Low Gene Expression of Bone Morphogenetic Protein 7 in Brainstem Astrocytes in Major Depression

The noradrenergic locus coeruleus (LC) is the principal source of brain norepinephrine, a neurotransmitter thought to play a major role in the pathology of major depressive disorder (MDD) and in the therapeutic action of many antidepressant drugs. The goal of this study was to identify potential mediators of brain noradrenergic dysfunction in MDD. Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor-β superfamily, is a critical mediator of noradrenergic neuron differentiation during development and has neurotrophic and neuroprotective effects on mature catecholaminergic neurons. Real-time PCR of reversed transcribed RNA isolated from homogenates of LC tissue from 12 matched pairs of MDD subjects and psychiatrically normal control subjects revealed low levels of BMP7 gene expression in MDD. No differences in gene expression levels of other members of the BMP family were observed in the LC, and BMP7 gene expression was normal in the prefrontal cortex and amygdala in MDD subjects. Laser capture microdissection of noradrenergic neurons, astrocytes, and oligodendrocytes from the LC revealed that BMP7 gene expression was highest in LC astrocytes relative to the other cell types, and that the MDD-associated reduction in BMP7 gene expression was limited to astrocytes. Rats exposed to chronic social defeat exhibited a similar reduction in BMP7 gene expression in the LC. BMP7 has unique developmental and trophic actions on catecholamine neurons and these findings suggest that reduced astrocyte support for pontine LC neurons may contribute to pathology of brain noradrenergic neurons in MDD

Molecular and morphological analyses of basal forebrain cholinergic neurons in mouse models of aging and Alzheimer's disease

Thesis (M.A.)--Boston UniversityBasal forebrain cholinergic neurons (BFCNs) of the medial septal nuclei, the diagonal bands of Broca and the nucleus basalis magnocellularis synthesize acetylcholine (ACh) and their projections extend to the cerebral cortex, hippocampus and the amygdala. ACh neurotransmission is essential for learning, attention, memory, arousal and sleep. BFCNs are dependent on a regulated neurochemical environment for the induction, development, maturation and maintenance of their phenotype and viability. However, events that compromise this neurochemical environment can contribute to BFCN dysfunction and/or degeneration, decreased ACh levels and disrupted brain function. During normal aging and Alzheimer’s disease (AD) BFCNs become more vulnerable to dysfunction due to trophic factor withdrawal, cell signaling impairments and other cytopathologic changes. AD is characterized by the deposition of Amyloid-beta (Aβ) plaques and neurofibrillary Tau tangles (NFTs) in the cortex and hippocampus. These pathological AD hallmarks overlap with cortical and hippocampal cholinergic dysfunction, implicating both as the drivers of cognitive and behavioral decline associated with AD. Since, BFCNs are highly vulnerable to AD pathophysiology, factors that support the BFCN phenotype may have practical use in preserving neuronal networks and cognitive function. There is now strong evidence that bone morphogenetic protein-9 (BMP9 also known as growth/differentiating factor 2, GDF2) acts as an induction and maintenance factor that regulates BFCN differentiation in-vitro and in-vivo. Here we used transgenic mice that express green fluorescent protein (GFP) in BFCNs to make observations concerning the BFCN phenotype in aging and AD. We found qualitative evidence that BFCNs of the 24-month old WT/ChAT-GFP mice were smaller and more rounded with shorter processes when compared to 6- month old mouse BFCNs. We analyzed the effects of intracerebroventricular infusion of BMP9 on BFCN projection fibers in the APPswe/PS1dE9 mouse model of AD using laser scanning confocal microscopy. BMP9 not only increased the density of cholinergic projection fibers in the hippocampus of wild type and AD model APPswe/PS1dE9 mice, but it also reduced the plaque burden in the hippocampus of the AD mouse model. These data indicate that BMP9 ameliorated two major pathophysiologic hallmarks of AD, observable in these transgenic APPswe/PS1dE9 mice. BMP9 reduced Aβ plaque burden in this AD model, and enhanced the outgrowth and viability of cholinergic fibers within the hippocampus of both wild-type and APPswe/PS1dE9 mice. [TRUNCATED

Complexity in Developmental Systems: Toward an Integrated Understanding of Organ Formation

During animal development, embryonic cells assemble into intricately structured organs by working together in organized groups capable of implementing tightly coordinated collective behaviors, including patterning, morphogenesis and migration. Although many of the molecular components and basic mechanisms underlying such collective phenomena are known, the complexity emerging from their interplay still represents a major challenge for developmental biology. Here, we first clarify the nature of this challenge and outline three key strategies for addressing it: precision perturbation, synthetic developmental biology, and data-driven inference. We then present the results of our effort to develop a set of tools rooted in two of these strategies and to apply them to uncover new mechanisms and principles underlying the coordination of collective cell behaviors during organogenesis, using the zebrafish posterior lateral line primordium as a model system. To enable precision perturbation of migration and morphogenesis, we sought to adapt optogenetic tools to control chemokine and actin signaling. This endeavor proved far from trivial and we were ultimately unable to derive functional optogenetic constructs. However, our work toward this goal led to a useful new way of perturbing cortical contractility, which in turn revealed a potential role for cell surface tension in lateral line organogenesis. Independently, we hypothesized that the lateral line primordium might employ plithotaxis to coordinate organ formation with collective migration. We tested this hypothesis using a novel optical tool that allows targeted arrest of cell migration, finding that contrary to previous assumptions plithotaxis does not substantially contribute to primordium guidance. Finally, we developed a computational framework for automated single-cell segmentation, latent feature extraction and quantitative analysis of cellular architecture. We identified the key factors defining shape heterogeneity across primordium cells and went on to use this shape space as a reference for mapping the results of multiple experiments into a quantitative atlas of primordium cell architecture. We also propose a number of data-driven approaches to help bridge the gap from big data to mechanistic models. Overall, this study presents several conceptual and methodological advances toward an integrated understanding of complex multi-cellular systems

Developmental Effects of Chronic Low-Level Arsenic Exposure in Mouse Embryonic Stem Cells and in Human Induced Pluripotent Stem Cells

Arsenic is an environmental contaminant commonly found in food and drinking water. Exposure to arsenic during embryonic development has been linked to reduced muscle growth, disrupted muscle development and locomotor activity, impaired neurodevelopment, reduced IQ, impaired memory and learning deficits. While the mechanisms responsible for developmental changes following in utero exposure to arsenic are not well known, one possibility is that arsenic might disrupt proper cellular differentiation. Therefore, we aimed to investigate the mechanisms by which arsenic exposure could alter stem cell differentiation into neurons. First, we continuously exposed P19 mouse embryonic stem (ES) cells to 0.1 μM (7.5 ppb) arsenic for 28 weeks to assess if chronic, low level arsenic exposure would delay cellular differentiation into neuronal cells. Importantly, this concentration is below the current drinking water standard of 10 ppb. The results show temporal changes of genes associated with pluripotency and cellular differentiation. Specifically, starting at week 12, transcript levels of the pluripotency markers Sox2 and Oct4 were increased by 1.9- to 2.5- fold in arsenic-exposed cells. By week 16, SOX2 protein expression was increased, and starting at week 20, the expression of a SOX2 target protein, N-cadherin, was also increased. Concurrently, by week 16, levels of the differentiation marker Gdf3 were decreased by 3.4- fold, along with the reduced phosphorylation of the GDF3 target protein SMAD2/3. To investigate the mechanisms responsible for maintaining pluripotency and hindering cellular differentiation into neurons, RNA sequencing was performed in control and arsenic-exposed cells at week 8, 16 and 24. This analysis revealed significant exposure-dependent changes in gene expression starting at week 16. Pathway analysis showed that arsenic exposure disrupts the Hippo signaling pathway, which is involved in pluripotency maintenance and embryonic development. Immunohistochemistry revealed that the ratios between nuclear (active) and cytoplasmic (inactive) expression of the main effector YAP and the main transcription factor TEAD were significantly increased in arsenic-exposed cells at week 16 and 28. Consistently, expression of the Hippo pathway target genes Ctgf and c-Myc were also significantly upregulated following arsenic exposure. These results indicate that chronic arsenic exposure impairs the Hippo signaling pathway resulting in increased YAP activation, thereby reducing neuronal differentiation. Previous studies have shown that P19 cells differentiate into sensory neurons, so we also wanted to investigate whether arsenic impaired differentiation into motor neurons. Thus, we switched to using human induced pluripotent stem (iPS) cells, which can differentiate into day 6 neuroepithelial progenitors (NEPs), day 12 motor neuron progenitors (MNPs), day 18 early motor neurons (MNs) and day 28 mature MNs. During this process, cells were exposed to arsenic concentrations up to 0.75 μM (56.25 ppb), and morphological alterations along with pluripotency and stage-specific neuronal markers were assessed. Day 6 NEPs exposed to arsenic had reduced levels of the neural progenitor/stem cell marker NES and neuroepithelial progenitor marker SOX1, while levels of these transcripts were increased in MNPs at day 12. Additionally, levels of the motor neuron progenitor marker OLIG2 were increased in day 12 MNPs while levels of the cholinergic neuron marker CHAT were reduced by 2.5- fold in MNPs exposed arsenic. RNA sequencing and pathway analysis showed that the cholinergic synapse pathway was impaired following exposure to 0.5 μM arsenic, and that transcript levels of genes involved in acetylcholine synthesis (CHAT), transport (SLC18A3 and SLC5A7) and degradation (ACHE) were all downregulated in early motor neurons at day 18. In mature motor neurons at day 28, expression of MAP2 and ChAT protein was significantly downregulated by 2.8- and 2.1- fold, respectively, concomitantly with a reduction in neurite length by 1.8- fold following exposure to 0.5 μM arsenic. Similarly, adult mice exposed to 100 ppb arsenic for five weeks had significantly reduced hippocampal ChAT levels. Taken all together, the results of the dissertation show that environmentally relevant levels of arsenic have detrimental effects on neuronal differentiation

Amélioration de l'image et la segmentation (applications en imagerie médicale)

Avancement dans l'acquisition d'image et le progrès dans les méthodes de traitement d'image ont apporté les mathématiciens et les informaticiens dans les domaines qui sont d'une importance énorme pour les médecins et les biologistes. Le diagnostic précoce de maladies (comme la cécité, le cancer et les problèmes digestifs) ont été des domaines d'intérêt en médecine. Développement des équipements comme microscope bi-photonique à balayage laser et microscope de fluorescence par réflexion totale interne fournit déjà une bonne idée des caractéristiques très intéressantes sur l'objet observé. Cependant, certaines images ne sont pas appropriés pour extraire suffisamment d'informations sur de cette image. Les méthodes de traitement d'image ont été fournit un bon soutien à extraire des informations utiles sur les objets d'intérêt dans ces images biologiques. Rapide méthodes de calcul permettent l'analyse complète, dans un temps très court, d'une série d'images, offrant une assez bonne idée sur les caractéristiques souhaitées. La thèse porte sur l'application de ces méthodes dans trois séries d'images destinées à trois différents types de diagnostic ou d'inférence. Tout d'abord, Images de RP-muté rétine ont été traités pour la détection des cônes, où il n'y avait pas de bâtonnets présents. Le logiciel a été capable de détecter et de compter le nombre de cônes dans chaque image. Deuxièmement, un processus de gastrulation chez la drosophile a été étudié pour observer toute la mitose et les résultats étaient cohérents avec les recherches récentes. Enfin, une autre série d'images ont été traités où la source était une vidéo à partir d'un microscopie photonique à balayage laser. Dans cette vidéo, des objets d'intérêt sont des cellules biologiques. L'idée était de suivre les cellules si elles subissent une mitose. La position de la cellule, la dispersion spatiale et parfois le contour de la membrane cellulaire sont globalement les facteurs limitant la précision dans cette vidéo. Des méthodes appropriées d'amélioration de l'image et de segmentation ont été choisies pour développer une méthode de calcul pour observer cette mitose. L'intervention humaine peut être requise pour éliminer toute inférence fausse.Advancement in Image Acquisition Equipment and progress in Image Processing Methods have brought the mathematicians and computer scientists into areas which are of huge importance for physicians and biologists. Early diagnosis of diseases like blindness, cancer and digestive problems have been areas of interest in medicine. Development of Laser Photon Microscopy and other advanced equipment already provides a good idea of very interesting characteristics of the object being viewed. Still certain images are not suitable to extract sufficient information out of that image. Image Processing methods have been providing good support to provide useful information about the objects of interest in these biological images. Fast computational methods allow complete analysis, in a very short time, of a series of images, providing a reasonably good idea about the desired characteristics. The thesis covers application of these methods in 3 series of images intended for 3 different types of diagnosis or inference. Firstly, Images of RP-mutated retina were treated for detection of rods, where there were no cones present. The software was able to detect and count the number of cones in each frame. Secondly, a gastrulation process in drosophila was studied to observe any mitosis and results were consistent with recent research. Finally, another series of images were treated where biological cells were observed to undergo mitosis. The source was a video from a photon laser microscope. In this video, objects of interest were biological cells. The idea was to track the cells if they undergo mitosis. Cell position, spacing and sometimes contour of the cell membrane are broadly the factors limiting the accuracy in this video. Appropriate method of image enhancement and segmentation were chosen to develop a computational method to observe this mitosis. Cases where human intervention may be required have been proposed to eliminate any false inference.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Developmental Origin of Patchy Axonal Connectivity in the Neocortex: A Computational Model

Injections of neural tracers into many mammalian neocortical areas reveal a common patchy motif of clustered axonal projections. We studied in simulation a mathematical model for neuronal development in order to investigate how this patchy connectivity could arise in layer II/III of the neocortex. In our model, individual neurons of this layer expressed the activator-inhibitor components of a Gierer-Meinhardt reaction-diffusion system. The resultant steady-state reaction-diffusion pattern across the neuronal population was approximately hexagonal. Growth cones at the tips of extending axons used the various morphogens secreted by intrapatch neurons as guidance cues to direct their growth and invoke axonal arborization, so yielding a patchy distribution of arborization across the entire layer II/III. We found that adjustment of a single parameter yields the intriguing linear relationship between average patch diameter and interpatch spacing that has been observed experimentally over many cortical areas and species. We conclude that a simple Gierer-Meinhardt system expressed by the neurons of the developing neocortex is sufficient to explain the patterns of clustered connectivity observed experimentall