Increased Post-Hypoxic Oxidative Stress and Activation of the PERK Branch of the UPR in Trap1-Deficient Drosophila melanogaster Is Abrogated by Metformin

Hypoxia is known to impair mitochondrial and endoplasmic reticulum (ER) homeostasis. Post-hypoxic perturbations of the ER proteostasis result in the accumulation of misfolded/unfolded proteins leading to the activation of the Unfolded Protein Response (UPR). Mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) is reported to preserve mitochondrial membrane potential and to impede reactive oxygen species (ROS) production thereby protecting cells from ER stress as well as oxidative stress. The first-line antidiabetic drug Metformin has been attributed a neuroprotective role after hypoxia. Interestingly, Metformin has been reported to rescue mitochondrial deficits in fibroblasts derived from a patient carrying a homozygous TRAP1 loss-of-function mutation. We sought to investigate a putative link between Metformin, TRAP1, and the UPR after hypoxia. We assessed post-hypoxic/reperfusion longevity, mortality, negative geotaxis, ROS production, metabolic activity, gene expression of antioxidant proteins, and activation of the UPR in Trap1-deficient flies. Following hypoxia, Trap1 deficiency caused higher mortality and greater impairments in negative geotaxis compared to controls. Similarly, post-hypoxic production of ROS and UPR activation was significantly higher in Trap1-deficient compared to control flies. Metformin counteracted the deleterious effects of hypoxia in Trap1-deficient flies but had no protective effect in wild-type flies. We provide evidence that TRAP1 is crucially involved in the post-hypoxic regulation of mitochondrial/ER stress and the activation of the UPR. Metformin appears to rescue Trap1-deficiency after hypoxia mitigating ROS production and downregulating the pro-apoptotic PERK (protein kinase R-like ER kinase) arm of the UPR.

The Drosophila melanogaster Levodopa-Induced Depression Model Exhibits Negative Geotaxis Deficits and Differential Gene Expression in Males and Females

More than 320 million people live with depression in the world, a disorder that severely limits psychosocial functioning and diminishes quality of life. The prevalence of major depression is almost two times higher in women than in men. However, the molecular mechanisms of its sex-specific pathophysiology are still poorly understood. Drosophila melanogaster is an established model for neurobiological research of depression-like states, as well as for the study of molecular and genetic sex differences in the brain. Here, we investigated sex-specific effects on forced-climbing locomotion (negative geotaxis) and gene expression of a fly model of depression-like phenotypes induced by levodopa administration, which was previously shown to impair normal food intake, mating frequency, and serotonin concentration. We observed that both males and females show deficits in the forced-climbing paradigm; however, modulated by distinct gene expression patterns after levodopa administration. Our results suggest that Drosophila models can be a valuable tool for identifying the molecular mechanisms underlying the difference of depressive disorder prevalence between men and women.

Phenotypical Effect of Phosphodiesterase 5 (PDE5) Inhibitor on Behavioral Activities of Fruit Fly Drosophila melanogaster

Phosphodiesterase 5 (PDE5) inhibitor is a class of drugs currently used to treat erectile dysfunction. Physiologically, inhibition of PDE5 may lead to vasodilation, blood flow increment, and penile erection. However, PDE5 inhibitors have been reported not only to modify the function of the male reproductive organ but also to influence other physiological systems. To explore the effect of PDE5 inhibitor on metazoan physiological systems, a fruit fly (Drosophila melanogaster) model organism is used since the catalytic domain of fruit fly PDE5/6 shares a high similarity of amino acid sequence (58%) with the PDE5 of humans. This study aimed to investigate whether the effect of PDE5 inhibition by sildenafil is phenotypically observable as changes in the behavioral states. Two behavioral phenotypes of D. melanogaster, negative geotaxis, and ethanol sensitivity, were used as test parameters in this explorative study. The results demonstrated that sildenafil had a significant effect on reducing locomotor activity, as reflected by negative geotaxis assay, but it had no influence on the fruit fly sensitivity to ethanol. Taken together, our results suggested that PDE5 inhibition might impair the physiological condition of the metazoan species. Also, an explorative study using D. melanogaster might offer valuable insight as a model organism in the discovery and repurposing approach of PDE5 inhibitor.

In utero exposure to protease inhibitor-based antiretroviral regimens delays growth and developmental milestones in mice

Antiretroviral therapy (ART) in pregnancy has dramatically reduced HIV vertical transmission rates. Consequently, there is a growing number of children that are HIV exposed uninfected (CHEUs). Studies suggest that CHEUs exposed in utero to ART may experience developmental delays compared to their peers. We investigated the effects of in utero ART exposure on perinatal neurodevelopment in mice, through assessment of developmental milestones. Developmental milestone tests (parallel to reflex testing in human infants) are reflective of brain maturity and useful in predicting later behavioral outcomes. We hypothesized that ART in pregnancy alters the in utero environment and thereby alters developmental milestone outcomes in pups. Throughout pregnancy, dams were treated with boosted-atazanavir combined with either abacavir/lamivudine (ATV/r/ABC/3TC), or tenofovir/emtricitabine (ATV/r/TDF/FTC), or water as control. Pups were assessed daily for general somatic growth and on a battery of tests for primitive reflexes including surface-righting, negative-geotaxis, cliff-aversion, rooting, ear-twitch, auditory-reflex, forelimb-grasp, air-righting, behaviors in the neonatal open field, and olfactory test. In utero exposure to either ART regimen delayed somatic growth in offspring and evoked significant delays in the development of negative geotaxis, cliff-aversion, and ear-twitch reflexes. Exposure to ATV/r/ABC/3TC was also associated with olfactory deficits in male and forelimb grasp deficits in female pups. To explore whether delays persisted into adulthood we assessed performance in the open field test. We observed no significant differences between treatment arm for males. In females, ATV/r/TDF/FTC exposure was associated with lower total distance travelled and less ambulatory time in the centre, while ATV/r/ABC/3TC exposure was associated with higher resting times compared to controls. In utero PI-based ART exposure delays the appearance of primitive reflexes that involve vestibular and sensory-motor pathways in a mouse model. Our findings suggest that ART could be disrupting the normal progress/maturation of the underlying neurocircuits and encourage further investigation for underlying mechanisms.

Effect of Methanolic Extract of Caryota no Seed on Geotactic Behaviour and Fecundity in Drosophila melanogaster

Aims: To investigate the effect of the methanolic extracts of Caryota no (CN) seeds negative geotaxis, fecundity and acetylcholinesterase (AChE) using Drosophila melanogaster (DM). Study Design: Experimental design. Place and Duration: Sample: African Centre of Excellence for Phytomedicine Research and Development, University of Jos, Jos Plateau State Nigeria between June 2018 and February 2019 Methodology: 50 flies were exposed in each vial to the following concentrations: 300 mg, 350 mg, 400 mg, 500 mg and 600 mg of methanolic extracts in 5 replicates for 7 days with daily recording of mortality. Total protein assays were carried out by Randox method from the supernatant from homogenized whole flies. In vivo antioxidant activity study was conducted by measuring level of acetylcholinesterase (AChE) enzyme activity from supernatants of whole fly homogenates using a spectrophotometer at specific wavelengths over a 2 minute duration. The values were derived as part of the total protein value. Negative geotaxis was done by the climbing assay and fecundity was examined by rate of emergence of larva after exposure of the flies to treatment. The statistical difference among test groups was presumed at P < .05. Results: The methanolic extract of CN caused nonsignificant (P = .33) decrease in total protein levels compared to the control. There were also nonsignificant decreases in AChE (P = .30) activity, negative geotactic (P = .80) behaviour and nonsignificant increase in fecundity (P = .17) in the methanolic extract-treated flies compared to the controls. Conclusion: It can therefore be concluded that the methanolic extract of Caryota no nonsignificantly improved fertitity and reduced negative geotaxis and AChE activity in DM.

Daughterless, the Drosophila orthologue of TCF4, is required for associative learning and maintenance of the synaptic proteome

ABSTRACTMammalian transcription factor 4 (TCF4) has been linked to schizophrenia and intellectual disabilities, such as Pitt–Hopkins syndrome (PTHS). Here, we show that similarly to mammalian TCF4, fruit fly orthologue Daughterless (Da) is expressed widely in the Drosophila brain. Furthermore, silencing of da, using several central nervous system-specific Gal4 driver lines, impairs appetitive associative learning of the larvae and leads to decreased levels of the synaptic proteins Synapsin (Syn) and Discs large 1 (Dlg1), suggesting the involvement of Da in memory formation. Here, we demonstrate that Syn and dlg1 are direct target genes of Da in adult Drosophila heads, as Da binds to the regulatory regions of these genes and the modulation of Da levels alter the levels of Syn and dlg1 mRNA. Silencing of da also affects negative geotaxis of the adult flies, suggesting the impairment of locomotor function. Overall, our findings suggest that Da regulates Drosophila larval memory and adult negative geotaxis, possibly via its synaptic target genes Syn and dlg1. These behavioural phenotypes can be further used as a PTHS model to screen for therapeutics.This article has an associated First Person interview with the first author of the paper.

Methanolic Leaves and Arils Extracts of Ackee (Blighia sapida)Plant Ameliorate Mercuric Chloride-Induced Oxidative Stress in Drosophila melanogaster

Ackee (Blighia sapida) has been used in traditional medicine for treating oxidative stress-induced diseases; diabetes and cancer. Ameliorative roles of Ackee leaves and arils methanolic extracts were evaluated on mercuric chloride (HgCl2) induced-oxidative stress in D. melanogaster. 1-3 days old D. melanogaster were orally exposed to different concentrations of Ackee leaves (AL), and Ackee arils (AS) extracts as well as HgCl2 in a diet for 7 days. Subsequently, survival and negative geotaxis assays, quantification of reactive oxygen species (ROS) generation, and oxidative biomarker enzymes were determined. 500 µM HgCl2 was selected based on the highest lethality, 0.2 mg/g for AL, and 0.4 mg/g for AS; they gave the highest antioxidant effects. Exposure to AL and AS plant extracts improved negative geotaxis behavior and extracts were able to ameliorate the effect of Hg2+ on catalase positively, acetylcholinesterase, glutathione-S transferase activities, increase total thiol and GSH levels and reverted increased nitric oxide and hydrogen peroxide production (P<0.05) when compared to the control. Our results suggest that the Hg2+ mechanism of toxicity is associated with oxidative damage, as evidenced by the alteration in the oxidative stress-antioxidant imbalance, and that extracts possess essential phytochemicals that could alleviate possibly effects of environmental pollutants such as HgCl2.

Changes in Neuronal Density of the Sensorimotor Cortex and Neurodevelopmental Behaviour in Neonatal Mice with Kaolin-Induced Hydrocephalus

<b><i>Introduction:</i></b> Hydrocephalus is a disorder in which the circulation of cerebrospinal fluid is altered in a manner that leads to its accumulation in the ventricles and subarachnoid space. Its impact on the neuronal density and networks in the overlying cerebral cortex in a time-dependent neonatal hydrocephalic process is largely unknown. We hypothesize that hydrocephalus will affect the cytoarchitecture of the cerebral cortical mantle of neonatal hydrocephalic mice, which will in turn modify sensorimotor processing and neurobehaviour. <b><i>Objective:</i></b> The purpose of this study is to probe the effect of hydrocephalus on 3 developmental milestones (surface righting reflex, cliff avoidance reflex, and negative geotaxis) and on cortical neuronal densities in neonatal hydrocephalic mice. <b><i>Methods:</i></b> Hydrocephalus was induced in 1-day-old mice by intracisternal injection of sterile kaolin suspension. The pups were tested for reflex development and sensorimotor ability using surface righting reflex (PND 5, 7, and 9), cliff avoidance (PND 6), and negative geotaxis (PND 10 and 12) prior to their sacrifice on PND 7, 14, and 21. Neuronal density and cortical thickness in the sensorimotor cortex were evaluated using atlas-based segmentation of the neocortex and boundary definition in 4-μm paraffin-embedded histological sections with hematoxylin and eosin as well as cresyl violet stains. <b><i>Results:</i></b> Surface righting and cliff avoidance activities were significantly impaired in hydrocephalic pups but no statistically significant difference was observed in negative geotaxis in both experimental and control pups. The neuronal density of the sensorimotor cortex was significantly higher in hydrocephalic mice than in age-matched controls on PND 14 and 21 (373.20 ± 21.54 × 10⁻⁶ μm² vs. 157.70 ± 21.88 × 10⁻⁶ μm²; 230.0 ± 44.1 × 10⁻⁶ μm² vs. 129.60 ± 3.72 × 10⁻⁶ μm², respectively; <i>p</i> &#x3c; 0.05). This was accompanied by reduction in the cortical thickness (µm) in the hydrocephalic mice on PND 7 (2,409 ± 43.37 vs. 3,752 ± 65.74, <i>p</i> &#x3c; 0.05), PND 14 (2,035 ± 322.10 vs. 4,273 ± 67.26, <i>p</i> &#x3c; 0.05), and PND 21 (1,676 ± 33.90 vs. 4,945 ± 81.79, <i>p</i> &#x3c; 0.05) compared to controls. <b><i>Conclusion:</i></b> In this murine model of neonatal hydrocephalus, the quantitative changes in the cortical neuronal population may play a role in the observed changes in neurobehavioural findings.

Alteration of the aggregation and spatial organization of the vector of Chagas disease, Triatoma infestans, by the parasite Trypanosoma cruzi

Triatominae insects are vectors of the parasite Trypanosoma cruzi, the etiological agent of Chagas disease affecting millions of people in Latin America. Some species, such as Triatoma infestans, live in the human neighborhood, aggregating in walls or roof cracks during the day and going out to feed blood at night. The comprehension of how sex and T. cruzi infection affect their aggregation and geotaxis is essential for understanding their spatial organization and the parasite dispersion. Experiments in laboratory-controlled conditions were carried out with groups of ten adults of T. infestans able to explore and aggregate on a vertical surface. The influence of the sex (male vs. female) and the proportion of infected insects in the group were tested (100% of infected insects vs. a small proportion of infected insects, named infected and potentially weakly infected groups, respectively). Therefore, four distinct groups of insects were tested: infected males, infected females, potentially weakly infected males, and potentially weakly infected females, with 12, 9, 15, and 16 replicates, respectively. The insects presented a high negative geotaxis and a strong aggregation behavior whatever the sex or their infection. After an exploration phase, these behaviors were stable in time. The insects exhibited a preferential vertical position, head toward the top of the setup. Males had a higher negative geotaxis and a higher aggregation level than females. Both behaviors were enhanced in groups of 100% infected insects, the difference between sexes being maintained. According to a comparison between experimental and theoretical results, geotaxis favors the aggregation that mainly results from the inter-attraction between individuals.