Pedro Martinez

Sex peptide of Drosophila melanogaster males is a global regulator of reproductive processes in females

Seminal fluid proteins (Sfps) alter female behaviour and physiology and can mediate sexual conflict. In Drosophila melanogaster, a single Sfp, the sex peptide (SP), triggers remarkable post-mating responses in females, including altered fecundity, feeding, immunity and sexual receptivity. These effects can favour the evolutionary interests of males while generating costs in females. We tested the hypothesis that SP is an upstream master-regulator able to induce diverse phenotypes through efficient induction of widespread transcriptional changes in females. We profiled mRNA responses to SP in adult female abdomen (Abd) and head+thorax (HT) tissues using microarrays at 3 and 6 h following mating. SP elicited a rich, subtle signature of temporally and spatially controlled mRNAs. There were significant alterations to genes linked to egg development, early embryogenesis, immunity, nutrient sensing, behaviour and, unexpectedly, phototransduction. There was substantially more variation in the direction of differential expression across time points in the HT versus Abd. The results support the idea that SP is an important regulator of gene expression in females. The expression of many genes in one sex can therefore be under the influence of a regulator expressed in the other. This could influence the extent of sexual conflict both within and between loci.

Insulin signalling regulates remating in female Drosophila

Mating rate is a major determinant of female lifespan and fitness, and is predicted to optimize at an intermediate level, beyond which superfluous matings are costly. In female Drosophila melanogaster, nutrition is a key regulator of mating rate but the underlying mechanism is unknown. The evolutionarily conserved insulin/insulin-like growth factor-like signalling (IIS) pathway is responsive to nutrition, and regulates development, metabolism, stress resistance, fecundity and lifespan. Here we show that inhibition of IIS, by ablation of Drosophila insulin-like peptide (DILP)-producing median neurosecretory cells, knockout of dilp2, dilp3 or dilp5 genes, expression of a dominant-negative DILP-receptor (InR) transgene or knockout of Lnk, results in reduced female remating rates. IIS-mediated regulation of female remating can occur independent of virgin receptivity, developmental defects, reduced body size or fecundity, and the receipt of the female receptivity-inhibiting male sex peptide. Our results provide a likely mechanism by which females match remating rates to the perceived nutritional environment. The findings suggest that longevity-mediating genes could often have pleiotropic effects on remating rate. However, overexpression of the IIS-regulated transcription factor dFOXO in the fat body—which extends lifespan—does not affect remating rate. Thus, long life and reduced remating are not obligatorily coupled.

Intestinal Parasites in Children from a Day Care Centre in Matanzas City, Cuba

Background Intestinal parasitic infections are widely distributed throughout the world and children are the most affected population. Day care centres are environments where children have proven to be more susceptible to acquiring IP. Methods and Principal Findings A cross-sectional study was carried to determine the prevalence of intestinal parasites in stool samples among children who attend to a day care centre in an urban area of Matanzas city, Cuba, from March to June 2012. 104 children under five years old were included on the study after informed consent form was signed by parents or legal guardians. Three fresh faecal samples were collected from each child in different days and were examined by direct wet mount, formalin-ether, and Kato- Katz techniques. Data relating to demography, socioeconomic status, source of drinking water, and personal hygiene habits were also collected using a standardized questionnaire. In total, 71.1% of children harbored at least one type of intestinal parasite and 47 (45.2%) were infected by more than one species. Giardia duodenalis and Blastocystis sp. were the most common parasites found, with prevalence rates of 54.8% and 38.5% respectively. Conclusions Despite public health campaigns, improvement in the level of education, and the availability of and access to medical services in Cuba infections by intestinal protozoan is high in this centre. Almost nothing is published regarding intestinal parasites in Matanzas province during the last 40 years so this work could also be the initial point to carry out other studies to clarify the IP status in this region.

Ageing increases vulnerability to aβ42 toxicity in Drosophila.

Age is the major risk factor for many neurodegenerative diseases, including Alzheimers Disease (AD), for reasons that are not clear. The association could indicate that the duration or degree of exposure to toxic proteins is important for pathology, or that age itself increases susceptibility to protein toxicity. Using an inducible Drosophila model of AD, we investigated these possibilities by varying the expression of an Aβ42 transgene in neurons at different adult ages and measuring the effects on Aβ42 levels and associated pathological phenotypes. Acute induction of Arctic Aβ42 in young adult flies resulted in rapid expression and clearance of mRNA and soluble Arctic Aβ42 protein, but in irreversible expression of insoluble Arctic Aβ42 peptide. Arctic Aβ42 peptide levels accumulated with longer durations of induction, and this led to a dose-dependent reduction in negative geotaxis and lifespan. For a standardised level of mRNA expression, older flies had higher levels of Arctic Aβ42 peptide and associated toxicity, and this correlated with an age-dependent reduction in proteasome activity. Equalising Aβ42 protein at different ages shortened lifespan in correlation with the duration of exposure to the peptide, suggesting that Aβ42 expression accumulates damage over time. However, the relative reduction in lifespan compared to controls was greater in flies first exposed to the peptide at older ages, suggesting that ageing itself also increases susceptibility to Aβ42 toxicity. Indeed older flies were more vulnerable to chronic Aβ42 toxicity even with a much lower lifetime exposure to the peptide. Finally, the persistence of insoluble Aβ42 in both young and old induced flies suggests that aggregated forms of the peptide cause toxicity in later life. Our results suggest that reduced protein turnover, increased duration of exposure and increased vulnerability to protein toxicity at later ages in combination could explain the late age-of-onset of neurodegenerative phenotypes.

Lithium suppresses Aβ pathology by inhibiting translation in an adult Drosophila model of Alzheimer's disease.

The greatest risk factor for Alzheimers disease (AD) is age, and changes in the ageing nervous system are likely contributors to AD pathology. Amyloid beta (Aβ) accumulation, which occurs as a result of the amyloidogenic processing of amyloid precursor protein (APP), is thought to initiate the pathogenesis of AD, eventually leading to neuronal cell death. Previously, we developed an adult-onset Drosophila model of AD. Mutant Aβ42 accumulation led to increased mortality and neuronal dysfunction in the adult flies. Furthermore, we showed that lithium reduced Aβ42 protein, but not mRNA, and was able to rescue Aβ42-induced toxicity. In the current study, we investigated the mechanism/s by which lithium modulates Aβ42 protein levels and Aβ42 induced toxicity in the fly model. We found that lithium caused a reduction in protein synthesis in Drosophila and hence the level of Aβ42. At both the low and high doses tested, lithium rescued the locomotory defects induced by Aβ42, but it rescued lifespan only at lower doses, suggesting that long-term, high-dose lithium treatment may have induced toxicity. Lithium also down-regulated translation in the fission yeast Schizosaccharomyces pombe associated with increased chronological lifespan. Our data highlight a role for lithium and reduced protein synthesis as potential therapeutic targets for AD pathogenesis.

Direct Keap1-Nrf2 disruption as a potential therapeutic target for Alzheimer's disease

Nrf2, a transcriptional activator of cell protection genes, is an attractive therapeutic target for the prevention of neurodegenerative diseases, including Alzheimer’s disease (AD). Current Nrf2 activators, however, may exert toxicity and pathway over-activation can induce detrimental effects. An understanding of the mechanisms mediating Nrf2 inhibition in neurodegenerative conditions may therefore direct the design of drugs targeted for the prevention of these diseases with minimal side-effects. Our study provides the first in vivo evidence that specific inhibition of Keap1, a negative regulator of Nrf2, can prevent neuronal toxicity in response to the AD-initiating Aβ42 peptide, in correlation with Nrf2 activation. Comparatively, lithium, an inhibitor of the Nrf2 suppressor GSK-3, prevented Aβ42 toxicity by mechanisms independent of Nrf2. A new direct inhibitor of the Keap1-Nrf2 binding domain also prevented synaptotoxicity mediated by naturally-derived Aβ oligomers in mouse cortical neurons. Overall, our findings highlight Keap1 specifically as an efficient target for the re-activation of Nrf2 in AD, and support the further investigation of direct Keap1 inhibitors for the prevention of neurodegeneration in vivo.

Water-independent effects of dietary restriction in Drosophila

Dietary restriction (DR) is a moderate reduction in food intake, without malnutrition, that extends the healthy life span in many organisms, including Drosophila (1). Although fruit flies have many practical advantages for studying DR, various technical complexities can have large effects on experimental outcomes (1). For DR, it is important that the basic food conditions are optimal. This ensures that increased longevity due to food restriction prolongs a healthy life span rather than returning sick animals to normal health by limiting access to a nutritionally inappropriate diet. We have systematically optimized our conditions for Drosophila DR, eliminated several non-nutritional explanations including water imbalance, and recommend a Brewers-yeast-based diet (2, 3). Ja et al. (4) report contradictory data. Here, we present additional data in support of our conclusions and point out a flaw in the data concerning the Brewers yeast diet presented by Ja et al. First, we developed a system that effectively hydrates flies under salt stress (Table 1, experiment 1). Adding 8 g·L−1 NaCl to our standard food [1.0 SYBrewers (2)] shortened median life span by 24%. Adding to each vial a 200-μl pipette tip filled with water (1% agar) restored normal life span. The rescue was not due to the tip itself because an identical tip filled with dry cotton wool had no effect. Furthermore, addition of a tip containing wet cotton wool also restored normal life span. This was reproducible when the food was made from a different yeast [SYBakers (2)] with salt added (Table 1, experiment 2). Thus, our technique is effective in delivering water to salt-stressed flies and in rescuing the associated shortening of the life span. Table 1. Providing a water source to flies rescues the life-shortening effect caused by addition of salt (NaCl) to food, but not life-span alterations due to dietary restriction Next, we established that the life-span change associated with DR was not rescued by water addition [Table 1, experiment 3; replicate of our published data (2)].These data demonstrate that DR in Drosophila under our conditions is not due to rescue of hydration stress. These results directly contradict data from an unreplicated experiment by Ja et al. using conditions ostensibly replicating ours, which reported that life-span extension by DR was eliminated by the addition of water, concluding that hydration stress explains DR (figure 2, G–I, in ref. 4). There are two problems with this conclusion. First, Ja et al. used more sugar (100 g·L−1) in their concentrated medium (CM) than did we, and this higher concentration causes a significant reduction in egg laying compared with the level we use (50 g·L−1) (2), perhaps because of water stress. [Furthermore, fly feeding behavior can be dramatically reduced as sugar concentration increases in this range, which may explain why Ja et al. observed lowered feeding in their CM (figure 1F in ref. 4) and we do not.] Second, the data in figure 2, G–I, from Ja et al. (4) demonstrate that water addition shortened DR life span to the level of the CM (Fig. 1). Thus the “rescue” of the DR effect by water addition could also be explained by water shortening the life span of DR flies, rather than increasing the life span of those on CM. Fig. 1. Life-span differences due to DR and water addition. Ja et al. (4) reported significant extension of life span when the concentration of nutrients in a Brewers yeast diet were diluted (DR vs. CM) and that this difference was eliminated by the addition ... These data presented from our laboratory, both here and elsewhere (2, 3), robustly demonstrate that hydration stress does not explain DR under the conditions that we use. Furthermore, we have discovered that DR in Drosophila is mediated by an amino acid imbalance in the food that is not modified by water addition (3).

Deletion of endogenous Tau proteins is not detrimental in Drosophila

Human Tau (hTau) is a highly soluble and natively unfolded protein that binds to microtubules within neurons. Its dysfunction and aggregation into insoluble paired helical filaments is involved in the pathogenesis of Alzheimer’s disease (AD), constituting, together with accumulated β-amyloid (Aβ) peptides, a hallmark of the disease. Deciphering both the loss-of-function and toxic gain-of-function of hTau proteins is crucial to further understand the mechanisms leading to neurodegeneration in AD. As the fruit fly Drosophila melanogaster expresses Tau proteins (dTau) that are homologous to hTau, we aimed to better comprehend dTau functions by generating a specific tau knock-out (KO) fly line using homologous recombination. We observed that the specific removal of endogenous dTau proteins did not lead to overt, macroscopic phenotypes in flies. Indeed, survival, climbing ability and neuronal function were unchanged in tau KO flies. In addition, we did not find any overt positive or negative effect of dTau removal on human Aβ-induced toxicity. Altogether, our results indicate that the absence of dTau proteins has no major functional impact on flies, and suggests that our tau KO strain is a relevant model to further investigate the role of dTau proteins in vivo, thereby giving additional insights into hTau functions.

Ageing increases vulnerability to Aβ42 toxicity in drosophila

a transgenic animal model of familial AD, namely the PD-APP mouse, over a time frame where Ab42 levels are known to rise. Methods: Experiments were performed in accordance with the Animal (Scientific Procedures) Act 1986. Adult male PD-APP transgenic mice and C57Bl/6NTac aged matched controls (weight range 29-45g, n1⁄4 15/group) were implanted with a telemeter i.p. and with a custom cranial implant. At seven months of age animals were evaluated in the SCORE2004 bioassay, which allows continual measurement of electro-encephalogram (EEG) and electromyogram (EMG) (cranial implant), locomotor activity and body temperature (telemetry). Animals were kept under 12/12 light/dark cycle with food and drink ad libitum. Results: At the first time point, data was compared between 10 PD-APP and 13 control animals. The time spent in REM throughout both the light and dark cycle was significantly reduced (light: PD-APP 27 6 6 minutes per 12 hour light cycle, control 476 5, P <0.05; dark: PD-APP 156 3, control 276 4, P <0.05). Additionally the average REM bout length was reduced during each light phase compared to control (light: PD-APP 0.56 0.1 minutes, control 1.06 0.1,P<0.01; dark: PD-APP 0.46 0.1, control 0.96 0.1,P<0.05). No change in the amount of time spent inNREMorwakewas observed and no significant changes in latency to NREM or REM were detected after animals were disturbed. Conclusions: In conclusion, preliminary data from this longitudinal study shows that male PD-APP mice aged 7 months have reduced time in REM and reduced average REM bout lengths in both light and dark environments. No changes in REM latency were observed in our male mice however increased latency has been reported in 3-5 month old female PDAPPmice (Huitron-Resendez et al 2002 Brain Res 928: 126-137). Evaluation of these mice, including spectral profiles, is planned to continue until animals reach 22 months of age.