Human Diseases Associated with Notch Signalling: Lessons from Drosophila melanogaster.

Drosophila melanogaster has been used as a model system to identify and characterize genetic contributions to development, homeostasis, and to investigate the molecular determinants of numerous human diseases. While there exist many differences at the genetic, structural, and molecular level, many signalling components and cellular machineries are conserved between Drosophila and humans. For this reason, Drosophila can and has been used extensively to model, and study human pathologies. The extensive genetic resources available make this model system a powerful one. Over the years, the sophisticated and rapidly expanding Drosophila genetic toolkit has provided valuable novel insights into the contribution of genetic components to human diseases. The activity of Notch signalling is crucial during development and conserved across the Metazoa and has been associated with many human diseases. Here we highlight examples of mechanisms involving Notch signalling that have been elucidated from modelling human diseases in Drosophila melanogaster that include neurodegenerative diseases, congenital diseases, several cancers, and cardiac disorders.

Notch1 Phase Separation Coupled Percolation facilitates target gene expression and enhancer looping.

The Notch receptor is a pleiotropic signaling protein that translates intercellular ligand interactions into changes in gene expression via the nuclear localization of the Notch intracellular Domain (NICD). Using a combination of immunohistochemistry, RNA in situ, Optogenetics and super-resolution live imaging of transcription in human cells, we show that the N1ICD can form condensates that positively facilitate Notch target gene expression. We determined that N1ICD undergoes Phase Separation Coupled Percolation (PSCP) into transcriptional condensates, which recruit, enrich, and encapsulate a broad set of core transcriptional proteins. We show that the capacity for condensation is due to the intrinsically disordered transcriptional activation domain of the N1ICD. In addition, the formation of such transcriptional condensates acts to promote Notch-mediated super enhancer-looping and concomitant activation of the MYC protooncogene expression. Overall, we introduce a novel mechanism of Notch1 activity in which discrete changes in nuclear N1ICD abundance are translated into the assembly of transcriptional condensates that facilitate gene expression by enriching essential transcriptional machineries at target genomic loci.

Notch1 Phase Separation Coupled Percolation facilitates target gene expression and enhancer looping.

The Notch receptor is a pleiotropic signaling protein that translates intercellular ligand interactions into changes in gene expression via the nuclear localization of the Notch intracellular Domain (NICD). Using a combination of immunohistochemistry, RNA in situ, Optogenetics and super-resolution live imaging of transcription in human cells, we show that the N1ICD can form condensates that positively facilitate Notch target gene expression. We determined that N1ICD undergoes Phase Separation Coupled Percolation (PSCP) into transcriptional condensates, which recruit, enrich, and encapsulate a broad set of core transcriptional proteins. We show that the capacity for condensation is due to the intrinsically disordered transcriptional activation domain of the N1ICD. In addition, the formation of such transcriptional condensates acts to promote Notch-mediated super enhancer-looping and concomitant activation of the MYC protooncogene expression. Overall, we introduce a novel mechanism of Notch1 activity in which discrete changes in nuclear N1ICD abundance are translated into the assembly of transcriptional condensates that facilitate gene expression by enriching essential transcriptional machineries at target genomic loci.

PlayBack cloning: simple, reversible, cost-effective cloning for the combinatorial assembly of complex expression constructs.

With advancements in multicomponent molecular biological tools, the need for versatile, rapid and cost-effective cloning that enables successful combinatorial assembly of DNA plasmids of interest is becoming increasingly important. Unfortunately, current cloning platforms fall short regarding affordability, ease of combinatorial assembly and, above all, the ability to iteratively remove individual cassettes at will. Herein we construct, implement and make available a broad set of cloning vectors, called PlayBack vectors, that allow for the expression of several different constructs simultaneously under separate promoters. Overall, this system is substantially cheaper than other multicomponent cloning systems, has usability for a wide breadth of experimental paradigms and includes the novel feature of being able to selectively remove components of interest at will at any stage of the cloning platform.

Molluscan RXR Transcriptional Regulation by Retinoids in a Drosophila CNS Organ Culture System.

Retinoic acid, the active metabolite of Vitamin A, is important for the appropriate development of the nervous system (e.g., neurite outgrowth) as well as for cognition (e.g., memory formation) in the adult brain. We have shown that many of the effects of retinoids are conserved in the CNS of the mollusc, Lymnaea stagnalis. RXRs are predominantly nuclear receptors, but the Lymnaea RXR (LymRXR) exhibits a non-nuclear distribution in the adult CNS, where it is also implicated in non-genomic retinoid functions. As such, we developed a CNS Drosophila organ culture-based system to examine the transcriptional activity and ligand-binding properties of LymRXR, in the context of a live invertebrate nervous system. The novel ligand sensor system was capable of reporting both the expression and transcriptional activity of the sensor. Our results indicate that the LymRXR ligand sensor mediated transcription following activation by both 9-cis RA (the high affinity ligand for vertebrate RXRs) as well as the vertebrate RXR synthetic agonist, SR11237. The LymRXR ligand sensor was also activated by all-trans RA, and to a much lesser extent by the vertebrate RAR synthetic agonist, EC23. This sensor also detected endogenous retinoid-like activity in the CNS of developing Drosophila larvae, primarily during the 3rd instar larval stage. These data indicate that the LymRXR sensor can be utilized not only for characterization of ligand activation for studies related to the Lymnaea CNS, but also for future studies of retinoids and their functions in Drosophila development.

Plant Extract of Limonium gmelinii Attenuates Oxidative Responses in Neurons, Astrocytes, and Cerebral Endothelial Cells In Vitro and Improves Motor Functions of Rats after Middle Cerebral Artery Occlusion.

There are numerous publications demonstrating that plant polyphenols can reduce oxidative stress and inflammatory processes in the brain. In the present study we have investigated the neuroprotective effect of plant extract isolated from the roots of L. gmelinii since it contains a rich source of polyphenols and other biologically active compounds. We have applied an oxidative and inflammatory model induced by NMDA, H2O2, and TNF-α in human primary neurons and astrocytes, and mouse cerebral endothelial cell (CECs) line in vitro. The levels of ROS generation, NADPH oxidase activation, P-selectin expression, and activity of ERK1/2 were evaluated by quantitative immunofluorescence analysis, confocal microscopy, and MAPK assay. In vivo, sensorimotor functions in rats with middle cerebral artery occlusion (MCAO) were assessed. In neurons NMDA induced overproduction of ROS, in astrocytes TNF-α initiated ROS generation, NADPH oxidase activation, and phosphorylation of ERK1/2. In CECs, the exposure by TNF-α induced oxidative stress and triggered the accumulation of P-selectin on the surface of the cells. In turn, pre-treatment of the cells with the extract of L. gmelinii suppressed oxidative stress in all cell types and pro-inflammatory responses in astrocytes and CECs. In vivo, the treatment with L. gmelinii extract improved motor activity in rats with MCAO.

The Effects of Mobile Phone Radiofrequency Electromagnetic Fields on ß-Amyloid-Induced Oxidative Stress in Human and Rat Primary Astrocytes.

Amyloid beta peptide (Aß) is implicated in the development of pathological reactions associated with Alzheimer's disease (AD), such as oxidative stress, neuro-inflammation and death of brain cells. Current pharmacological approaches to treat AD are not able to control the deposition of Aß and suppression of Aß-induced cellular response. There is a growing body of evidence that exposure to radiofrequency electromagnetic field (RF-EMF) causes a decrease of beta-amyloid deposition in the brains and provides cognitive benefits to Alzheimer's Tg mice. Herein, we investigated the effects of mobile phone radiofrequency EMF of 918 MHz on reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP), activity of NADPH-oxidase, and phosphorylation of p38MAPK and ERK1/2 kinases in human and rat primary astrocytes in the presence of Aß42 and H2O2. Our data demonstrate that EMF is able to reduce Aß42- and H2O2-induced cellular ROS, abrogate Aß42-induced production of mitochondrial ROS and the co-localization between the cytosolic (p47-phox) and membrane (gp91-phox) subunits of NADPH oxidase, while increasing MMP, and inhibiting H2O2-induced phosphorylation of p38MAPK and ERK1/2 in primary astrocytes. Yet, EMF was not able to modulate alterations in the phosphorylation state of the MAPKs triggered by Aß42. Our findings provide an insight into the mechanisms of cellular and molecular responses of astrocytes on RF-EMF exposure and indicate the therapeutic potential of RF-EMF for the treatment of Alzheimer's disease.