easily shocked: Biological Overview | References
Gene name - easily shocked
Cytological map position - 14B7-14B7
Function - enzyme
Keywords - cell proliferation, brain, phospholipid biosynthesis
Symbol - eas
FlyBase ID: FBgn0000536
Genetic map position - X:16,172,360..16,179,654 [+]
Classification - Ethanolamine kinase
Cellular location - cytoplasmic
|Recent literature||Saras, A. and Tanouye, M. A. (2016). Seizure suppression by high temperature via cAMP modulation in Drosophila. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27558668
Bang Sensitive (BS) Drosophila mutants display characteristic seizure like activity (SLA) and paralysis after mechanical shock. After high frequency electrical stimulation (HFS) of the brain, they generate robust seizures at very low threshold voltage. This paper reports an important phenomenon, which effectively suppresses SLA in BS mutants. High temperature causes seizure suppression in all BS mutants (parabss1, eas, sda) examined in this study. This effect is fully reversible and flies show complete recovery from BS paralysis once the temperature effect is nullified. High temperature induces an increase in seizure threshold after a brief pulse of heat shock (HS). By genetic screening, the involvement of cAMP was identified in the suppression of seizures by high temperature. It is proposed that HS induces adenylyl cyclase which in turn increases cAMP concentration which eventually suppresses seizures in mutant flies. In summary, this study describes an unusual phenomenon, where high temperature can suppress SLA in flies by modulating cAMP concentration.
The Drosophila mushroom bodies (MBs), paired brain structures composed of vertical and medial lobes, achieve their final organization at metamorphosis. The alpha lobe absent (ala) mutant randomly lacks either the vertical lobes or two of the median lobes. This study characterize the ala axonal phenotype at the single-cell level, and showed that the ala mutation affects Drosophila ethanolamine (Etn) kinase activity and induces Etn accumulation. Etn kinase is overexpressed in almost all cancer cells. This enzymatic activity is required in MB neuroblasts to allow a rapid rate of cell division at metamorphosis, linking Etn kinase activity with mitotic progression. Tight control of the pace of neuroblast division is therefore crucial for completion of the developmental program in the adult brain (Pascual, 2005).
In all species, organogenesis entails a precisely regulated temporal and spatial pattern of cell proliferation. In this respect, the question of how a neural progenitor cell can generate different types of neurons and glia is an outstanding problem in developmental biology. Two sets of determinative factors, external cues and internal cell-autonomous responses, interplay to define cell fate. Thus, the position and time of birth of a neuron in the central nervous system allows it to receive specific and transient signals from surrounding cells. The mushroom bodies (MBs) are insect brain structures highly relevant to this issue, as their highly specialized organization is elaborated in several discrete developmental steps (Pascual, 2005).
Adult MB cells (Kenyon cells) send their dendrites into the calyx, where they receive input from the antennal lobes. Their axons extend anteriorly and ventrally into the peduncle and terminate in one of several groups of lobes that are composed of several classes of neurons. Three of these, γ, α′/β′, and α/β neurons, have been particularly well studied. The MBs receive multimodal sensory information and have been implicated in higher-order brain functions, including olfactory learning and short-term memory, olfactory long-term memory, courtship behavior, and elementary cognitive functions, such as visual context generalization. The individual MB lobes are functionally specialized. In particular, specific lobes have been implicated in short-term memory, while the vertical MB lobes play a role in long-term memory. How this neural diversity is generated during development remains poorly understood (Pascual, 2005).
Four neuroblasts (Nbs) give rise to each MB. These progenitor cells are among the first to delaminate from the procephalic embryonic ectoderm, and they begin to proliferate from embryonic stage 9 onward. During embryogenesis, the four MB Nbs give rise to between 100 and 300 γ neurons, whose axons branch to form a medial and a dorsal lobe. Most other embryonic Nbs stop dividing transiently in the late embryo. However, MB Nbs continue proliferating through the postembryogenic stages, and they are actively dividing at the time of larval hatching. About 12 h after hatching, some scattered Nbs in the central brain resume division. Neurogenesis proceeds at an accelerating rate in the central brain through the remainder of larval life and puparium formation. Nb proliferation ceases about 20 to 30 h after puparium formation (APF except for the MB Nbs, which continue to divide almost until the end of metamorphosis. Thus, MB Nbs are distinctive in that they divide continuously throughout development (Pascual, 2005 and references therein).
During metamorphosis, many larva-specific neurons are definitively removed by programmed cell death, while most of the remaining cells withdraw larva-specific projections and extend new processes. Some immature neurons differentiate during metamorphosis to produce adult-specific networks. Clonal analysis has demonstrated that all MB neurons generated from the time of larval hatching until the mid third-instar larval stage give rise to branched γ neurons. In mid third-instar larvae, the progeny of the MB Nbs undergo a sharp change in cell fate and start to generate branched α′/β′ neurons. The larval projections of these neurons remain relatively unchanged during metamorphosis. In contrast, γ projections undergo pruning by glial cells at metamorphosis to give rise to adult γ lobes that project only medially. Finally, all MB neurons born after puparium formation are α/β neurons (Pascual, 2005).
With the aim of identifying genes involved in brain metamorphosis, enhancer trap lines displaying specific patterns of expression in the central brain at the third-instar larval stage were screened. This work led to the recovery of six mutants showing central brain defects in the adult. One of these, alpha lobes absent (ala) presents a peculiar MB phenotype. ala MBs completely lack α′ and α or β′ and β lobes in a random pattern (Pascual, 2001). In contrast, γ lobes appear normal. This phenotype proved useful in ascribing to dorsal MB lobes a role in Drosophila long-term memory (Isabel, 2004; Pascual, 2005).
This study shows that ala corresponds to easily shocked (eas), a previously described gene that encodes ethanolamine (Etn) kinase, the first enzyme of the Kennedy pathway (Pavlidis, 1994). This study shows that eas mutants display a brain phenotype similar to that of ala mutants. Etn kinase is expressed in MB Nbs, where it controls the rapid mitoses that occur just before and during metamorphosis (Pascual, 2005).
To identify the gene responsible for the ala brain phenotype, genomic DNA from the ala locus was recovered by plasmid rescue, sequenced, and compared to sequences in the Drosophila database. The P-element lies at nucleotide 38 of the previously described easily shocked gene (Pavlidis, 1994). This gene encodes two isoforms of the Drosophila Etn kinase, which catalyzes the first step of the synthesis of phosphatidylethanolamine (PE) via the Kennedy pathway (Kennedy, 1957). The P insertion lies in a DNA region corresponding to an exon region that is shared by the two known eas mRNAs (Pascual, 2005).
Polyclonal antibodies were raised against the Eas protein. Western blot analysis of larval protein extracts from strains carrying the easalaP and easalaE13 alleles (Boquet, 2000) or an EMS-induced allele (eas2) (Pavlidis, 1994) revealed reduced levels of Eas protein in comparison with extracts from wild-type strains. The amount of Eas protein detected on Western blots inversely correlates with the severity of brain phenotype (Pascual, 2005).
The eas mutant was first isolated as a 'bang-sensitive' paralytic strain (Benzer, 1971; Ganetzky, 1982). This defect in eas2 animals but neither the easalaP nor the easalaE13 mutant displays this phenotype, as homozygotes or as heterozygotes with the eas2 allele or the Df(1)4b18 deficiency, which uncovers the eas region (Boquet, 2000). This result confirms that the easalaP and easalaE13 mutations are hypomorphic (Pascual, 2005).
A previous work had shown a slightly altered PE/phosphatidylcholine (PC) ratio in eas flies (Pavlidis, 1994). Moreover, expression of the Drosophila Etn kinase in NIH 3T3 fibroblasts generates a significant increase in phosphorylethanolamine (PEtn) synthesis but only a modest increase in the level of PE (Kiss, 1997). It was asked whether the lack of Etn kinase activity correlated with an accumulation of Etn. Indeed, high levels of Etn are detected in eas2 larvae, suggesting that the primary biochemical defect of the mutant is related to the accumulation of Etn (or to the lack of PEtn) rather than to an indirect effect on phospholipid composition (Pascual, 2005).
Amorphic eas2 flies show strong MB lobe defects. easalaP and easalaE13 flies present a MB brain defect (Boquet, 2000; Pascual, 2001). 10.5% of easalaP individuals possess all five lobes of each MB in both hemispheres, 36% lack β′ and β lobes in both hemispheres, and 4.5% lack α′ and α vertical lobes in both hemispheres. The remaining flies show different lobe configurations in the left and right hemispheres. Brain analysis of eas2 flies revealed that they have a similar phenotype but with a stronger penetrance, since fewer than 1% of eas2 individuals possess all five lobes in both hemispheres, 29.6% lack the β′ and β lobes in both hemispheres, and 14.8% lack the α′ and α vertical lobes in both hemispheres. In some cases (5.5%), α′/β′ and α/β fibers do not exit the peduncle at the branching point and continue to grow until they reach the antennal lobes. eas2 is considered as an amorphous allele given (1) the molecular nature of the mutation, which creates a premature stop codon (Pavlidis, 1994); (2) the absence of protein, as revealed by Western blot analysis; and (3) the extreme severity of the brain phenotype (Pascual, 2005).
γ neurons appear to be normal in eas2 adults. This observation is reinforced by the observation that second-instar larval eas2 mutants possess vertical and medial γ projections indistinguishable from those of wild-type MB γ neurons (Pascual, 2005).
To determine whether the absence of vertical or median lobes in mutants is linked to a failure of axonal branching by α′/β′ and α/β neurons or to the misprojection of both branches to the same lobe, MB-GFP clones were generated in easalaE13 flies using the MARCM system to allow the trajectories of individual axons to be followed. Confocal analysis of small MB clones generated during the first 48 h APF revealed two different morphologies for α′/β′ and α/β neurons. About 50% of eas MB clones do not divide when invading the dorsal (α′ or α) or medial (β′ or β) lobes, while in the remaining clones axons branch and project into the same lobe. The observation that both branched and unbranched α′/β′ and α/β axons are found in eas mutants displaying an identical missing-lobes phenotype suggested that the failure to branch is not the primary cellular defect of eas MBs (Pascual, 2005).
To directly determine the effect of the eas mutation on MB cells, clones homozygous for the easalaE13 mutation were generated in an easalaE13/+ background with the MARCM system. This experiment was performed at various developmental stages, and the Gal4-OK107 enhancer-trap line was used to specifically follow MB clones. No MB axonal guidance defects were found for any clone, either large clones affecting the entire progeny of a single Nb or small clones. This result suggested that Eas is not required in the MBs themselves or that abnormal MB fibers can follow a correct pathway as long as some normal eas/+ neurons are correctly positioned within the same MB. To distinguish between these two possibilities, eas+ was expressed in differentiating MBs of easalaE13 animals using the UAS-eas+ transgene driven by the Gal4-OK107 insertion. The expression of the eas+ gene allowed almost complete rescue of the eas brain phenotype. This result confirms that eas is autonomously required for the proper development of differentiating cells in MBs. Thus, these data rule out the possibility that the eas mutation affects signals external to the MBs (Pascual, 2005).
Analysis of the third-instar larval brain allowed identification of several regions with strong Eas expression, such as Nb proliferating centers in the optic lobes. In MBs, Eas expression is restricted to Nbs and to the first layers of the post-mitotic cells surrounding them. The protein is detected mainly in newly differentiated MB neurons. Throughout MB development, a central core of actin-rich thin fibers is visible, which is first constituted of γ axons that arise during embryonic and larval stages. These new axons extend into the inner layer of the central core and are shifted to surrounding layers as they differentiate. The time of appearance of Eas-positive neurons at the end of the third larval instar indicates that newly born α′/β′ neurons also send projections into the MB core. Expression of Eas in Nbs is still detectable at 24 h APF, suggesting that young α/β neurons also express the enzyme (Pascual, 2005).
Using the P(Gal4) insertion (easalaP) to drive a P(UAS-mCD8::GFP) reporter, an expression profile was detected similar to that obtained with the Eas antibody. No Eas expression was detected in MB neuroblasts of first-instar larvae. As expected, Eas protein was not detected in the eas2 larval brain (Pascual, 2005).
Previous studies showed that overexpression of the Drosophila eas gene in human fibroblasts promotes mitosis and allows survival in cell culture (Kiss, 1997; Malewicz, 1998). Taken together with the expression of Eas in MB Nbs, this observation prompted an analysis of Nb cell division during eas development. Since MB Nbs are the only Nbs observed to continue dividing 48 h APF, they can be readily studied using the BrdU incorporation technique. Examination of MB cell clusters after 1 h of BrdU incorporation clearly showed reduced numbers of BrdU-positive eas2 clusters, as compared to wild-type pupae, suggesting either that mitosis is slowed in eas MB Nbs or that some Nbs die in the mutant. To differentiate between these two hypotheses, longer BrdU incorporation times were used. If MB Nbs exhibited normal viability in the eas mutant, an increase in the number of labeled MB cell clusters was predicted, as expected for the wild type. In contrast, dead neuroblasts cannot be labeled after longer BrdU exposure. Indeed, a 3-h incubation yielded an increase in the number of labeled MB clusters in both mutant and wild-type strains, indicating that Nbs are still alive in the eas mutant. Again, a significant decrease in the number of BrdU-positive clusters was observed in eas as compared to wild-type pupae. The number of labeled cells per cluster is also lower in eas pupae confirming that the rate of mitosis is affected in the eas mutant (Pascual, 2005).
To determine if the defect in eas Nbs mitosis seen in MBs at the pupal stage has a global effect on MB formation, MB calyx size was measured in adult brains . Calyces in eas2 flies are 30% smaller than those in wild-type flies. Thus, the reduced mitotic rate is not compensated for by a prolonged phase of Nb division. Similar results were obtained for the easalaP allele by following BrdU incorporation and by measuring calyx size (data not shown) (Pascual, 2005).
MB-GFP clones generated in easalaE13 white puparia were used to estimate the overall effect of the eas mutation on mitotic activity. It was reasoned as follows: a clone can be generated with the MARCM system if the DNA is undergoing replication while the Flp recombinase (Flipase) is present. For a clone to be visualized after a mitotic recombination event, the cells must have divided at least once. Thus, the mitotic activity of Flipase-targeted cells can be estimated by measuring their capacity to generate detectable clones. Interestingly, the number of MB clones generated in the eas mutant is severely decreased as compared to the wild type. This effect is ascribed to a general deceleration in the rate of Nb mitosis in eas MBs. This interpretation is reinforced by the observation of many more large clones (Nb clones with more than two cells) in wild-type pupae than in eas mutants and a corresponding increase in the number of small clones (two-cell/single-cell clones) in eas pupae. Thus, it is likely that in some eas clones, which normally would have generated a large number of progeny, the rate of mitosis is dramatically reduced, thereby yielding a smaller number of descendants (Pascual, 2005).
To determine when the Eas protein is required for MB development, easalaE13/Y; hs-eas+/+ transgenic animals were heat shocked for various periods of time. Heat induction of hs-eas+ animals for 30 min daily from the embryonic stage until the adult stage allows complete rescue of the eas MB axonal defect. Expression of Eas initiated at the first day of the third-instar larval stage provides almost complete rescue, while induction from the late third-instar larval stage leads to only partial rescue. Later induction of Eas expression during development fails to rescue the eas brain phenotype. Taken together with the observation that strong Eas expression is detected in MBs during the later stages of larval life, these results indicate that the requirement for Eas activity in axonal MB development begins just before metamorphosis (Pascual, 2005).
This study has shown that ala MB mutations (Boquet, 2000; Pascual, 2001) affect the eas gene. Conversely, the original eas2 allele (Pavlidis, 1994) confers a MB defect similar to that found for easala flies (Pascual, 2005).
eas was originally isolated as a behavioral mutant that belongs to a family of bang-sensitive paralytic mutants (Benzer, 1971; Ganetzky, 1982). These flies become paralyzed when vortexed for 10 s. A brief bang causes a period of hyperactivity lasting 1-2 s (Ganetzky, 1982). The eas bang sensitivity is thought to be due to an excitability defect caused by altered membrane lipid composition (Pavlidis, 1994). This behavioral phenotype is found only in eas2 flies, which bear a null allele. In contrast, this study shows that genetic combinations of the eas2 allele with the hypomorphic eas alleles do not lead to a paralytic phenotype (Pascual, 2005).
Both the anatomical brain phenotype and the paralytic phenotype are rescued by the ectopic expression of eas+, but for each phenotype expression is needed at different times: developmental expression is required to rescue the MB phenotype, while transient adult expression allows the behavioral phenotype to be rescued (Pavlidis, 1994). Taken together, these results argue for distinct roles of the Etn kinase during development and in adult flies and exclude the hypothesis that the MB defect accounts for the bang-sensitive phenotype (Pascual, 2005).
Etn kinase catalyzes the first step of the synthesis of PE, one of the three major membrane phospholipids, via the Kennedy pathway (Kennedy, 1957). This pathway is one of several synthetic pathways for PE. The next enzyme in the Kennedy pathway, a cytidyltransferase, is thought to be the major regulator of PE synthesis (Bladergroen, 1997). Phospholipid analysis of eas flies revealed a slight decrease in the PE/phosphatidylcholine (PC) ratio (Pavlidis, 1994), and a recent study using a different phospholipid measurement technique found a small decrease in the level of PE and phosphatidylserine (PS) (Nyako, 2001). These results clearly indicate that eas flies are not grossly impaired in PE synthesis, and it seems likely that other pathways (e.g., decarboxylation of PS) are capable of providing most of the PE in eas flies (Pavlidis, 1994). This is in agreement with the results obtained for yeast eki1 mutants, which lack Etn kinase activity but are not altered in overall phospholipid composition (Kim, 1998). In addition, overexpression of the Drosophila eas gene in NIH 3T3 fibroblasts leads to only a modest increase in the synthesis of PE but a strong increase in PEtn formation (Kiss, 1997). The current results show that Etn accumulates in eas larvae. Thus, it is possible that eas developmental defects are directly due to the accumulation of Etn or to the lack of PEtn rather than to a lower rate of PE synthesis (Pascual, 2005).
What is the original cellular defect in eas mutants? At the developmental stage at which Etn accumulates in eas mutants, it was found that the Eas protein is strongly expressed in wild-type MB Nbs. In the absence of Etn kinase activity, the rate of Nb mitosis in MBs is reduced, as shown by a decrease in the incorporation of BrdU by MB Nbs in eas pupae, and by the reduced number of MARCM MB-GFP clones in eas flies. The hypothesis that abnormal cell death occurs in eas mutants can be ruled out based on two observations: (1) MB clones could be generated in eas flies at least until 48 h APF; second, an increase in the BrdU incorporation time from 1 to 3 h leads to an increase in the number of cell clusters labeled in wild-type as well as in eas pupae (Pascual, 2005).
The MB Nbs are, together with a lateral Nb, the only Nbs that continue to proliferate after larval hatching. Also, while other Nbs proliferate for about 10 h in embryos and for about 100 h from the second-instar larval to first-day pupal stages, MB Nbs continuously divide for an extraordinarily long period, more than 200 h from the early embryonic to late pupal stages. Consequently, the MB Nbs are the only Nbs that produce new neurons after metamorphic reorganization of the Drosophila brain has taken place. The differences between the time course of MB Nb proliferation and that of other Nbs raise the possibility that a specific genetic mechanism controls the proliferation of MB Nbs. For example, the mushroom body defect (mud) mutant has a higher number of dividing Nbs in the MB cortex, and MB clones homozygous for enoki mushroom (enok) present a defect in MB proliferation. In contrast, enok clones generated in wing discs do not have this phenotype (Scott, 2001), although the gene is expressed in these discs (Pascual, 2005).
The results presented in this study indicate that eas is necessary for MB Nb proliferation, especially at the end of larval life and at the start of metamorphosis, developmental times at which the rate of MB Nb division is maximal. Altogether, these results point to a role of Etn or PEtn in controlling MB Nb cell proliferation. The mechanisms by which these molecules control the cell cycle remain an open question, but an intriguing clue comes from the observation that PEtn strongly inhibits the activities of some decarboxylases (Gilad, 1984). These enzymes are involved in the synthesis of polyamines, molecules that have been proposed as regulators of cell division (Thomas, 2001). It will be interesting to see how mutations in polyamine anabolic pathways interact with the eas mutation in the control of cell division (Pascual, 2005).
Cells in many human tumors have intracellular concentrations of phosphorylcholine and PEtn that are well above normal levels, and this characteristic is a useful diagnostic tool. The levels of these water-soluble phospholipid intermediates may also be elevated in actively proliferating normal tissues (Granata, 2000). Increases in PEtn in dividing cells have been linked to an enhanced activity of Etn kinase, but it is unclear whether these phenomena cause or result from proliferation. The present work suggests that these molecules do indeed play a central role in the control of cell division (Pascual, 2005).
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 leads 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).
easily shocked (eas) is a Drosophila 'bang-sensitive' paralytic mutant. Electrophysiological recordings from flight muscles in the giant fiber pathway of adult eas flies reveal that induction of paralysis with electrical stimulation results in a brief seizure, followed by a failure of the muscles to respond to giant fiber stimulation. Molecular cloning, germline transformation, and biochemical experiments show that eas mutants are defective in the gene for ethanolamine kinase, which is required for a pathway of phosphatidylethanolamine synthesis. Assays of phospholipid composition reveal that total phosphatldylethanolamine is decreased in eas mutants. The data suggest that eas bang sensitivity is due to an excitability defect caused by altered membrane phospholipid composition (Pavlidis, 1994).
eas is the first member of the choline/ethanolamine kinase family to be cloned that apparently lacks significant choline kinase activity. This specificity was shown by gene dosage experiments in which an increase in the dosage of eas does not cause a change in total choline kinase activity and does not change in an attempt to inhibit ethanolamine kinase activity with choline. However, without studying the protein in isolation, it is difficult to be sure that eas does not have any choline kinase activity, though it would be a very small fraction of the total in the fly (Pavlidis, 1994).
There is a precedent for a separation of ethanolamine and choline kinases in some organisms and in dipterans in particular. The two activities are separable in mosquito and are at least partially separable in the blowfly. In budding yeast, there is evidence for a minor ethanolamine-specific kinase as well as a combined cholinelethanolamine kinase. In contrast, the two activities are associated with the same molecule in most other organisms, though this has been a point of some controversy. On the basis of the sequence homology of Eas to the mammalian and yeast choline/ethanolamine kinases, it is not possible to determine which regions of the protein are responsible for the difference in substrate specificity (Pavlidis, 1994).
The ethanolamine kinase defect in eas has a dramatic effect on nervous system excitability. The combination of extreme hyperactivity (what was termed seizure) and failure suggests a model for the phenotype in which the intense activity of the seizure, triggered by a brief electrical buzz or mechanical bang, results in inactivation of a labile site in the nervous system. Specifically, the hyperactivity phase of the phenotype might inactivate action potential ion channels or deplete readily releasable neurotransmitter stores, blocking transmission until recovery occurs (Pavlidis, 1994).
How does a buzz or bang trigger a seizure? One possibility is that eas neurons are inherently hyperexcitable, but in a way that is not apparent until 'set off' by a relatively strong stimulus. Alternatively, the seizure might result from an imbalance between excitatory and inhibitory pathways following the buzz. The latter possibility is suggested by the observation that pharmacological antagonists of inhibitory transmitters cause convulsions in many organisms, including insects, and that decreased inhibition is thought to contribute to the formation of seizures in epilepsy. Such an imbalance could result from abnormal lability of eas neurons, rather than increased excitability; such lability might also result in the inactivation of excitatory pathways by the seizure (Pavlidis, 1994).
Despite the uncertain etiology of the seizure, data from other studies are currently more supportive of a hyperexcitability defect in eas. First, a recently isolated Na+-K+ ATPase mutant, which suffers from behavioral hyperexcitability, can be briefly paralyzed by mechanical stimuli. Second, the bang-senseless larval electrophysiological phenotype is characterized by nerve hyperexcitability and abnormal facilitation. Third, the bang-sensitive defect of eas is suppressed by mlenapt8 (maleless-no action potential-temperature sensitive) which also suppresses the hyperexcitability defects in Shaker and seizure mutants. Fourth, the tko mutation is proposed to affect mitochondrial function. This could cause hyperexcitability by decreasing ATP synthesis (and therefore ion pump activity) or perhaps by a failure of the mitochondria to buffer calcium in presynaptic terminals, resulting in increased transmitter release. Taken together with the data, these studies suggest that eas paralysis is due to a hyperexcitability defect that results in failure in the nervous system (Pavlidis, 1994).
A determination of the cause of the seizure will be critical in testing this hypothesis. It will also be of interest to determine the site of failure. It has been proposed that synaptic transmission is affected in bang-sensitive mutants. The decrease in the amplitude of the DLM activity during the seizure is consistent with the failure of the dorsal longitudinal muscle motor neuron synapses, as opposed to the all-or-none failure that might be expected if action potential propagation were affected (Pavlidis, 1994).
Ethanolamine kinase is required for the synthesis of PE via the CDP-ethanolamine pathway. Ethanolamine kinase phosphorylates ethanolamine, which is further modified by CTP-phosphoethanolamine cytidylyltransferase before it is incorporated into PE. Consistent with its role in this pathway, eas mutants have decreased levels of PE. However, the deviations from wild type found in whole flies were small, and it is unknown whether these changes, if uniformly distributed in the fly, could explain the excitability defects. It is possible that the small overall change in PC and PE levels reflects larger changes within certain cell types orsubcellular membrane compartments. Also, there may be changes in the levels of other lipids that were not assayed. Regardless, it is clear that the eas mutant does not have a gross inability to synthesize PE. This is interesting because the CDP-ethanolamine pathway is thought normally to be the major source of PE in most animals, including insects. Although the possiblility cannot be ruled out of trace ethanolamine kinase activity in eas mutants, it seems likely that other pathways (e.g., decarboxylation of phosphatidylserine are capable of providing most of the PE in flies. It would be interesting to see how the eas mutation affects the activity of other lipid synthetic pathways (Pavlidis, 1994).
The altered membrane phospholipid composition in eas suggests that the eas behavioral defects arise from the effects of the mutation on lipid metabolism. There are several ways in which this might generate changes in excitability. First, the generation of lipid-derived second messenger signals might be impaired or altered in eas flies. This could lead to altered ion channel or neurotransmitter receptor function via changes in phosphorylation state, for example. Although it is considered relatively unlikely that second messenger generation plays a direct role in the rapid events following a bang, there might be chronic effects of eas on membrane excitability or synaptic function that result in a susceptibility to paralysis. Another general way in which eas might cause excitability defects is by altering the activity of membrane proteins required for normal excitability. Lipid environment has been shown to affect the activity of many membrane proteins in vitro. This mechanism has been proposed for the alteration of ion channel function in a Paramecium mutant with an abnormal membrane composition. A third mechanism is suggested by recent findings showing that the regulation of lipid metabolism and composition is important to membrane and protein traffic in cells. Additionally, lipid composition has significant effects on processes such as membrane fusion in vitro. Thus, the eas mutation might result in altered organelle integrity, membrane protein traffic, or neurotransmitter secretion (Pavlidis, 1994).
In considering all these possible mechanisms, the specificity of the eas defect is stressed. Under most conditions, eas behavior and electrophysiology are apparently largely normal, as are development and viability. This suggests a certain level of specificity of the eas defect at the cellular level. This may mean that the relevant effect of eas on cell function is restricted to particular cells or parts of cells or to specific proteins (Pavlidis, 1994).
The easily shocked gene of Drosophila encodes ethanolamine kinase (EK), the first step in phosphatidylethanolamine (PE) synthesis via the CDP-ethanolamine pathway. Flies mutant for eas display a complex neurological phenotype. This paper looked at the contribution of EK to lipid metabolism during Drosophila development with the goal of linking the eas biochemical defect with the organismal phenotype. Using a chromatography-based assay, EK activity was detected in wild-type flies throughout development. Most of the activity in the adult was present in heads, which is primarily tissue of neural origin. Flies mutant for eas showed severely reduced levels of activity at each stage assayed. Using standard extraction methods and thin layer chromatography, phospholipid composition was assayed in whole flies and in heads. While PE levels were decreased significantly in both tissues, heads also had significantly less phosphatidylserine (PS). Therefore, decreases in both phospholipids may play a role in producing the aberrant phenotype in eas flies (Nyako, 2001).
Search PubMed for articles about Drosophila Easily shocked
Benzer, S. (1971). From the gene to behavior. JAMA 218: 1015-1022. PubMed ID: 4942064
Bladergroen, B A. and van Golde, L. M. (1997). CTP: phosphoethanolamine cytidylyltransferase. Biochim. Biophys. Acta 1348: 91-99. PubMed ID: 9370320
Boquet, I., et al. (2000). Central brain postembryonic development in Drosophila: implication of genes expressed at the interhemispheric junction. J. Neurobiol. 42: 33-48. PubMed ID: 10623899
Ganetzky, B. and Wu, C. F. (1982). Indirect suppression involving behavioral mutants with altered nerve excitability in Drosophila melanogaster. Genetics 100: 597-614. PubMed ID: 17246073
Gilad, G. M. and Gilad, V. H. (1984). Inhibition of ornithine decarboxylase and glutamic acid decarboxylase activities by phosphorylethanolamine and phosphorylcholine. Biochem. Biophys. Res. Commun. 122: 277-282. PubMed ID: 6743332
Granata, F., et al. (2000). Phosphocholine and phosphoethanolamine during chick embryo myogenesis: a (31)P-NMR study. Biochim. Biophys. Acta 1483: 334-342. PubMed ID: 10666568
Isabel, G., Pascual, A. and Preat, T. (2004). Exclusive consolidated memory phases in Drosophila. Science 304: 1024-1027. PubMed ID: 15143285
Kennedy, E. P. (1957). Metabolism of lipides. Annu. Rev. Biochem. 26: 119-148
Kim, K., et al. (1998). Isolation and characterization of the Saccharomyces cerevisiae EKI1 Gene Encoding Ethanolamine Kinase. J. Biol. Chem. 274: 14857-14866. PubMed ID: 10329685
Kiss, Z., et al. (1997). Ethanolamine, but not phosphoethanolamine, potentiates the effects of insulin, phosphocholine, and ATP on DNA synthesis in NIH 3T3 cells. Role of mitogen-activated protein-kinase-dependent and protein-kinase-independent mechanisms. Eur. J. Biochem. 250: 395-402. PubMed ID: 9428690
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
Malewicz, B., et al. (1998). Phosphorylation of ethanolamine, methylethanolamine, and dimethylethanolamine by overexpressed ethanolamine kinase in NIH 3T3 cells decreases the co-mitogenic effects of ethanolamines and promotes cell survival. Eur. J. Biochem. 253: 10-19. PubMed ID: 9578455
Nyako, M., et al. (2001). Tissue-specific and developmental effects of the easily shocked mutation on ethanolamine kinase activity and phospholipid composition in Drosophila melanogaster. Biochem. Genet. 39: 339-349. PubMed ID: 11758729
Pascual, A. and Preat, T. (2001). Localization of long-term memory within the Drosophila mushroom body. Science 294: 1115-1117. PubMed ID: 11691997
Pascual, A., Chaminade, M. and Préat, T. (2005). Ethanolamine kinase controls neuroblast divisions in Drosophila mushroom bodies. Dev. Biol. 280(1): 177-86. PubMed ID: 15766757
Pavlidis, P., Ramaswami, M. and Tanouye, M. A. (1994). The Drosophila easily shocked gene: a mutation in a phospholipid synthetic pathway causes seizure, neuronal failure, and paralysis. Cell 79: 23-33. PubMed ID: 7923374
Scott, E. K., Lee, T. and Luo, L. (2001). enok encodes a Drosophila putative histone acetyltransferase required for mushroom body neuroblast proliferation. Curr. Biol. 11: 99-104. PubMed ID: 11231125
Thomas, T. and Thomas, T. J. (2001). Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications. Cell. Mol. Life Sci. 58: 244-258. PubMed ID: 11289306
date revised: 20 October 2008
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