Human neurodevelopment requires the coordinated expression of thousands of genes, exquisitely regulated in both spatial and temporal dimensions, to achieve the proper specialization and inter-connectivity of brain regions. Consequently, the dysregulation of complex gene networks in the developing brain is believed to underlie many neurodevelopmental disorders, such as autism spectrum disorders (ASD). Autism has a significant genetic etiology, but there are hundreds of genes implicated, and their functions are heterogeneous and complex. Therefore, an understanding of shared molecular and cellular pathways underlying the development ASD has remained elusive, hampering attempts to develop common diagnostic biomarkers or treatments for this disorder. I hypothesized that analyzing functional genomics relationships among ASD candidate genes during normal human brain development would provide insight into common cellular and molecular pathways that are affected in autistic individuals, and may help elucidate how hundreds of diverse genes can all be linked to a single clinical phenotype. This thesis describes a coordinated set of bioinformatics experiments that first (i) assessed for gene expression and co-expression properties among ASD candidates and other non-coding RNAs during normal human brain development to discover potential shared mechanisms; and then (ii) directly assessed for changes in these pathways in autistic post-mortem brain tissue. The results demonstrated that when examined in the context of normal human brain gene expression during early development, autism candidate genes appear to be strongly related to the neurodevelopmental pathways of synaptogenesis, mitochondrial function, glial cytokine signaling, and transcription/translation regulation. Furthermore, the known sex bias in ASD prevalence appeared to relate to differences in gene expression between the developing brains of males and females. Follow up studies in autistic brain tissue confirmed that changes in mitochondrial gene expression networks, glial pathways, and gene expression regulatory mechanisms are all altered in the brains of autistic individuals. Together, these results show that the heterogeneous set of autism candidate genes are related to each other through shared transcriptional networks that funnel into common molecular mechanisms, and that these mechanisms are aberrant in autistic brains.

The classical cell cycle regulatory pathway is well appreciated as a key regulator of cell fate determination during neurogenesis; however, the extent of pRB/E2F function in neural stem and progenitor cells is not fully understood, and insight into the mechanisms underlying its connection with cell fate regulation are lacking. The E2F3 transcription factor has emerged as an important regulator of neural precursor cell (NPC) proliferation in the embryonic and adult forebrain, and we demonstrate here that it also influences the self-renewal potential of NPCs. Using knockout mouse models of individual E2F3 isoforms, we demonstrate the surprising result that the classical transcriptional activator E2F3a represses NPC self-renewal and promotes neuronal differentiation, while E2F3b promotes the expansion of the NPC pool and inhibits differentiation. We attribute these opposing activities to a unique mechanism of transcriptional regulation at the Sox2 locus, a key regulator of stem cell pluripotency, whereby E2F3a recruits transcriptional repressors to this site, and E2F3b promotes Sox2 activation. Importantly, E2F3a-mediated Sox2 regulation is necessary for cognitive function in the adult. Additionally, through the determination of genome-wide promoter binding sites for E2f3 isoforms as well as E2F4, another key regulator of NPC self-renewal, we determined that E2Fs are poised to regulate an extensive set of target genes with key roles in regulating diverse cell fate choices in NPCs, including self-renewal, cell death, progenitor expansion, maintenance of the precursor state, and differentiation. Together, these results reveal a diversity of function for E2Fs in the control of neural precursor cell fate, and identify E2F3 isoforms as important regulators of the pluripotency and stem cell maintenance gene Sox2.

Success For Life (SFL) is a brain research-based program for children, birth through age six. This research examined the development and implementation of SFL in 13 early childhood settings. Participants were 24 female early childhood teachers and 146 (73 male) children. Teachers included seven infant, four toddler, nine preschool and four kindergarten teachers. Children included infants(n=29), toddlers(n=27), and prek/kindergartners (n=90). A Request for Proposals was disseminated to identify possible implementation sites. After participation was confirmed, teachers attended a full day's training which included a description of brain development/function, the latest brain research, how to implement SFL and other logistics of the study. Program implementation occurred over approximately four months. A field site coordinator visited each site bimonthly to provide on-going technical assistance. This was an intervention project with a pre and post implementation design. Four instruments were used: a teacher questionnaire, a classroom environment measure, a child measure and teacher journals. Results suggested that teachers became more knowledgeable about brain development research and about how children grow and learn. Teachers were better able to make connections between brain research findings and how to apply these findings to their programs and daily activities. Likewise, the environment measure indicated that teachers were better able to arrange environments for learning. They reported that children showed significant increases in skills development and performance in the following areas: physical mastery, social relations/interactions, cognitive development, and language/communications. Additionally, teachers reported improvements in emotional expression and well-being among infants and toddlers. Toddlers and preschoolers showed significant increases in creative/ artistic expression. Finally, teachers indicated that preschoolers showed increases in initiative, use of logic/mathematics skills, and musical coordination and movement. Research findings suggest that Success For Life is able to bridge the gap between theory and practice and benefits children, teachers and programs.

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Parmi les innovations technologiques récentes, la téléphonie mobile a connu une progression fulgurante. Les expositions aux champs électromagnétiques radiofréquences (CEM RF) apparaissent de plus en plus tôt, lors de l’adolescence, voire dès l’enfance. Parallèlement la littérature scientifique rapporte des effets des CEM RF (GSM 900 MHz) à forts niveaux d’exposition sur l’expression de la glial fibrillary acidic protein (GFAP), le principal filament intermédiaire des astrocytes. Ces cellules jouent un rôle dans la transmission synaptique et dans la réparation des lésions cérébrales. Ce contexte nous a amené à poser l’hypothèse d’une perturbation des fonctions astrocytaires et cérébrales par des expositions aux CEM RF à niveaux élevés réalisées durant les stades de maturation cérébrale du foetus ou de l’adolescent. Nous avons posé une seconde hypothèse selon laquelle la fragilisation des organismes en développement par un épisode inflammatoire les rendrait plus vulnérables aux expositions environnementales, favorisant ainsi l’expression des effets neuro-biologiques des CEM RF.Afin de tester ces hypothèses, nous avons mimé les effets foeto-toxiques consécutifs à un état pathologique de la mère dans un modèle d’inflammation gestationnelle de rat utilisant des injections intra-péritonéales de lipopolysaccharides (LPS). Ce modèle a été exposé aux CEM RF, soit durant toute la gestation (co-exposition avec le LPS), soit durant le stade adolescent. Dans un autre groupe expérimental, nous avons mimé une astrogliose réactive consécutive à une infection ou un état neuro-pathologique au stade adolescent par micro-perfusion de LPS dans les ventricules cérébraux. Dans ce modèle, les rats adolescents ont été co-exposés au LPS aux CEM RF. Les variables d’intérêt ont été mesurées chez le jeune adulte et, dans le cas des co-expositions gestationnelles, au cours des stades juvénile et adolescent. Des paradigmes comportementaux ont été utilisés pour examiner les états émotionnels, la perception et l’adaptation à la nouveauté. Les niveaux de GFAP ont été quantifiés dans le cortex préfrontal, l’hippocampe, le striatum et l’amygdale. Nos résultats indiquent des perturbations comportementales (notamment en réponse à la nouveauté) chez le jeune adulte antérieurement exposé durant la gestation (et non pas durant l’adolescence) aux CEM RF. Une seule interaction du LPS et des CEM RF a été montrée dans le cas d’une co-exposition chez l’adolescent, par une plus faible augmentation des niveaux de GFAP à 1,5W/kg. D’un point de vue de santé publique, ces résultats sont obtenus avec des niveaux d’exposition aux CEM RF bien supérieurs (10 à 50 fois) à ceux environnementaux induits par le port du téléphone portable à proximité du foetus par la femme enceinte ou proche de l’oreille par un appel téléphonique. Dans un premier temps, il sera important de reproduire ces effets avant d’envisager des hypothèses mécanistiques d’interaction des CEM RF sur le développement foetal et sur le processus neuro-inflammatoire au stade adolescent. Il conviendra par ailleurs d’identifier si ces effets sont induits à des niveaux de CEM RF environnementaux afin de contribuer à l’évaluation du risque neuro-toxicologique des CEM RF. / The widespread use of mobile phones raises the question of the possible health effects of radiofrequency electromagnetic fields (RF EMF, GSM 900 MHz) on the brain. Acquisition of the first cell phone occurs predominantly before adolescence. Scientific literature reports effects of high levels of RF EMF exposure on the expression of the glial fibrillary acidic protein (GFAP). The GFAP is the principal intermediate filament of the astrocytes. These cells play a role in the synaptic transmission and brain damages repair. In this context, we hypothesized a disturbance of the astrocytes and brain functions by the exposure of high RF EMF levels carried out during foetal or adolescent cerebral maturation. A second assumption is made that the organisms under development sensitised by an inflammatory episode would be more vulnerable to the environmental exposures and lead the expression of the neuro-biological effects of RF EMF. To test these hypotheses, we mimicked the foetotoxic effects of a pathological state of the mother. We used a gestational inflammation model of rat obtained with intra-peritoneal injections of lipopolysaccharides (LPS). This model was exposed to RF EMF, either during all gestation (co-exposure with LPS), or during the adolescent stage. In another experimental group, we mimicked a reactive astrogliosis consecutive to an infection or a neuro-pathological state at the adolescent stage by micro-perfusion of LPS in the cerebral ventricle. In this model, adolescent rats were co-exposed to LPS and RF EMF. The different endpoints were measured in the young adult. In gestational co-exposure, endpoints were measured during juvenile and adolescent stages. Behavioural paradigms were used to examine the emotional states, the perception and the adaptation to novelty. The GFAP levels were quantified in the prefrontal cortex, the hippocampus, striatum and amygdala. Our results indicate effects on behavioural endpoints (particularly in novelty perception) in the young adult previously exposed to RF EMF during gestation (and not during adolescence). Only one interaction between the LPS and RF EMF was shown in co-exposure during adolescence. A weaker increase of the GFAP levels was shown after a 1,5W/kg exposure. These results were obtained with levels of RF EMF exposure which were much higher (10 to 50 times) than those induced by the mobile phone held near the foetus by the pregnant woman or near the ear during a phone call. It will be important to reproduce these effects before considering mechanistic interactions of RF EMF on the foetal development and the neuro-inflammatory process at the adolescent stage. In addition, it will be necessary to identify if these effects are induced at environmental RF EMF levels in order to contribute to the neuro-toxicological risk evaluation of RF EMF.

Advances in Down syndrome (DS) research depend on the availability of mouse models that replicate the genetic landscape and resulting phenotypes of DS which allow for experimental manipulation to correlate cellular and molecular changes with behavior, in a way that is not possible with human studies alone. These models have been a critical component in understanding the underlying mechanism of the intellectual disability in people with Down syndrome. The Ts(1716)65Dn (Ts65Dn) mouse is one of the most commonly used models as it recapitulates many of the phenotypes seen in individuals with Down syndrome, including neuroanatomical changes and impaired learning and memory. Although Ts65Dn exhibits a number of traits also present in DS, studies have produced variable results across time that call into question the validity of Ts65Dn and its use as a tool for studying Down syndrome. As Ts65Dn is the platform employed to gather preclinical evidence for treatments of DS, a critical assessment regarding the validity of the model over time is necessary. In this study, we conduct a rigorous and comprehensive, comparative analysis of multiple cohorts from the Ts65Dn line to assess the stability and reproducibility of neuroanatomical and behavioral characteristics. We measured gross anatomical brain and body size, neuronal density in the hippocampus and cerebellum, alterations to oligodendrocyte maturation and myelination, acquisition of developmental milestones, and learning and memory performance using the Morris water maze. Our results show a significant amount of variability in Ts65Dn, both across as well as within cohorts. Inconsistent phenotypes in Ts65Dn mice highlight specific cautions and caveats for use of these mice when studying Down syndrome and suggest it is not always the most appropriate model system to use. In addition to phenotypic variability, a major pitfall of the Ts65Dn model is the unintended triplication of 60 non-DS-related genes and the unknown consequences on resulting phenotypes. Recent advances in gene editing strategies have allowed for the gene dosage normalization of those 60 genes and the generation of a new mouse model of Down syndrome, Ts66YAH. As this newly developed line has not yet been characterized, we conducted an analysis complimentary to our study of Ts65Dn to investigate the utility of Ts66YAH for Down syndrome research. We found Ts66YAH mice show no measurable neuroanatomical changes, developmental delays, or learning and memory deficits suggesting that the deleted non-DS-related genes do influence the phenotypes seen in Ts65Dn. Although Ts66YAH is a more genetically representative model of Down syndrome compared to Ts65Dn, it does not exhibit disease relevant phenotypes and therefore, may not be a suitable model for studying DS. The various downfalls identified in the present study may be impacting other mouse models of DS as well and thus, our analysis of Ts65Dn across time and comparative study of Ts66YAH illustrate the need for careful use and increased rigor to ensure translational and reproducible results when working with all mouse models of Down syndrome.

<p>PUFAs serve many important biological and physiological functions within the body and are key for the structure and function of the brain. Omega-6 and omega-3 PUFAs are found in abundance in phospholipids of neuronal membranes that impart structure and function of neurons. Omega-6 PUFAs are instrumental for neurotransmission, neuronal elongation, and neuritogenesis; whereas, omega-3 PUFAs promote neuronal maturation through synaptogenesis. The types of PUFAs incorporated into neuronal membranes is especially important in determining the progression of development. The processes of neurogenesis, neuritogenesis and elongation require large amounts of PUFAs to be incorporated into the membrane phospholipids. To accommodate for the high PUFA needs, maternal dietary PUFA, especially EPA and DHA, recommendations, mobilization of fatty acids into maternal circulation increases, and the accretion rate of PUFA are increased. If maternal nutritional inadequacy of PUFAs occurs during gestation, this can result in impaired cognition, behavioral abnormalities, reduced number of neurons, decreased dendric arborization, altered myelin sheath, and a reduction in brain size. </p> <p> Even though the essentiality of PUFAs in neuronal development is widely accepted, the mechanism is not well understood. There is a lack of consensus in the current literature on the effects of individual PUFAs on each stage of neuronal development and the molecular pathways involved. Despite the inconsistent evidence, the results of numerous studies have consistently suggested that neuronal membrane PUFA composition is associated with neuronal development outcomes, such as number of neurons and neurites, neurite length, and neurotransmitter release. The varying results may be the result of methodological discrepancies with PUFA composition and concentrations, as well as the models used for neuronal development. Additionally, very few studies have taken into consideration the competitive relationship of omega-6 and omega-3 PUFAs in the body when assessing neurodevelopment. </p> <p> This thesis was focused on addressing the role of PUFAs in neuronal development and to address some of the inconsistencies in the literature. attempt to elucidate the individual roles of ALA, ARA, and EPA on neuronal membrane composition and neuronal development. The aim of the thesis research project was to assess the impact of individual PUFAs on neuronal membrane PUFA composition, the membrane n-6:n-3 ratio, and the morphology of SH-SY5Y cells during differentiation. The results of this study demonstrated that supplementation of individual PUFAs alters membrane PUFA composition and the n-6:n-3 ratio. However, there wasn’t a significant effect on neurite number with ALA, ARA, and EPA treatment. Lastly, ARA treatment decreased cell viability compared to the other treatments and the BSA control. Furthermore, additional research needs to be conducted to address other morphological measures and functional outcomes, such as neurotransmitter production and release.</p>

In the adult nervous system, neurotransmitters mediate cellular communication within neuronal circuits. In developing tissues and primitive organisms, neurotransmitters subserve growth regulatory and morphogenetic functions. Accumulated evidence suggests that acetylcholine, (ACh), released from growing axons, regulates growth, differentiation, and plasticity of developing central nervous system neurons. In addition to intrinsic cholinergic neurons, the cerebral cortex and hippocampus receive extensive innervation from cholinergic neurons in the basal forebrain, beginning prenatally and continuing throughout the period of active growth and synaptogenesis. Acute exposure to ethanol in early gestation (which prevents formation of basal forebrain cholinergic neurons) or neonatal lesioning of basal forebrain cholinergic neurons, significantly compromises cortical development and produces persistent impairment of cognitive functions. Neonatal visual deprivation alters developmental expression of muscarinic acetylcholine receptors (mAChR) in visual cortex, whereas local infusion of mAChR antagonists impairs plasticity of visual cortical neurons. These findings raise the possibility that exposure to environmental neurotoxins that affect cholinergic systems may seriously compromise brain development and have long-lasting morphologic, neurochemical, and functional consequences.

The anterograde horseradish peroxidase method was used to determine if prenatal exposure to ethanol affected the development of the characteristic afferent lamination pattern of the commissural projection to the dentate gyrus. Mean ethanol consumption for the ethanol-consuming dams was 12.7 g/kg ± 0.3 g per day. Adult offspring of rats that consumed a liquid diet containing 35% ethanol-derived calories during days 1-21 of gestation, and both pair-fed and normal controls were examined. Brain weights and volumes of the ethanol and pair-fed control rats did not differ significantly from normal controls. However, body weights of ethanol-exposed rats were significantly reduced compared to normal controls. Computer-assisted image analysis of the HRP-labeling revealed that in spite of the heavy ethanol exposure there was no evidence of alterations in the spatial distribution of the commissural terminal field.