The role of Nuclear Factor One transcription factors in cerebellar development

The cerebellum is a unique and often overlooked component of the central nervous system, with a lobular and multi-layered structure. It consists of a dense interwoven network of interacting neurons and glia with only one major output; through Purkinje neuron afferent projections.\ua0 Upwards of 80% of the cells in the cerebellum are cerebellar granule neurons (CGNs). CGNs arise from highly proliferative, MATH1+ progenitors (CGNPs) in the embryonic brain, found in a germinal region called the rhombic lip (RL). RL progenitors respond to mitogenic signals and cues initiated by transcription factors and migrate along the cerebellar anlage, forming the external granule layer (EGL). Mitogens such as Sonic Hedgehog (SHH), encourage these progenitors to proliferate in the EGL, before they differentiate and migrate inwards forming the internal granule layer (IGL). This migration results in eventual disintegration of the EGL early in postnatal development. One family of transcription factors identified to play a role in self-renewal and differentiation of stem and progenitor populations is the Nuclear Factor One (NFI) family.NFIA, NFIB and NFIX are expressed throughout the central nervous system (CNS) and are essential for normal development. \ua0NFI-mutant mice are embryonic or postnatal lethal, with a number of developmental defects. In the cerebellum, loss of NFIX has been shown to cause delays in the development of both neurons and glia. As the cerebellum consists of almost a dozen different cell types, the first step of this thesis was to examine the cell-type specific expression of NFIX. Immunohistochemistry and co-immunofluorescence analysis revealed that NFIX is strongly expressed in proliferative progenitors in the RL, embryonic and postnatal EGL, as well as in post-mitotic CGNs in the IGL. NFIX was also expressed in Bergmann glia, and GFAP expressing astrocytes in the IGL and white matter. NFIX was expressed in stellate and basket interneurons, as well as in subpopulations of unipolar brush cells, microglia and oligodendrocytes. Interestingly, NFIX was not expressed in Purkinje neurons, despite strong NFIX expression in the ventricular and nuclear transitory zone of the embryonic cerebellum. Lastly, analysis revealed the presence of four NFIX transcript variants in isolated CGNPs. Together these findings suggest NFIX may play an extensive role in cerebellar development, especially in the maturation of CGNs.Nfix-/- mice display delays in cerebellar granule neuron progenitor (CGNP) differentiation, yet the mechanisms behind this delay are yet to be determined. Using co-immunofluorescence staining with granule neuron and proliferative markers, we determined that a delay EGL differentiation is present at P15, with a higher number of proliferative and mitotically active CGNPs. Furthermore, in vitro analysis revealed increased proliferation of Nfix-/- neurospheres, compared to wild-type controls. As CGNPs are the cell of origin of some medulloblastomas (MB), and loss of NFIA expression has led to poorer outcomes in mouse models of MB, we decided to look at NFIX expression in human MB cell lines. NFIX expression was markedly reduced, suggesting that NFIX is crucial for CGNP differentiation. Lastly, to identify mechanistic determinants of this developmental delay, we performed both ChIP-seq for NFIX and RNA-seq on Nfix-/- and control CGNPs at P7. Combining these data with a DNase I hypersensitive site dataset (accessible chromatin) and an RNA-seq dataset from Math1-/- CGNPs, we revealed 578 directly regulated gene targets of NFIX in CGNPs, of which 90 were co-ordinately regulated by Math1. \ua0One of these downstream targets was the Reelin-pathway scaffold protein Itsn1. We showed that like Nfix, loss of Itsn1 results in a delay in CGNP differentiation, potentially exacerbated Nfix-mediated manipulation of Itsn1 binding partner Dab1.\ua0 Lastly, we also found that ITSN1 expression was downregulated in human MB cell lines. Collectively, this showed that NFI-mediation of downstream targets genes is a crucial in the regulation of CGNP differentiation,Previous analysis has shown that NFI expression overlaps in progenitor populations in the developing CNS, suggesting a redundant, compensatory or synergistic mechanism may direct progenitor cell differentiation. Indeed, using co-immunofluorescence staining we found that NFIA and X expression overlaps in both CGNPs, postnatal CGNs and Bergmann glia throughout postnatal development. As co-expression suggests the potential for an overlapping role in development, we performed RNA-seq in Nfiafl/fl; Math1-cre+ CGNPs, and compared this with our Nfix-/- RNA-seq dataset from the previous chapter, to reveal co-ordinately mis-regulated gene targets of NFIA and NFIX. Then, combining these data with a DNase I hypersensitive site dataset, and NFIX, NFIA and NFIB ChIP-seq datasets in P7 CGNPs, we reveal 304 directly regulated gene targets of NFIA and NFIX, of which 283 show co-ordinate regulation, Additionally, 282 of these contain an NFIB-associated ChIP-peak. Further examination of these targets using Gene Ontology reveal co-ordinate regulation of genes involved in nervous system development and cell differentiation, as well as a suite of other transcription factors.Collectively, this thesis presents an in-depth characterisation of both the expression and multifaceted roles of NFI transcription factors in the postnatal cerebellum

Neurodevelopment under the prism of environmental challenges

Prenatal development affects adult health. Exposures to a variety of prenatal environ-mental factors have important effects on fetal development and, in turn, are extensively associated with neurobehavioral, structural and functional phenotypes after birth. Developmental processes are in part promoted by orchestrated levels of glucocorticoids, which are steroid hormones involved in fetal organ maturation. Glucocorticoids also mediate the hormonal stress response of the organism as part of the hypothalamic-pituitary-adrenal axis. During pregnancy levels of glucocorticoids outside of the normal range, either due to maternal pathology including stress-related psychiatric disorders or to antenatal synthetic glucocorticoid treatments, have been associated with altered brain structural and neurobehavioral phenotypes after birth. Interestingly, developmental time-windows seem to interplay with the exposure to influence the direction of post-natal phenotypes. Exposures later in gestation are mainly associated with adverse out-comes while exposures earlier in gestation are additionally associated with potentially beneficial outcomes. While many studies have investigated the effects of glucocorticoids on late developmental time-windows, so far little evidence is available on their effects on early human cortical development and especially during the neurogenic period, which is when neurons are produced. Thus, the potential cellular and molecular underpinnings of the timing dependent divergent effects of glucocorticoids on postnatal phenotypes are not known. To investigate these processes in a complex model of early human neurodevelopment that is reactive to environmental stimuli, I used induced Pluripotent Stem Cells-derived 3-dimensional cerebral organoids and combined them with in vivo mouse neurodevelopment. I found that application of glucocorticoids during neurogenesis increases neurogenic processes that are enriched in species with a gyrified brain, like humans, while are rare in species with a smooth brain, like rodents. These processes contribute to the increased neuronal production and cortical expansion seen in gyrencephalic species. More specifically, at the molecular level this effect is mediated by the glucocorticoid receptor, a transcription factor, which in turn activates ZBTB16 by altering its methylation landscape in specific DNA regulatory elements. Subsequently ZBTB16, a transcription factor itself, increases the expression of PAX6, a key driver of neurogenesis, by activating its promoter. This results in increased numbers of progenitor cells expressing PAX6 and EOMES (a marker of more mature progenitors) in the basal regions of the germinal zones in both organoids and mice. PAX6- and EOMES- positive progenitors are enriched in gyrified species while they are rare in species with smooth brains. The increased numbers of these highly proliferative and neurogenic progenitors lead to an extended neurogenic period and ultimately to increased production of deep layer neurons (BCL11B- positive). Finally, the altered cellular architecture due to glucocorticoids and ZBTB16 potentially mediates beneficial postnatal outcomes as indicated by causal associations with higher educational attainment and increased postnatal cortical thick-ness. This work highlights the importance of early neurodevelopment and specifically of the neurogenic period as a sensitive time-window for glucocorticoid effects. In addition, the molecular and cellular mechanisms as well as the pathways identified could have pro-found implications for our understanding of glucocorticoid effects during early brain development that potentially mediate postnatal outcomes

The role of myeloid cells in neurodegenerative diseases. Studies on cellular phenotype and communication.

Neurological disorders are listed as the second leading cause of death worldwide. Stroke and Alzheimer’s disease (AD) are the major contributors to neurological disorders of deaths. Every year, around 14 million new people are affected by stoke and nearly 10 million new cases of AD are diagonosed worldwide. The number of deaths and disabilities due to these disorders have increased considerably. Neuroinflammation is a major pathological feature appearing in the central nervous system (CNS) in these disorders. The central cellular components for neuroinflammation in CNS is microglia. The multifaceted roles of microglia under pathological conditions have been investigated by several research groups. Even if the knowledge about these cells is extensive, many aspects of their role in diseases have not been well elucidated. One of them is how microglia contribute to the pathology. Thus, I investigated how microglia communicate with other cells through extracellular vesicles (EVs) and how microglia cross-talk with the periphery under AD pathology. Firstly, upon LPS activation of microglia, we found that larger sizes of EVs were released and the levels of proinflammatory cytokines, TNF and IL-6, were increased in EVs. The proteomic profile was shifted to ribosomal assembly and translation. Complete ablation of TNF expression alleviated the inflammation and lead to reduced EVs secretion in a mouse model of stroke, as well as in vitro with LPS stimulation. Microglia are highly dynamic cells and can alter their phenotypes in response to diverse conditions. Therefore, we wanted to elucidate the activation profile of microglia under pathogenic conditions. In the early phase of AD pathogenesis, there is little known about how microglia contribute to the pathology. Thus, the 5xFAD mouse model of AD was used to investigate the effects before the formation of amyloid-β (Aβ) plaques in the CNS. Using a proteomic approach, early inflammatory activation of microglia was found before the formation of plaques.Last but not least, we studied the cross-talk between CNS and periphery under Aβ pathology. There are conflicting findings from animal studies on the contribution of peripheral inflammation on AD due to the variations in the degree of inflammation, timepoint of the stimulation, and the duration. Therefore, we designed our study to have two timepoints wherein we modulate the innate immunity, one was at the pre-plaque stage and the other was at the postnatal stage. Strikingly, a reduced number of plaques was found in hippocampus accompanied by less microglial activation in 5xFAD mice 4.5 months after the peripheral LPS challenge during the pre-plaque state. Subsequently, single-cell sequencing demonstrated that the early inflammation at the postnatal phase affected microglia and bone marrow resident monocytes (BM-Mo) similar to how AD affects these cell types. Interestingly, unique subpopulations of microglia were identified that were enriched in genes associated with lysosomal function (Lyz2) and a detrimental inflammatory marker (Galectin-3). These particular subsets carried a BM-Mo-like phenotype and appeared in response to acute peripheral inflammation and chronic inflammation caused by AD. We also observed that some subsets of myeloid cells were provoked by Aβ pathology but not systemic inflammation and vice versa.To summarize, microglia have multiple ways of contributing to the pathology of AD. Systemic inflammatory challenge can have pronounced effects on innate immunity in the CNS and the underlying mechanism remains to be elucidated. The findings of this thesis also provide potential candidates for use in early diagnostics and pharmacological targets to prevent the progression of AD

Top ten discoveries of the year: Neurodevelopmental disorders

Developmental brain disorders, a highly heterogeneous group of disorders with a prevalence of around 3% of worldwide population, represent a growing medical challenge. They are characterized by impaired neurodevelopmental processes leading to deficits in cognition, social interaction, behavior and motor functioning as a result of abnormal development of brain. This can include developmental brain dysfunction, which can manifest as neuropsychiatric problems or impaired motor function, learning, language or non-verbal communication. Several of these phenotypes can often co-exist in the same patient and characterize the same disorder. Here I discuss some contributions in 2019 that are shaking our basic understanding of the pathogenesis of neurodevelopmental disorders. Recent developments in sophisticated in-utero imaging diagnostic tools have raised the possibility of imaging the fetal human brain growth, providing insights into the developing anatomy and improving diagnostics but also allowing a better understanding of antenatal pathology. On the other hand, advances in our understanding of the pathogenetic mechanisms reveal a remarkably complex molecular neuropathology involving a myriad of genetic architectures and regulatory elements that will help establish more rigorous genotype-phenotype correlations

The role of Tenascin-R in human neurodevelopmental disorders associated with cerebellar dysfunctions

Environ 500 000 enfants au Canada souffrent de maladies génétiques rares. Chacune de ces pathologies causant divers problèmes de santé et touchant un nombre restreint d'individus, nos connaissances des mécanismes sous-jacents et des possibles approches thérapeutiques sont ainsi limitées. Néanmoins, les progrès actuels des technologies de séquençage de l'ADN permettent désormais de découvrir efficacement de nouveaux gènes impliqués dans les maladies neuronales. Grâce à cette approche, le gène de la Tenascin R (TNR) a récemment été identifié comme étant à l'origine d'une maladie neurologique rare. Jusqu'ici, il a été montré chez un enfant souffrant de troubles du développement neurologique que des mutations de la TNR sont associées à une ataxie cérébelleuse et un retard de développement global. TNR est une glycoprotéine de la matrice extracellulaire exclusivement exprimée dans le système nerveux central. Elle participe à la régulation de l'extension et la régénération de l'axone, mais également à la synaptogenèse, la croissance et la migration neuronales. Néanmoins, nos connaissances du rôle de la TNR dans les processus neurodéveloppementaux se basent sur des travaux réalisés chez des rongeurs, et la fonction de cette protéine au cours du développement du cerveau humain demeure inconnue. L'objectif de mon projet de recherche est d'investiguer le profil développemental de cellules progénitrices neuronales humaines (NPCs) issues du patient mentionné ci-dessus, et de déterminer si les anomalies observées au sein du cerveau humain présentant une mutation de TNR sont liées à une altération de la migration, maturation ou encore intégration fonctionnelle des neurones. Grâce à ces travaux, il sera possible d'acquérir des informations importantes sur la fonction de la TNR dans la migration et la maturation des neurones humains. Ce programme de recherche approfondira également notre compréhension des mécanismes fondamentaux régulant le développement neuronal des NPCs issues de patients, ceci étant essentiel à la conception de stratégies thérapeutiques ainsi qu'à la validation de médicaments.Approximately 500,000 children in Canada are affected by rare genetic disorders. Each specific disorder causes several health problems and affects a small number of individuals, therefore our knowledge about mechanisms underlying the disease and possible therapeutic interventions are strongly limited. However, the progress in DNA sequencing technologies now provides an effective way to discover new genes involved in neuronal diseases. Using this innovative approach, Tenascin R (TNR) gene has been recently identified as novel rare neurological disease-causing gene. So far, it has been showed, in a child affected by neurodevelopmental disorder, that mutations in TNR correlate with cerebellar ataxia and global development delay. TNR is a member of extracellular matrix glycoproteins and is exclusively expressed in the central nervous system. TNR contributes to the regulation of axon extension and regeneration, but also to synaptogenesis, neuronal growth and migration. However, our knowledge about the role of TNR in different neurodevelopmental processes is based on experimental work performed in rodents, and the function of this protein in human brain development remains unknown. The aim of this research project is to study the developmental profile of human neuronal progenitor cells (NPCs) derived from the above-mentioned patient and control subjects and to determine whether abnormalities observed in the human brain with TNR mutation are linked to affected neuronal migration, maturation or functional integration. This work will provide crucial information on TNR function during migration and maturation of human neurons. This research project will also deepen our understanding of fundamental mechanisms regulating neuronal development of patient-derived NPCs which will be crucial for designing treatment strategies and drug testing/validation

Neurodevelopmental Consequences of Prenatal Alcohol Exposure: Behavioural and Transcriptomic Alterations in a Mouse Model

Fetal Alcohol Spectrum Disorder (FASD) is an umbrella term referring to a range of physical, behavioural, and cognitive deficits resulting from prenatal alcohol exposure. The resulting abnormalities are heterogeneous and often attributed to timing and dosage of alcohol exposure. However, the specific effects of developmental timing are not well-known. This research used C57BL/6J (B6) as an animal model for early (human trimester one) and mid-gestation (human trimester two) alcohol exposure. Pregnant B6 mice were injected with 2.5 g/kg ethanol on gestational day (GD) 8 and 11 (trimester one equivalent), or on GD 14 and 16 (trimester two equivalent). Resulting pups were followed from birth to adulthood using FASD-relevant behavioural tests. At postnatal day (PD) 70, whole brain tissues were extracted. A third group of dams were injected on GD 16 (short-term). Two hours post injection, fetal brains were removed. Brains were used for genome-wide expression analysis, including microRNAs. Downstream analyses were completed using software packages and online databases. All ethanol-treated pups showed motor skill delays, increased activity, and spatial learning deficits. Gene expression analysis resulted in altered expression of 48 short-term genes between ethanol and control mice treated during the second trimester. Fifty-five and 68 genes were differentially-expressed in the long-term analyses of mice treated during trimester one and two, respectively. Genes involved in immune system response were disrupted across all treatments. Disrupted short-term processes included cytoskeleton development and immunological functions. Processes altered in long-term exposures included stress signaling, DNA stability, and cellular proliferation. MicroRNA analyses returned eight and 20 differentially-expressed miRNAs in trimesters one and two, respectively. Target filtering of trimester one microRNAs and mRNAs resulted in inverse relationships between miR-532-5p and Atf1, Itpripl2, and Stxbp6. Trimester two target filtering resulted in miR-302c targeting Ccdc6. Gene expression and microRNA results demonstrate the stage-specific genes and processes altered during neurodevelopment upon ethanol exposure. Certain cellular processes are disrupted no matter the timing of ethanol exposure. Given that microRNAs are fine-tuners of gene expression, they may play an important role in the maintenance of FASD. Furthermore, transcriptomic changes in the brain may explain the observed behavioural effects of prenatal ethanol exposure