Automatic Robust Neurite Detection and Morphological Analysis of Neuronal Cell Cultures in High-content Screening

Cell-based high content screening (HCS) is becoming an important and increasingly favored approach in therapeutic drug discovery and functional genomics. In HCS, changes in cellular morphology and biomarker distributions provide an information-rich profile of cellular responses to experimental treatments such as small molecules or gene knockdown probes. One obstacle that currently exists with such cell-based assays is the availability of image processing algorithms that are capable of reliably and automatically analyzing large HCS image sets. HCS images of primary neuronal cell cultures are particularly challenging to analyze due to complex cellular morphology. Here we present a robust method for quantifying and statistically analyzing the morphology of neuronal cells in HCS images. The major advantages of our method over existing software lie in its capability to correct non-uniform illumination using the contrast-limited adaptive histogram equalization method; segment neuromeres using Gabor-wavelet texture analysis; and detect faint neurites by a novel phase-based neurite extraction algorithm that is invariant to changes in illumination and contrast and can accurately localize neurites. Our method was successfully applied to analyze a large HCS image set generated in a morphology screen for polyglutaminemediated neuronal toxicity using primary neuronal cell cultures derived from embryos of a Drosophila Huntington’s Disease (HD) model.National Institutes of Health (U.S.) (Grant

Target Identification : A Challenging Step in Forward Chemical Genetics

Investigation of the genetic functions in complex biological systems is a challenging step in recent year. Hence, several valuable and interesting research projects have been developed with novel ideas to find out the unknown functions of genes or proteins. To validate the applicability of their novel ideas, various approaches are built up. To date, the most promising and commonly used approach for discovering the target proteins from biological system using small molecule is well known a forward chemical genetics which is considered to be more convenient than the classical genetics. Although, the forward chemical genetics consists of the three basic components, the target identification is the most challenging step to chemical biology researchers. Hence, the diverse target identification methods have been developed and adopted to disclose the small molecule bound protein. Herein, in this review, we briefly described the first two parts chemical toolbox and screening, and then the target identifications in forward chemical genetics are thoroughly described along with the illustrative real example case study. In the tabular form, the different biological active small molecules which are the successful examples of target identifications are accounted in this research review.22Yothe

Dynein Modifiers in C. elegans: Light Chains Suppress Conditional Heavy Chain Mutants

Cytoplasmic dynein is a microtubule-dependent motor protein that functions in mitotic cells during centrosome separation, metaphase chromosome congression, anaphase spindle elongation, and chromosome segregation. Dynein is also utilized during interphase for vesicle transport and organelle positioning. While numerous cellular processes require cytoplasmic dynein, the mechanisms that target and regulate this microtubule motor remain largely unknown. By screening a conditional Caenorhabditis elegans cytoplasmic dynein heavy chain mutant at a semipermissive temperature with a genome-wide RNA interference library to reduce gene functions, we have isolated and characterized twenty dynein-specific suppressor genes. When reduced in function, these genes suppress dynein mutants but not other conditionally mutant loci, and twelve of the 20 specific suppressors do not exhibit sterile or lethal phenotypes when their function is reduced in wild-type worms. Many of the suppressor proteins, including two dynein light chains, localize to subcellular sites that overlap with those reported by others for the dynein heavy chain. Furthermore, knocking down any one of four putative dynein accessory chains suppresses the conditional heavy chain mutants, suggesting that some accessory chains negatively regulate heavy chain function. We also identified 29 additional genes that, when reduced in function, suppress conditional mutations not only in dynein but also in loci required for unrelated essential processes. In conclusion, we have identified twenty genes that in many cases are not essential themselves but are conserved and when reduced in function can suppress conditionally lethal C. elegans cytoplasmic dynein heavy chain mutants. We conclude that conserved but nonessential genes contribute to dynein function during the essential process of mitosis

Glycogen Synthase Kinase-3: a Role in Ageing and Metabolism

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Modelos Animales en Biotecnología y BiomedicinaClave Programa: DBICódigo Línea: 22Prohibitins (PHBs) are a class of conserved mitochondrial proteins that profoundly influence ageing. PHB depletion shortens the lifespan of wild type animals, while it causes a dramatic extension in metabolically compromised daf-2(e1370) mutants. This opposing lifespan phenotype is attributed to alterations in mitochondrial function and metabolism, but the exact function of PHBs is yet to be deciphered. This project was developed to better understand the function of the essential mitochondrial prohibitins in the regulation of ageing. To elucidate novel signalling mechanisms mediating the metabolic adjustments that lead to opposite ageing outcomes in response to PHB depletion, we performed a kinase RNAi screen using prohibitin deletion mutants. First, we characterized prohibitin deletion mutants. As these mutants are sterile, they are maintained balanced heterozygous. We accomplished a sorting protocol for selection of homozygous PHB mutants. We used vital Nile Red (NR) staining as a read-out as PHB depletion reduces NR staining. In order to quantify the intensity of NR staining, we developed an image analysis protocol. From the screen, we identified the conserved Glycogen Synthase Kinase-3 (GSK-3), as a strong suppressor of the reduced NR staining phenotype caused by prohibitin deletion mutants. Beyond its role as a regulator of insulin-dependent glycogen synthesis, GSK-3 also controls critical cellular functions. We investigated how GSK-3 influences longevity in conditions of compromised insulin signalling and mitochondrial impairment. We demonstrate that GSK-3 depletion decreases wild type lifespan but does not affect phb-2 mutants. However, the long lived daf-2 and phb-2;daf-2 mutants show strong suppression in lifespan upon loss of GSK-3. We show that GSK-3 is ubiquitously expressed via CRISPR-Cas9 endogenous gene tagging. We examined several parameters, including alterations in energy stores - glycogen/triglycerides, mitochondrial respiration and lipid composition to deduce how metabolic alterations upon GSK-3 depletion influence lifespan and found that these varied in a genetic background specific manner. Additionally, we also prove that the activity of GSK-3 is essential in the intestine for normal ageing and especially for the long lived daf-2 mutants. Our data thus, delineates a novel role for GSK-3 in metabolism and its interplay with IIS and mitochondrial metabolism in ageing regulation.Universidad Pablo de Olavide de Sevilla. Departamento de Biología Molecular e Ingeniería BioquímicaPostprin

Unravelling the Role of Long Noncoding RNAs in the Context of Cell-growth and Regeneration

[eng] Long noncoding RNAs (lncRNAs) have proven biological roles in plethora cellular contexts. Nonetheless, only a handful have been clearly characterized, leaving thousands of newly discovered lncRNAs without an associated function, and sometimes considered as transcriptional by-products. To this end, this thesis work had focused on exploring lncRNA functionality in two scenarios. First, in order to discern between lncRNAs affecting cell-growth rate (lncRNA-hits) and lncRNA-not-hits, we built a tree-based classifier based on high-throughput CRISPRi functional screen data in seven human cell lines, as well as, cell-specific ENCODE transcription factor ChIP-seq data; finding that the genomic features used in our study showed small effects and tend to be transcript-specific. Our classifier outperformed previ- ous algorithms, displayed balanced sensitivity and specificity values, and uncovered a lncRNA (LINC00879) involved in cell-growth. Additionally, we unveiled a list of 40 lncRNAs as candidates for experimental validation. Second, we characterized the lncRNA profile during regeneration, using Drosophila wing imaginal disc as a regeneration-model. We selected a candidate lncRNA (CR40469) and evaluated its role in regeneration at the early stage of cell-damage. Subsequently, using RNA- seq data, we observed significant transcriptomic alterations in consequence of the CR40469 genetic deletion, suggesting its role in regeneration. In this study we have generated a list of lncRNAs whose possible biological role in cell-growth and in re- generation can be further studied

Refining prognosis capacity and implementing priciples of personalized therapies for Neurofibromatosis Type 2

[eng] Neurofibromatosis type 2 (NF2) is an autosomal dominant condition caused by loss of function variants in the NF2 gene. The pathognomonic feature of the disease is the development of bilateral vestibular schwannomas (VS), benign tumours that result from the overgrowth of Schwann cells (SC) on the vestibulocochlear nerves. Affected individuals are at high risk of developing schwannomas in other cranial, spinal and peripheral nerves, as well meningiomas and ependymomas, tumours that are responsible for significant morbidity. The clinical management of these patients is complex due to the limited treatment options and the broad and variable clinical spectrum of the disease, strongly associated with the type of variant inherited in the NF2 gene. The general objective of this thesis was to contribute to the development of strategies for a personalized medicine for Neurofibromatosis Type 2. Specifically, the three aims were to assess and improve the prognosis capacity of the disease, to evaluate the use of antisense gene therapy approaches in vitro for pathogenic variants causing NF2 and finally, to develop an induced pluripotent stem cell-based model for further studies on the NF2 gene role and the development of targeted therapies. In 2017, the UK NF2 Reference Group established a Genetic Severity Score (GSS) to predict the severity of the disease considering the NF2 pathogenic variant. In the present work, we validated the GSS in a cohort of NF2 patients from the Spanish National Reference Centre (CSUR) of Phacomatoses. In addition, data from the identification of potential novel prognostic molecular markers were considered to suggest a revision of the GSS, called the Functional Genetic Severity Score (FGSS). The FGSS includes data from functional assays and the predicted effect of pathogenic variants on merlin, the NF2 protein. With the aim to develop a personalized therapeutic approach for NF2 patients, we evaluated the use of antisense oligonucleotides for NF2 loss of function variants in patients’ primary fibroblast cell cultures. First, we tested the use of phosphorodiamidate morpholino oligomers (PMOs) to modulate the effect of NF2 splice site variants, although the approach proved unsuccessful to restore merlin levels. The second strategy consisted of reducing the severe effect of truncating variants affecting the NF2 gene. We used PMOs to induce the skipping of an in-frame exon harbouring a nonsense or frameshift variant to generate novel, and potentially functional, merlin forms. Encouraging results emerged for germline truncating variants in exon 11 of NF2, for which it was possible to generate a hypomorphic merlin (merlin-e11) that partially rescued the NF2 phenotype in primary fibroblasts cultures. This result constitutes an in vitro proof of concept of the use of PMOs as a personalized therapy for NF2 patients. Finally, due to the limited preclinical models available for NF2, we generated induced pluripotent stem cell (iPSC) lines with single or bi-allelic loss of function of NF2 through combining direct reprogramming of VS cells with the use of CRISPR/Cas9 genome editing. In our experience, the NF2 gene could be essential for reprogramming and maintaining pluripotency. Despite difficulties encountered in maintaining pure merlin-deficient pluripotent cultures, iPSCs lines were characterized and differentiated towards the Neural Crest – Schwann cell axis. The application of a 3D Schwann cell differentiation protocol led to the successful generation of NF2 (+/-) and NF2 (-/-) spheroids that co-expressed the classical lineage markers p75 and S100B, potentially representing a genuine VS model

Modélisations de maladies des motoneurones en utilisant le poisson zébré

Les paraplégies spastiques familiales (PSF) sont un groupe de maladie neurodégénératives hétérogènes affectant les neurones moteurs supérieurs et causant une faiblesse musculaire progressive des membres inférieurs entrainant des problèmes de marche. Plus de 60 gènes ont été lies à cette maladie, leur nombre augmentant régulièrement. La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative à déclenchement tardif qui affecte les neurones moteurs supérieurs et inférieurs, entrainant une atrophie musculaire accompagnée de spasticité. La mort, causée par une insuffisance respiratoire survient dans les 2 à 5 ans après le début de la maladie. Ces deux maladies de neurones moteurs, bien que différentes, ont des gènes et des mécanismes pathologiques en commun. Ainsi, accroitre notre connaissance de leurs similarités et de leurs différences pourra nous aider à mieux comprendre chacune individuellement. Pour étudier ces deux maladies, nous avons utilisé des modèles de poisson zébré précédemment caractérisés et en avons développé de nouveaux pour approfondir nos connaissances sur les mécanismes physiopathologiques. Dans la première partie de cette thèse, nous avons identifié le stress du réticulum endoplasmique (RE) comme un nouveau mécanisme pathologique induit par la perte de fonction de spastin, un gène impliqué dans la PSF, et avons montré que des modulateurs du stress du RE sont capables de renverser le phénotype locomoteur. Nous avons aussi identifié un nouveau gène causatif de la PSF, CAPN1 (SPG76), et avons validé in vivo la pathogénicité de sa perte de fonction en identifiant une désorganisation des réseaux de microtubules comme phénotype principal. Dans la deuxième partie de cette thèse, nous avons généré plusieurs nouveaux modèles de poisson zébré de la SLA. Deux lignées transgéniques exprimant la protéine humaine de type sauvage ou mutante sous le contrôle d’un promoteur inductible nous ont permis de reproduire des résultats obtenus précédemment par l’injection d’ARNm et d’identifier des changements transcriptomiques similaires à ceux obtenus récemment avec des modèles de souris. Nous ii avons aussi généré deux nouvelles lignées en introduisant des mutations ponctuelles liées à la SLA dans les gènes tardbp et fus du poisson zébré en utilisant la technologie CRISPR/Cas9. Ces résultats soulignent la valeur du poisson zébré comme modèle pour étudier les maladies des neurones moteurs et leurs mécanismes physiopathologiques, et suggèrent de nouvelles approches thérapeutiques.Hereditary spastic paraplegias (HSP) are a group of heterogeneous neurodegenerative diseases affecting upper motor neurons, causing progressive gait dysfunction and more than 60 genes have been linked to this disease. On the other hand, amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disorder that affects both upper and lower motor neurons, leading to muscle atrophy with spasticity and death in two to five years due to respiratory failure. These two motor neuron disorders, while separate, share common genes and pathological mechanisms and as such, increasing our knowledge about their similarities and differences can help us have a better understanding of each of them individually. In order to study these two diseases, we used previously characterized zebrafish models and developed new ones to deepen our understanding of the pathophysiological mechanisms of HSP and ALS. In the first part of this thesis, we identified ER stress as a new pathological mechanism at play in HSP due to spastin loss-of-function and showed that ER stress modulators are able to rescue the locomotor phenotype. We also identified a new gene causative of HSP, CAPN1 (SPG76), provided in vivo validation of its loss-of-function pathogenicity and identified microtubule networks disorganization as one of the main defects. In the second part of this thesis, we generated several new zebrafish models to study ALS. Two transgenic lines expressing either a wild-type or a mutant TDP-43 protein under the control of an inducible promoter allowed us to recapitulate previous findings obtained with mRNA injections and identify transcriptomic changes due to the mutant protein that are in line with recent transcriptomic data obtained in mouse models. We also generated two new lines with knock-in of ALS-causative point mutations in the tardbp and fus zebrafish endogenous genes using the CRISPR/Cas9 technology. These results underscore the value of the zebrafish model to study motor neuron disorders and their pathophysiological mechanisms as well as open new therapeutic avenues