InteractiveFly: GeneBrief

Acyl-CoA synthetase long-chain: Biological Overview | References

Nervous system development and function are tightly regulated by metabolic processes, including the metabolism of lipids such as fatty acids (FAs). Mutations in long-chain acyl-CoA synthetase 4 (ACSL4) are associated with non-syndromic intellectual disabilities. A previous study reported that Acsl, the Drosophila ortholog of mammalian ACSL3 and ACSL4, inhibits neuromuscular synapse growth by suppressing transforming growth factor-beta/bone morphogenetic protein (BMP) signaling. This study reports that Acsl regulates the composition of FAs and membrane lipid, which in turn affect neuromuscular junction (NMJ) synapse development. Acsl mutant brains had decreased abundance of C16:1 fatty acyls; restoration of Acsl expression abrogated NMJ overgrowth and the increase in BMP signaling. A lipidomic analysis revealed that Acsl suppressed the levels of three lipid raft components in the brain, including mannosyl glucosylceramide (MacCer), phosphoethanolamine ceramide, and ergosterol. MacCer level was elevated in Acsl mutant NMJs and along with sterol promoted NMJ overgrowth but was not associated with the increase in BMP signaling in the mutants. These findings suggest that Acsl inhibits NMJ growth by stimulating C16:1 and concomitantly suppressing raft-associated lipid levels (Huang, 2016).

Lipids are essential membrane components that have crucial roles in neural development and function. Dysregulation of lipid metabolism underlies a wide range of human neurological diseases including neurodegeneration and intellectual disability. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is the first gene in fatty acid metabolism associated with non-syndromic intellectual disability. ACSL4 protein has two variants: a ubiquitously expressed short form, and a brain-specific long form that is highly expressed in the hippocampus, a crucial region for memory. Indeed, ACSL4 has been shown to play an important role in synaptic spine formation. However, it is unclear how mutations in ACSL4 lead to intellectual disability (Huang, 2016).

There are 26 genes encoding acyl-CoA synthetases (ACSs) in humans. Each of the enzymes has distinct substrate preferences for fatty acid with various lengths of aliphatic carbon chains. In contrast, there are 13 ACS genes in the Drosophila genome (Watkins, 2007). ACSs convert free fatty acids into acyl-CoAs for lipid synthesis, fatty acid degradation or membrane lipid remodeling (Mashek, 2007). For example, ACSL4 converts long-chain fatty acids (LCFAs; aliphatic tails longer than 12 carbons), preferentially arachidonic acid (C20:4), into LCFA-CoAs that are incorporated into glycerol-phospholipids (GPLs) and neutral lipids in non-neuronal cells. The mechanism of how fatty acids and fatty-acid-modifying enzymes affect lipid composition, and thereby modulate development processes, is beginning to be understood in lower model organisms (Kniazeva, 2012; Zhang, 2011; Zhu, 2013; Huang, 2016 and references therein).

Synaptic growth is required for normal brain function such as learning and memory. Many neurological disorders including intellectual disability are associated with synaptic defects. The Drosophila neuromuscular junction (NMJ) is a powerful system for studying the mechanisms that regulate synaptic growth. Drosophila Acsl, also known as dAcsl, is the ortholog of mammalian ACSL3 and ACSL4 (Zhang, 2009). It has been reported that Acsl affects axonal transport of synaptic vesicles and inhibits NMJ growth by inhibiting bone morphogenetic protein (BMP) signaling (Liu, 2011; Liu, 2014). However, how Acsl affects lipid metabolism and the role of Acsl-regulated lipid metabolism in synapse development are largely unknown (Huang, 2016).

This study demonstrates that Acsl positively regulates the abundance of the LCFA palmitoleic acid (C16:1) in the brain. Reduced levels of C16:1 in Acsl mutants led to NMJ overgrowth and enhanced BMP signaling. A lipidomic analysis revealed that mannosyl glucosylceramide (MacCer), phosphoethanolamine ceramide (CerPE, the Drosophila analog of sphingomyelin) and ergosterol levels were increased in Acsl mutant brains. Genetic and pharmacological analyses further showed that the increased level of MacCer and sterol underlie the NMJ overgrowth in Acsl mutants in a pathway parallel to BMP signaling. These results indicate that Acsl regulates fatty acid and sphingolipid levels to modulate growth signals and NMJ growth, providing insight into the pathogenesis of ACSL4-related intellectual disability (Huang, 2016).

Like ACSL4, Acsl primarily associates with the ER and facilitates fatty acid incorporation into lipids (Golej, 2011; Küch, 2014; Meloni, 2009; Zhang, 2009); impairment of Acsl activity reduces the abundance of its substrate LCFAs in lipids. Although this study did not directly determine the substrate preference of Acsl, fatty acid analysis suggests that both Acsl and ACSL4 positively regulate C16:1 abundance in Drosophila brain. The two proteins also have conserved functions in other processes, such as lipid storage (Zhang, 2009), axonal transport and synaptic development (Liu, 2014; Liu, 2011; Huang, 2016 and references therein).

The similar NMJ overgrowth in desat1 and Acsl mutants suggests that the normal fatty acid composition is essential for proper development of synapses. The rescue effect of C16:1, together with the genetic interaction between Acsl and desat1, indicates that reduced C16:1 contributes to NMJ overgrowth in Acsl mutants. It has been previously reported that the synaptic overgrowth in Acsl mutants is due in part to an elevation of BMP signaling resulting from defects in endocytic recycling and BMP receptor inactivation (Liu, 2014). Endosomes are membrane compartments that are regulated by various membrane lipids, particularly the conversion between different PtdIns species, such as PI(3)P, PI(4,5)P₂, PI(3,4,5)P₃ and so on. The current findings suggest that increased BMP signaling in Acsl mutants is associated with an imbalance in fatty acid composition, specifically, a decrease in C16:1. It is thus possible that proper fatty acid composition is necessary for the normal conversion and localization of endosomal lipids (e.g., PtdIns species), affecting endosomal recycling and BMP receptor inactivation. Future studies will examine the regulation of specific fatty acids such as C16:1 in endosomal recycling and BMP signaling (Huang, 2016).

Acsl primarily associates with the ER in multiple cell types including motor neurons and participates in lipid synthesis (O'Sullivan, 2012; Zhang, 2009). In Drosophila, most of acyl chains in GPLs are C16 and C18 LCFAs without VLCFAs. In contrast, sphingolipids contain higher levels of VLCFAs than LCFAs as acyl chains. Thus, most LCFA-CoAs are channeled into GPLs whereas VLCFA-CoAs are mainly incorporated into sphingolipids in Drosophila. This study found that Acsl positively regulates the production of C16:1-containing GPLs, as well as the level of PtdEth, the most abundant GPL in the brain. Presumably, fatty acids that are less preferred by Acsl could be channeled into lipids by other ACSs, and might show increased abundance because of a compensatory mechanism in Acsl mutants, which could contribute to the elevation in VLCFA-containing sphingolipids (Huang, 2016).

In addition to ER localization, ACSL4 and Acsl also localize to peroxisomes in a few non-neuronal cells, suggesting a role for these proteins in the activation of VLCFAs for peroxisomal degradation. Indeed, in animal models and patients with impaired peroxisomal function, accumulation of VLCFAs or increased levels of lipid species with longer fatty acid chains is observed. Thus, a defect in peroxisomal VLCFA degradation might underlie the elevation in sphingolipid species with VLCFA chains in Acsl mutant brains (Huang, 2016).

Alternatively, Acsl might affect lipid composition through a pathway that is independent of fatty acid incorporation. For example, as degradation of sphingolipids occurs primarily within lysosomes, a defect in lysosomal degradation might lead to an accumulation of sphingolipids and sterols in Acsl mutants. In addition, fatty acids and acyl-CoAs are ligands of many transcription factors, and ACSL3 activates the transcription of lipogenic genes in rat hepatocytes. Thus, Acsl might transcriptionally regulate genes encoding enzymes involved in the metabolism of lipids such as MacCer, CerPE and PtdEth. However, the detailed mechanism of how Acsl affects lipid class composition, especially the downregulation of raft-related MacCer and CerPE by Acsl in the nervous system, remains to be elucidated (Huang, 2016).

The data showed that elevation of the raft-related lipids MacCer and sterol facilitate NMJ overgrowth in Acsl mutants. Moreover, MacCer promotes bouton formation in a pathway parallel to BMP signaling, at least in part. It is unclear how these raft-related lipids regulate synaptic growth. It is likely that MacCer and sterol might interact with raft-associated growth signaling pathways. Larval NMJ development is mediated by multiple growth factors and downstream signaling cascades. For instance, Wingless (Wg) (Wnt1 in mammals) is a raft-associated protein that activates signaling pathways essential for NMJ growth and synaptic differentiation. It is therefore possible that the level or activity of some raft-associated growth factors is increased in Acsl mutants, thereby promoting NMJ overgrowth. Further investigation is needed to dissect the regulatory mechanisms of raft-related lipids, particularly MacCer, in promoting synaptic growth and bouton formation. It also would be of interest to address how Acsl-regulated lipids regulate neurotransmission in conjunction with synapse development (Huang, 2016).

dAcsl, the Drosophila ortholog of acyl-CoA synthetase long-chain family member 3 and 4, inhibits synapse growth by attenuating bone morphogenetic protein signaling via endocytic recycling

Fatty acid metabolism plays an important role in brain development and function. Mutations in acyl-CoA synthetase long-chain family member 4 (ACSL4), which converts long-chain fatty acids to acyl-CoAs, result in nonsyndromic X-linked mental retardation. ACSL4 is highly expressed in the hippocampus, a structure critical for learning and memory. However, the underlying mechanism by which mutations of ACSL4 lead to mental retardation remains poorly understood. This study reports that dAcsl, the Drosophila ortholog of ACSL4 and ACSL3, inhibits synaptic growth by attenuating BMP signaling, a major growth-promoting pathway at neuromuscular junction (NMJ) synapses. Specifically, dAcsl mutants exhibited NMJ overgrowth that was suppressed by reducing the doses of the BMP pathway components, accompanied by increased levels of activated BMP receptor Thickveins (Tkv) and phosphorylated Mothers against decapentaplegic (Mad), the effector of the BMP signaling at NMJ terminals. In addition, Rab11, a small GTPase involved in endosomal recycling, was mislocalized in dAcsl mutant NMJs, and the membrane association of Rab11 was reduced in dAcsl mutant brains. Consistently, the BMP receptor Tkv accumulated in early endosomes but reduced in recycling endosomes in dAcsl mutant NMJs. dAcsl was also required for the recycling of photoreceptor rhodopsin in the eyes, implying a general role for dAcsl in regulating endocytic recycling of membrane receptors. Importantly, expression of human ACSL4 rescued the endocytic trafficking and NMJ phenotypes of dAcsl mutants. Together, these results reveal a novel mechanism whereby dAcsl facilitates Rab11-dependent receptor recycling and provide insights into the pathogenesis of ACSL4-related mental retardation (Liu, 2014).

Endocytosis of BMP receptors is considered to be an efficient mechanism for limiting the amount of surface receptors. Thus, a possible explanation for the elevated BMP signaling in dAcsl mutants is that dAcsl inhibits endocytosis of BMP receptors. However, this study found no defect in synaptic vesicle (SV) endocytosis as detected by FM1-43 uptake. Additionally, dAcsl boutons lack the typical ultrastructural features of endocytic mutants, including a low density of SVs, enlarged SVs, and an accumulation of endocytic intermediates. Thus, dAcsl does not regulate SV endocytosis, although the possibility of a defect in receptor-mediated endocytosis at dAcsl NMJ synapses cannot be formally ruled out (Liu, 2014).

BMP signaling can also be attenuated by targeting active receptors to lysosomes for degradation. However, in contrast to mutants with defects in lysosomal degradation, which show enlarged early or late endosomal compartments, the distribution of endosomal markers was normal at dAcsl NMJ synapses. Thus, endosomal trafficking from early endosomes to lysosomes appears unaffected in dAcsl mutant NMJs. Furthermore, in contrast to the studies in cultured human cells, where knockdown of ACSL4 results in delayed degradation of EGFR, the Tkv protein level at dAcsl synapses was comparable with the control, suggesting that dAcsl may not affect Tkv degradation (Liu, 2014).

Instead, multiple lines of evidence support a model in which dAcsl attenuates BMP signaling by promoting Rab11-dependent recycling and inactivation of BMP receptors. First, the level of activated but not the total Tkv receptors was increased in dAcsl NMJs. In addition, the colocalization of Tkv receptors with the recycling endosome marker Rab11 was reduced, whereas that with early endosomal markers Rab5 and Avl/Syx7 was increased in dAcsl mutants, suggesting that the trafficking of BMP receptors from early endosomes to recycling endosomes is impaired. Second, the localization of Rab11 at synaptic boutons and the membrane-cytosol cycle of Rab11 in the nervous system were altered in dAcsl mutants. It is speculated that the expanded staining pattern of Rab11 at NMJs may be caused by the increased level of cytosolic Rab11. Consistent with this possibility, a mutated Rab5A, which lacks the membrane anchor, completely loses endosomal localization and instead shows a cytosolic and nuclear distribution in Cos-7 cells, and dissociation of Rab11 from membrane redistributes Rab11 to the cytoplasm of CHO cells. The membrane association of Rab proteins is mediated by multiple interacting proteins, such as Rab escort proteins and Rab GDP-dissociation inhibitors, and by post-translational covalent attachment of prenyl groups to Rabs. Prenylation of Rabs leads to a mobility change in PAGE. However, the mobility of Rab11 in polyacrylamide gels was normal, suggesting that the prenylation of Rab11 is normal in dAcsl mutants. Thus, it is possible that a regulatory step involved in protein-protein interactions, protein-lipid interactions, or conformational changes of Rab11 might be altered in dAcsl mutants. Third, dAcsl synergistically interacts with rab11 to restrict synaptic overgrowth by attenuating BMP signaling. Finally, dAcsl also promotes recycling of the photoreceptor rhodopsin to the rhabdomere in the eye. Together, these data indicate that dAcsl regulates Rab11-dependent recycling and inactivation of the BMP receptor Tkv in different neuronal cell types (Liu, 2014).

How does dAcsl affect the Rab11-mediated endocytic recycling of membrane receptors? ACSL4 converts long-chain fatty acids to acyl-CoA esters. The synthetase activity of disease-causing mutations in ACSL4 is significantly decreased, suggesting that reduction of the enzyme activity of ACSL4 is responsible for the pathogenesis of ACSL4-related mental retardation. Indeed, expression of these mutated ACSL4 failed to rescue the mistargeting of the retinal axons in the brains, the NMJ growth defects, and the altered Rab11 localization in dAcsl mutants. These data suggest that the acyl-CoA synthetase activity may be responsible for the endocytic recycling defects. ACSL4 has a strong preference for polyunsaturated fatty acids, such as arachidonic acid (C20:4) and eicosatetraenoic acid (C20:5). Reduced incorporation of C20:4 and C20:5 into phospholipids may change membrane properties, such as bilayer fluidity and membrane protein targetingh, and may underlie the decreased membrane association of Rab11. The altered membrane association of Rab11, but not Rab5, suggests that dAcsl might regulate lipid composition of specific membrane compartments. Indeed, lipid composition specifically affects membrane association of different Rabs. In addition to the enrichment of dAcsl in ER, the major site for lipid biosynthesis, colocalization was observed between dAcsl and Tkv-GFP, as well as the endosomal markers Rab5 and Rab11 at NMJs, indicating that dAcsl might act locally in regulating endocytic trafficking. The exact mechanisms by which dAcsl regulates Rab11-mediated endocytic recycling, however, remain to be determined (Liu, 2014).

Despite the obvious overgrowth of NMJs in the anterior segments, the NMJs in the posterior segments were dystrophic. This distinct bidirectional NMJ phenotype in dAcsl mutants has not been previously reported. The overgrown NMJs in the anterior segments can be readily explained by increased BMP signaling. But how are the dystrophic NMJs in the posterior segments of dAcsl mutants brought about? It is observed that the levels of pMad and activated Tkv receptors were increased, and the staining pattern of Rab11 was altered in the posterior NMJs of dAcsl mutants. Thus, the Rab11-dependent trafficking of the BMP receptor Tkv in the posterior NMJs of dAcsl mutants was also defective. However, as a result of the axonal transport defects (Liu, 2011), the increased levels of activated Tkv and pMad at the posterior NMJs and other retrograde cargoes could not be efficiently transported back to the cell bodies of motoneurons, or the anterograde transport of lipids, proteins, and organelles to the synaptic terminals is impaired, or both, resulting in malnourished posterior NMJs in dAcsl mutants. Consistent with this scenario, it has been previously shown that the longer the axons, the more susceptible the NMJ growth to axonal transport defects. Thus, the upregulated BMP signaling at NMJs across different segments and the increased susceptibility of posterior NMJs to axonal transport defects may cause the unique overgrowth-to-undergrowth phenotype of NMJs in dAcsl mutants (Liu, 2014).

In the larval brains of dAcsl mutants, the production of decapentaplegic (Dpp, a BMP-like molecule) is diminished, although dAcsl is not intrinsically required for Dpp expression. Consistent with the Dpp reduction, the number of glial cells and neurons is decreased, and the retinal axons are mistargeted in the visual cortex. In contrast to the reduced production of Dpp in the brains, elevated BMP signaling was observed at the peripheral NMJ synapses of dAcsl mutants. Thus, dAcsl regulates BMP signaling differentially in different cellular texts. The underlying mechanisms for the differential regulation of BMP pathway by dAcsl await further investigations (Liu, 2014).

In conclusion, this study provides experimental evidence implicating dAcsl in synaptic development and endocytic trafficking of BMP receptors via recycling endosomes and offers novel insight into the pathogenesis of mental retardation caused by mutations in ACSL4. Given that several genes involved in the synthesis and turnover of long-chain fatty acids affect brain development and cognitive function, these findings may also help understand the neurodevelopmental defects caused by mutations in other genes involved in lipid metabolism pathways (Liu, 2014).

Drosophila Acyl-CoA synthetase long-chain family member 4 regulates axonal transport of synaptic vesicles and is required for synaptic development and transmission

Acyl-CoA synthetase long-chain family member 4 (ACSL4) converts long-chain fatty acids to acyl-CoAs that are indispensable for lipid metabolism and cell signaling. Mutations in ACSL4 cause nonsyndromic X-linked mental retardation. Drosophila dAcsl has been shown to be functionally homologous to human ACSL4, and is required for axonal targeting in the brain. This study reports that Drosophila dAcsl mutants exhibited distally biased axonal aggregates that are immunopositive for the synaptic-vesicle proteins synaptotagmin (Syt) and cysteine-string protein, the late endosome/lysosome marker lysosome-associated membrane protein 1, the autophagosomal marker Atg8, and the multivesicular body marker Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate). In contrast, the axonal distribution of mitochondria and the cell adhesion molecule Fas II (fasciclin II) is normal. Electron microscopy revealed accumulation of prelysomes and multivesicle bodies. These aggregates appear as retrograde instead of anterograde cargos. Live imaging analysis revealed that dAcsl mutations increases the velocity of anterograde transport but reduces the flux, velocity, and processivity of retrograde transport of Syt-enhanced green fluorescent protein-labeled vesicles. Immunohistochemical and electrophysiological analyses showed significantly reduced growth and stability of neuromuscular synapses, and impaired glutamatergic neurotransmission in dAcsl mutants. The axonal aggregates and synaptic defects in dAcsl mutants were fully rescued by neuronal expression of human ACSL4, supporting a functional conservation of ACSL4 across species in the nervous system. Together, these findings demonstrate that dAcsl regulates axonal transport of synaptic vesicles and is required for synaptic development and function. Defects in axonal transport and synaptic function may account, at least in part, for the pathogenesis of ACSL4-related mental retardation (Liu 2011).

Light and electron microscopic analysis showed prominent axonal aggregates of SVs in dAcsl mutants. One possibility for the aggregate formation is a targeting or fusion defect of SVs to the synaptic terminals in dAcsl mutants. However, alterations in the expressions of mammalian β-catenin and scribble, which are required for clustering SVs at presynaptic sites, result in no axonal SV aggregates. Similarly, mutations in Drosophila exocyst components Sec5 and Sec15 involved in the secretory pathway lead to no axonal accumulations either. Therefore, it is unlikely that the axonal aggregates in dAcsl mutants result from a defect in SV targeting or fusion. Another possibility for the aggregates is a defect in protein turnover or the formation of membrane organelles in dAcsl mutants. However, it has been reported that mutants of the autophagy pathway do not show axonal aggregates. Mutants of spinster, encoding a multipass transmembrane protein involved in late endosome/lysosome function, show accumulations of membrane structures in the soma and NMJ synapses. Furthermore, this study found that LAMP1-GFP, RFP-Atg8, and Hrs were localized normally in both the cell bodies and the NMJ terminals, but accumulated specifically in the axons of dAcsl mutant motoneurons. Together, it seems unlikely that the axonal aggregates in dAcsl mutants are caused by a defect in SV targeting, protein degradation, or the formation of membrane organelles (Liu 2011).

Four independent lines of evidence support the idea that dAcsl primarily regulates retrograde transport of SVs. First, there was a distally biased accumulation of aggregates, a phenotype shared by the mutants of roadblock, which encodes a dynein-associated protein mediating retrograde transport. Consistently, motoneurons innervating the anterior muscles with shorter axons showed well developed NMJ synapses with fewer axonal aggregates. Second, immunohistochemical analysis showed that the CSP-positive aggregates in dAcsl mutants were mostly positive for the late endosome/lysosome marker LAMP1 and partly positive for the autophagosome marker Atg8. Consistently, EM analysis revealed that there were conspicuous prelysosomal bodies (PLBs) and multivesicular bodies (MVBs) in dAcsl mutant axons reminiscent of the accumulations of retrograde cargos observed on the distal side of ligated axons. These data demonstrated an accumulation of mostly retrograde organelles in dAcsl mutants. Third, the dynein subunit Dlic also accumulated and colocalized with the CSP-positive aggregates in dAcsl axons. The limited availability of Dlic could inhibit the retrograde transport of CSP-positive SVs. It would be interesting to test whether overexpression of retrograde motors such as Dlic would rescue the axonal and synaptic defects in dAcsl mutants. Finally, live imaging of Syt-eGFP-tagged vesicles in motoneurons showed that the flux, velocity, and processivity of retrograde transport were all significantly reduced in dAcsl mutants. Defective retrograde transport may contribute to the distally biased accumulation of the various axonal cargos (Liu 2011).

Axonal vesicles can associate with both anterograde kinesins and retrograde dyneins, and anterograde and retrograde transport show interdependence. Two hypotheses have been proposed to explain bidirectional transport. One is the 'tug-of-war' hypothesis in which both simultaneously active anterograde and retrograde motors move the same cargo with different efficacy. The other is the 'coordination' hypothesis in which additional factors inhibit one of the opposing motors for direction-selective transport. The unprecedented reduced retrograde velocity concomitant with enhanced anterograde transport of SVs in dAcsl mutants favors the model of tug-of-war, as the coordination model predicts a transport defect in only one direction (Liu 2011).

How dAcsl regulates axonal transport is currently unknown. The finding that transport of mitochondria was unaffected in dAcsl mutants indicates that the microtubules along which mitochondria move and the energy supply are normal, though the microtubules detected by anti-Futsch staining in the synaptic terminals retract or disappear in dAcsl mutants . There are robust genetic interactions among motors and their interacting proteins. However, no obvious genetic interaction was found between dAcsl and motor genes such as Khc, roadblock, or p150^glued in regulating the axonal aggregates, suggesting that motors and their interacting proteins were not substantially compromised in dAcsl mutants. Still, there are at least two possible explanations for the transport defects observed in dAcsl mutants. dAcsl mutations that reduce levels of neutral lipids could alter the lipid composition of the cargo membranes, resulting in abnormal anchorage of motors to membranous cargos. Previous studies showed that binding of dynein-dynactin to the acidic phospholipids phosphatidic acid or phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] in vesicle membranes requires spectrin. Knockdown of spectrin or expression of a mutated spectrin in motoneurons caused axonal aggregates and slowed and erratic transport of SVs in Drosophila. It would be interesting to test whether dAcsl and spectrin interact in regulating axonal transport. Alternatively, dAcsl, acting in the first step of lipid metabolism, could alter the levels of certain lipid-related signaling molecules, which in turn regulate axonal transport (Liu 2011).

Analyses of mental dysfunction-related ACSl4 in Drosophila reveal its requirement for Dpp/BMP production and visual wiring in the brain

Long-chain acyl-CoA synthetases (ACSLs) convert long-chain fatty acids to acyl-CoAs, the activated substrates essential in various metabolic and signaling pathways. Mutations in ACSL4 are associated with non-syndromic X-linked mental retardation (MRX). However, the developmental functions of ACSL4 and how it is involved in the pathogenesis of MRX remain largely unknown. The Drosophila ACSL-like protein is highly homologous to human ACSL3 and ACSL4, and it has been designated as dAcsl. This study demonstrated that dAcsl and ACSL4 are highly conserved in terms of ACSL4's ability to substitute the functions of dAcsl in organismal viability, lipid storage and the neural wiring in visual center. In neurodevelopment, decapentaplegic (Dpp, a BMP-like molecule) production diminished specifically in the larval brain of dAcsl mutants. Consistent with the Dpp reduction, the number of glial cells and neurons dramatically decreased and the retinal axons mis-targeted in the visual cortex. All these defects in Drosophila brain were rescued by the wild-type ACSL4 but not by the mutant products found in MRX patients. Interestingly, expression of an MRX-associated ACSL4 mutant form in a wild-type background led to the lesions in visual center, suggesting a dominant negative effect. These findings validate Drosophila as a model system to reveal the connection between ACSL4 and BMP pathway in neurodevelopment, and to infer the pathogenesis of ACSL4-related MRX (Zhang, 2009).

Using various analyses in Drosophila development, this study illustrated the functional conservation between ACSL4 and its fly homolog. The larval optic lobe, in particular, provides an accessible, sensitive and relevant readout to reveal the potential functions of ACSL4 in neural development. The Drosophila homolog of ACSL4 was found to be required in the brain optic lobe for the Dpp/BMP production, for the formation of properly aligned glia and neurons, and for the accurate targeting of retinal axons. The correlation between the MRX-associated molecular changes in ACSL4 and their disruption of ACSL4 activity in these assays implies how this protein functions in development, and thus how the mutant products may lead to mental disorder as a result of impaired development (Zhang, 2009).

How is dAcsl involved in the Dpp regulation during the development of visual center? The reduction of Dpp domain in dAcsl mutant brain can be attributed to the changes in expression levels, cell number and/or cell fate. Besides the Dpp reporters, cellular markers specific for this domain are currently lacking. Thus, the possibilities that Dpp reduction in dAcsl mutant is due to reduced cell number and/or change in cell fate, or decreased Dpp expression in each cell cannot be distinguished. This issue remains to be clarified with additional markers for Dpp-expressing cells in the larval brain (Zhang, 2009).

As indicated by the mutant clonal analysis, dAcsl is not intrinsically required for Dpp expression. This suggests that factors extrinsic to the Dpp-expressing cells may be affected in dAcsl mutants. Clusters of Dpp- and Wg-positive cells are adjacent to each other, and Wg has been demonstrated to be the upstream signal required for Dpp activation in the larval optic lobe. Because Wg protein in the larval brain did not seem to decrease in dAcsl mutants, it is speculated that Wg signaling could be compromised at any step post-translationally (Zhang, 2009).

Palmitate, a 16-carbon saturated fatty acid, is attached to the Wg in the secretory process, and palmitoylation is required for Wg secretion and signaling activity. This study and others have shown that ACSL4 subfamily enzymes are enriched in ER. As well as the fact that palmitate is a fatty acid of the length preferred by ACSL-family enzymes, it is conceivable that dAcsl could be involved in Wg palmitoylation. Additionally, Wg and lipoprotein particles can be co-purified and the latter is required for Wg signaling in Drosophila wing primordia. It is possible that dAcsl contributes to the make-up of the lipid components in lipoprotein particles, and thus gets involved in Wg signaling. In any case, whether or not Wg acylation, trafficking and/or signaling are altered in dAcsl mutants requires further investigations (Zhang, 2009).

From fly to mammal, BMP and WNT morphogens are crucial for cell growth, differentiation and patterning of the whole organism or various tissues including nervous system. In mammal, BMP and WNT are expressed at the dorsomedial edge of each cerebral cortical hemisphere, and serve as the patterning/differentiation signals and axon guidance cues. WNT-3A is required for hippocampus formation, and BMP helps to maintain WNT signaling and hippocampal developmen. Moreover, a deletion of frizzled, one of the WNT receptors, leads to hippocampal and visuospatial learning defects in mice. Analogously, Wg and Dpp act together to pattern the visual center of Drosophila brain Analysis of human ACSL4 in Drosophila visual cortex implicates that ACSL4 could play a role in BMP activation during neurodevelopment. If the close analogy exists between fly and mammal, the reduced ACSL4 activities cause the decrease of BMP expression which would result in un-sustained WNT signaling and hippocampus malformation (Zhang, 2009).

In the families inheriting MRX, some of the female carriers heterozygous for ACSL4 mutations also display mild mental disabilities, although skewed X-inactivation was detected. This may be explained by the potential dominant negative effect of the mutations in the cells of incomplete gene silencing upon X-inactivation and/or in a tissue mosaic for different X chromosomes. In support of the former possibility, expression of ACSL4(P375L) in a wild-type background exhibited dominant negative effect and phenocopied the defects of dAcsl mutants observed in larval brain. Nevertheless, all speculations on ACSL4 and neurodevelopment ought to be tested in mammalian models (Zhang, 2009).

An interesting discrepancy observed in this study is that the defects in visual cortex formation are more severe in dAcsl mutants than in Dpp pathway mutants. It could be a combined effect of Dpp reduction and disrupted lipid metabolism in dAcsl mutants. Neurodegeneration and fatty acids accumulation were observed in the mutants of bubblegum, encoding very long-chain fatty acid CoA synthetase. Although no obvious change was detected in neutral lipids accumulation in dAcsl mutant brain, the possibility that the homeostasis of other lipids was disturbed or that critical but subtle changes occurred and exacerbated the existing lesion caused by Dpp reduction cannot be excluded (Zhang, 2009).

Applying various cellular markers in diverse tissues of Drosophila, this study has clearly illustrated the functional conservation between ACSL4 and its fly homolog, and has contrasted the wild-type and the MRX-associated ACSL4 variants in every assay ranging from lethality rescue, lipid storage, to the Dpp-induced events in visual cortex development. These findings promise the functional dissection of human ACSL4 in neurodevelopment and lipid metabolism using Drosophila experimental system. These assays can be further explored to screen for other genes involved in the process, to assess the functions of related but different mammalian products, and possibly to be utilized for drug discovery (Zhang, 2009).

Search PubMed for articles about Drosophila Acsl

Golej, D. L., Askari, B., Kramer, F., Barnhart, S., Vivekanandan-Giri, A., Pennathur, S. and Bornfeldt, K. E. (2011). Long-chain acyl-CoA synthetase 4 modulates prostaglandin E2 release from human arterial smooth muscle cells. J. Lipid. Res. 52: 782-793. PubMed ID: 21242590

Huang, Y., Huang, S., Lam, S. M., Liu, Z., Shui, G. and Zhang, Y. Q. (2016). Acsl, the Drosophila ortholog of intellectual disability-related ACSL4, inhibits synaptic growth by altered lipids. J Cell Sci 129(21):4034-4045. PubMed ID: 27656110

Kniazeva, M., Shen, H. L., Euler, T., Wang, C. and Han, M. (2012). Regulation of maternal phospholipid composition and IP3-dependent embryonic membrane dynamics by a specific fatty acid metabolic event in C. elegans. Gene Dev. 26: 554-566. PubMed ID: 22426533

Küch, E.-M., Vellaramkalayil, R., Zhang, I., Lehnen, D., Brügger, B., Sreemmel, W., Ehehalt, R., Poppelreuther, M. and Füllekrug, J. (2014). Differentially localized acyl-CoA synthetase 4 isoenzymes mediate the metabolic channeling of fatty acids towards phosphatidylinositol. Biochim. Biophys. Acta 1841: 227-239. PubMed ID: 24201376

Liu, Z., Huang, Y., Zhang, Y., Chen, D. and Zhang, Y. Q. (2011). Drosophila Acyl-CoA synthetase long-chain family member 4 regulates axonal transport of synaptic vesicles and is required for synaptic development and transmission. J Neurosci 31(6): 2052-2063. PubMed ID: 21307243

Liu, Z., Huang, Y., Hu, W., Huang, S., Wang, Q., Han, J. and Zhang, Y. Q. (2014). dAcsl, the Drosophila ortholog of acyl-CoA synthetase long-chain family member 3 and 4, inhibits synapse growth by attenuating bone morphogenetic protein signaling via endocytic recycling. J Neurosci 34(8): 2785-2796. PubMed ID: 24553921

Mashek, D. G., Li, L. O. and Coleman, R. A. (2007). Long-chain acyl-CoA synthetases and fatty acid channeling. Future Lipidol. 2: 465-476. PubMed ID: 20354580

Meloni, I., Parri, V., De Filippis, R., Ariani, F., Artuso, R., Bruttini, M., Katzaki, E., Longo, I., Mari, F., Bellan, C. (2009). The XLMR gene ACSL4 plays a role in dendritic spine architecture. Neuroscience 159: 657-669. PubMed ID: 19166906

O'Sullivan, N. C., Jahn, T. R., Reid, E. and O'Kane, C. J. (2012). Reticulon-like-1, the Drosophila orthologue of the Hereditary Spastic Paraplegia gene reticulon 2, is required for organization of endoplasmic reticulum and of distal motor axons. Hum. Mol. Genet. 21: 3356-3365. PubMed ID: 22543973

Watkins, P. A., Maiguel, D., Jia, Z. and Pevsner, J. (2007). Evidence for 26 distinct acyl-coenzyme A synthetase genes in the human genome. J. Lipid Res. 48: 2736-2750. PubMed ID: 17762044

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date revised: 10 February 2017

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