Assessing genotoxic effects of plastic leachates in Drosophila melanogaster.

Plastic polymers were largely added with chemical substances to be utilized in the items and product manufacturing. The leachability of these substances is a matter of concern given the wide amount of plastic waste, particularly in terrestrial environments, where soil represents a sink for these novel contaminants and a possible pathway of human health risk. In this study, we integrated genetic, molecular, and behavioral approaches to comparatively evaluate toxicological effects of plastic leachates, virgin and oxodegradable polypropylene (PP) and polyethylene (PE), in Drosophila melanogaster, a novel in vivo model organism for environmental monitoring studies and (eco)toxicological research. The results of this study revealed that while conventional toxicological endpoints such as developmental times and longevity remain largely unaffected, exposure to plastic leachates induces chromosomal abnormalities and transposable element (TE) activation in neural tissues. The combined effects of DNA damage and TE mobilization contribute to genome instability and increase the likelihood of LOH events, thus potentiating tumor growth and metastatic behavior ofRasV12 clones. Collectively, these findings indicate that plastic leachates exert genotoxic effects in Drosophila thus highlighting potential risks associated with leachate-related plastic pollution and their implications for ecosystems and human health.

WiFi Related Radiofrequency Electromagnetic Fields Promote Transposable Element Dysregulation and Genomic Instability in Drosophila melanogaster.

Exposure to artificial radio frequency electromagnetic fields (RF-EMFs) has greatly increased in recent years, thus promoting a growing scientific and social interest in deepening the biological impact of EMFs on living organisms. The current legislation governing the exposure to RF-EMFs is based exclusively on their thermal effects, without considering the possible non-thermal adverse health effects from long term exposure to EMFs. In this study we investigated the biological non-thermal effects of low-level indoor exposure to RF-EMFs produced by WiFi wireless technologies, using Drosophila melanogaster as the model system. Flies were exposed to 2.4 GHz radiofrequency in a Transverse Electromagnetic (TEM) cell device to ensure homogenous controlled fields. Signals were continuously monitored during the experiments and regulated at non thermal levels. The results of this study demonstrate that WiFi electromagnetic radiation causes extensive heterochromatin decondensation and thus a general loss of transposable elements epigenetic silencing in both germinal and neural tissues. Moreover, our findings provide evidence that WiFi related radiofrequency electromagnetic fields can induce reactive oxygen species (ROS) accumulation, genomic instability, and behavioural abnormalities. Finally, we demonstrate that WiFi radiation can synergize with RasV12 to drive tumor progression and invasion. All together, these data indicate that radiofrequency radiation emitted from WiFi devices could exert genotoxic effects in Drosophila and set the stage to further explore the biological effects of WiFi electromagnetic radiation on living organisms.

Cytological heterogeneity of heterochromatin among 10 sequenced Drosophila species.

In Drosophila chromosomal rearrangements can be maintained and are associated with karyotypic variability among populations from different geographic localities. The abundance of variability in gene arrangements among chromosomal arms is even greater when comparing more distantly related species and the study of these chromosomal changes has provided insights into the evolutionary history of species in the genus. In addition, the sequencing of genomes of several Drosophila species has offered the opportunity to establish the global pattern of genomic evolution, at both genetic and chromosomal level. The combined approaches of comparative analysis of syntenic blocks and direct physical maps on polytene chromosomes have elucidated changes in the orientation of genomic sequences and the difference between heterochromatic and euchromatic regions. Unfortunately, the centromeric heterochromatic regions cannot be studied using the cytological maps of polytene chromosomes because they are underreplicated and therefore reside in the chromocenter. In Drosophila melanogaster, a cytological map of the heterochromatin has been elaborated using mitotic chromosomes from larval neuroblasts. In the current work, we have expanded on that mapping by producing cytological maps of the mitotic heterochromatin in an additional 10 sequenced Drosophila species. These maps highlight 2 apparently different paths, for the evolution of the pericentric heterochromatin between the subgenera Sophophora and Drosophila. One path leads toward a progressive complexity of the pericentric heterochromatin (Sophophora) and the other toward a progressive simplification (Drosophila). These maps are also useful for a better understanding how karyotypes have been altered by chromosome arm reshuffling during evolution.

Neuroprotective Effects of PARP Inhibitors in Drosophila Models of Alzheimer's Disease.

Alzheimer's disease (AD) is an irreversible age-related neurodegenerative disorder clinically characterized by severe memory impairment, language deficits and cognitive decline. The major neuropathological hallmarks of AD include extracellular deposits of the ß-amyloid (Aß) peptides and cytoplasmic neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein. The accumulation of plaques and tangles in the brain triggers a cascade of molecular events that culminate in neuronal damage and cell death. Despite extensive research, our understanding of the molecular basis of AD pathogenesis remains incomplete and a cure for this devastating disease is still not available. A growing body of evidence in different experimental models suggests that poly(ADP-ribose) polymerase-1 (PARP-1) overactivation might be a crucial component of the molecular network of interactions responsible for AD pathogenesis. In this work, we combined genetic, molecular and biochemical approaches to investigate the effects of two different PARP-1 inhibitors (olaparib and MC2050) in Drosophila models of Alzheimer's disease by exploring their neuroprotective and therapeutic potential in vivo. We found that both pharmacological inhibition and genetic inactivation of PARP-1 significantly extend lifespan and improve the climbing ability of transgenic AD flies. Consistently, PARP-1 inhibitors lead to a significant decrease of Aß42 aggregates and partially rescue the epigenetic alterations associated with AD in the brain. Interestingly, olaparib and MC2050 also suppress the AD-associated aberrant activation of transposable elements in neuronal tissues of AD flies.

Transposable element activation promotes neurodegeneration in a Drosophila model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant disorder with progressive motor dysfunction and cognitive decline. The disease is caused by a CAG repeat expansion in the IT15 gene, which elongates a polyglutamine stretch of the HD protein, Huntingtin. No therapeutic treatments are available, and new pharmacological targets are needed. Retrotransposons are transposable elements (TEs) that represent 40% and 30% of the human and Drosophila genomes and replicate through an RNA intermediate. Mounting evidence suggests that mammalian TEs are active during neurogenesis and may be involved in diseases of the nervous system. Here we show that TE expression and mobilization are increased in a Drosophila melanogaster HD model. By inhibiting TE mobilization with Reverse Transcriptase inhibitors, polyQ-dependent eye neurodegeneration and genome instability in larval brains are rescued and fly lifespan is increased. These results suggest that TE activation may be involved in polyQ-induced neurotoxicity and a potential pharmacological target.

Unravelling HP1 functions: post-transcriptional regulation of stem cell fate.

Heterochromatin protein 1 (HP1) is a non-histone chromosomal protein first identified in Drosophila as a major component of constitutive heterochromatin, required for stable epigenetic gene silencing in many species including humans. Over the years, several studies have highlighted additional roles of HP1 in different cellular processes including telomere maintenance, DNA replication and repair, chromosome segregation and, surprisingly, positive regulation of gene expression. In this review, we briefly summarize past research and recent results supporting the unexpected and emerging role of HP1 in activating gene expression. In particular, we discuss the role of HP1 in post-transcriptional regulation of mRNA processing because it has proved decisive in the control of germline stem cells homeostasis in Drosophila and has certainly added a new dimension to our understanding on HP1 targeting and functions in epigenetic regulation of stem cell behaviour.

The Hsp70 chaperone is a major player in stress-induced transposable element activation.

Previous studies have shown that heat shock stress may activate transposable elements (TEs) in Drosophila and other organisms. Such an effect depends on the disruption of a chaperone complex that is normally involved in biogenesis of Piwi-interacting RNAs (piRNAs), the largest class of germline-enriched small noncoding RNAs implicated in the epigenetic silencing of TEs. However, a satisfying picture of how chaperones could be involved in repressing TEs in germ cells is still unknown. Here we show that, in Drosophila, heat shock stress increases the expression of TEs at a posttranscriptional level by affecting piRNA biogenesis through the action of the inducible chaperone Hsp70. We found that stress-induced TE activation is triggered by an interaction of Hsp70 with the Hsc70-Hsp90 complex and other factors all involved in piRNA biogenesis in both ovaries and testes. Such interaction induces a displacement of all such factors to the lysosomes, resulting in a functional collapse of piRNA biogenesis. This mechanism has clear evolutionary implications. In the presence of drastic environmental changes, Hsp70 plays a key dual role in increasing both the survival probability of individuals and the genetic variability in their germ cells. The consequent increase of genetic variation in a population potentiates evolutionary plasticity and evolvability.

A role of the Trx-G complex in Cid/CENP-A deposition at Drosophila melanogaster centromeres.

Centromeres are epigenetically determined chromatin structures that specify the assembly site of the kinetochore, the multiprotein machinery that binds microtubules and mediates chromosome segregation during mitosis and meiosis. The centromeric protein A (CENP-A) and its Drosophila orthologue centromere identifier (Cid) are H3 histone variants that replace the canonical H3 histone in centromeric nucleosomes of eukaryotes. CENP-A/Cid is required for recruitment of other centromere and kinetochore proteins and its deficiency disrupts chromosome segregation. Despite the many components that are known to cooperate in centromere function, the complete network of factors involved in CENP-A recruitment remains to be defined. In Drosophila, the Trx-G proteins localize along the heterochromatin with specific patterns and some of them localize to the centromeres of all chromosomes. Here, we show that the Trx, Ash1, and CBP proteins are required for the correct chromosome segregation and that Ash1 and CBP mediate for Cid/CENP-A recruitment at centromeres through post-translational histone modifications. We found that centromeric H3 histone is consistently acetylated in K27 by CBP and that nej and ash1 silencing respectively causes a decrease in H3K27 acetylation and H3K4 methylation along with an impairment of Cid loading.

Heterochromatin protein 1 (HP1) is intrinsically required for post-transcriptional regulation of Drosophila Germline Stem Cell (GSC) maintenance.

A very important open question in stem cells regulation is how the fine balance between GSCs self-renewal and differentiation is orchestrated at the molecular level. In the past several years much progress has been made in understanding the molecular mechanisms underlying intrinsic and extrinsic controls of GSC regulation but the complex gene regulatory networks that regulate stem cell behavior are only partially understood. HP1 is a dynamic epigenetic determinant mainly involved in heterochromatin formation, epigenetic gene silencing and telomere maintenance. Furthermore, recent studies have revealed the importance of HP1 in DNA repair, sister chromatid cohesion and, surprisingly, in positive regulation of gene expression. Here, we show that HP1 plays a crucial role in the control of GSC homeostasis in Drosophila. Our findings demonstrate that HP1 is required intrinsically to promote GSC self-renewal and progeny differentiation by directly stabilizing the transcripts of key genes involved in GSCs maintenance.

Anti-Inflammatory Activity of A Polyphenolic Extract from Arabidopsis thaliana in In Vitro and In Vivo Models of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder and the primary form of dementia in the elderly. One of the main features of AD is the increase in amyloid-beta (Aß) peptide production and aggregation, leading to oxidative stress, neuroinflammation and neurodegeneration. Polyphenols are well known for their antioxidant, anti-inflammatory and neuroprotective effects and have been proposed as possible therapeutic agents against AD. Here, we investigated the effects of a polyphenolic extract of Arabidopsis thaliana (a plant belonging to the Brassicaceae family) on inflammatory response induced by Aß. BV2 murine microglia cells treated with both Aß25⁻35 peptide and extract showed a lower pro-inflammatory (IL-6, IL-1ß, TNF-α) and a higher anti-inflammatory (IL-4, IL-10, IL-13) cytokine production compared to cells treated with Aß only. The activation of the Nrf2-antioxidant response element signaling pathway in treated cells resulted in the upregulation of heme oxygenase-1 mRNA and in an increase of NAD(P)H:quinone oxidoreductase 1 activity. To establish whether the extract is also effective against Aß-induced neurotoxicity in vivo, we evaluated its effect on the impaired climbing ability of AD Drosophila flies expressing human Aß1⁻42. Arabidopsis extract significantly restored the locomotor activity of these flies, thus confirming its neuroprotective effects also in vivo. These results point to a protective effect of the Arabidopsis extract in AD, and prompt its use as a model in studying the impact of complex mixtures derived from plant-based food on neurodegenerative diseases.

Stress-induced strain and brain region-specific activation of LINE-1 transposons in adult mice.

Transposable elements (TEs) are conserved mobile genetic elements that are highly abundant in most eukaryotic genomes. Although the exact function of TEs is still largely unknown, it is increasingly clear that they are significantly modulated in response to stress in a wide range of organisms, either directly or indirectly through regulation of epigenetic silencing. We investigated the effect of repeated restraint stress (2 h a day, for 5 d) on transcription levels of LINE-1 (L1) retrotransposon in the brain of inbred BALB/c, DBA/2, C57BL/6N, and outbred CD1 mice. Repeated restraint stress induced strain and brain region-specific modulation of L1 activity. We observed a significant derepression of L1 transcription in the hippocampus (HIPP) of BALB/c mice and a significant downregulation in the hippocampus of C57BL/6N mice. No significant change in L1 expression was found in the other strains and brain regions. These findings indicate in mice the control of transposons expression as an additional mechanism in stress-induced pathophysiological responses, demonstrating that their regulation is highly dependent on the strain genetic background and the brain region. Lay summary Hippocampal expression of the transposon L1 is significantly altered by repeated restraint stress in mice. L1 modulation is not only region specific, but also strain dependent, suggesting that the genetic background is an important determinant of L1 response to environmental stimuli.

Drosophila CG3303 is an essential endoribonuclease linked to TDP-43-mediated neurodegeneration.

Endoribonucleases participate in almost every step of eukaryotic RNA metabolism, acting either as degradative or biosynthetic enzymes. We previously identified the founding member of the Eukaryotic EndoU ribonuclease family, whose components display unique biochemical features and are flexibly involved in important biological processes, such as ribosome biogenesis, tumorigenesis and viral replication. Here we report the discovery of the CG3303 gene product, which we named DendoU, as a novel family member in Drosophila. Functional characterisation revealed that DendoU is essential for Drosophila viability and nervous system activity. Pan-neuronal silencing of dendoU resulted in fly immature phenotypes, highly reduced lifespan and dramatic motor performance defects. Neuron-subtype selective silencing showed that DendoU is particularly important in cholinergic circuits. At the molecular level, we unveiled that DendoU is a positive regulator of the neurodegeneration-associated protein dTDP-43, whose downregulation recapitulates the ensemble of dendoU-dependent phenotypes. This interdisciplinary work, which comprehends in silico, in vitro and in vivo studies, unveils a relevant role for DendoU in Drosophila nervous system physio-pathology and highlights that DendoU-mediated neurotoxicity is, at least in part, contributed by dTDP-43 loss-of-function.