Modeling Mucopolysaccharidosis Type II in the Fruit Fly by Using the RNA Interference Approach.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder that occurs due to the deficit of the lysosomal enzyme iduronate 2-sulfatase (IDS) that leads to the storage of the glycosaminoglycan heparan- and dermatan-sulfate in all organs and tissues. It is characterized by important clinical features and the severe form presents with a heavy neurological involvement. However, almost nothing is known about the neuropathogenesis of MPS II. To address this issue, we developed a ubiquitous, neuronal, and glial-specific knockdown model in Drosophila melanogaster by using the RNA interference (RNAi) approach. Knockdown of the Ids/CG12014 gene resulted in a significant reduction of the Ids gene expression and enzymatic activity. However, glycosaminoglycan storage, survival, molecular markers (Atg8a, Lamp1, Rab11), and locomotion behavior were not affected. Even strongly reduced, IDS-activity was enough to prevent a pathological phenotype in a MPS II RNAi fruit fly. Thus, a Drosophila MPS II model requires complete abolishment of the enzymatic activity.

Ceramide Synthase Schlank Is a Transcriptional Regulator Adapting Gene Expression to Energy Requirements.

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.

Nuclear Drosophila CerS Schlank regulates lipid homeostasis via the homeodomain, independent of the lag1p motif.

Drosophila Ceramide Synthase (CerS) Schlank regulates both ceramide synthesis and fat metabolism. Schlank contains a catalytic lag1p motif and, like many CerS in other species, a homeodomain of unknown function. Here, we show that the Drosophila CerS Schlank is imported into the nucleus and requires two nuclear localization signals (NLSs) within its homeodomain and functional Importin-ß import machinery. Expression of Schlank variants containing the homeodomain without functional lag1p motif rescued the fat metabolism phenotype of schlank mutants whereas a variant with a mutated NLS site did not rescue. Thus, the homeodomain of Schlank is involved in the regulation of lipid metabolism independent of the catalytic lag1p motif.

Schlank, a member of the ceramide synthase family controls growth and body fat in Drosophila.

Ceramide synthases are highly conserved transmembrane proteins involved in the biosynthesis of sphingolipids, which are essential structural components of eukaryotic membranes and can act as second messengers regulating tissue homeostasis. However, the role of these enzymes in development is poorly understood due to the lack of animal models. We identified schlank as a new Drosophila member of the ceramide synthase family. We demonstrate that schlank is involved in the de novo synthesis of a broad range of ceramides, the key metabolites of sphingolipid biosynthesis. Unexpectedly, schlank mutants also show reduction of storage fat, which is deposited as triacylglyerols in the fat body. We found that schlank can positively regulate fatty acid synthesis by promoting the expression of sterol-responsive element-binding protein (SREBP) and SREBP-target genes. It further prevents lipolysis by downregulating the expression of triacylglycerol lipase. Our results identify schlank as a new regulator of the balance between lipogenesis and lipolysis in Drosophila. Furthermore, our studies of schlank and the mammalian Lass2 family member suggest a novel role for ceramide synthases in regulating body fat metabolism.

Gap junction channel protein innexin 2 is essential for epithelial morphogenesis in the Drosophila embryo.

Direct communication of neighboring cells by gap junction channels is essential for the development of tissues and organs in the body. Whereas vertebrate gap junctions are composed of members of the connexin family of transmembrane proteins, in invertebrates gap junctions consist of Innexin channel proteins. Innexins display very low sequence homology to connexins. In addition, very little is known about their cellular role during developmental processes. In this report, we examined the function and the distribution of Drosophila Innexin 2 protein in embryonic epithelia. Both loss-of-function and gain-of-function innexin 2 mutants display severe developmental defects due to cell death and a failure of proper epithelial morphogenesis. Furthermore, immunohistochemical analyses using antibodies against the Innexins 1 and 2 indicate that the distribution of Innexin gap junction proteins to specific membrane domains is regulated by tissue specific factors. Finally, biochemical interaction studies together with genetic loss- and gain-of-function experiments provide evidence that Innexin 2 interacts with core proteins of adherens and septate junctions. This is the first study, to our knowledge, of cellular distribution and protein-protein interactions of an Innexin gap junctional channel protein in the developing epithelia of Drosophila.

The Drosophila gap junction channel gene innexin 2 controls foregut development in response to Wingless signalling.

In invertebrates, the direct communication of neighbouring cells is mediated by gap junctions, which are composed of oligomers of the innexin family of transmembrane proteins. Studies of the few known innexin mutants in Drosophila and C. elegans have shown that innexin proteins, which are structurally analogous to the connexins in vertebrates, play a major structural role as gap junctional core components in electric signal transmission. We show that Drosophila innexin 2 mutants display a feeding defect that originates from a failure of epithelial cells to migrate and invaginate during proventriculus organogenesis. The proventriculus is a valve-like organ that regulates food passage from the foregut into the midgut. Immunohistological studies indicate that innexin 2 is functionally required to establish a primordial structure of the proventriculus, the keyhole, during the regionalisation of the embryonic foregut tube, which is under the control of Wingless and Hedgehog signalling. Our genetic lack- and gain-of-function studies, and experiments in Dorsophila tissue culture cells provide strong evidence that innexin 2 is a target gene of Wingless signalling in the proventricular cells. This is the first evidence, to our knowledge, that an invertebrate gap junction gene controls epithelial tissue and organ morphogenesis in response to the conserved WNT signalling cascade.