Characterisation of dysfunctional Wnt/β-Catenin signalling in the Down syndrome brain

Down syndrome (DS) is the most common human aneuploidy. It results from the presence of three copies, or trisomy, of human chromosome 21 (Hsa21). DS is associated with a plethora of characteristic clinical features, most notably learning disability, altered body morphology, congenital heart disease and early-onset Alzheimer’s disease (AD-DS). Despite knowledge of its primary cause, pathological mechanisms underlying DS are poorly understood, including potential deficits in essential signalling processes. This thesis investigates Wnt/β-catenin signalling dysfunction in DS. The Wnt signalling pathway is a fundamental transduction cascade with key roles in development, cancer and neurodegeneration. Particularly, mounting evidence suggests that AD neuropathology may be underscored by critical dysfunction of canonical Wnt signalling. Given the close relationship between AD and DS, it is proposed in this thesis that Wnt abnormalities may also be present in the DS brain. This hypothesis is thus investigated, combining bioinformatics with RNA and protein analysis in DS mouse models and humans. The evidence gathered here suggests canonical Wnt signalling is dysfunctional in the DS brain. Most importantly, Wnt signalling activity is suppressed in the adult DS hippocampus. Furthermore, this thesis identifies the Hsa21-encoded kinase DYRK1A, an essential contributor to DS, as a novel, bimodal Wnt signalling regulator. DYRK1A may both suppress and enhance Wnt activity, depending on the activation state of the pathway. It is proposed that, in DS, dosage imbalance of DYRK1A may substantially affects Wnt signalling, with a complex array of resulting transcriptional changes to Wnt target genes. This mechanism may contribute to several developmental and adult features of DS, particularly learning disability and AD-DS. Overall, these findings may provide key evidence for the global understanding of this condition, and targeting Wnt signalling may open unexplored avenues for therapeutic development

Immunity Against Malaria: an Atlas of the Mosquito Cellular Immune System at Single Cell Resolution

Malaria is a deadly, worldwide disease, yearly responsible for 219 million cases and over four hundred thousand deaths. The Anopheles gambiae species complex is the main African vector for the most virulent malaria parasite: Plasmodium falciparum. Mosquitos are not mere bystanders however, and rely on both humoral and cellular innate immune divisions to defeat invading pathogens. These efforts are coordinated by hemocytes, the insect equivalent to vertebrate’s white blood cells, circulating in the hemolymph within the insects’ body cavity. Yet, hemocyte biology is largely unknown, mainly due to the low number and fragility of mosquito immune cells. Here we dissect the Anopheles immune system under baseline and challenged conditions with single-cell RNA sequencing to identify previously unknown cell types, their gene signatures, and spatial-temporal localization in the mosquito. We profiled 5,292 individual Anopheles hemocytes 1,3 and 7 days after sugar-feeding, blood-feeding, or infection with Plasmodium berghei, as well as 3123 A. aegypti hemocytes. We identified 9 cell sub-types, including novel effector, phagocytic, and anti-microbial cell subtypes, in addition to dividing progenitor cells, validating the main cell types via correlative microscopy and morphology. And we described four lineages of hemocytes, showing them to be divided into two branches: oenocytoids and prohemocyte-granulocyte. We also found both blood-feeding and malaria infection to dramatically shift the composition of a mosquito’s immune system, activating effector and proliferating cells at days 1 and 3 before returning to baseline by day 7. Conversely, human P. falciparum appears to inactivate an important local effector subtype. Our work is the first comprehensive transcriptomic study of a whole insect immune system. It demonstrates hemocytes are a dynamic, diverse class of insect cells which complexity far exceeds what is currently described in the literature. Our methods and results will hopefully serve as a resource for many entomologists, and could prove useful in developing novel vector control strategies. Our website will ease data access and provide an intuitive way to visualise hemocyte information: https://hemocytes.cellgeni.sanger.ac.uk/NIH Oxford-Cambridge scholarship, the UCLA-Caltech MSTP, and the NIGMS T32 GM00804

Control of T-cell development and function in health and disease

T lymphocytes are at the core of the adaptive immune system. Fitness of T cells defines susceptibility to infections, autoimmune diseases or cancer. Inherited failure in T-cell generation and/or further differentiation might lead to primary immunodeficiencies (PIDs). The generation of healthy T cells is a complex, multistep process that essentially continues throughout the life of an organism. Therefore, its holistic understanding is a prerequisite for the development of therapeutic strategies. In the works summarised here, T-cell development served as a model to address several fundamental questions in immunology. It starts from the earliest events during the thymus settling and T-cell development. Both depend on minute numbers of precursors that enter this organ from the blood. It was not known precisely how many precursors enter the thymus per day and how this process is regulated. Using fate-mapping experiments and mathematical modelling, we quantified the number of precursors entering the thymus and cellular feedback which regulates this process. This is a very important finding, especially in the context of thymus reconstitution and transplantation. Three further studies demonstrate how fine-tuning of T cell development by single micro RNA (miRNA), miR-181a-1, critically influences the emergence and function of T cells which require strong TCR-signals during their selection, namely invariant NKT (iNKT) cells and regulatory T (Treg) cells. Of note, post-transcriptional changes mediated by miR-181a-1 can imprint long-lasting changes on the protein levels, which has not been yet reported for any other miRNA. Finally, through studying human primary immunodeficiencies, we discovered that gene previously solely associated with the initiation of clathrin-mediated endocytosis (CME) – FCHo1 (F-BAR domain only protein 1) – is essential for human T cell development and activation. This initial genetic discovery followed by experiments on human cellular models and high-resolution confocal microscopy allowed us to show broader principles governing the immune system: endocytosis of T cell receptor (TCR) and in consequence signalling, activation and subsequent lymphocyte selection depend on CME – processes initiated by FCHo1. This opened so far unexplored avenues of research.T-Lymphozyten bilden den Kern des adaptiven Immunsystems. Die Fitness von T-Zellen definiert die Anfälligkeit für Infektionen, Autoimmunerkrankungen oder Krebs. Vererbtes Versagen bei der Erzeugung von T-Zellen und / oder weitere Differenzierungen können zu primären Immundefekten (PIDs) führen. Die Erzeugung gesunder T-Zellen ist ein komplexer, mehrstufiger Prozess, der sich im Wesentlichen während des gesamten Lebens eines Organismus fortsetzt. Daher ist sein ganzheitliches Verständnis eine Voraussetzung für die Entwicklung therapeutischer Strategien. In den hier zusammengefassten Arbeiten diente die T-Zellen-Entwicklung als Modell zur Beantwortung mehrerer grundlegender Fragen der Immunologie. Es beginnt mit den frühesten Ereignissen während der Thymussiedlung und der T-Zellen-Entwicklung. Beides hängt von der winzigen Anzahl von Vorläuferzellen ab, die aus dem Blut in das Organ gelangen. Es war bislang nicht genau bekannt, wie viele Vorläuferzellen pro Tag in den Thymus gelangen und wie dieser Prozess reguliert wird. Mithilfe von fate-mapping Experimenten und mathematischen Modellen haben wir die Anzahl der Vorläuferzellen quantifiziert, die in den Thymus eintreten und die zelluläre Rückkopplung, die diesen Prozess reguliert. Dies ist ein sehr wichtiger Befund, insbesondere im Zusammenhang mit der Rekonstitution und Transplantation des Thymus. Drei weitere Studien zeigen, wie die Feinabstimmung der T-Zellen-Entwicklung durch einzelne Mikro-RNA (miRNA), miR-181a-1, die Entstehung und Funktion von T-Zellen, die während ihrer Selektion starke TCR-Signale benötigen, nämlich die invariante NKT (iNKT), Zellen und regulatorische T (Treg) - Zellen entscheidend beeinflusst. Bemerkenswerterweise können durch miR-181a-1 vermittelte post-transkriptionelle Veränderungen langanhaltende Veränderungen des Proteinspiegels erwirken, über die bisher für keine andere miRNA berichtet wurde. Schließlich entdeckten wir durch Untersuchung der primären Immundefekte beim Menschen, dass das Gen, das zuvor ausschließlich mit der Initiierung der Clathrin-vermittelten Endozytose (CME) assoziiert war - FCHo1 (nur Protein 1 der F-BAR-Domäne) - für die Entwicklung und Aktivierung menschlicher T-Zellen essentiell ist. Diese erste genetische Entdeckung, gefolgt von Experimenten an menschlichen Zellmodellen und hochauflösender konfokaler Mikroskopie, ermöglichte es uns, umfassendere Prinzipien für das Immunsystem aufzuzeigen: Die Endozytose des T-Zellen-Rezeptors (TCR) und folglich die Signalübertragung, Aktivierung und anschließende Selektion der Lymphozyten hängen von CME ab - von FCHo1 initiierten Prozessen. Dies eröffnete bisher unerforschte Forschungswege

Global and Local Regulation of Gene Expression in the Human Brain

Neuropsychiatric disorders are behavioral conditions marked by intellectual, social, or emotional deficits that can be linked to diseases of the nervous system. Autism spectrum disorder (ASD), schizophrenia (SCZ), bipolar disorder (BP), major depressive disorder (MDD), and attention deficit and hyperactivity disorder (ADHD) are common, heritable diseases each with a prevalence exceeding 1% of the population, none of which can be characterized by discernable anatomical or neurological pathologies. Genetic association studies have identified mutations in hundreds of genes that contribute to risk for at least one of these disorders, and have shown that a substantial fraction of the genetic liability is shared between many of these neuropsychiatric diseases. It has long been hoped that with enough genetic evidence we will identify the biological pathways, developmental time points, and brain regions that, when disrupted, give rise to neuropsychiatric disorders. However, the cellular and functional complexity of the human brain, as well as the genetic complexity of neuropsychiatric disease, make it difficult to search for such convergence. In this thesis, I investigate global and local transcriptional regulation within and across 12 regions of the human brain in order to investigate the regional specificity of neuropsychiatric disorders. I develop novel bioinformatics methods – ranging from data processing to network construction – to identify whether the transcriptional regulation of a set of genes is shared or specific. I hypothesize that local, region-specific transcriptional regulation corresponds directly to cell types and processes that are specific to, or far more prevalent in, a given region; that cross-regional transcriptional regulation corresponds to cell types that show little heterogeneity across brain regions; and that genetic disruption of region-specific transcriptional programs results in regional susceptibility. I use a systems-biology approach to summarize transcriptional regulation into reproducibly co-expressed gene sets (“co-expression modules”), which can be analyzed statistically to identify common functions, pathways, and cell types. I then integrate data from genetic association studies to ascertain gene sets conferring outsized risk for neuropsychiatric disorders, thereby implicating the corresponding pathways for further investigation in disease etiology. Finally, I use the network structure itself to investigate the genetic architecture of ASD and SCZ in terms of omnigenics and network polygenics. Chapter 1 presents the biological background for the studies and summarizes some of the major studies of neuropsychiatric disorders along with their principal methods and conclusions. In chapter 2, utilizing my multi-regional co-expression approach, I identify 12 brain-wide, 114 region-specific, and 50 cross-regional co-expression modules. Nearly 40% of expressed genes fall into brain-wide modules and correspond to major cell classes and conserved biological processes, while region-specific modules comprise 25% of expressed genes and correspond to region-specific cell types. The detailed study in chapter 3 demonstrates that neuropsychiatric risk concentrates in both brain wide and multi-regional modules, implicating major core cell types in disease etiology but not region-specific susceptibility. Chapter 4 presents a new and more general framework for defining genetic networks. Using this framework, I show that the network pattern of ASD-associated rare loss-of-function mutations, as well as the large number of significant targets for trans master regulators in BP and SCZ, support a classical polygenic architecture with thousands of directly causal genes. These results suggest that a nontrivial component of risk for neuropsychiatric disease comes from the global polygenic disruption of neuronal function and neuronal maturation

FROM microRNAS TO MITOCHONDRIA IN THE MACROPHAGE RESPONSE TO MYCOBACTERIUM TUBERCULOSIS: AND INFLAMMASOME ACTIVATION IN COVID-19

Tuberculosis (TB) and COVID-19 are two major infectious disease problems. While TB is caused by a slow growing bacterium, Mycobacterium tuberculosis (Mtb), and COVID-19 is caused by a virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, SARS-COV-2), both diseases have widespread impact on human health and share many common pathologies. Outcome of infection with both pathogens is heavily influenced by the response of host macrophages. Here we use a combination of unbiased and targeted approaches, including transcriptomics, methylomics, and cytokine analysis to evaluate immunological responses in human macrophages exposed to Mtb and SARS-CoV-2. Using in vitro macrophage exposure models and both unbiased and targeted analysis approaches, we find that the macrophage response to Mtb is shaped by changes in the production of small non-coding RNAs, including microRNAs (miRNAs) and tRNA-derived fragments (tRFs), gene expression, methylation, mitochondrial responses, while host responses to SARS-CoV-2 are shaped by macrophage-mediated viral sensing and inflammasome activation. Using next generation sequencing, we show that certain miRNAs are consistently dysregulated in Mtb infection. These miRNAs target a number of differentially expressed genes involved in processes central to the anti-TB response, including immune cell activation, macrophage lipid metabolism, and blood vessel development. Many genes involved in immune cell activation and metabolic reprogramming were also subject to changes in methylation. Additionally, we investigate dysregulation of tRFs, a novel form of small non-coding RNA that have never before been studied in the context of bacterial infections. We find that tRFs are significantly dysregulated in infection with Mtb and that dysregulated tRFs derive primarily from the host mitochondrial genome. Fluorescent imaging shows that increased abundance of mitochondria-biased tRFs is linked to recruitment of a tRF cleaving enzyme Angiogenin (ANG) and the apoptotic suppressor x-linked inhibitor of apoptosis protein (XIAP) to host mitochondria. Finally, we investigate the role of the inflammasome in SARS-COV-2 infection and find that SARS-COV-2 stimulates activation of the NLRP3 inflammasome through MyD88-mediated direct sensing of extracellular virus in macrophages, but not nasal or lung epithelial cells. Taken together, our studies show that the macrophage plays a central role in the host response to both Mtb and SARS-COV-2 infection and that macrophage responses are shaped by a network of pre- and post-transcriptional molecular regulatory factors