Flight to insight: maximizing the potential of Drosophila models of C9orf72-FTD.

Advancements in understanding the pathogenesis of C9orf72-associated frontotemporal dementia (C9orf72-FTD) have highlighted the role of repeat-associated non-ATG (RAN) translation and dipeptide repeat proteins (DPRs), with Drosophila melanogaster models providing valuable insights. While studies have primarily focused on RAN translation and DPR toxicity, emerging areas of investigation in fly models have expanded to neuronal dysfunction, autophagy impairment, and synaptic dysfunction, providing potential directions for new therapeutic targets and mechanisms of neurodegeneration. Despite this progress, there are still significant gaps in Drosophila models of C9orf72-FTD, namely in the areas of metabolism and circadian rhythm. Metabolic dysregulation, particularly lipid metabolism, autophagy, and insulin signaling, has been implicated in disease progression with findings from animal models and human patients with C9orf72 repeat expansions. Moreover, circadian disruptions have been observed in C9of72-FTD, with alterations in rest-activity patterns and cellular circadian machinery, suggesting a potential role in disease pathophysiology. Drosophila models offer unique opportunities to explore these aspects of C9orf72-FTD and identify novel therapeutic targets aimed at mitigating neurodegeneration.

Drosophila TRIM32 cooperates with glycolytic enzymes to promote cell growth.

Cell growth and/or proliferation may require the reprogramming of metabolic pathways, whereby a switch from oxidative to glycolytic metabolism diverts glycolytic intermediates towards anabolic pathways. Herein, we identify a novel role for TRIM32 in the maintenance of glycolytic flux mediated by biochemical interactions with the glycolytic enzymes Aldolase and Phosphoglycerate mutase. Loss of Drosophila TRIM32, encoded by thin (tn), shows reduced levels of glycolytic intermediates and amino acids. This altered metabolic profile correlates with a reduction in the size of glycolytic larval muscle and brain tissue. Consistent with a role for metabolic intermediates in glycolysis-driven biomass production, dietary amino acid supplementation in tn mutants improves muscle mass. Remarkably, TRIM32 is also required for ectopic growth - loss of TRIM32 in a wing disc-associated tumor model reduces glycolytic metabolism and restricts growth. Overall, our results reveal a novel role for TRIM32 for controlling glycolysis in the context of both normal development and tumor growth.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains.

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community's investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer's two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

A novel ex vivo method for measuring whole brain metabolism in model systems.

BACKGROUND: Many neuronal and glial diseases have been associated with changes in metabolism. Therefore, metabolic reprogramming has become an important area of research to better understand disease at the cellular level, as well as to identify targets for treatment. Model systems are ideal for interrogating metabolic questions in a tissue dependent context. However, while new tools have been developed to study metabolism in cultured cells there has been less progress towards studies in vivo and ex vivo. NEW METHOD: We have developed a method using newly designed tissue restraints to adapt the Agilent XFe96 metabolic analyzer for whole brain analysis. These restraints create a chamber for Drosophila brains and other small model system tissues to reside undisrupted, while still remaining in the zone for measurements by sensor probes. RESULTS: This method generates reproducible oxygen consumption and extracellular acidification rate data for Drosophila larval and adult brains. Single brains are effectively treated with inhibitors and expected metabolic readings are observed. Measuring metabolic changes, such as glycolytic rate, in transgenic larval brains demonstrates the potential for studying how genotype affects metabolism. COMPARISON WITH EXISTING METHODS AND CONCLUSIONS: Current methodology either utilizes whole animal chambers to measure respiration, not allowing for targeted tissue analysis, or uses technically challenging MRI technology for in vivo analysis that is not suitable for smaller model systems. This new method allows for novel metabolic investigation of intact brains and other tissues ex vivo in a quick, and simplistic way with the potential for large-scale studies.

Tetrathiomolybdate inhibits mitochondrial complex IV and mediates degradation of hypoxia-inducible factor-1α in cancer cells.

Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that triggers adaptive responses upon low oxygen conditions and plays a crucial role in cancer metabolism and therapy resistance. Tetrathiomolybdate (TM), a therapy option for copper overload disorder, has also been shown to be capable of limiting tumor angiogenesis, although its underlying mechanism remains unclear. Using ovarian and endometrial cancer cell lines, we observed that TM downregulates HIF-1α protein levels and HIF-transcriptional targets involved in tumor angiogenesis and glycolysis, but did not affect HIF-1α protein synthesis. TM-mediated HIF-1α downregulation was suppressed when HIF-prolyl hydroxylase activity was pharmacologically inhibited using deferoxamine or dimethyloxaloylglycine, and also when the oxygen-dependent degradation domains of HIF-1α, which are responsible for the interaction with HIF-prolyl hydroxylase, were deleted. These findings suggest that TM causes HIF-1α downregulation in a HIF-prolyl hydroxylase-dependent manner. Our studies showed that TM inhibits the activity of the copper-dependent mitochondrial complex IV and reduces mitochondrial respiration, thereby possibly increasing oxygen availability, which is crucial for HIF-prolyl hydroxylase activity. Pimonidazole staining also showed that TM elevates oxygen tension in hypoxic cells. Our studies provide mechanistic evidence for TM-mediated HIF-1α regulation and suggest its therapeutic potential as a method of blocking angiogenesis in ovarian and endometrial tumors.

The GTPase regulatory proteins Pix and Git control tissue growth via the Hippo pathway.

The Salvador-Warts-Hippo (Hippo) pathway is a conserved regulator of organ size and is deregulated in human cancers. In epithelial tissues, the Hippo pathway is regulated by fundamental cell biological properties, such as polarity and adhesion, and coordinates these with tissue growth. Despite its importance in disease, development, and regeneration, the complete set of proteins that regulate Hippo signaling remain undefined. To address this, we used proteomics to identify proteins that bind to the Hippo (Hpo) kinase. Prominent among these were PAK-interacting exchange factor (known as Pix or RtGEF) and G-protein-coupled receptor kinase-interacting protein (Git). Pix is a conserved Rho-type guanine nucleotide exchange factor (Rho-GEF) homologous to Beta-PIX and Alpha-PIX in mammals. Git is the single Drosophila melanogaster homolog of the mammalian GIT1 and GIT2 proteins, which were originally identified in the search for molecules that interact with G-protein-coupled receptor kinases. Pix and Git form an oligomeric scaffold to facilitate sterile 20-like kinase activation and have also been linked to GTPase regulation. We show that Pix and Git regulate Hippo-pathway-dependent tissue growth in D. melanogaster and that they do this in parallel to the known upstream regulator Fat cadherin. Pix and Git influence activity of the Hpo kinase by acting as a scaffold complex, rather than enzymes, and promote Hpo dimerization and autophosphorylation of Hpo's activation loop. Therefore, we provide important new insights into an ancient signaling network that controls the growth of metazoan tissues.

Drosophila as a model for context-dependent tumorigenesis.

Drosophila can exhibit classic hallmarks of cancer, such as evasion of apoptosis, sustained proliferation, metastasis, prolonged survival, genome instability, and metabolic reprogramming, when cancer-related genes are perturbed. In the last two decades, studies in flies have identified several tumor suppressor and oncogenes. However, the greatest strength of the fly lies in its ability to model cancer hallmarks in a variety of tissue types, which enables the study of context-dependent tumorigenesis. We review the organs and tissues that have been used to model tumor formation, and propose new strategies to maximize the potential of Drosophila in cancer research.

Riquiqui and minibrain are regulators of the hippo pathway downstream of Dachsous.

The atypical cadherins Fat (Ft) and Dachsous (Ds) control tissue growth through the Salvador-Warts-Hippo (SWH) pathway, and also regulate planar cell polarity and morphogenesis. Ft and Ds engage in reciprocal signalling as both proteins can serve as receptor and ligand for each other. The intracellular domains (ICDs) of Ft and Ds regulate the activity of the key SWH pathway transcriptional co-activator protein Yorkie (Yki). Signalling from the FtICD is well characterized and controls tissue growth by regulating the abundance of the Yki-repressive kinase Warts (Wts). Here we identify two regulators of the Drosophila melanogaster SWH pathway that function downstream of the DsICD: the WD40 repeat protein Riquiqui (Riq) and the DYRK-family kinase Minibrain (Mnb). Ds physically interacts with Riq, which binds to both Mnb and Wts. Riq and Mnb promote Yki-dependent tissue growth by stimulating phosphorylation-dependent inhibition of Wts. Thus, we describe a previously unknown branch of the SWH pathway that controls tissue growth downstream of Ds.

A screen for conditional growth suppressor genes identifies the Drosophila homolog of HD-PTP as a regulator of the oncoprotein Yorkie.

Mammalian cancers depend on "multiple hits," some of which promote growth and some of which block apoptosis. We screened for mutations that require a synergistic block in apoptosis to promote tissue overgrowth and identified myopic (mop), the Drosophila homolog of the candidate tumor-suppressor and endosomal regulator His-domain protein tyrosine phosphatase (HD-PTP). We find that Myopic regulates the Salvador/Warts/Hippo (SWH) tumor suppressor pathway: Myopic PPxY motifs bind conserved residues in the WW domains of the transcriptional coactivator Yorkie, and Myopic colocalizes with Yorkie at endosomes. Myopic controls Yorkie endosomal association and protein levels, ultimately influencing expression of some Yorkie target genes. However, the antiapoptotic gene diap1 is not affected, which may explain the conditional nature of the myopic growth phenotype. These data establish Myopic as a Yorkie regulator and implicate Myopic-dependent association of Yorkie with endosomal compartments as a regulatory step in nuclear outputs of the SWH pathway.

ß-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development.

ß-Arrestins have been implicated in the regulation of multiple signalling pathways. However, their role in organism development is not well understood. In this study, we report a new in vivo function of the Drosophila ß-arrestin Kurtz (Krz) in the regulation of two distinct developmental signalling modules: MAPK ERK and NF-κB, which transmit signals from the activated receptor tyrosine kinases (RTKs) and the Toll receptor, respectively. Analysis of the expression of effectors and target genes of Toll and the RTK Torso in krz maternal mutants reveals that Krz limits the activity of both pathways in the early embryo. Protein interaction studies suggest a previously uncharacterized mechanism for ERK inhibition: Krz can directly bind and sequester an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. A simultaneous dysregulation of different signalling systems in krz mutants results in an abnormal patterning of the embryo and severe developmental defects. Our findings uncover a new in vivo function of ß-arrestins and present a new mechanism of ERK inhibition by the Drosophila ß-arrestin Krz.

Common-path multimodal optical microscopy.

We have developed a common-path multimodal optical microscopy system that is capable of using a single optical source and a single camera to image amplitude, phase, and fluorescence features of a biological specimen. This is achieved by varying either contrast enhancement filters at the Fourier plane and/or neutral density/fluorescence filters in front of the CCD camera. The feasibility of the technique is demonstrated by obtaining brightfield, fluorescence, phase-contrast, spatially filtered, brightfield+fluorescence, phase+fluorescence, and edge-enhanced+fluorescence images of the same Drosophila embryo without the need for image registration and fusion. This comprehensive microscope has the capability of providing both structural and functional information and may be used for applications such as studying live-cell dynamics and in high throughput microscopy and automated microscopy.

Tandem affinity purification in Drosophila: the advantages of the GS-TAP system.

Tandem affinity purification (TAP) has been widely used for the analysis of protein complexes. We investigated the parameters of the recently developed TAP method (GS-TAP) and its application in Drosophila. This new tag combination includes two Protein G modules and a streptavidin binding peptide (SBP), separated by one or two TEV protease cleavage sites. We made pMK33-based GS-TAP vectors to allow for generation of stable cell lines using hygromycin selection and inducible expression from a metallothionein promoter, as well as pUAST-based vectors that can be used for inducible expression in flies. Rescue experiments in flies demonstrated that the GS-TAP tag preserves the function of the tagged protein. We have done parallel purifications of proteins tagged with the new GS-TAP tag or with the conventional TAP tag (containing the Protein A and calmodulin binding peptide domains) at the amino terminus, using both cultured cells and embryos. A major difference between the two tags was in the levels of contaminating proteins, which were significantly lower in the GS-TAP purifications. The GS-TAP procedure also resulted in higher yield of the bait protein. Overall, GS-TAP is an improved method of protein complex purification because it provides a superior signal-to-noise ratio of the bait protein relative to contaminants in purified material.