufficiency of fatty acids. For mammalian cells that cannot activate SREBP or for dsrebp- mutant flies, survival is restored if the appropriate lipids are added to the culture medium (cholesterol and unsaturated fatty acids in mammalian cells, fatty acids in Drosophila). In contrast to mammalian cells, flies lacking dS2P (Matthews, 2009) or dScap are viable and may be readily maintained as homozygous stocks. Thus, while dsrebp is essential to larvae, components of the classical processing machinery are not. The present work shows that dScap is dispensable and, in a subset of larval tissues, cleavage of dSREBP continues in its absence. The tissue-specific pattern of dSREBP activation differs in mutant versus wild type animals. This may be explained if different tissues employ different mechanisms to activate dSREBP with only some of the tissues in which dSREBP is normally active (e.g. the fat body) relying on dScap. In the absence of dScap some, but not all, tissues would retain the ability to activate dSREBP and this is what was observed (Matthews, 2010).

The consequences of complete loss of Scap or S2P are not known in whole mammals. Therefore it is also unknown whether any mammalian tissues also utilize alternate means of bypassing the classical processing machinery to facilitate normal metabolic responses or whether these phenomena are restricted to insects (in mammals, cleavage of SREBPs by caspases has only been observed during apoptosis. The fact that cultured mammalian cells require Scap and S2P does not imply that every mammalian cell type has such a requirement. When Drosophila S2 cells are made deficient for dScap via an RNAi strategy, dSREBP is not cleaved. These cells display reduced accumulation of transcripts of dSREBP target genes just as do S2 cells treated with RNAi against dSREBP itself (Seegmiller, 2002). Yet the situation in the whole fly is different; some dSREBP continues to be activated in dscap mutants, which exhibit deficits in the transcription of dSREBP target genes as compared to wild-type larvae. dSREBP is one of the transcription factors responsible for the upregulation of transcription of genes involved in fatty acid synthesis (e.g. ACS, ACC, and FAS). Their transcription also depends on other factors in addition to dSREBP. This is indicated by the clear, yet notably reduced accumulation of their transcripts in dsrebp null larvae. This may be similar to the case in mammalian systems where well-established SREBP targets such as FAS and ACC are also the direct targets of several other transcription factors. Thus the kinetics of transcript accumulation for ACS, ACC, and FAS are not as simple as seen for CG6295, the gene whose expression is known to be most strongly dependent on dSREBP. Its expression is consistently very low in dscap larvae but somewhat greater than in dsrebp larvae. For the ds2p mutants, cleavage by the caspase Drice activates dSREBP in larvae and is necessary for their survival (Matthews, 2010).

Drice-dependent dSREBP activation in ds2p^- larvae is predominately detected in the fat body but no activation is observed in oenocytes (Matthews, 2009) indicating that caspase activation does not occur in these cells. Accordingly, it was observed that dSREBP is predominately activated in oenocytes, but significantly reduced in the fat body, of dscap larvae. Caspase activation may, however, explain the variable activation of dSREBP that was observed in the fat body or anterior midgut of dscap larvae. These are tissues where dSREBP continues to be activated by Drice in ds2p mutants and the same process may be active to some extent in dscap larvae as well. However, Drice is dispensable to dscap mutants and thus Drice cleavage does not explain the survival of the dscap mutants. These data indicate that Drosophila larvae harbor multiple alternative mechanisms that enable activation of dSREBP. The alternative mechanism at work in the dscap nulls involves cleavage of dSREBP by dS1P and dS2P. In dscap mutants, dS1P continues to cleave dSREBP as indicated by the accumulation of the intermediate form in the membranes of dscap ds2p double mutants. The lack of the intermediate form (I) in membranes from dscap mutants indicates that in these mutants, the intermediate form is efficiently cleaved by dS2P, just as in wild type flies. In larvae, cleavage of the precursor by S1P and S2P and the accumulation dSREBP and CG6295 transcripts remain responsive to the lipid content of the diet. Owing to differences in feeding behavior, it was not possible to employ precisely parallel nutritional regimens in adults and larvae. Therefore, a starvation/refeeding protocol was devised for adults that exhibits effects similar to lipid supplementation in larvae; in adults, accumulation of nuclear dSREBP and dSREBP transcripts is likewise responsive to the nutritional state. Some or all of the nutritional responsiveness of dSREBP-mediated transcription in dscap larvae may be due to transcriptional regulation of dsrebp itself rather than cleavage of the precursor by S1P and S2P. In the absence of dScap, flies may no longer be able to regulate the mechanism responsible for bringing dSREBP and dS1P together. How does membrane-bound dSREBP encounter dS1P and dS2P, which are localized to the Golgi apparatus? One tissue which consistently shows activation of dSREBP in larvae lacking dScap is the oenocytes. Insect oenocytes are thought to be involved in the synthesis and secretion of cuticular hydrocarbons. They also exhibit some hepatocyte-like features. They possess a highly elaborated endomembrane system replete with lipid and their ultrastructural morphology is reminiscent of steroidogenic cells in mammals. Identifying subcellular compartments based on morphology alone is probably insufficient for oenocytes owing to their ultrastructural complexity. Oenocytes might conceivably experience some admixture of ER and Golgi components resulting in dSREBP being accessible to dS1P without the need for dScap-mediated vesicular transport. Alternatively, in these cells, dSREBP might be packaged into COPII vesicles, perhaps via itself interacting with COPII components. Another possibility is that for dSREBP to move to dS1P (in the Golgi) in the absence of dScap, a different escort factor acting analogously to dScap could be required. The restricted activation of dSREBP in dscap mutants, compared to wild type larvae, would then be due to tissue-specific expression of this putative escort factor or of specific COPII components interacting with dSREBP (e.g. in oenocytes). Data are currently being sought that will permit discrimination among these several hypotheses (Matthews, 2010).

The current results, together with those from ds2p mutants (Amarneh 2009; Matthews, 2009), show that flies possess at least two alternative means of activating dSREBP that differ from the classical Scap-S1P-S2P mechanism known from mammals. The first, requiring cleavage of dSREBP by the caspase Drice, explains the survival of flies lacking dS2P. A second novel mode of activation permits dSREBP to be cleaved by dS1P and dS2P in a subset of tissues in the absence of dScap and this is sufficient to support the survival of dscap mutants (Matthews, 2010).

Phospholipid homeostasis regulates lipid metabolism and cardiac function through SREBP signaling in Drosophila

The epidemic of obesity and diabetes is causing an increased incidence of dyslipidemia-related heart failure. While the primary etiology of lipotoxic cardiomyopathy is an elevation of lipid levels resulting from an imbalance in energy availability and expenditure, increasing evidence suggests a relationship between dysregulation of membrane phospholipid homeostasis and lipid-induced cardiomyopathy. The present study reports that the Drosophila easily shocked (eas) mutants that harbor a disturbance in phosphatidylethanolamine (PE) synthesis display tachycardia and defects in cardiac relaxation and are prone to developing cardiac arrest and fibrillation under stress. The eas mutant hearts exhibit elevated concentrations of triglycerides, suggestive of a metabolic, diabetic-like heart phenotype. Moreover, the low PE levels in eas flies mimic the effects of cholesterol deficiency in vertebrates by stimulating the Drosophila sterol regulatory element-binding protein (dSREBP) pathway. Significantly, cardiac-specific elevation of dSREBP signaling adversely affects heart function, reflecting the cardiac eas phenotype, whereas suppressing dSREBP or lipogenic target gene function in eas hearts rescues the cardiac hyperlipidemia and heart function disorders. These findings suggest that dysregulated phospholipid signaling that alters SREBP activity contributes to the progression of impaired heart function in flies and identifies a potential link to lipotoxic cardiac diseases in humans (Lim, 2011).

This study used Drosophila genetic approaches to identify a novel metabolic cardiomyopathy that exhibits striking features of obesity- and diabetes-related heart failure in humans. Specifically, it was shown that a genetically dysregulated phospholipid metabolism leads to chronic stimulation of the transcription factor dSREBP and its lipogenic target genes, which in turn leads to cardiac fat accumulation associated with electrical and functional signatures of heart failure. This study highlights a regulatory relationship between the PE phospholipid and TG metabolism that could play a major role in eliciting cardiac steatosis and dysfunction, and identifies the dSREBP signaling pathway as the key metabolic pathway that underlies the increased synthesis and accumulation of TG upon the disruption of PE homeostasis (Lim, 2011).

The current data lead to a model that describes how the dysregulation of membrane PE homeostasis could promote the pathogenesis of lipotoxic cardiomyopathy. In wild-type flies, a decrease in membrane PE level triggers the proteolytic release of a transcriptionally active form of dSREBP (m-dSREBP) and induces the biosynthesis of fatty acids in a manner similar to that in mammals. Upon the subsequent use of these fatty acids in PE synthesis, and the restoration of normal PE concentrations in cellular membranes, further processing of dSREBP is blocked and overall lipid synthesis is reduced. The presence of such a feedback inhibitory loop ensures that PE homeostasis can be achieved under physiological conditions. In flies harboring a genetic perturbation of the CDP-ethanolamine pathway, the failure to produce PE and the ensuing low levels of PE disrupt the homeostatic negative feedback loop, resulting in the continuous activation of the dSREBP pathway. Prolonged stimulation of lipogenesis and the oversupply of lipid intermediates such as acyl coA and DAG could lead to increased production of TG, resulting in hypertriglyceridemia, cardiac steatosis, and the progressive development of lipotoxic cardiomyopathy (Lim, 2011).

It is possible that the above phenomenon, although identified in a fly model, also occurs in mammals. In fact, in mice, elimination of the CDP-ethanolamine pathway resulting in the absence of PE synthesis induced a significant elevation of TG levels. Along with hypertriglyceridemia, it was also observed in these studies that the expression of key fatty acid biosynthetic genes such as ACC and FAS is up-regulated in PE-deficient mice. It has been proposed that the elevated TG concentration is due to an increased availability of DAG arising from its underutilization by the CDP-ethanolamine pathway that leads to a redirection of DAG to TG formation. However, this proposal fails to explain how the passive accumulation of DAG in the PE-deprived state could induce an upstream event such as the expression of the lipogenic genes. The mechanism proposed in this model based on the eas² fly studies could reconcile to some extent this dilemma in the mammalian system. The model posits that constitutively low levels of PE drive a compensatory hyperactivation of the SREBP pathway. Once activated, SREBP can induce de novo lipogenesis and the active generation of intermediates such as acyl coA and DAG, a sequence of steps that culminates in the heightened production of TG. Indeed, in mice lacking the capacity to generate PE, the expression of one of the mammalian SREBP isoforms, SREBP-1c, was found to be up-regulated. Furthermore, the PE-deficient mice also develop metabolic disorders such as hepatic steatosis and insulin resistance. However, it remains to be seen whether SREBP signaling might similarly be regulated by PE homeostasis in mammals such that a deficit in PE levels elicits an activation of the SREBP pathway to generate increased amounts of fatty acids and DAG/TG. It would be interesting to test whether the enhanced levels of TGs, as well as the severity of these phenotypes, would be significantly ameliorated upon the down-regulation of SREBP expression or activity in these mice, indicating a primary role of SREBP signaling in mediating the development of hypertriglyceridemia and its related metabolic disorders upon the perturbation of PE synthesis in the mammalian context (Lim, 2011).

This model, based on studies in Drosophila eas mutants, provides insights into the potential role of the dSREBP signaling pathway in coupling membrane phospholipid homeostasis with lipid metabolism and its associated metabolic functions. These findings also support the notion that Drosophila shares many of the basic metabolic functions found in vertebrates, and that the genetic dissection of the metabolic and transcriptional responses in a less complex model organism such as Drosophila facilitates understanding of fundamental aspects of metabolic control, cardiac physiology, and associated disease mechanisms (Lim, 2011).

Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration

Reactive oxygen species (ROS) and mitochondrial defects in neurons are implicated in neurodegenerative disease. This study finds that a key consequence of ROS and neuronal mitochondrial dysfunction is the accumulation of lipid droplets (LD) in glia. In Drosophila, ROS triggers c-Jun-N-terminal Kinase (JNK) and Sterol Regulatory Element Binding Protein (SREBP) activity in neurons leading to LD accumulation in glia prior to or at the onset of neurodegeneration. The accumulated lipids were peroxidated in the presence of ROS. Reducing LD accumulation in glia and lipid peroxidation via targeted lipase overexpression and/or lowering ROS significantly delayed the onset of neurodegeneration. Furthermore, a similar pathway led to glial LD accumulation in Ndufs4 mutant mice with neuronal mitochondrial defects, suggesting that LD accumulation following mitochondrial dysfunction is an evolutionarily conserved phenomenon, and represents an early, transient indicator and promoter of neurodegenerative disease (Liu, 2015).

This study shows that neuronal mitochondrial defects that lead to elevated levels of ROS, induce activation of JNK and SREBP, which in turn elevate lipid synthesis in neurons and formation of LD in glial cells. These LDs contribute to and promote ND through elevated levels of lipid peroxidation. LDs form in glia prior to or at the onset of the appearance of obvious degenerative histological features in Drosophila and mice. Reducing the number and size of LD pharmacologically or genetically delays ND in the fly. This is the first indication that SREBP, lipid droplet biogenesis, and lipid metabolism play a role in the pathogenesis of several neurodegenerative diseases (Liu, 2015).

A growing body of evidence points to the importance of glial health and function in nervous system energy metabolism and homeostasis. Nevertheless, given the number and prevalence of different types of neurodegenerative diseases, very few reports have documented the presence of LDs in either neuron or glia in patients and in animal models. LD accumulation in the brain has been reported in cells that line the ventricles in the globus pallidus and substantia nigra in mutant mice lacking both subunits of the liver X receptor, apolipoprotein E, or a peroxisomal biogenesis factor (Pex5) . In addition, in vitro studies using immortalized cell lines and explants show that LD may form and accumulate in glia under conditions of nutrient deprivation or lipopolysaccharide induced stress. However, LDs have not been shown to play an active role in neurodegenerative processes. Furthermore, LD accumulation has not been reported in patients with or animal models of Leigh syndrome (NDUFS4/Ndufs4, NDUFAF6/sicily), CMT-2A2 or HMSN6 (MFN2/Marf), and ARSAL (MARS2/Aats-met). The lack of neuropathological reports of LDs in animal models or in patients with ND may be attributed to the fact that LD accumulation is transient and mostly occur during presymptomatic stages of the disease (Liu, 2015).

Although these genes/mutants are implicated in very different mitochondrial processes, they exhibit a common phenotype of elevated levels of ROS, leading to LD accumulation. Similar morphological changes of glia have been reported under stress conditions. Interestingly, mid- and late-stage Ndufs4-/- mice exhibit CNS lesions in the same brain regions where the LD accumulate in early stage animals, showing a strong correlative relationship. Similarly, LD accumulation in Drosophila mutants occurs prior to or at the onset of physical signs of ND. Importantly, the delivery of AD4 is able to significantly ameliorate LD accumulation in Drosophila and delay the onset of ND in flies and mice. Hence, the molecular mechanisms underlying these phenotypes are likely to be conserved between these species and potentially also in higher organisms (Liu, 2015).

In the clinical setting, the prescription of antioxidants toward treatment of neurodegenerative diseases has been tested repeatedly on patients with neurodegenerative disorders, without compelling results. The LD accumulation phenotype in these mutants occurs prior to histopathological and physical signs of ND. A brief period of AD4 delivery prior to the onset of symptoms in mutant mice is effective in delaying onset of clinical signs. Thus, therapy with an effective antioxidant that penetrates the blood-brain barrier should be started early and sustained over long periods. In addition, pharmacological manipulation of JNK or lipid levels in the brain may serve as a potential therapy to delay the onset of ND. However, similar to antioxidant treatment, this may need to be administered at an early stage. Hence, early identification of potential ROS related neurological disease based on genetic/genomic diagnosis or by biomarkers may be critical. Since LD accumulation is one of the earliest presymptomatic changes that occurs in the nervous system, detection of LD itself or changes in neurometabolism may be a promising biomarker (Liu, 2015).

In summary, this study provides evidence for the role of altered lipid metabolism and a neuron-glia interplay that promotes ND. In some mitochondrial mutants, an upregulation of SREBP was observed, as well as lipid biogenesis and glial LD formation. The accumulation of LD is not sufficient to promote the ND process itself. However, in the presence of ROS the accumulated lipids are peroxidated and promote ND, possibly by promoting the release of lipids from LD, elevating the cytoplasmic load, and causing a progressive loss of LD. Hence, the synergistic effects of increased lipid synthesis and/or LD accumulation in combination with elevated ROS and lipid peroxidation promote ND. Finally, it was shown that LD accumulation occurs at the onset or precedes ND in flies and mice, suggesting that LD and changes in lipid metabolism in the nervous system may be a promising biomarker to identify brain regions susceptible to but not yet exhibiting symptoms of ND (Liu, 2015).

CDK8-Cyclin C mediates nutritional regulation of developmental transitions through the Ecdysone receptor in Drosophila

EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval-pupal transition (Xie, 2015).

In animals, the amount of juvenile growth is controlled by the coordinated timing of maturation and growth rate, which are strongly influenced by the environmental factors such as nutrient availability. This is particularly evident in arthropods, such as insects, arachnids and crustaceans, which account for over 80% of all described animal species on earth. Characterized by their jointed limbs and exoskeletons, juvenile arthropods have to replace their rigid cuticles periodically by molting. In insects, the larval-larval and larval-pupal transitions are controlled by the interplay between juvenile hormone (JH) and steroid hormone ecdysone. Drosophila has been a powerful system for deciphering the conserved mechanisms that regulate hormone signaling, sugar and lipid homeostasis, and the molecular mechanisms underlying the nutritional regulation of development. In Drosophila, all growth occurs during the larval stage when larvae constantly feed, and as a result their body mass increases approximately 200-fold within 4 d, largely due to de novo lipogenesis. At the end of the third instar, pulses of ecdysone, combined with a low level of JH, trigger the larval-pupal transition and metamorphosis. During this transition, feeding is inhibited, and after pupariation, feeding is impossible, thus the larval-pupal transition marks when energy metabolism is switched from energy storage by lipogenesis in larvae to energy utilization by lipolysis in pupae (Xie, 2015).

The molecular mechanisms of ecdysone-regulated metamorphosis and developmental timing have been studied extensively in Drosophila. Ecdysone binds to the Ecdysone Receptor (EcR), which heterodimerizes with Ultraspiracle (USP), an ortholog of the vertebrate Retinoid X Receptor (RXR). By activating the expression of genes whose products are required for metamorphosis, ecdysone and EcR-USP are essential for the reorganization of flies' body plans before emerging from pupal cases as adults. Despite the tremendous progress in understanding of the physiological and developmental effects of EcR-USP signaling, the molecular mechanism of how the EcR-USP transcription factor interacts with the general transcription machinery of RNA polymerase II (Pol II) and stimulates its target gene expression remains mysterious. EcR is colocalized with Pol II in Bradysia hygida and Chironomus tentans. Although a number of proteins, such as Alien, Bonus, Diabetes and Obesity Regulated (dDOR), dDEK, Hsc70, Hsp90, Rigor mortis (Rig), Smrter (Smr), Taiman, and Trithorax-related (TRR), have been identified as regulators or cofactors of EcR-mediated gene expression, it is unknown how these proteins communicate with the general transcription machinery and whether additional cofactors are involved in EcR-mediated gene expression. In addition, it remains poorly understood how EcR activates transcription correctly after integrating nutritional and developmental cues (Xie, 2015).

The multisubunit Mediator complex serves as a molecular bridge between transcriptional factors and the core transcriptional machinery, and is thought to regulate most (if not all) of Pol II-dependent transcription. Biochemical analyses have identified two major forms of the Mediator complexes: the large and the small Mediator complexes. In addition to a separable 'CDK8 submodule', the large Mediator complex contains all but one (MED26) of the subunits of the small Mediator complex. The CDK8 submodule is composed of MED12, MED13, CDK8, and CycC. CDK8 is the only enzymatic subunit of the Mediator complex, and CDK8 can both activate and repress transcription depending on the transcription factors with which it interacts. Amplification and mutation of genes encoding CDK8, CycC, and other subunits of Mediator complex have been identified in a variety of human cancers, however, the function and regulation of CDK8-CycC in non-disease conditions remain poorly understood. CDK8 and CycC are highly conserved in eukaryotes, thus analysis of the functional regulation of CDK8-CycC in Drosophila is a viable approach to understand their activities (Xie, 2015).

Previous, work has shown that CDK8-CycC negatively regulates the stability of sterol regulatory element-binding proteins (SREBPs) by directly phosphorylating a conserved threonine residue. This study now reports that CDK8-CycC also regulates developmental timing in Drosophila by linking nutrient intake with EcR-activated gene expression. Homozygous cdk8 or cycC mutants resemble EcR mutants in both pupal morphology and retarded developmental transitions. Despite the elevation of both EcR and USP proteins in cdk8 or cycC mutants, genome-wide gene expression profiling analyses reveal systematic down-regulation of EcR-target genes, suggesting the CDK8-CycC defect lies between the receptor complex and transcriptional activation. CDK8-CycC is required for EcR-USP transcription factor binding to EcR target genes. Mass spectrometry analysis for proteins that co-immunoprecipitate with EcR and USP has identified multiple Mediator subunits, including CDK8 and CycC, and yeast two-hybrid assays have revealed that CDK8 and Med14 can directly interact with the EcR-AF1 domain. Furthermore, the dynamic changes of CDK8, EcR, USP, and SREBP correlated with the fundamental roles of SREBP in regulating lipogenesis and EcR-USP in regulating metamorphosis during the larval–pupal transition. Importantly, it was shown that starving the early third instar larvae causes precocious increase of CDK8, EcR and USP proteins, as well as premature inactivation of SREBP; whereas refeeding of the starved larvae reduces CDK8, EcR, and USP proteins, but potently stimulates SREBP activity. These results suggest a dual role of CDK8-CycC, linking nutrient intake to de novo lipogenesis (by inhibiting SREBP) and developmental signaling (by regulating EcR-dependent transcription) during the larval–pupal transition (Xie, 2015).

Through EcR-USP, ecdysone plays pivotal roles in controlling developmental timing in Drosophila. This study shows that cdk8 or cycC mutants resemble EcR-B1 mutants and CDK8-CycC is required for proper activation of EcR-target genes. Molecular and biochemical analyses suggest that CDK8-CycC and the Mediator complexes are directly involved in EcR-dependent gene activation. In addition, the protein levels of CDK8 and CycC are up-regulated at the onset of the wandering stage, closely correlated with the activation of EcR-USP and down-regulation of SREBP-dependent lipogenesis during the larval–pupal transition. Remarkably, starvation of the feeding larvae leads to premature up-regulation of CDK8 and EcR-USP, and precocious down-regulation of SREBP, while refeeding of the starved larvae results in opposite effects on the CDK8-SREBP/EcR network. Thus, it is proposed that CDK8-CycC serves as a key mediator linking food consumption and nutrient intake to EcR-dependent developmental timing and SREBP-dependent lipogenesis during the larval–pupal transition (Xie, 2015).

The Mediator complex is composed of up to 30 different subunits, and biochemical analyses of the Mediator have identified the small Mediator complex and the large Mediator complex, with the CDK8 submodule being the major difference between the two complexes. Several reports link EcR and certain subunits of the Mediator complex. For example, Med12 and Med24 were shown to be required for ecdysone-triggered apoptosis in Drosophila salivary glands. It was recently reported that ecdysone and multiple Mediator subunits could regulate cell-cycle exit in neuronal stem cells by changing energy metabolism in Drosophila, and specifically, EcR was shown to co-immunoprecipitate with Med27. However, exactly how Mediator complexes are involved in regulating EcR-dependent transcription remains unknown. The current data suggest that CDK8 and CycC are required for EcR-activated gene expression. Loss of either CDK8 or CycC reduced USP binding to EcR target promoters, diminished EcR target gene expression, and delayed developmental transition, which are reminiscent of EcR-B1 mutants. Importantly, mass spectrometry analysis for proteins that co-immunoprecipitate with EcR or USP has identified multiple Mediator subunits, including all four subunits of the CDK8 submodule (Xie, 2015).

Taken together, previous works and the present work highlight a critical role of the Mediator complexes including CDK8-CycC in regulating EcR-dependent transcription. How does CDK8-CycC regulate EcR-activated gene expression? Biochemical analyses show that CDK8 can interact with EcR and USP in vivo and that CDK8 can directly interact with EcR-AF1. These observations, together with the current understanding of how nuclear receptors and Mediator coordinately regulate transcription, suggest that CDK8-CycC may positively and directly regulate EcR-dependent transcription. Yeast two-hybrid analysis indicates that the recruitment of CDK8-CycC to EcR-USP can occur via interactions between CDK8 and the AF1 domain of EcR. Interestingly, this assay also revealed a direct interaction between EcR-AF1 and a fragment of Med14 that contains the LXXLL motif. In future work, it will be interesting to determine whether CDK8 and Med14 compete with each other in binding with the EcR-AF1, whether they interact with EcR-AF1 sequentially in activating EcR-dependent transcription, and how the Mediator complexes coordinate with other known EcR cofactors in regulating EcR-dependent gene expression (Xie, 2015).

In cdk8 or cycC mutants, the binding of USP to the promoters of the EcR target genes is significantly compromised, even though nuclear protein levels of both EcR and USP are increased. It is unclear how CDK8-CycC positively regulates EcR-USP binding to EcREs near promoters. CDK8 can directly phosphorylate a number of transcription factors, such as Notch intracellular domain, E2F1, SMADs, SREBP, STAT1, and p53. Interestingly, the endogenous EcR and USP are phosphorylated at multiple serine residues, and treatment with 20E enhances the phosphorylation of USP. Protein kinase C has also been proposed to phosphorylate USP. It will be interesting to determine whether CDK8 can also directly phosphorylate either EcR or USP, thereby potentiating expression of EcR target genes and integrating signals from multiple signaling pathways (Xie, 2015).

Although a direct role for CDK8-CycC to regulate EcR-USP activated gene expression is favored, it was not possible to exclude the potential contribution of impaired biosynthesis of 20E to the developmental defects in cdk8 or cycC mutants. For example, the expression of genes involved in synthesis of 20E, such as sad and spok, is significantly reduced in cdk8 or cycC mutant larvae. Indeed, the ecdysteroid titer is significantly lower in cdk8 mutants than control from the early L3 to the WPP stages, and feeding the cdk8 mutant larvae with 20E can partially reduce the retardation in pupariation. Nevertheless, impaired ecdysone biosynthesis alone cannot explain developmental defects that were characterized in this report for the following reasons. First, feeding cdk8 or cycC mutants with 20E cannot rescue the defects in pupal morphology, developmental delay, and the onset of pupariation. Second, the expression of EcRE-lacZ reporter in cdk8 or cycC mutant salivary glands cannot be as effectively stimulated by 20E treatment as in control. Third, knocking down of either cdk8 or cycC in PG did not lead to obvious defects in developmental timing. Therefore, the most likely scenario is that the cdk8 or cycC mutants are impaired not only in 20E biosynthesis in the PG, but also in EcR-activated gene expression in peripheral tissues. Defects in either ecdysone biosynthesis or EcR transcriptional activity will generate the same outcome: diminished expression of the EcR target genes, thereby delayed onset of pupariation (Xie, 2015).

How CDK8-CycC regulates biosynthesis of ecdysone in PG remains unknown. Several signaling pathways have been proposed to regulate ecdysone biosynthesis in Drosophila PG, including PTTH and Drosophila insulin-like peptides (dILPs)-activated receptor tyrosine kinase pathway and Activins/TGFβ signaling pathway. Interestingly, CDK8 has been reported to regulate the transcriptional activity of SMADs, transcription factors downstream of the TGFβ signaling pathway, in both Drosophila and mammalian cells. Thus, it is conceivable that the effect of cdk8 or cycC mutation on ecdysone biosynthesis may due to dysregulated TGFβ signaling in the PG (Xie, 2015).

An effort to explore the potential role of food consumption and nutrient intake on CDK8-CycC has resulted an unexpected observation that the protein level of CDK8 is strongly influenced by starvation and refeeding: starvation potently increased CDK8 level, while refeeding has opposite effect, and both occur post-transcriptionally. The importance of this observation is highlighted in two aspects. First, considering the generally repressive role of CDK8 on Pol II-dependent gene expression, up-regulation of CDK8 may provide an efficient way to quickly tune down most of the Pol II-dependent transcription in response to starvation; while down-regulation of CDK8 in response to refeeding may allow many genes to express when nutrients are abundant. Second, it will be necessary to test whether the effects of nutrient intake on CDK8-CycC is conserved in mammals. If so, considering that both CDK8 and CycC are dysregulated in a variety of human cancers, the effects of nutrient intake on CDK8 may have important implications in not only understanding of the effects of nutrients on tumorigenesis, but also providing nutritional guidance for patients with cancer (Xie, 2015).

Major dietary components including carbohydrates, lipids, and proteins, can strongly influence the developmental timing in Drosophila. Excessive dietary carbohydrates repress growth and potently retard the onset of pupariation. One elegant model proposed to explain how high sugar diet delays developmental timing is that high sugar diet reduces the activity of the Target of Rapamycin (TOR) in the PG, thereby reducing the secretion of ecdysone and delaying the developmental transition. Previously, it was reported that insulin signaling could down-regulate CDK8-CycC, and that ectopic expression of CycC could antagonize the effect of insulin stimulation in mammalian cells, as well as the effect of refeeding on the expression of dFAS in Drosophila (Zhao, 2012). Although the mRNA levels of TOR and insulin receptor (InR) are not significantly affected in cdk8 or cycC mutants, it is necessary to further study whether and how different dietary components may regulate CDK8-CycC in the future (Xie, 2015).

Developmental genetic analyses of the cdk8 and cycC mutants have revealed major defects in fat metabolism and developmental timing. De novo lipogenesis, which is stimulated by insulin signaling, contributes significantly to the rapid increase of body mass during the constant feeding larval stage. This process is terminated by pulses of ecdysone that trigger the wandering behavior at the end of the L3 stage, followed by the onset of the pupariation. Insulin and ecdysone signaling are known to antagonize each other, and together determine body size of Drosophila. The genetic interaction is established, but the detailed molecular mechanisms are not. The SREBP family of transcription factors controls the expression of lipogenic enzymes in metazoans and the expression of cholesterogenic enzymes in vertebrates. Previous work shows that CDK8 directly phosphorylates the nuclear SREBP proteins on a conserved threonine residue and promotes the degradation of nuclear SREBP proteins. Consistent with the lipogenic role of SREBP and the inhibitory role of insulin to CDK8-CycC, the transcriptional activity of SREBP is high while the levels of CDK8-CycC and EcR-USP are low prior to the onset of wandering stage. Subsequently during the wandering and non-mobile, non-feeding pupal stage, the transcriptional activity of SREBP is dramatically reduced, accompanied by the significant accumulation of CDK8-CycC and EcR-USP (Xie, 2015).

The causal relationship of these phenomena was further tested by starvation and refeeding experiments. On the one hand, it was observed that the levels of CDK8, EcR and USP are potently induced by starvation, while the mature SREBP level and the transcriptional activity of SREBP are reduced by starvation. Starvation of larvae prior to the two nutritional checkpoints in early L3, known as minimum viable weight and critical weight, which are reached almost simultaneously in Drosophila, will lead to larval lethality; while starvation after larvae reach the critical weight will lead to early onset of pupariation and formation of small pupae. Thus, this nutritional checkpoint ensures the larvae have accumulated sufficient growth before metamorphosis initiation. If the status with high CDK8, EcR, and USP is regarded as an older or later stage, these results indicate that starvation shifts the regulatory network precociously (see Model for the CDK8-SREBP/EcR regulatory network). On the other hand, the current analyses of refed larvae show that refeeding potently reduced the levels of CDK8, EcR and USP. If the status with low CDK8, EcR, and USP is considered as a younger or earlier stage, these results indicate that refeeding delays the activation of this network, which is consistent with the model and delayed pupariation as observed. Taken together, these results based on starved and refed larvae suggest that CDK8-CycC is a key regulatory node linking nutritional cues with de novo lipogenesis and developmental timing (Xie, 2015).

The larval-pupal transition is complex and dynamic. Although the expression of SREBP target genes fit well with the predicted effects of starvation and refeeding, the expression of EcR targets during the stage that was analyzed in this study does not reflect the changes in the protein levels of EcR and USP. It is reasonable to consider that CDK8-CycC and EcR-USP are necessary, but not sufficient, for the activation of EcR target genes. One possibility is that there is a delay on synthesis of 20E or other cofactors that are required for EcR-activated gene expression in response to starvation. Indeed, the 20E levels were measured during the first 16 hr of starvation, and no significant difference was observed between fed and starved larvae. It will be necessary to further analyze the effect of starvation on 20E synthesis at later time points in the future (Xie, 2015).

Taken together, a model is proposed whereby CDK8-CycC functions as a regulatory node that coordinates de novo lipogenesis during larval stage and EcR-dependent pupariation in response to nutritional cues. It is likely that pulses of 20E synthesized in the PG, and subsequent behavioral change from feeding to wandering, ultimately trigger the transition from SREBP-dependent lipogenesis to EcR-dependent pupariation. The opposite effects of CDK8-CycC on SREBP- and EcR-dependent gene expression suggest that the role of CDK8 on transcription is context-dependent (Xie, 2015).

In conclusion, this study illustrates how CDK8-CycC regulates EcR-USP-dependent gene expression, and the results suggest that CDK8-CycC may function as a regulatory node linking fat metabolism and developmental timing with nutritional cues during Drosophila development (Xie, 2015).

Phospholipid homeostasis regulates dendrite morphogenesis in Drosophila sensory neurons

Disruptions in lipid homeostasis have been observed in many neurodevelopmental disorders that are associated with dendrite morphogenesis defects. However, the molecular mechanisms of how lipid homeostasis affects dendrite morphogenesis are unclear. This study found that easily shocked (eas), which encodes a kinase with a critical role in phospholipid phosphatidylethanolamine (PE) synthesis, and two other enzymes in this synthesis pathway are required cell autonomously in sensory neurons for dendrite growth and stability. Furthermore, this study shows that the level of Sterol Regulatory Element-Binding Protein (SREBP) activity is important for dendrite development. SREBP activity increases in eas mutants, and decreasing the level of SREBP and its transcriptional targets in eas mutants largely suppresses the dendrite growth defects. Furthermore, reducing Ca(2+) influx in neurons of eas mutants ameliorates the dendrite morphogenesis defects. This study uncovers a role for EAS kinase and reveals the in vivo function of phospholipid homeostasis in dendrite morphogenesis (Meltzer, 2017).

A growing body of evidence suggests that lipid metabolism and homeostasis is important for nervous system development. This study provides evidence that EAS kinase and SREBP signaling pathway are required for dendrite growth during development. The results do not rule out the possibility that a reduction in PE levels could also independently contribute to the eas dendritic phenotype, due to a lack of PE supply for expanding the plasma membrane during dendrite growth. Interestingly, although both reduced and increased SREBP activity led to the same dendrite reduction phenotype, the underlying mechanism may be different. Reduced lipid production caused by low SREBP activity might impair the lipid supply for growing dendrites, while aberrantly increased lipid production by SREBP might lead to lipotoxicity. Since the homologues of both genes are highly conserved, it will be interesting to determine their roles in dendrite morphogenesis within the developing mammalian nervous system (Meltzer, 2017).

It was observed that terminal dendrite dynamics, but not initial establishment of the dendritic territory, is perturbed in eas mutants. It is possible that terminal dendrite growth and stability rely more on PE, while the primary and secondary branches do not. It is also possible that although PE synthesis is impaired in eas mutants, neurons still have enough PE to support initial dendrite growth. As the animal grows, neurons cannot supply enough PE for the exuberant terminal dendrite growth. It would be of interest to identify how impairment in PE specifically affects terminal dendrite dynamics in future studies (Meltzer, 2017).

Reducing the level of cacophony, a subunit of the Cav2 voltage-gated Ca2+ channel, ameliorates the dendrite morphogenesis defects in eas^KO mutants. In contrast, reducing SREBP signaling largely rescues the dendrite phenotypes but does not suppress the adult seizure-like phenotype. It is conceivable that the part of the neural circuit that heavily relies on SREBP signaling for dendrite morphogenesis during development does not contribute to the seizure-like behavior. Consistent with this hypothesis, expressing eas acutely in neurons in adult eas flies suppresses the seizure-like phenotype. It thus appears that alterations in neuronal Ca2+ influx contribute to not only seizure-like phenotypes but also dendrite morphogenesis defects during larval development (Meltzer, 2017).

In summary, this study uncovers an important role for phospholipid PE synthesis in neural development, and they further reveal a molecular pathway involving conserved molecules for phospholipid homeostasis as being important in the regulation of dendrite morphogenesis (Meltzer, 2017).

Transcriptional Feedback Links Lipid Synthesis to Synaptic Vesicle Pools in Drosophila Photoreceptors

Neurons can maintain stable synaptic connections across adult life. However, the signals that regulate expression of synaptic proteins in the mature brain are incompletely understood. This study describes a transcriptional feedback loop between the biosynthesis and repertoire of specific phospholipids and the synaptic vesicle pool in adult Drosophila photoreceptors. Mutations that disrupt biosynthesis of a subset of phospholipids cause degeneration of the axon terminal and loss of synaptic vesicles. Although degeneration of the axon terminal is dependent on neural activity, activation of sterol regulatory element binding protein (SREBP) is both necessary and sufficient to cause synaptic vesicle loss. These studies demonstrate that SREBP regulates synaptic vesicle levels by interacting with tetraspanins, critical organizers of membranous organelles. SREBP is an evolutionarily conserved regulator of lipid biosynthesis in non-neuronal cells; these studies reveal a surprising role for this feedback loop in maintaining synaptic vesicle pools in the adult brain (Tsai, 2019).

These studies demonstrate that disrupting the biosynthesis of specific membrane phospholipids causes adult-onset degeneration of R cells and loss of synaptic vesicles. These two phenotypes arise via distinct molecular mechanisms that can be doubly dissociated using genetic and physiological manipulations. Degeneration of the axon terminal is an activity-dependent process that requires calcium-mediated vesicle fusion. Conversely, loss of synaptic vesicles is driven by activation of the transcription factor SREBP. Thus, in these cells, SREBP is activated by alterations in the levels of specific phospholipids. Here, SREBP affects the expression of a specific subset of genes that are largely not directly involved in lipid regulation, thus defining a previously unknown SREBP function. Rather, SREBP activation leads to reduced expression of four tetraspanins. Restoring expression of either of two of these tetraspanins suppresses the effects of SREBP activation, demonstrating that tetraspanins are functional effectors of SREBP in photoreceptors. Thus, a specialized feedback loop from the synaptic terminal to the nucleus links the levels of specific phospholipids to photoreceptor function and synaptic vesicle number. It is proposed that this feedback loop matches the vesicular demand for phospholipids to their production. As SREBP is evolutionarily conserved, and recent studies have linked SREBP to neuronal damage in several contexts, it is speculated that this feedback loop plays a central role in maintaining synaptic vesicle pools in the healthy aging brain (Tsai, 2019).

In Drosophila, mutations that disrupt phospholipid biosynthesis cause broad defects in brain function, including increased seizure activity and photoreceptor degeneration. However, these and other studies examining phospholipid composition in flies have either not quantified phospholipid levels or have not differentiated different phospholipid species. These studies using a high-resolution lipidomic approach demonstrate that biosynthesis of specific PE and PC species is required for maintaining synaptic vesicle pools and the axon terminal in adult photoreceptors. Moreover, the biosynthetic enzyme Pect is found at the axon terminal. It is speculated that the production of specific phospholipids can occur locally, coupling precise levels of phospholipids to the cellular processes that require them in the axonal compartment. Finally, recent work has demonstrated that derivatives of very long chain (VLC) PC species are neuroprotective in vertebrate photoreceptors and neurons. Although this study detected only one VLC PC precursor, PC c44:12, representing 0.01% of the total PC species in the fly retina, future studies will determine the extent to which derivatives of this or other PC or PE species play roles in maintaining adult photoreceptor axons and synapses in Drosophila. This work suggests the following model. The ultrastructural analysis of pect mutants reveals phenotypes in the axon terminal that are strongly reminiscent of those in endocytic mutants. Consistent with this, blocking exocytosis in pect mutants by either reducing light exposure or by genetic means suppresses axon terminal degeneration. It is therefore inferred that the inability to retrieve synaptic vesicles from the plasma membrane is sufficient to cause neuronal degeneration and that the availability of specific phospholipids can be rate limiting for endocytosis. These results are consistent with previous studies in C. elegans that demonstrated that a phospholipid desaturase causes defects in endocytosis through effects on synaptojanin, a critical component in endocytosis. At the same time, altering phospholipid production may also impair vesicle biogenesis, in which case blocking synaptic transmission could suppress neuronal degeneration by removing the demand for vesicle biogenesis via an as-yet-unknown mechanism (Tsai, 2019).

SREBP is a central regulator of genes involved in lipid biosynthesis in many cell types. The current data support the notion that SREBP plays an additional role in Drosophila photoreceptors. As the levels of only a few phospholipids are altered in pect mutants, SREBP activation appears linked to the detection of changes in levels in these PE and PC species. Moreover, although activation of SREBP does upregulate a small number of genes involved in lipid biosynthesis, it also downregulates many genes involved in phototransduction and synaptic function. Among these, genetic interaction studies demonstrate that tetraspanins are functionally critical SREBP effectors. Tetraspanins are transmembrane proteins that have been linked to synapse development, lysosomal function in R cells, and to outer segment structure and function in the vertebrate retina. Moreover, recent work has demonstrated that they can serve as cholesterol-binding proteins, further implicating this family in the regulation of membrane function. Although unraveling the specific molecular mechanisms that link tetraspanin function to synaptic vesicle pools remains a challenge for future studies, the current model for this role of SREBP represents an extension of SREBP's long-standing role in regulating lipid biosynthesis. In particular, a central role for phospholipids that is unique to neurons is as a critical component of synaptic vesicles. It is hypothesized that, when SREBP is activated and tetraspanin expression is reduced, either the biogenesis of synaptic vesicles is downregulated or their turnover and degradation is increased, shrinking the synaptic vesicle pool in an activity-independent manner. As a result, the cellular demand for the specific phospholipids found in synaptic vesicles is reduced. More broadly, these studies suggest that SREBP might complement its long-standing role in lipid biosynthesis with an additional role in controlling phospholipid utilization. Finally, by combining the high-resolution lipidomic approach this study developed to work with small populations of labeled cells with the powerful genetic tools available in this system, future work may shed further light on the regulation of SREBP activity and phospholipid levels (Tsai, 2019).

SREBP has been linked to both neurodegenerative disease and stroke. Recent studies in flies have demonstrated that reactive oxygen species can activate SREBP to cause lipid droplet formation in glia. However, the molecular mechanisms by which SREBP might act in these contexts are unknown. In addition, mutations in a human tetraspanins have been linked to intellectual disability. The demonstration that SREBP acts through tetraspanins to regulate synaptic vesicle pools and negatively regulates other genes required for synaptic function suggests a unifying mechanism for these seemingly disparate observations. Taken together, these studies argue that SREBP plays an evolutionarily conserved role in regulating neuronal and synaptic function, suggesting a link between the neuronal phospholipid repertoire and synapse maintenance in the adult brain (Tsai, 2019).

Search PubMed for articles about Drosophila SREBP

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Dobrosotskaya, I. Y., et al (2002). Regulation of SREBP Processing and Membrane Lipid Production by Phospholipids in Drosophila. Science 296: 879-83. PubMed ID: 11988566

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Goldstein, J. L., Rawson, R. B. and Brown, M. S. (2002). Mutant mammalian cells as tools to delineate the sterol regulatory element-binding protein pathway for feedback regulation of lipid synthesis. Arch. Biochem. Biophys. 397: 139-48. PubMed ID: 11795864

Higgins, M. E. and Ioannou, Y. A. (2001). Apoptosis-induced release of mature sterol regulatory element-binding proteins activates sterol-responsive genes. J. Lipid Res. 42: 1939-1946. PubMed ID: 11734566

Kunte, A. S., Matthews. K. A. and Rawson, R. B. (2006). Fatty acid auxotrophy in Drosophila larvae lacking SREBP. Cell Metab. 3: 439-448. PubMed ID: 16753579

Lim, H. Y., et al. (2011). Phospholipid homeostasis regulates lipid metabolism and cardiac function through SREBP signaling in Drosophila. Genes Dev. 25(2): 189-200. PubMed ID: 21245170

Liu, L., Zhang, K., Sandoval, H., Yamamoto, S., Jaiswal, M., Sanz, E., Li, Z., Hui, J., Graham, B.H., Quintana, A. and Bellen, H.J. (2015). Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration. Cell 160: 177-190. PubMed ID: 25594180

Matsuda, M., et al. (2001). SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation. Genes Dev. 15: 1206-1216. PubMed ID: 11358865

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Meltzer, S., Bagley, J. A., Perez, G. L., O'Brien, C. E., DeVault, L., Guo, Y., Jan, L. Y. and Jan, Y. N. (2017). Phospholipid homeostasis regulates dendrite morphogenesis in Drosophila sensory neurons. Cell Rep 21(4): 859-866. PubMed ID: 29069593

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Tsai, J. W., Kostyleva, R., Chen, P. L., Rivas-Serna, I. M., Clandinin, M. T., Meinertzhagen, I. A., Clandinin, T. R. (2019). Transcriptional Feedback Links Lipid Synthesis to Synaptic Vesicle Pools in Drosophila Photoreceptors. Neuron, 101(4):721-737 e724 PubMed ID: 30737130

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date revised: 25 October 2023

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