A Novel Drosophila-based Drug Repurposing Platform Identified Fingolimod As a Potential Therapeutic for TDP-43 Proteinopathy.

Pathogenic changes to TAR DNA-binding protein 43 (TDP-43) leading to alteration of its homeostasis are a common feature shared by several progressive neurodegenerative diseases for which there is no effective therapy. Here, we developed Drosophila lines expressing either wild type TDP-43 (WT) or that carrying an Amyotrophic Lateral Sclerosis /Frontotemporal Lobar Degeneration-associating G384C mutation that recapitulate several aspects of the TDP-43 pathology. To identify potential therapeutics for TDP-43-related diseases, we implemented a drug repurposing strategy that involved three consecutive steps. Firstly, we evaluated the improvement of eclosion rate, followed by the assessment of locomotive functions at early and late developmental stages. Through this approach, we successfully identified fingolimod, as a promising candidate for modulating TDP-43 toxicity. Fingolimod exhibited several beneficial effects in both WT and mutant models of TDP-43 pathology, including post-transcriptional reduction of TDP-43 levels, rescue of pupal lethality, and improvement of locomotor dysfunctions. These findings provide compelling evidence for the therapeutic potential of fingolimod in addressing TDP-43 pathology, thereby strengthening the rationale for further investigation and consideration of clinical trials. Furthermore, our study demonstrates the utility of our Drosophila-based screening pipeline in identifying novel therapeutics for TDP-43-related diseases. These findings encourage further scale-up screening endeavors using this platform to discover additional compounds with therapeutic potential for TDP-43 pathology.

Gene expression of PLAT and ATS3 proteins increases plant resistance to insects.

MAIN CONCLUSION: Genes of the PLAT protein family, including PLAT and ATS3 subfamilies of higher plants and homologs of liverwort, are involved in plant defense against insects. Laticifer cells in plants contain large amounts of anti-microbe or anti-insect proteins and are involved in plant defense against biotic stresses. We previously found that PLAT proteins accumulate in laticifers of fig tree (Ficus carica) at comparable levels to those of chitinases, and the transcript level of ATS3, another PLAT domain-containing protein, is highest in the transcriptome of laticifers of Euphorbia tirucalli. In this study, we investigated whether the PLAT domain-containing proteins are involved in defense against insects. Larvae of the lepidopteran Spodoptera litura showed retarded growth when fed with Nicotiana benthamiana leaves expressing F. carica PLAT or E. tirucalli ATS3 genes, introduced by agroinfiltration using expression vector pBYR2HS. Transcriptome analysis of these leaves indicated that ethylene and jasmonate signaling were activated, leading to increased expression of genes for PR-1, ß-1,3-glucanase, PR5 and trypsin inhibitors, suggesting an indirect mechanism of PLAT- and ATS3-induced resistance in the host plant. Direct cytotoxicity of PLAT and ATS3 to insects was also possible because heterologous expression of the corresponding genes in Drosophila melanogaster caused apoptosis-mediated cell death in this insect. Larval growth retardation of S. litura occurred when they were fed radish sprouts, a good host for agroinfiltration, expressing any of nine homologous genes of dicotyledon Arabidopsis thaliana, monocotyledon Brachypodium distachyon, conifer Picea sitchensis and liverwort Marchantia polymorpha. Of these nine genes, the heterologous expression of A. thaliana AT5G62200 and AT5G62210 caused significant increases in larval death. These results indicated that the PLAT protein family has largely conserved anti-insect activity in the plant kingdom (249 words).

Control of tissue size and development by a regulatory element in the yorkie 3'UTR.

Regulation of the Hippo pathway via phosphorylation of Yorkie (Yki), the Drosophila homolog of human Yes-associated protein 1, is conserved from Drosophila to humans. Overexpression of a non-phosphorylatable form of Yki induces severe overgrowth in adult fly eyes. Here, we show that yki mRNA associates with microsomal fractions and forms foci that partially colocalize to processing bodies in the vicinity of endoplasmic reticulum. This localization is dependent on a stem-loop (SL) structure in the 3' untranslated region of yki. Surprisingly, expression of SL deleted yki in eye imaginal discs also results in severe overgrowth phenotypes. When the structure of the SL is disrupted, Yki protein levels increase without a significant effect on RNA levels. When the SL is completely removed, protein levels drastically increase, but in this case, due to increased RNA stability. In the latter case, we show that the increased RNA accumulation is due to removal of a putative miR-8 seed sequence in the SL. These data demonstrate the function of two novel regulatory mechanisms, both controlled by the yki SL element, that are essential for proper Hippo pathway mediated growth regulation.

The Histone Deacetylase Gene Rpd3 Is Required for Starvation Stress Resistance.

Epigenetic regulation in starvation is important but not fully understood yet. Here we identified the Rpd3 gene, a Drosophila homolog of histone deacetylase 1, as a critical epigenetic regulator for acquiring starvation stress resistance. Immunostaining analyses of Drosophila fat body revealed that the subcellular localization and levels of Rpd3 dynamically changed responding to starvation stress. In response to starvation stress, the level of Rpd3 rapidly increased, and it accumulated in the nucleolus in what appeared to be foci. These observations suggest that Rpd3 plays a role in regulation of rRNA synthesis in the nucleolus. The RT-qPCR and ChIP-qPCR analyses clarified that Rpd3 binds to the genomic region containing the rRNA promoters and activates rRNA synthesis in response to starvation stress. Polysome analyses revealed that the amount of polysomes was decreased in Rpd3 knockdown flies under starvation stress compared with the control flies. Since the autophagy-related proteins are known to be starvation stress tolerance proteins, we examined autophagy activity, and it was reduced in Rpd3 knockdown flies. Taken together, we conclude that Rpd3 accumulates in the nucleolus in the early stage of starvation, upregulates rRNA synthesis, maintains the polysome amount for translation, and finally increases stress tolerance proteins, such as autophagy-related proteins, to acquire starvation stress resistance.

Structural characterization and subcellular localization of Drosophila organic solute carrier partner 1.

BACKGROUND: Organic solute carrier partner 1 (OSCP1) is known to facilitate the transport of various organic solutes into cells and reported to play a role in cell growth and cell differentiation. Moreover, OSCP1 is known as a tumor suppressor gene that is frequently down-expressed in nasopharyngeal carcinomas and acute myeloid leukemia. However, the underlying mechanisms of action remain unclear and the subcellular localization of OSCP1 has yet to be determined in detail. RESULTS: Drosophila contains a single orthologue of OSCP1 (dOSCP1) that shares 58% homology with its human counterpart. To study the expression pattern and subcellular localization of dOSCP1, we prepared a specific antibody. Subcellular localization analyses of dOSCP1 with these revealed localization in the plasma membrane, endoplasmic reticulum, Golgi apparatus and mitochondria, but no detection in cytosol. dOSCP1 signals were also detected in the nucleus, although at weaker intensity than in plasma membranes and subcellular organelles. In addition, native polyacrylamide gel electrophoresis analysis with and without ß-mercaptoethanol treatment revealed that recombinant dOSCP1 forms dimers and trimers in solution. The dimer form of dOSCP1 could also be detected by Western immunoblot analyses in third instar larval extracts. CONCLUSIONS: The data revealed that dOSCP1 localizes not only in the plasma membrane but also in the nucleus, ER, Golgi apparatus and mitochondria. It is therefore conceivable that this protein may interact with various partners or form multimeric complexes with other proteins to play multiple roles in cells, providing clues to understanding the functions of dOSCP1 during Drosophila development.