Brain development and function rely on vascular features that ensure adequate supply of oxygen and nutrients from the blood stream. These features consist of a well-established vascular network, a functional blood-brain barrier, as well as cerebral blood flow regulation. Early life impairments in these features can lead to neurodevelopmental defects. Very few studies have considered the contribution of the brain vasculature to autism spectrum disorders (ASD). A recent postmortem study in young ASD brains suggested an impairment in angiogenesis, a process through which new vessels are formed. A possible link between ASD and altered cerebral perfusion has also been suggested by functional imaging studies. Yet, contribution of cerebrovascular deficits to ASD physiopathology remains elusive, hence a detailed analysis of these deficits is needed. ASD are viewed as neurodevelopmental conditions associated with genetic origins. Mutations identified as a possible cause for ASD include the common 16p11.2 deletion, which leads to the haploinsufficiency of approximately 30 highly-conserved genes. In this thesis, we are using a multidisciplinary approach in order to decipher the cerebrovascular underpinnings of ASD in a mouse model of the 16p11.2 deletion syndrome (16p11.2df/+ mice). We have identified functional and structural cerebrovascular deficits during postnatal development in constitutive 16p11.2df/+ mutants. In particular, 16p11.2df/+ mice display a significant decrease in microvascular branching and density in the cerebral cortex at P14 when compared to age-matched WT littermates. In addition, 16p11.2df/+ mice display a collection of functional abnormalities at P50 when compared to WT mice, such as altered neurovascular coupling in vivo and altered vascular reactivity ex vivo. Notably, we demonstrated a defective endothelium-dependent vasodilation in 16p11.2df/+ mice, while smooth muscle function is unaffected. Furthermore, we generated mice harboring the endothelial-specific 16p11.2 haploinsufficiency (Cdh5-Cretg/+;16p11.2flox/+) in order to dissect the endothelial contribution to ASD phenotypes. These mice underwent behavioral testing to assess whether they display 16p11.2 syndrome -related characteristics. We demonstrated that these conditional mutant mice show home cage hyperactivity in the beam break test, repetitive behaviors in the marble burying test, as well as motor coordination deficits in the rotarod test. Our findings thus establish endothelial cells as key contributors to the pathophysiology of the 16p11.2 deletion syndrome, and provide novel insight into how the cerebral endothelium fine-tunes brain maturation.

The 16p11.2 copy-number variant (CNV) represents a well-characterized, high-risk factor for autism spectrum disorder that additionally predisposes deletion carriers (16pdel) to increased head circumference, known as macrocephaly. The 16p11.2 CNV consists of 29 known genes, many of which are associated with neurobiological processes relevant for macrocephaly such as cell proliferation and apoptosis, differentiation and cell growth. Our lab’s previous work has demonstrated that induced pluripotent stem cell (iPSC)-derived neurons from 16pdel carriers show altered cellular morphology related to growth, which include increased soma size, total dendritic length and dendritic complexity. However, specific CNV genes responsible for these phenotypes have not been established. Here, we investigate the relationship between three 16p11.2 genes and the observed cellular phenotypes. We differentiated neurons from control iPSC-derived neural progenitor cells (NPCs) and used short hairpin RNA (shRNA) to reduce the expression of these CNV genes: KCTD13, MAPK3 and C16ORF53. We then assessed neuronal morphology by evaluating soma size, total dendritic length and dendritic complexity. We demonstrate that knocking down KCTD13 and C16ORF53 increases soma size and total dendrite length, respectively, similar to that observed in 16pdel iPSC-derived neurons. For this reason, we speculate that these genes may have a role in cell growth and might underlie macrocephaly. Thus, our study investigates genes in the 16p11.2 CNV that contribute to neuronal morphology, which may have a role in influencing brain size.

Background: Prevalence of metabolic disturbances is higher among individuals with neurodevelopmental disorders (NDDs), yet this association has been poorly studied. Investigation into human disease remains challenging, as a complete pathophysiological understanding relies on accurate modeling and highly controlled variables. As such, genetically engineered mouse models are increasingly used to gain insight into the biology of human NDDs, but preclinical research focus has been mainly on behavioral and neurophysiological abnormalities. Mouse models engineered to embody human-equivalent genetic variations can display discrepancies to human phenotypes, therefore a thorough characterization of mouse phenotypes must be conducted in order to evaluate how accurately a mouse model embodies a human phenotype. Also, mouse models can help discover unsuspected abnormalities that can be further validated in humans. Objective: In this study, we sought to investigate the metabolic alterations derived from NDD-associated genetic polymorphisms in previously-validated genetic mouse models. Due to the similarities in NDD-associated phenotypic expression, we hypothesized that our NDDs of interest would express similar metabolic signatures. Further, we anticipated that we might uncover unknown metabolic anomalies, and that sex may alter these differences. Methods: We used the Comprehensive Lab Animal Monitoring System coupled to EchoMRI, as well as quantification of key plasma metabolites by liquid chromatography-mass spectrometry to characterize and compare basal metabolism in three mouse models of NDDs, namely Down syndrome (Dp(16)Yey/+ mice), 16p11.2 deletion syndrome (16p11.2df/+ mice) and Fragile X syndrome (Fmr1-/- KO mice) and their wild-type (WT) counterparts. Results: Our study reveals that each mouse model expresses a unique metabolic signature that is sex-specific, independent of the amount of food consumed and minimally influenced by physical activity. We found striking differences in body composition, respiratory exchange ratio, caloric expenditure and concentrations of circulating plasma metabolites related to mitochondrial function. Conclusion: Providing novel insight into NDD-associated metabolic alterations provides a basis for future studies aimed at understanding physiological mechanisms and provides a point of reference for research aimed at detecting changes in response to intervention.

Die 3D Struktur von Chromosomen im Zellkern reguliert verschiedene Funktionen in der Zelle und Fehler in der 3D Faltung des Genoms können pathogen sein. 3D Genomfaltung kann mit verschiedenen Methoden untersucht werden um Chromatinkontakte, sowie die Position von DNA in Relation zu sub-nuklearen Bereichen oder der Kernmembran zu detektieren. Hier verwende ich GAM und Hi-C um zwei Aspekte der 3D Genomtopologie zu untersuchen, die Allelspezifität von Chromatinkontakten und Kontakte zwischen Chromosomen. Ich untersuche allelspezifische Kontakte in murinen embryonalen Stammzellen und Interaktionen zwischen Chromosomen im Zusammenhang mit Autismus Spektrum Störung auf ihre Relevanz in der Regulation von Genen. Zur allelspezifischen Detektion von Chromatinkontakten generierte ich einen GAM Datensatz der tausende von nuklearen Cryodünnschnitten enthält. Die Generierung dieser Daten beinhaltete die Entwicklung einer verbesserten Version der GAM Methode zur Produktion von großen Datensätzen in Hochdurchsatz. Hier zeige ich, dass GAM effizient Haplotyp-spezifische Chromatinkontakte bestimmen kann. Erste Untersuchungen von allelspezifischer 3D Genomtopologie zeigten weitreichende Unterschiede zwischen den Allelen, welche „A/B compartments“ und spezifische Chromatinkontakte beinhalten, wie zum Beispiel am Imprinting Locus H19/Igf2. Zur Untersuchung von interchromosomalen Kontakten detektierte ich Chromatinkontakte mit Hi-C im Kontext einer genomischen Deletion am humanen 16p11.2 Locus, assoziiert mit Autismus Spektrum Störung. Ich zeige hier, dass die Deletion am 16p11.2 Locus zu der Reorganisation von spezifischen interchromosomalen Kontakten zwischen 16p11.2 und Chromosom 18 führt, und stelle eine Hypothese auf wie diese interchromosomalen Kontakte zur ektopischen Aktivierung von Pcdh Genen auf Chromosom 18 führen. Protocadherins haben wichtige Funktionen in neuronaler Konnektivität, ein Prozess dessen Störung zur Manifestierung von Autismus Spektrum Störung beitragen könnte. / The 3D folding of interphase chromosomes inside the nucleus regulates important nuclear functions and once disrupted can lead to the manifestation of disease. Different techniques can be used to map 3D genome folding and detect pairwise and multiway interactions of the genome, or map the positions of DNA with respect to subnuclear compartments or the nuclear lamina. Here, I use GAM and Hi-C to explore two aspects of 3D genome topology, the allele specificity of chromatin contacts and long-range contacts between chromosomes, respectively. I detect specific contacts of the parental alleles in mouse embryonic stem cells and interactions between chromosomes in the context of congenital disease and study them with regard to their functionality and importance in mammalian gene regulation. For detecting chromatin contacts with allele specificity, I produced a GAM dataset containing thousands of nuclear slices. The collection of this data was accompanied by the development of a high-throughput version of GAM that allows the generation of large datasets. I show that GAM can determine haplotype-specific chromatin contacts with high efficiencies. First explorations of allele-specific chromatin topologies reveal many differences between the parental alleles, including allele-specific compartments A and B, and specific chromatin contacts, for example at the imprinted H19/Igf2 locus. For the exploration of inter-chromosomal contacts in disease, I mapped chromatin interactions with Hi-C in the context of a CNV at the human 16p11.2 locus, associated with autism spectrum disorders. Here, I show that the deletion at the 16p11.2 locus results in the rearrangement of specific inter-chromosomal contacts between the 16p11.2 locus and chromosome 18 and propose a role for these inter-chromosomal contact changes in the upregulation of the nearby Pcdhb gene cluster, which comprises protocadherin genes with important functions in neuronal connectivity during development.