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The endoplasmic reticulum (ER) and mitochondria interact and exchange material in healthy neurons. Mutants for the ER protein Creld show strong, Parkinson’s disease-like locomotion deficits. We found that Creld facilitates phospholipid flux at the ER-mitochondria interface, thereby enabling the formation of hydrogen peroxide at respiratory complex I, which is required for dopaminergic neuron activity.

Science Advances 8(29), eabo0155 (2022). doi:10.1126/sciadv.abo0155, Published by American Association for the Advancement of Science, Washington, DC [u.a.]

The human LIPA gene encodes for the enzyme lysosomal acid lipase, which hydrolyzes cholesteryl ester and triacylglycerol. Lysosomal acid lipase deficiency results in Wolman disease and cholesteryl ester storage disease. The Drosophila genome encodes for two LIPA orthologs, Magro and Lipase 3. Magro is a gut lipase that hydrolyzes triacylglycerides, while Lipase 3 lacks characterization based on mutant phenotypes. We found previously that Lipase 3 transcription is highly induced in mutants with defects in peroxisome biogenesis, but the conditions that allow a similar induction in wildtypic flies are not known. Here we show that Lipase 3 is drastically upregulated in starved larvae and starved female flies, as well as in aged male flies. We generated a lipase 3 mutant that shows sex-specific starvation resistance and a trend to lifespan extension. Using lipidomics, we demonstrate that Lipase 3 mutants accumulate phosphatidylinositol, but neither triacylglycerol nor diacylglycerol. Our study suggests that, in contrast to its mammalian homolog LIPA, Lipase 3 is a putative phospholipase that is upregulated under extreme conditions like prolonged nutrient deprivation and aging.

Drosophila melanogaster is the most successful genetic model organism to study different human disease with a recent increased popularity to study neurological disorders. Drosophila melanogaster has a complex yet well-defined brain with defined anatomical regions with specific functions. The neuronal network in the adult brain has a structural organization highly similar to human neurons, but in a brain that is much more amenable for complex analyses. The availability of sophisticated genetic tools to study neurons permits to examine neuronal functions at the single cell level in the whole brain by confocal imaging, which does not require sections. Thus, Drosophila has been used to successfully study many neurological disorders such as Parkinson's disease and has been recently adopted to understand the complex networks leading to neurological disorders with metabolic origins such as Leigh disease and X-linked adrenoleukodystrophy (X-ALD).In this review, we will describe the genetic tools available to study neuronal structures and functions and also illustrate some limitations of the system. Finally, we will report the experimental efforts that in the past 10 years have established Drosophila melanogaster as an excellent model organism to study neurodegenerative disorders focusing on X-ALD.

The gut has a central role in digestion and nutrient absorption, but it also serves in defending against pathogens, engages in mutually beneficial interactions with commensals, and is a major source of endocrine signals. Gut homeostasis is necessary for organismal health and changes to the gut are associated with conditions like obesity and diabetes and inflammatory illnesses like Crohn’s disease. We report that peroxisomes, organelles involved in lipid metabolism and redox balance, are required to maintain gut epithelium homeostasis and renewal in Drosophila and for survival and development of the organism. Dysfunctional peroxisomes in gut epithelial cells activate Tor kinase-dependent autophagy that increases cell death and epithelial instability, which ultimately alter the composition of the intestinal microbiota, compromise immune pathways in the gut in response to infection, and affect organismal survival. Peroxisomes in the gut effectively function as hubs that coordinate responses from stress, metabolic, and immune signaling pathways to maintain enteric health and the functionality of the gut–microbe interface.

Inherited peroxisomal biogenesis disorders (PBDs) are characterized by the absence of functional peroxisomes. They are caused by mutations of peroxisomal biogenesis factors encoded by Pex genes, and result in childhood lethality. Owing to the many metabolic functions fulfilled by peroxisomes, PBD pathology is complex and incompletely understood. Besides accumulation of peroxisomal educts (like very-long-chain fatty acids [VLCFAs] or branched-chain fatty acids) and lack of products (like bile acids or plasmalogens), many peroxisomal defects lead to detrimental mitochondrial abnormalities for unknown reasons. We generated Pex19 Drosophila mutants, which recapitulate the hallmarks of PBDs, like absence of peroxisomes, reduced viability, neurodegeneration, mitochondrial abnormalities, and accumulation of VLCFAs. We present a model of hepatocyte nuclear factor 4 (Hnf4)-induced lipotoxicity and accumulation of free fatty acids as the cause for mitochondrial damage in consequence of peroxisome loss in Pex19 mutants. Hyperactive Hnf4 signaling leads to up-regulation of lipase 3 and enzymes for mitochondrial β-oxidation. This results in enhanced lipolysis, elevated concentrations of free fatty acids, maximal β-oxidation, and mitochondrial abnormalities. Increased acid lipase expression and accumulation of free fatty acids are also present in a Pex19-deficient patient skin fibroblast line, suggesting the conservation of key aspects of our findings.

Maintenance of metabolic homeostasis requires adaption of gene regulation to the cellular energy state via transcriptional regulators. Here, we identify a role of ceramide synthase (CerS) Schlank, a multiple transmembrane protein containing a catalytic lag1p motif and a homeodomain, which is poorly studied in CerSs, as a transcriptional regulator. ChIP experiments show that it binds promoter regions of lipases lipase3 and magro via its homeodomain. Mutation of nuclear localization site 2 (NLS2) within the homeodomain leads to loss of DNA binding and deregulated gene expression, and NLS2 mutants can no longer adjust the transcriptional response to changing lipid levels. This mechanism is conserved in mammalian CerS2 and emphasizes the importance of the CerS protein rather than ceramide synthesis. This study demonstrates a double role of CerS Schlank as an enzyme and a transcriptional regulator, sensing lipid levels and transducing the information to the level of gene expression.

Drosophila melanogaster is the most successful genetic model organism to study different human disease with a recent increased popularity to study neurological disorders. Drosophila melanogaster has a complex yet well-defined brain with defined anatomical regions with specific functions. The neuronal network in the adult brain has a structural organization highly similar to human neurons, but in a brain that is much more amenable for complex analyses. The availability of sophisticated genetic tools to study neurons permits to examine neuronal functions at the single cell level in the whole brain by confocal imaging, which does not require sections. Thus, Drosophila has been used to successfully study many neurological disorders such as Parkinson’s disease and has been recently adopted to understand the complex networks leading to neurological disorders with metabolic origins such as Leigh disease and X-linked adrenoleukodystrophy (X-ALD).

Free or non-esterified fatty acids are the product of lipolysis of storage fat, i.e. triacylglyceroles. When the amount of fat exceeds the capacity of lipid-storing organs, free fatty acids affect and damage other non-lipid-storing organs. This process is termed lipotoxicity. Within a cell, free fatty acids can damage mitochondria, and lipotoxicity-induced mitochondrial damage has been associated recently with Peroxisomal Biogenesis Disorders. Drosophila melanogaster has a rising popularity as a model organism for metabolic diseases, but an optimized assay for measuring free fatty acids in Drosophila tissue samples is missing. Here we present a detailed protocol highlighting technical requirements and pitfalls to determine free fatty acids in samples of Drosophila tissue. The colorimetric assay allows the reproducible and cost-efficient measurement of free fatty acids in a 96 well plate format. We used our assay to determine changes in free fatty acid levels in different developmental stages and feeding conditions, and found that larvae and adults have different patterns of free fatty acid formation during starvation. Our assay is a valuable tool in the modeling of metabolic diseases with Drosophila melanogaster.

Inherited peroxisomal biogenesis disorders (PBDs) are characterized by the absence of functional peroxisomes. They are caused by mutations of peroxisomal biogenesis factors encoded by

Biochemical experiments in mammalian cells have linked Src family kinase activity to the insulin signaling pathway. To explore the physiological link between Src and a central insulin pathway effector, we investigated the effect of different Src signaling levels on the Drosophila transcription factor dFOXO in vivo. Ectopic activation of Src42A in the starved larval fatbody was sufficient to drive dFOXO out of the nucleus. When Src signaling levels were lowered by means of loss-of-function mutations or pharmacological inhibition, dFOXO localization was shifted to the nucleus in growing animals, and transcription of the dFOXO target genes d4E-BP and dInR was induced. dFOXO loss-of-function mutations rescued the induction of dFOXO target gene expression and the body size reduction of Src42A mutant larvae, establishing dFOXO as a critical downstream effector of Src signaling. Furthermore, we provide evidence that the regulation of FOXO transcription factors by Src is evolutionarily conserved in mammalian cells., Scientific Reports, 4, ISSN:2045-2322

Antimicrobial peptides (AMPs) are conserved cationic peptides which act both as defense molecules of the host immune system and as regulators of the commensal microbiome. Expression of AMPs is induced in response to infection by the Toll and Imd pathway. Under non-infected conditions, the transcription factor dFOXO directly regulates a set of AMP expression at low levels when nutrients are limited. Here we have analyzed whether target of rapamycin (TOR), another major regulator of growth and metabolism, also modulates AMP responses in Drosophila. We found that downregulation of TOR by feeding the drug rapamycin or by overexpressing the negative TOR regulators TSC1/TSC2, resulted in a specific induction of the AMPs Diptericin (Dpt) and Metchnikowin (Mtk). In contrast, overexpression of Rheb, which positively regulates TOR led to a repression of the two AMPs. Genetic and pharmacological experiments indicate that Dpt and Mtk activation is controlled by the transcription factor Forkhead (FKH), the founding member of the FoxO family. Shuttling of FKH from the cytoplasm to the nucleus is induced in the fat body and in the posterior midgut in response to TOR downregulation. The FKH-dependent induction of Dpt and Mtk can be triggered in dFOXO null mutants and in immune-compromised Toll and IMD pathway mutants indicating that FKH acts in parallel to these regulators. Together, we have discovered that FKH is the second conserved member of the FoxO family cross-regulating metabolism and innate immunity. dFOXO and FKH, which are activated upon downregulation of insulin or TOR activities, respectively, act in parallel to induce different sets of AMPs, thereby modulating the immune status of metabolic tissues such as the fat body or the gut in response to the oscillating energy status of the organism.

Metabolic homeostasis is fundamental for the development and the survival of animals. It requires the proper functioning of pathways that control the sensing and processing of nutrients, the storage and mobilization of energy. Recent data demonstrate that energy homeostasis and immune responses are tightly connected and that inaccurate metabolic regulation can adversely influence immune functions. Dysfunctions of the immune system have been demonstrated to underlie many chronic metabolic diseases, including diabetes, metabolic syndrome and atherosclerosis. The molecular mechanisms behind the cross-regulation of metabolism and immunity in health and disease are only beginning to emerge. We use the model organism Drosophila melanogaster to gain insights into evolutionary conserved mechanisms by which energy homeostasis and innate immunity interact.

Forkhead transcription factors of the FoxO subfamily regulate gene expression programs downstream of the insulin signaling network. It is less clear which proteins mediate transcriptional control exerted by Target of rapamycin (TOR) signaling, but recent studies in nematodes suggest a role for FoxA transcription factors downstream of TOR. In this study we present evidence that outlines a similar connection in Drosophila, in which the FoxA protein Fork head (FKH) regulates cellular and organismal size downstream of TOR. We find that ectopic expression and targeted knockdown of FKH in larval tissues elicits different size phenotypes depending on nutrient state and TOR signaling levels. FKH overexpression has a negative effect on growth under fed conditions, and this phenotype is not further exacerbated by inhibition of TOR via rapamycin feeding. Under conditions of starvation or low TOR signaling levels, knockdown of FKH attenuates the size reduction associated with these conditions. Subcellular localization of endogenous FKH protein is shifted from predominantly cytoplasmic on a high-protein diet to a pronounced nuclear accumulation in animals with reduced levels of TOR or fed with rapamycin. Two putative FKH target genes, CG6770 and cabut, are transcriptionally induced by rapamycin or FKH expression, and silenced by FKH knockdown. Induction of both target genes in heterozygous TOR mutant animals is suppressed by mutations in fkh. Furthermore, TOR signaling levels and FKH impact on transcription of the dFOXO target gene d4E-BP, implying a point of crosstalk with the insulin pathway. In summary, our observations show that an alteration of FKH levels has an effect on cellular and organismal size, and that FKH function is required for the growth inhibition and target gene induction caused by low TOR signaling levels., PLoS ONE, 5 (12), ISSN:1932-6203