24 research outputs found
Molecular and functional characterization of the Odorant Receptor2 (OR2) in the tiger mosquito Aedes albopictus.
No full textIn mosquitoes, olfactory system plays a crucial role in many behaviors, including nectar feeding, host preference selection, searching for the right place to lay eggs. A.albopicus, known also as tiger mosquito, is an anthropophilic species which in the last years, due to a strong ecological plasticity, has spread throughout the world and all over Italy with a high abundance in man-made environments. Although long considered a secondary vector of viruses, the potentiality of its vectorial capacity is very dangerous and may constitute the foundation for a public health alert. Nevertheless, to date, for this mosquito nothing is known at molecular level. Based on the idea that an improved understanding of the olfactory system of mosquitoes may help in developing control methods that interfere with its behavior, recently we have undertaken a study aimed to characterize the A. albopictus Odorant Receptors. During my PhD work, I focused my attention on the identification, cloning and functional characterization of the A. albopictus OR2 ortholog. My data indicate that A. albopictus OR2 (AalOR2) shares a high degree of identity with the other mosquito OR2 orthologs characterized to date, confirming that OR2 is one of the most conserved mosquito ORs; furthermore, AalOR2 is expressed in the olfactory appendages of larvae and adults and its expression increases after a blood meal, as determined by a semi-quantitative RT-PCR.
Interestingly, this is the first report of an up-regulation of an OR in response to a blood meal; this increase could suggest a role of AalOR2 in searching oviposition right places. AalOR2, such as the other orthologs, is narrowly tuned to indole, a ubiquitous volatile compound that has been linked to host seeking, and oviposition. The de-orphaning of AalOR2 has been obtained, with same results, through Ca2+ imaging assay in HEK293 cells, and “in vivo” experiments using the Single Sensillum Recording (SSR) in an engineered neuron of the fruitfly Drosophila melanogaster that express AalOR2.
Furthermore, by using this technique, I was able to identify also a molecule, (-)Menthone, that produced an inhibitory effect on this Odorant Receptor. In summary, this work led to the cloning and de-orphaning of the first Odorant Receptor in A. albopictus, that may be used as potential molecular target for developing environmentally friendly strategies to control mosquito populations
Methods to Test Endocrine Disruption in Drosophila melanogaster
No full textIn recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-α-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed
The Reversible Carnitine Palmitoyltransferase 1 Inhibitor (Teglicar) Ameliorates the Neurodegenerative Phenotype in a Drosophila Huntington’s Disease Model by Acting on the Expression of Carnitine-Related Genes
No full textHuntington’s disease (HD) is a dramatic neurodegenerative disorder caused by the abnormal expansion of a CAG triplet in the huntingtin gene, producing an abnormal protein. As it leads to the death of neurons in the cerebral cortex, the patients primarily present with neurological symptoms, but recently metabolic changes resulting from mitochondrial dysfunction have been identified as novel pathological features. The carnitine shuttle is a complex consisting of three enzymes whose function is to transport the long-chain fatty acids into the mitochondria. Here, its pharmacological modification was used to test the hypothesis that shifting metabolism to lipid oxidation exacerbates the HD symptoms. Behavioural and transcriptional analyses were carried out on HD Drosophila model, to evaluate the involvement of the carnitine cycle in this pathogenesis. Pharmacological inhibition of CPT1, the rate-limiting enzyme of the carnitine cycle, ameliorates the HD symptoms in Drosophila, likely acting on the expression of carnitine-related genes
The Discovery of Highly Potent THP Derivatives as OCTN2 Inhibitors: From Structure-Based Virtual Screening to In Vivo Biological Activity
No full textA mismatch between -oxidation and the tricarboxylic acid cycle (TCA) cycle flux in mitochondria produces an accumulation of lipid metabolic intermediates, resulting in both blunted metabolic flexibility and decreased glucose utilization in the affected cells. The ability of the cell to switch to glucose as an energy substrate can be restored by reducing the reliance of the cell on fatty acid oxidation. The inhibition of the carnitine system, limiting the carnitine shuttle to the oxidation of lipids in the mitochondria, allows cells to develop a high plasticity to metabolic rewiring with a decrease in fatty acid oxidation and a parallel increase in glucose oxidation. We found that 3-(2,2,2-trimethylhydrazine)propionate (THP), which is able to reduce cellular carnitine levels by blocking both carnitine biosynthesis and the cell membrane carnitine/organic cation transporter (OCTN2), was reported to improve mitochondrial dysfunction in several diseases, such as Huntington’s disease (HD). Here, new THP-derived carnitine-lowering agents (TCL), characterized by a high affinity for the OCTN2 with a minimal effect on carnitine synthesis, were developed, and their biological activities were evaluated in both in vitro and in vivo HD models. Certain compounds showed promising biological activities: reducing protein aggregates in HD cells, ameliorating motility defects, and increasing the lifespan of HD Drosophila melanogaster
L-Carnitine in Drosophila: A Review
No full textL-Carnitine is an amino acid derivative that plays a key role in the metabolism of fatty acids, including the shuttling of long-chain fatty acyl CoA to fuel mitochondrial β-oxidation. In addition, L-carnitine reduces oxidative damage and plays an essential role in the maintenance of cellular energy homeostasis. L-carnitine also plays an essential role in the control of cerebral functions, and the aberrant regulation of genes involved in carnitine biosynthesis and mitochondrial carnitine transport in Drosophila models has been linked to neurodegeneration. Drosophila models of neurodegenerative diseases provide a powerful platform to both unravel the molecular pathways that contribute to neurodegeneration and identify potential therapeutic targets. Drosophila can biosynthesize L-carnitine, and its carnitine transport system is similar to the human transport system; moreover, evidence from a defective Drosophila mutant for one of the carnitine shuttle genes supports the hypothesis of the occurrence of β-oxidation in glial cells. Hence, Drosophila models could advance the understanding of the links between L-carnitine and the development of neurodegenerative disorders. This review summarizes the current knowledge on L-carnitine in Drosophila and discusses the role of the L-carnitine pathway in fly models of neurodegeneration
Application of the 3C Method to Study the Developmental Genes in Drosophila Larvae
No full textA transition from one developmental stage to another is accompanied by activation of developmental programs and corresponding gene ensembles. Changes in the spatial conformation of the corresponding loci are associated with this activation and can be investigated with the help of the Chromosome Conformation Capture (3C) methodology. Application of 3C to specific developmental stages is a sophisticated task. Here, we describe the use of the 3C method to study the spatial organization of developmental loci in Drosophila larvae. We critically analyzed the existing protocols and offered our own solutions and the optimized protocol to overcome limitations. To demonstrate the efficiency of our procedure, we studied the spatial organization of the developmental locus Dad in 3rd instar Drosophila larvae. Differences in locus conformation were found between embryonic cells and living wild-type larvae. We also observed the establishment of novel regulatory interactions in the presence of an adjacent transgene upon activation of its expression in larvae. Our work fills the gap in the application of the 3C method to Drosophila larvae and provides a useful guide for establishing 3C on an animal model
Meldonium improves Huntington’s disease mitochondrial dysfunction by restoring peroxisome proliferator‐activated receptor γ coactivator 1α expression
No full textMitochondrial dysfunction seems to play a fundamental role in the pathogenesis of
neurodegeneration in Huntington’s disease (HD). We assessed possible neuroprotective
actions of meldonium, a small molecule affecting mitochondrial fuel
metabolism, in in vitro and in vivo HD models. We found that meldonium was able
to prevent cytotoxicity induced by serum deprivation, to reduce the accumulation of
mutated huntingtin (mHtt) aggregates, and to upregulate the expression of
peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α) in mHTT‐
expressing cells. The PGC‐1α increase was accompanied by the increment of
mitochondrial mass and by the rebalancing of mitochondrial dynamics with a
promotion of the mitochondrial fusion. Meldonium‐induced PGC‐1α significantly
alleviated motor dysfunction and prolonged the survival of a transgenic HD
Drosophila model in which mHtt expression in the nervous system led to progressive
motor performance deficits. Our study strongly suggests that PGC‐1α, as a master
coregulator of mitochondrial biogenesis, energy homeostasis, and antioxidant
defense, is a potential therapeutic target in HD
Truncated Analogues of a G-Quadruplex-Forming Aptamer Targeting Mutant Huntingtin: Shorter Is Better!
No full textTwo analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington’s disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer
Fighting the Huntington’s Disease with a G-Quadruplex-Forming Aptamer Specifically Binding to Mutant Huntingtin Protein: Biophysical Characterization, In Vitro and In Vivo Studies
No full textA set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington’s disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer
