Human in vitro reporter model of neuronal development and early differentiation processes

<p>Abstract</p> <p>Background</p> <p>During developmental and adult neurogenesis, doublecortin is an early neuronal marker expressed when neural stem cells assume a neuronal cell fate. To understand mechanisms involved in early processes of neuronal fate decision, we investigated cell lines for their capacity to induce expression of doublecortin upon neuronal differentiation and develop <it>in vitro </it>reporter models using doublecortin promoter sequences.</p> <p>Results</p> <p>Among various cell lines investigated, the human teratocarcinoma cell line NTERA-2 was found to fulfill our criteria. Following induction of differentiation using retinoic acid treatment, we observed a 16-fold increase in doublecortin mRNA expression, as well as strong induction of doublecortin polypeptide expression. The acquisition of a neuronal precursor phenotype was also substantiated by the establishment of a multipolar neuronal morphology and expression of additional neuronal markers, such as Map2, βIII-tubulin and neuron-specific enolase. Moreover, stable transfection in NTERA-2 cells of reporter constructs encoding fluorescent or luminescent genes under the control of the doublecortin promoter allowed us to directly detect induction of neuronal differentiation in cell culture, such as following retinoic acid treatment or mouse Ngn2 transient overexpression.</p> <p>Conclusion</p> <p>Induction of doublecortin expression in differentiating NTERA-2 cells suggests that these cells accurately recapitulate some of the very early events of neuronal determination. Hence, the use of reporter genes under the control of the doublecortin promoter in NTERA-2 cells will help us to investigate factors involved early in the course of neuronal differentiation processes. Moreover the ease to detect the induction of a neuronal program in this model will permit to perform high throughput screening for compounds acting on the early neuronal differentiation mechanisms.</p

Разработка схемы очистки сточных вод от нефтепродуктов

В дипломной работе рассмотрены происхождение, состав, показатели качества сточных вод, методы очистки сточных вод от нефтепродуктов. Проведены исследования качества сточных вод. Разработаны наиболее эффективные методы очистки сточных вод от нефтепродуктов.The thesis examines the origin, composition, quality indicators of wastewater, methods of wastewater treatment from petroleum products. Research wastewater quality water The most effective methods for treating wastewater from oil products have been developed

The Nuclear Protein Sge1 of Fusarium oxysporum Is Required for Parasitic Growth

Dimorphism or morphogenic conversion is exploited by several pathogenic fungi and is required for tissue invasion and/or survival in the host. We have identified a homolog of a master regulator of this morphological switch in the plant pathogenic fungus Fusarium oxysporum f. sp. lycopersici. This non-dimorphic fungus causes vascular wilt disease in tomato by penetrating the plant roots and colonizing the vascular tissue. Gene knock-out and complementation studies established that the gene for this putative regulator, SGE1 (SIX Gene Expression 1), is essential for pathogenicity. In addition, microscopic analysis using fluorescent proteins revealed that Sge1 is localized in the nucleus, is not required for root colonization and penetration, but is required for parasitic growth. Furthermore, Sge1 is required for expression of genes encoding effectors that are secreted during infection. We propose that Sge1 is required in F. oxysporum and other non-dimorphic (plant) pathogenic fungi for parasitic growth

In Vivo Optical Imaging of Neurogenesis: Watching New Neurons in the Intact Brain

Adult neurogenesis is a highly dynamic process modulated by several pathologic and environmental factors, as well as by various compounds. So far, available techniques to study neurogenesis are lengthy and personnel and cost intensive. We developed a new tool based on the doublecortin promoter driving the expression of the luciferase reporter gene (DCX-promo-luciferase) in transgenic mice to perform in vivo imaging of neurogenesis. Indeed, the DCX-promo-luciferase mice allowed optical in vivo imaging of the onset of and increase in neurogenesis in developing fetal brains, as well as imaging of neurogenesis in the intact adult mouse central nervous system. Moreover, the capacity to specifically detect a small number of migrating neuronal precursors in vivo after transplantation is for the first time feasible using this DCX-promo-luciferase transgenic tool. The present imaging approach offers several crucial advantages over methods currently available, such as bromodeoxyuridine incorporation or labeling using iron oxide nanoparticles. Hence, it allows longitudinal study of neurogenesis in intact animals without the requirement of cellular prelabeling. Moreover, it guarantees that detection is specific for neuronal precursors and restricted to viable cells. Hence, our DCX-promo-luciferase transgenic model constitutes an effective tool that answers the pressing need for rapid investigation of the impact on neurogenesis of a large number of candidate compounds waiting to be tested

An alternative splicing modulator decreases mutant HTT and improves the molecular fingerprint in Huntington’s disease patient neurons

Krach et al. dissect the molecular mechanism of the alternative splicing modulator Branaplam in Huntington's disease. They show that the drug lowers mutant HTT protein levels and ameliorates alternative splicing pathology in an iPSC disease model. Huntington's disease (HD) is a neurodegenerative disorder caused by poly-Q expansion in the Huntingtin (HTT) protein. Here, we delineate elevated mutant HTT (mHTT) levels in patient-derived cells including fibroblasts and iPSC derived cortical neurons using mesoscale discovery (MSD) HTT assays. HD patients' fibroblasts and cortical neurons recapitulate aberrant alternative splicing as a molecular fingerprint of HD. Branaplam is a splicing modulator currently tested in a phase II study in HD (NCT05111249). The drug lowers total HTT (tHTT) and mHTT levels in fibroblasts, iPSC, cortical progenitors, and neurons in a dose dependent manner at an IC50 consistently below 10 nM without inducing cellular toxicity. Branaplam promotes inclusion of non-annotated novel exons. Among these Branaplam-induced exons, there is a 115 bp frameshift-inducing exon in the HTT transcript. This exon is observed upon Branaplam treatment in Ctrl and HD patients leading to a profound reduction of HTT RNA and protein levels. Importantly, Branaplam ameliorates aberrant alternative splicing in HD patients' fibroblasts and cortical neurons. These findings highlight the applicability of splicing modulators in the treatment of CAG repeat disorders and decipher their molecular effects associated with the pharmacokinetic and -dynamic properties in patient-derived cellular models

Human in vitro reporter model of neuronal development and early differentiation processes-2

could induce the expression of the DCX-promoter-EGFP reporter (A-H). NTERA-2cells inducing the expression of EGFP upon retinoic acid treatment (I), also expressed DCX (J) and frequently Map2 (K). Parallel upregulation of the EGFP reporter and DCX mRNAs (M) and proteins (N) upon retinoic acid differentiation could also be detected in NTERA-2 clones, but not in HeLa clones. Induction of a neurogenic differentiation program following transient transfection of mouse Ngn2 also resulted in a significant induction of DCX-promoter-luciferase reporter in NTERA-2 clones, but not in HeLa clones (O).<p><b>Copyright information:</b></p><p>Taken from "Human in vitro reporter model of neuronal development and early differentiation processes"</p><p>http://www.biomedcentral.com/1471-2202/9/31</p><p>BMC Neuroscience 2008;9():31-31.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2270856.</p><p></p

Human in vitro reporter model of neuronal development and early differentiation processes-3

ERA-2 cells (A . G) and D283 cells (B . H) showed a morphological response to retinoic acid. PC12 cells treated with NGF developed a complex network of cellular processes (I inset). Scale bar in L = 100 μm.<p><b>Copyright information:</b></p><p>Taken from "Human in vitro reporter model of neuronal development and early differentiation processes"</p><p>http://www.biomedcentral.com/1471-2202/9/31</p><p>BMC Neuroscience 2008;9():31-31.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2270856.</p><p></p