The Impact of Drosophila Awd/NME1/2 Levels on Notch and Wg Signaling Pathways.

Awd, the Drosophila homologue of NME1/2 metastasis suppressors, plays key roles in many signaling pathways. Mosaic analysis of the null awdJ2A4 allele showed that loss of awd gene function blocks Notch signaling and the expression of its target genes including the Wingless (Wg/Wnt1) morphogen. We also showed that RNA interference (RNAi)-mediated awd silencing (awdi) in larval wing disc leads to chromosomal instability (CIN) and to Jun amino-terminal kinases (JNK)-mediated cell death. Here we show that this cell death is independent of p53 activity. Based on our previous finding showing that forced survival of awdi-CIN cells leads to aneuploidy without the hyperproliferative effect, we investigated the Wg expression in awdi wing disc cells. Interestingly, the Wg protein is expressed in its correct dorso-ventral domain but shows an altered cellular distribution which impairs its signaling. Further, we show that RNAi-mediated knock down of awd in wing discs does not affect Notch signaling. Thus, our analysis of the hypomorphic phenotype arising from awd downregulation uncovers a dose-dependent effect of Awd in Notch and Wg signaling.

Extracellular NME proteins: a player or a bystander?

The Nm23/NME gene family has been under intensive study since Nm23H1/NME1 was identified as the first metastasis suppressor. Inverse correlation between the expression levels of NME1/2 and prognosis has indeed been demonstrated in different tumor cohorts. Interestingly, the presence of NME proteins in the extracellular environment in normal and tumoral conditions has also been noted. In many reported cases, however, these extracellular NME proteins exhibit anti-differentiation or oncogenic functions, contradicting their canonical anti-metastatic action. This emerging field thus warrants further investigation. In this review, we summarize the current understanding of extracellular NME proteins. A role in promoting stem cell pluripotency and inducing development of central nervous system as well as a neuroprotective function of extracellular NME have been suggested. Moreover, a tumor-promoting function of extracellular NME also emerged at least in some tumor cohorts. In this complex scenario, the secretory mechanism through which NME proteins exit cells is far from being understood. Recently, some evidence obtained in the Drosophila and cancer cell line models points to the involvement of Dynamin in controlling the balance between intra- and extracellular levels of NME. Further analyses on extracellular NME will lead to a better understanding of its physiological function and in turn will allow understanding of how its deregulation contributes to carcinogenesis.

Drosophila 4EHP is essential for the larval-pupal transition and required in the prothoracic gland for ecdysone biosynthesis.

Maternal expression of the translational regulator 4EHP (eIF4E-Homologous Protein) has an established role in generating protein gradients essential for specifying the Drosophila embryonic pattern. We generated a null mutation of 4EHP, which revealed for the first time that it is essential for viability and for completion of development. In fact, 4EHP null larvae, and larvae ubiquitously expressing RNAi targeting 4EHP, are developmentally delayed, fail to grow and eventually die. In addition, we found that expressing RNAi that targets 4EHP specifically in the prothoracic gland disrupted ecdysone biosynthesis, causing a block of the transition from the larval to pupal stages. This phenotype can be rescued by dietary administration of ecdysone. Consistent with this, 4EHP is highly expressed in the prothoracic gland and it is required for wild type expression levels of steroidogenic enzymes. Taken together, these results uncover a novel essential function for 4EHP in regulating ecdysone biosynthesis.

The ecdysone receptor signalling regulates microvilli formation in follicular epithelial cells.

Epithelial morphogenesis contributes greatly to the development and homeostasis of the organs and body parts. Here, we analysed the consequences of impaired ecdysone receptor (EcR) signalling in the Drosophila follicular epithelium. Besides governing cell growth, the three EcR isoforms act redundantly in controlling follicle cell positioning. Flattening of the microvilli and an aberrant actin cytoskeleton arise from defective EcR signalling in follicle cells, and these defects impact on the organisation of the oocyte membrane. We found that this signalling governs a complex molecular network since its impairment affects key molecules as atypical protein kinase C and activated Moesin. Interestingly, the activity of the transcription factor Tramtrack69 isoform is required for microvilli and their actin core morphogenesis as well as for follicle cell positioning. In conclusion, our findings provide evidence of novel roles for EcR signalling and Tramtrack69 transcription factor in controlling stage-specific differentiation events that take place in the follicular epithelium.

Neonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees.

Large-scale losses of honey bee colonies represent a poorly understood problem of global importance. Both biotic and abiotic factors are involved in this phenomenon that is often associated with high loads of parasites and pathogens. A stronger impact of pathogens in honey bees exposed to neonicotinoid insecticides has been reported, but the causal link between insecticide exposure and the possible immune alteration of honey bees remains elusive. Here, we demonstrate that the neonicotinoid insecticide clothianidin negatively modulates NF-κB immune signaling in insects and adversely affects honey bee antiviral defenses controlled by this transcription factor. We have identified in insects a negative modulator of NF-κB activation, which is a leucine-rich repeat protein. Exposure to clothianidin, by enhancing the transcription of the gene encoding this inhibitor, reduces immune defenses and promotes the replication of the deformed wing virus in honey bees bearing covert infections. This honey bee immunosuppression is similarly induced by a different neonicotinoid, imidacloprid, but not by the organophosphate chlorpyriphos, which does not affect NF-κB signaling. The occurrence at sublethal doses of this insecticide-induced viral proliferation suggests that the studied neonicotinoids might have a negative effect at the field level. Our experiments uncover a further level of regulation of the immune response in insects and set the stage for studies on neural modulation of immunity in animals. Furthermore, this study has implications for the conservation of bees, as it will contribute to the definition of more appropriate guidelines for testing chronic or sublethal effects of pesticides used in agriculture.

Notch signaling during development requires the function of awd, the Drosophila homolog of human metastasis suppressor gene Nm23.

BACKGROUND: The Drosophila abnormal wing discs (awd) belongs to a highly conserved family of genes implicated in metastasis suppression, metabolic homeostasis and epithelial morphogenesis. The cellular function of the mammalian members of this family, the Nm23 proteins, has not yet been clearly defined. Previous awd genetic analyses unraveled its endocytic role that is required for proper internalization of receptors controlling different signaling pathways. In this study, we analyzed the role of Awd in controlling Notch signaling during development. RESULTS: To study the awd gene function we used genetic mosaic approaches to obtain cells homozygous for a loss of function allele. In awd mutant follicle cells and wing disc cells, Notch accumulates in enlarged early endosomes, resulting in defective Notch signaling. Our results demonstrate that awd function is required before γ-secretase mediated cleavage since over-expression of the constitutively active form of the Notch receptor in awd mutant follicle cells allows rescue of the signaling. By using markers of different endosomal compartments we show that Notch receptor accumulates in early endosomes in awd mutant follicle cells. A trafficking assay in living wing discs also shows that Notch accumulates in early endosomes. Importantly, constitutively active Rab5 cannot rescue the awd phenotype, suggesting that awd is required for Rab5 function in early endosome maturation. CONCLUSIONS: In this report we demonstrate that awd is essential for Notch signaling via its endocytic role. In addition, we identify the endocytic step at which Awd function is required for Notch signaling and we obtain evidence indicating that Awd is necessary for Rab5 function. These findings provide new insights into the developmental and pathophysiological function of this important gene family.

Drosophila VHL tumor-suppressor gene regulates epithelial morphogenesis by promoting microtubule and aPKC stability.

Mutations in the human von Hippel-Lindau (VHL) genes are the cause of VHL disease, which displays multiple benign and malignant tumors. The VHL gene has been shown to regulate angiogenic potential and glycolic metabolism via its E3 ubiquitin ligase function against the alpha subunit of hypoxia-inducible factor (HIF). However, many other HIF-independent functions of VHL have been identified and recent evidence indicates that the canonical function cannot fully explain the VHL mutant cell phenotypes. Many of these functions have not been verified in genetically tractable systems. Using an established follicular epithelial model in Drosophila, we show that the Drosophila VHL gene is involved in epithelial morphogenesis via stabilizing microtubule bundles and aPKC. Microtubule defects in VHL mutants lead to mislocalization of aPKC and subsequent loss of epithelial integrity. Destabilizing microtubules in ex vivo culture of wild-type egg chambers can also result in aPKC mislocalization and epithelial defects. Importantly, paclitaxel-induced stabilization of microtubules can rescue the aPKC localization phenotype in Drosophila VHL mutant follicle cells. The results establish a developmental function of the VHL gene that is relevant to its tumor-suppressor activity.

Effect of aminergic signaling on the humoral innate immunity response of Drosophila.

Biogenic amines are crucial signaling molecules that modulate various physiological life functions both in vertebrates and invertebrates. In humans, these neurotransmitters influence the innate and adaptive immunity systems. In this work, we analyzed whether the aminergic neurotransmission of dopamine, serotonin, and octopamine could have an impact on the humoral innate immune response of Drosophila melanogaster. This is a powerful model system widely used to uncover the insect innate immunity mechanisms which are also conserved in mammals. We found that the neurotransmission of all these amines positively modulates the Toll-responsive antimicrobial peptide (AMP) drosomycin (drs) gene in adult flies infected with the Micrococcus luteus bacterium. Indeed, we showed that either blocking the neurotransmission in their specific aminergic neurons by expressing shibirets (Shits) or silencing the vesicular monoamine transporter gene (dVMAT) by RNAi caused a significantly reduced expression of the Toll-responsive drs gene. However, upon M. luteus infection, the block of aminergic transmission did not alter the expression of AMP attacin genes responding to the immune deficiency (Imd) and Toll pathways. Overall, our results not only reveal a neuroimmune function for biogenic amines in humoral immunity but also further highlight the complexity of the network controlling AMP gene regulation.

Role of neuronal and non-neuronal acetylcholine signaling in Drosophila humoral immunity.

Acetylcholine (ACh) is one the major neurotransmitters in insects, whose role in mediating synaptic interactions between neurons in the central nervous system is well characterized. It also plays largely unexplored regulatory functions in non-neuronal tissues. Here we demonstrate that ACh signaling is involved in the modulation of the innate immune response of Drosophila melanogaster. Knockdown of ACh synthesis or ACh vesicular transport in neurons reduced the activation of drosomycin (drs), a gene encoding an antimicrobial peptide, in adult flies infected with a Gram-positive bacterium. drs transcription was similarly affected in Drosophila α7 nicotinic acetylcholine receptor, nAChRalpha7 (Dα7) mutants, as well as in flies expressing in the nervous system a dominant negative form (Dα7DN) of this specific receptor subunit. Interestingly, Dα7DN elicited a comparable response when it was expressed in non-neuronal tissues and even when it was specifically produced in the hemocytes. Consistently, full activation of the drs gene required Dα7 expression in these cells. Moreover, knockdown of ACh synthesis in non-neuronal cells affected drs expression. Overall, these findings uncover neural and non-neural cholinergic signals that modulate insect immune defenses and shed light on the role of hemocytes in the regulation of the humoral immune response.

Intravenous volume expansion to prevent contrast-associated acute kidney injury.

OBJECTIVES: Several volume expansion protocols have been proposed to prevent contrast-associated acute kidney injury (CA-AKI). The aim of our study was to seek the ideal intravenous volume expansion to prevent CA-AKI in patients with chronic kidney disease (CKD) undergoing invasive cardiovascular procedures. METHODS: We analyzed 1927 CKD patients enrolled in 6 studies that took place from September 15, 2000 to June 6, 2019. Four volume expansion regiments were included: (1) conventional group (n=625); (2) bicarbonate group (n=255); (3) left ventricular end-diastolic pressure-guided group (n=355); and (4) urine flow rate-guided group (n=500). RESULTS: CA-AKI (serum creatinine increase ≥0.3 mg/dL at 48 hours) occurred in 224 (11%) patients. In patients with CA-AKI, volume expansion was lower (2090 ± 1382 mL vs 2551 ± 1716 mL; P less than .001) and acute pulmonary edema occurred more often (3.5% vs 0.29%; P less than .001). By ROC curve analysis, an absolute volume expansion greater than or equal to 1430 mL (AUC = 0.70) and a volume expansion to contrast media volume ratio greater than or equal to 17 (AUC = 0.57) were the best thresholds for freedom from CA-AKI. CONCLUSIONS: In our comprehensive pooled analysis, an absolute volume expansion greater than or equal to 1430 mL and a volume expansion to contrast media volume ratio greater than or equal to 17 are the best dichotomous thresholds for CA-AKI prevention. These cutoffs should be formally tested in a dedicated trial as a pragmatic means to prevent CA-AKI.

The impact on microtubule network of a bracovirus IkappaB-like protein.

Polydnavirus-encoded IkappaB-like proteins are similar to insect and mammalian IkappaB, and an immunosuppressive function in the host cells has been inferred to these proteins. Here we show that the expression of one of these IkappaB-like viral genes, the TnBVank1, in the Drosophila germline affects the localization of gurken, bicoid, and oskar mRNAs whose gene products are relevant for proper embryonic patterning. The altered localization of these mRNAs is suggestive of general defects in the intracellular, microtubule-based, trafficking routes. Analysis of microtubule motor proteins components such as the dynein heavy chain and the kinesin heavy chain revealed defects in the polarized microtubule network. Interestingly, the TnBVANK1 viral protein is uniformly distributed over the entire oocyte cortex, and appears to be anchored to the microtubule ends. Our data open up a very interesting issue on novel function(s) played by the ank gene family by interfering with cytoskeleton organization.

Retrotransposons Down- and Up-Regulation in Aging Somatic Tissues.

The transposon theory of aging hypothesizes the activation of transposable elements (TEs) in somatic tissues with age, leading to a shortening of the lifespan. It is thought that TE activation in aging produces an increase in DNA double-strand breaks, contributing to genome instability and promoting the activation of inflammatory responses. To investigate how TE regulation changes in somatic tissues during aging, we analyzed the expression of some TEs, as well as a source of small RNAs that specifically silence the analyzed TEs; the Drosophila cluster named flamenco. We found significant variations in the expression levels of all the analyzed TEs during aging, with a trend toward reduction in middle-aged adults and reactivation in older individuals that suggests dynamic regulation during the lifespan.

EcR-B1 and Usp nuclear hormone receptors regulate expression of the VM32E eggshell gene during Drosophila oogenesis.

Ecdysone signaling plays key roles in Drosophila oogenesis, as its activity is required at multiple steps during egg chamber maturation. Recently, its involvement has been reported on eggshell production by controlling chorion gene transcription and amplification. Here, we present evidence that ecdysone signaling also controls the expression of the eggshell gene VM32E, whose product is a component of vitelline membrane and endochorion layers. Specifically blocking the function of the different Ecdysone receptor (EcR) isoforms we demonstrate that EcR-B1 is responsible for ecdysone-mediated VM32E transcriptional regulation. Moreover, we show that the EcR partner Ultraspiracle (Usp) is also necessary for VM32E expression. By analyzing the activity of specific VM32E regulatory regions in usp(2) clones we identify the promoter region mediating ecdysone-dependent VM32E expression. By in vitro binding assay and site-directed mutagenesis we demonstrate that this region contains a Usp binding site necessary for VM32E regulation. Our results further support the crucial role of ecdysone signaling in controlling transcription of eggshell structural genes and suggest that the heterodimeric complex EcR-B1/Usp mediates the ecdysone-dependent VM32E transcriptional activation in the main body follicle cells.

Cell survival and polarity of Drosophila follicle cells require the activity of ecdysone receptor B1 isoform.

Proper assembly and maintenance of epithelia are critical for normal development and homeostasis. Here, using the Drosophila ovary as a model, we identify a role for the B1 isoform of the ecdysone receptor (EcR-B1) in this process. We performed a reverse genetic analysis of EcR-B1 function during oogenesis and demonstrate that silencing of this receptor isoform causes loss of integrity and multilayering of the follicular epithelium. We show that multilayered follicle cells lack proper cell polarity with altered distribution of apical and basolateral cell polarity markers including atypical-protein kinase C (aPKC), Discs-large (Dlg), and Scribble (Scrib) and aberrant accumulation of adherens junctions and F-actin cytoskeleton. We find that the EcR-B1 isoform is required for proper follicle cell polarity both during early stages of oogenesis, when follicle cells undergo the mitotic cell cycle, and at midoogenesis when these cells stop dividing and undergo several endocycles. In addition, we show that the EcR-B1 isoform is required during early oogenesis for follicle cell survival and that disruption of its function causes apoptotic cell death induced by caspase.

Silencing of Euchromatic Transposable Elements as a Consequence of Nuclear Lamina Dysfunction.

Transposable elements (TEs) are mobile genomic sequences that are normally repressed to avoid proliferation and genome instability. Gene silencing mechanisms repress TEs by RNA degradation or heterochromatin formation. Heterochromatin maintenance is therefore important to keep TEs silent. Loss of heterochromatic domains has been linked to lamin mutations, which have also been associated with derepression of TEs. In fact, lamins are structural components of the nuclear lamina (NL), which is considered a pivotal structure in the maintenance of heterochromatin domains at the nuclear periphery in a silent state. Here, we show that a lethal phenotype associated with Lamin loss-of-function mutations is influenced by Drosophilagypsy retrotransposons located in euchromatic regions, suggesting that NL dysfunction has also effects on active TEs located in euchromatic loci. In fact, expression analysis of different long terminal repeat (LTR) retrotransposons and of one non-LTR retrotransposon located near active genes shows that Lamin inactivation determines the silencing of euchromatic TEs. Furthermore, we show that the silencing effect on euchromatic TEs spreads to the neighboring genomic regions, with a repressive effect on nearby genes. We propose that NL dysfunction may have opposed regulatory effects on TEs that depend on their localization in active or repressed regions of the genome.

The role of transposable elements activity in aging and their possible involvement in laminopathic diseases.

Eukaryotic genomes contain a large number of transposable elements, part of which are still active and able to transpose in the host genome. Mobile element activation is repressed to avoid deleterious effects, such as gene mutations or chromosome rearrangements. Control of transposable elements includes a variety of mechanisms comprising silencing pathways, which are based on the production of small non-coding RNAs. Silencing can occur either through transposable element RNA degradation or through the targeting of DNA sequences by heterochromatin formation and consequent transcriptional inhibition. Since the important role of the heterochromatin silencing, the gradual loss of heterochromatin marks in constitutive heterochromatin regions during the aging process promotes derepression of transposable elements, which is considered a cause of the progressive increase in genomic instability and of the activation of inflammatory responses. This review provides an overview of the effects of heterochromatin loss on the activity of transposable elements during the aging process and the possible impact on genome function. In this context, we discuss the possible role of the nuclear lamina, a major player in heterochromatin dynamics, in the regulation of transposable element activity and potential implications in laminopathic diseases.

Vasa protein is localized in the germ cells and in the oocyte-associated pyriform follicle cells during early oogenesis in the lizard Podarcis sicula.

The vasa gene, first identified in Drosophila, is a key determinant for germline formation in eukaryotes. Homologs of vasa have been identified and linked to germline development, in many invertebrates and vertebrates. Here, we analyze the distribution of Vasa in early germ cells (oogonia and oocytes) and previtellogenic ovarian follicles of the lizard Podarcis sicula. During most of its previtellogenic growth, the oocyte in this lizard species is structurally and functionally integrated through intercellular bridges with special follicle cells called pyriform cells. The pyriform cells function similarly to Drosophila nurse cells, but are somatic in origin. In the oogenesis of P. sicula, Vasa is initially highly detected in the oogonia, but its levels decrease in early stage oocytes before the onset of pyriform cell differentiation. In the later stages of oogenesis, the high level of Vasa is related with the nurse function of the pyriform follicle cells. These observations suggest that cells of somatic origin are engaged in the synthesis of Vasa in the oogenesis of this lizard.

Vps28 Is Involved in the Intracellular Trafficking of Awd, the Drosophila Homolog of NME1/2.

The Awd (abnormal wing discs) gene is the Drosophila homolog of human NME1 and NME2 metastasis suppressor genes. These genes play a key role in tumor progression. Extensive studies revealed that intracellular NME1/2 protein levels could be related to either favorable or poor prognosis depending on tissue context. More recently, extracellular activities of NME1/2 proteins have also been reported, including a tumor- promoting function. We used Drosophila as a genetic model to investigate the mechanism controlling intra- and extracellular levels of NME1/2. We examined the role of several components of the ESCRT (endosomal sorting complex required for transport) complex in controlling Awd trafficking. We show that the Vps28 component of the ESCRT-I complex is required for maintenance of normal intracellular level of Awd in larval adipocytes. We already showed that blocking of Shibire (Shi)/Dynamin function strongly- lowers Awd intracellular level. To further investigate this down regulative effect, we analyzed the distribution of endosomal markers in wild type and Shi-defective adipocytes. Our results suggest that Awd does not enter CD63-positive endosomes. Interestingly, we found that in fat body cells, Awd partly- colocalizes with the ESCRT accessory component ALiX, the ALG-2 (apoptosis-linked gene 2)-interacting protein X. Moreover, we show that the intracellular levels of both proteins are downregulated by blocking the function of the Dynamin encoded by the shibire gene.

A polydnavirus-encoded ANK protein has a negative impact on steroidogenesis and development.

Polydnaviruses (PDV) are viral symbionts associated with ichneumonid and braconid wasps parasitizing moth larvae, which are able to disrupt the host immune response and development, as well as a number of other physiological pathways. The immunosuppressive role of PDV has been more intensely investigated, while very little is known about the PDV-encoded factors disrupting host development. Here we address this research issue by further expanding the functional analysis of ankyrin genes encoded by the bracovirus associated with Toxoneuron nigriceps (Hymenoptera, Braconidae). In a previous study, using Drosophila melanogaster as experimental model system, we demonstrated the negative impact of TnBVank1 impairing the ecdysone biosynthesis by altering endocytic traffic in prothoracic gland cells. With a similar approach here we demonstrate that another member of the viral ank gene family, TnBVank3, does also contribute to the disruption of ecdysone biosynthesis, but with a completely different mechanism. We show that its expression in Drosophila prothoracic gland (PG) blocks the larval-pupal transition by impairing the expression of steroidogenic genes. Furthermore, we found that TnBVank3 affects the expression of genes involved in the insulin/TOR signaling and the constitutive activation of the insulin pathway in the PG rescues the pupariation impairment. Collectively, our data demonstrate that TnBVANK3 acts as a virulence factor by exerting a synergistic and non-overlapping function with TnBVANK1 to disrupt the ecdysone biosynthesis.