La maladie d’Alzheimer (MA) est caractérisée par l’accumulation de protéines β-amyloïde (Aβ) et Tau malconformées. L’hypothèse que la MA soit transmissible de manière similaire à celles des maladies à prion est un sujet d’intense recherche. L’objectif de cette thèse est d’étudier la transmission des endophénotypes de la maladie d’Alzheimer par l’inoculation intracérébral d’homogénats de patients souffrant de MA.Tout d’abord, nous avons montré que la transmission expérimentale de la MA accélère l’amyloïdose dans des modèles murins d’amyloïdose génétique précoce et tardive. Ensuite, nous avons observé le développement d’altérations fonctionnelles et morphologiques semblables à celles observées dans la MA chez le primate microcèbe (Microcebus murinus) et accompagnées d’une amyloïdose subtile sans pathologie Tau. Une telle transmission en l’absence de sévères lésions neuropathologiques a été rapportée dans les maladies à prions mais jamais dans le contexte de la MA. Nos résultats suggèrent que les agents responsables des altérations observées puissent être des formes d’Aβ et/ou Tau non détectées en immunohistochimie et pouvant être transmises expérimentalement. En conclusion, nos résultats supportent l’hypothèse de type prion de la MA et le consensus actuel sur la toxicité des formes solubles d’Aβ et Tau. Pour finir, ils soutiennent la possibilité que l’amyloïdose soit transmissible chez l’Homme sous certaines conditions et appellent à l’évaluation des impacts fonctionnels chez les sujets à risque de contamination. / Alzheimer's disease (AD) is characterized by the accumulation of misfolded β-amyloid (Aβ) and Tau proteins. There has been longstanding interest as to whether AD might be transmissible similarly to prion diseases. Our objective was to study the transmissibility of AD endophenotypes after AD brain intracerebral inoculation in mice and primates.First, we showed that AD experimental transmission accelerated Aβ pathology in two rodent models of early or late genetic β-amyloidosis. Then, we focused on a primate model of sporadic AD, the mouse lemur (Microcebus murinus). AD-inoculated adult lemurs progressively developed cognitive impairments, neuronal activity alterations and cerebral atrophy. AD-inoculated mouse lemurs also developed subtle β-amyloidosis in the absence of Tau pathology, 18 months after inoculation. The transmission of an AD-like pathology in the absence of severe neuropathological lesions is striking. Such observations have already been reported for prion diseases but never in the context of AD. Our results suggest that agents leading to AD-like alterations may be not immunohistopathological-detectable forms of Aβ or Tau proteins and transmitted experimentally.In conclusion, our results support the “prion-like” hypothesis of AD and provide further arguments for a dichotomy between the toxicity of deposited and soluble assemblies of Aβ or Tau proteins. Finally, they complement recent evidence supporting iatrogenic β-amyloidosis in humans and provide strong arguments to evaluate functional outcomes in potentially contaminated individuals.

The study of protein synthesis, and the study of gene expression in general, has accelerated in recent years. Following the advent of next-generation RNA sequencing, powerful library preparation paradigms were developed to capture regulatory activity on a genome-wide scale. In particular, ribosome profiling has emerged as a widely-used measurement of translation. In this method, the state of ribosome association across the transcriptome is obtained by isolation and sequencing of the regions of RNA bound by ribosomes, revealing a snapshot of ribosome positions from which gene-specific densities can be calculated. In combination with RNA sequencing for a measurement of baseline transcription in the same samples, ribosome profiling offers a metric of “translation efficiency”, or TE, corresponding to the average ribosome load per given transcript. Ribosome profiling has advanced the study of translation considerably. However, low throughput in the generation of ribosome profiling and RNA sequencing libraries limits the scale of the experiments that can be performed, while issues in the interpretation of aligned ribosome-protected footprints complicate their analysis, especially in systems of complex regulation. The analysis of such regulatory systems would be greatly aided by a high-throughput sequencing method that can capture translational regulation, but current methods of measuring genome-wide translation are inherently limited in scale. This thesis addresses the key issues presented above in separate chapters. Chapter 2 discusses the analysis of elongation and initiation from ribosome profiling and RNA sequencing data in a mouse model of Fragile X Syndrome. In this chapter, several methods of measuring and modeling variability in the distribution of ribosomes along a coding sequence are used alongside analyses of differential RPF and RNA abundances and their ratio, RFApm, which we distinguish from TE to emphasize its dependence on factors other than initiation rate. The chapter summarizes current information regarding the observed effects of FMRP, and proposes a model congruent with these observations and more-recently published studies. Chapters 3 and 4 present approaches to modeling or inferring translational regulatory networks, either by a novel library preparation paradigm or computational inference from publicly-available data. Chapter 3 presents riboPLATE-seq, a high-throughput RNA-seq library construction method based on the existing PLATE-seq method. The method recapitulates significant findings from ribosome profiling and RNA sequencing at a fraction of the per-sample cost, with further advantages in scalability, and could be implemented in a large-scale screen of translational regulators to create a network of their specific targets. Chapter 4 presents an approach to inferring translational regulation from integrative analysis of public ribosome profiling and RNA sequencing data, tailoring the powerful inference engine ARACNe to measure translational interactions. This yields a comprehensive network of translational regulation, assigning target genes to the set of RNA-binding proteins.

Alopecia areata (AA) is a complex autoimmune disease in which autoreactive T cell-mediated attack of the hair follicle (HF) leads to non-scarring hair loss. Although AA is one of the most prevalent autoimmune diseases, the development of novel effective therapeutics has been limited. Standard of care remains observation for mild cases and steroids for moderate-to-severe cases, which have demonstrated only limited efficacy. The skin is a highly heterogeneous tissue at baseline, comprised of a diverse array of immune and non-immune cell types whose coordinated crosstalk is essential for homeostasis. The skin microenvironment becomes markedly altered as a result of disease-associated inflammation in AA. A pathognomonic histopathologic feature of AA is an intense lymphocytic infiltrate surrounding the lower portion of the HF in the growth phase of the hair cycle, known as anagen. We previously established that CD8+ T cells comprise the majority of this infiltrate in AA skin, and that they are necessary and sufficient to drive disease via JAK/STAT activation. While this discovery led to the pioneering use of JAK inhibitors as a novel class of therapeutics in AA, JAK inhibition is not a curative solution, since patients often experience relapse upon discontinuation of treatment. This not only underscores the continued need for translational drug discovery research in AA, but also reflects an incomplete understanding of the mechanisms that govern disease pathophysiology. Recent advances in single-cell RNA sequencing (scRNAseq) present an unprecedented opportunity to dissect the heterogeneity of complex tissues and disorders. Since its emergence, scRNAseq has proven to be a powerful tool for the discovery of rare cell types and novel therapeutic targets in a variety of contexts that range from cancer to autoimmunity. In this thesis, we leveraged scRNAseq to interrogate the cellular and molecular mechanisms underlying disease pathogenesis in AA at single-cell resolution, together with validation and functional experiments, with the goal of uncovering novel cell types and pathways that can guide the development of innovative therapeutic strategies. In Chapter 2, we performed scRNAseq of skin-infiltrating CD45+ immune cells to dissect lymphocyte heterogeneity in both murine and human AA. Our scRNAseq analyses informed a series of antibody-mediated cell depletion experiments that assessed the in vivo function of specific lymphocyte subsets in murine AA. Our results established CD8+ T cells as the predominant disease-driving cell type in AA. We identified shared mechanisms underlying CD8+ T cell heterogeneity in murine and human AA skin, in which CD8+ T cells form an “effectorness gradient” comprised of interrelated transcriptional states that culminate in increased expression of inflammatory cytokines and T cell effector function. We also demonstrated a role for CD4+ T helper cells in disease initiation, and determined that regulatory T cells possess intact immunosuppressive capacity in AA. In Chapter 3, we expanded upon the studies described in Chapter 2 and performed scRNAseq of skin-infiltrating CD45+ cells at various timepoints throughout disease course (from 3 to 24 weeks post-disease induction) in AA to analyze the temporal dynamics of lymphocyte heterogeneity in AA skin and skin-draining lymph nodes. In conjunction with scRNAseq, we also performed single-cell TCR sequencing to assess the dynamics of T cell clonality alongside changes in T cell transcriptional profiles. We observed a striking increase in CD8+ T cell clonal expansion during disease onset, which increased throughout disease progression and subsequently decreased in chronic AA, when the preclinical mouse model exhibits total body hair loss. Our single-cell analyses suggested that CD8+ T cell clonality and pathogenicity are closely linked, which we validated in vivo by demonstrating that a single expanded clonotypic population of CD8+ T cells is sufficient to induce disease in mice. In Chapter 4, we analyzed single-cell transcriptomic profiles obtained from full-thickness skin in mice with chronic AA to investigate the contributions of the HF and other non-T cell populations in disease. In this study, we also used a network biology-based approach to infer single-cell protein activity, which together with single-cell mRNA gene expression profiles uncovered a multitude of novel findings in AA. Our results revealed a role for necroptosis as a potential HF-intrinsic mechanism of pro-inflammatory signaling in AA, and also identified an MHC Class II signature specific to basal keratinocytes in AA skin. Furthermore, we uncovered a novel, rare population of disease-associated Arg1+ macrophages, which prompted us to revisit our immune-specific scRNAseq datasets described in Chapters 2 and 3 and perform an integrative analysis of this novel cell type in AA. Our preliminary in vivo studies suggested that targeting Arg1+ macrophages and/or arginine metabolism may ameliorate disease in AA. Taken together, this thesis presents a comprehensive, systematic interrogation of AA pathogenesis at single-cell resolution. Importantly, the validation and functional studies that were informed by our scRNAseq data demonstrate proof-of-concept of the use of single-cell technology to accelerate the discovery and translation of novel therapeutic targets in complex diseases. While we undertook a hypothesis-driven approach to design our studies, the data presented in this thesis was also profoundly hypothesis-generating, and has informed a number of ongoing projects in the laboratory with the shared goal of advancing our understanding of disease pathology in AA.

Indiana University-Purdue University Indianapolis (IUPUI) / Fetal alcohol spectrum disorders (FASD) is the leading neurodevelopment deficit in children born to women who drink alcohol during pregnancy. The hippocampus and cortex are among brain regions vulnerable to alcohol-induced neurotoxicity, and are key regions underlying the cognitive impairment, learning and memory deficits shown in FASD individuals. Hippocampal and cortical neuronal differentiation and maturation are highly influenced by both intrinsic transcriptional signaling and extracellular cues. Epigenetic mechanisms, primarily DNA methylation and histone modifications, are hypothesized to be involved in regulating key neural development events, and are subject to alcohol exposure. Alcohol is shown to modify DNA methylation and histone modifications through altering methyl donor metabolisms. Recent studies in our laboratory have shown that alcohol disrupted genome-wide DNA methylation and delayed early embryonic development. However, how alcohol affects DNA methylation in fetal hippocampal and cortical development remains elusive, therefore, will be the theme of this study. We reported that, in a dietary alcohol-intake model of FASD, prenatal alcohol exposure retarded the development of fetal hippocampus and cortex, accompanied by a delayed cellular DNA methylation program. We identified a programed 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC) cellular and chromatic re-organization that was associated with neuronal differentiation and maturation spatiotemporally, and this process was hindered by prenatal alcohol exposure. Furthermore, we showed that alcohol disrupted locus-specific DNA methylation on neural specification genes and reduced neurogenic properties of neural stem cells, which might contribute to the aberration in neurogenesis of FASD individuals. The work of this dissertation suggested an important role of DNA methylation in neural development and elucidated a potential epigenetic mechanism in the alcohol teratogenesis.

Indiana University-Purdue University Indianapolis (IUPUI) / Glucocorticoid excess is a leading cause of osteoporosis. The loss of bone mass and strength corresponds to the increase in fractures exhibited after three months of glucocorticoid therapy. Glucocorticoids induce the bone cellular responses of deceased bone formation, increased osteoblast/osteocyte apoptosis, and transient increased bone resorption, which result in rapid bone loss and degradation of bone microarchitecture. The current standard of care for osteoporosis is bisphosphonate treatment; however, these agents further suppress bone formation and increase osteonecrosis and low energy atypical fracture risks. Thus, there is an unmet need for interventions that protect from glucocorticoid therapy. The purpose of these studies was to investigate novel mechanisms that potentially interfere with glucocorticoid-induced bone loss. We chose to explore pathways that regulate endoplasmic reticulum stress, the canonical Wnt pathway, and Pyk2 activity. Pharmacologic reduction of endoplasmic reticulum stress through salubrinal administration protected against glucocorticoid-induced bone loss by preservation of bone formation and osteoblast/osteocyte viability. In contrast, inhibition of Wnt antagonist Sost/sclerostin and inhibition of Pyk2 signaling did not prevent glucocorticoid-induced reductions in bone formation; however, both Sost/sclerostin and Pyk2 deficiency protected against bone loss through inhibition of increases in resorption. Overall, these studies demonstrate the significant contributions of reductions in bone formation, increased osteoblast/osteocyte apoptosis, and elevations in resorption to the rapid 6-12% bone loss exhibited during the first year of glucocorticoid therapy. However, glucocorticoid excess also induces skeletal muscle weakness, which is not reversed by bisphosphonate treatment or the interventions reported here of salubrinal, Sost/sclerostin inhibition, or Pyk2 deficiency. Further, the novel finding of increased E3 ubiquitin ligase atrophy signaling induce by glucocorticoids in both bone and muscle, by tissue-specific upstream mechanisms, provides opportunities for therapeutic combination strategies. Thus, future studies are warranted to investigate the role of E3 ubiquitin ligase signaling in the deleterious glucocorticoid effects of bone and muscle.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is a genetic disorder that results from triplication of human chromosome 21 (Hsa21) and occurs in around 1 in 1000 live births. All individuals with DS present with skeletal abnormalities typified by craniofacial features, short stature and low bone mineral density (BMD). Differences between males and females with DS suggest a sexual dimorphism in how trisomy affects skeletal deficits associated with trisomy 21 (Ts21). Previous investigations of skeletal abnormalities in DS have varied methodology, sample sizes and ages making the underlying causes of deficits uncertain. Mouse models of DS were used to characterize skeletal abnormalities, but the genetic and developmental origin remain unidentified. Over-expression Dyrk1a, found on Hsa21 and mouse chromosome 16 (Mmu16) has been linked to cognitive deficits and skeletal deficiencies. Dp1Tyb mice contain three copies of all of the genes on Mmu16 that are homologous to Hsa21, males and females are fertile, and therefore are an excellent model to test the hypothesis that gene dosage influences the sexual dimorphism of bone abnormalities in DS. Dp1Tyb at 6 weeks 16 weeks showed distinctive abnormalities in BMD, trabecular architecture, and reduced bone strength over time that occur generally through an interaction between sex and genotype. Increased gene dosage and sexual dimorphism in Dp1Tyb mice revealed distinct phenotypes in bone formation and resorption. To assess how Dyrk1a influences the activity and function of osteoblasts Ts65Dn female trisomic mice, female mice with a floxed Dyrk1a gene (Ts65Dn, Dyrk1afl/+) were be bred to Osx1-GFP::Cre+ mice to generate Ts65Dn animals with a reduced copy of Dyrk1a in mature osteoblast cells. Female Ts65Dn,Dyrk1a+/+/+ and Ts65Dn,Dyrk1a+/+/-displayed significant defects in both trabecular architecture and cortical geometry. Ultimate force was reduced in trisomic animals, suggesting whole bone and tissue level properties are not adversely affected by trisomy. Reduction of Dyrk1a functional copy number in female mice did not improve skeletal deficits in an otherwise trisomic animal. Dyrk1a may not alter osteoblast cellular activity in an autonomous manner in trisomic female mice. These data establish sex, gene dosage, skeletal site and age as important factors in skeletal development of the skeleton in DS mice, potentially paving the way for identification of the causal dosage-sensitive genes in both male and female animals.

Midbrain dopamine neurons play a critical role in motor function, motivation, reward, and cognition by providing modulatory input to cortical and basal ganglia circuits. Given the importance of dopamine neurotransmission and its dysregulation in disease, mechanistic insight into the molecular underpinnings of dopaminergic neuronal function is needed. This thesis seeks to advance our understanding of dopamine neuronal cell biology by developing and applying cutting edge molecular profiling methods to study the subcellular translatome and proteome of dopamine neurons in mice. Chapter 1 provides an overview of the anatomy and cell biology of midbrain dopamine systems, with a particular emphasis on dopamine neurotransmission, neuronal heterogeneity, and selective vulnerability in Parkinson’s disease. Chapter 2 focuses on methods for studying local translation in neurons and describes newly discovered artifacts associated with two of these methods. Chapter 3 describes a global analysis of ribosome and mRNA localization in dopamine neurons; the results suggest that local translation in dopaminergic dendrites, but not axons, regulates dopamine release. Chapter 4 presents a method for subcellular proteomic profiling of dopamine neurons in the mouse brain, revealing the somatodendritic and axonal polarization of proteins encoded by Parkinson’s disease-linked genes. Emerging data are presented on Synaptotagmin 17, a novel axonal protein identified in midbrain dopamine neurons. Finally, I synthesize key findings regarding the molecular organization underlying dopamine neuronal cell biology and highlight promising areas for future investigation.

Brain metastasis (BM), the most common tumor of the central nervous system, occurs in 20-36% of primary cancers. In particular, 20-40% of patients with non-small cell lung cancer (NSCLC) develop brain metastases, with a dismal survival of approximately 4-11 weeks without treatment, and 16 months with treatment. This highlights a large unmet need to develop novel targeted therapies for the treatment of lung-to-brain metastases (LBM). Genomic interrogation of LBM using CRISPR technology can inform preventative therapies targeting genetic vulnerabilities in both primary and metastatic tumors. Loss-of-function studies present limitations in metastasis research, as knocking out genes essential for survival in the primary tumor cells can thwart the metastatic cascade prematurely. However, transcriptional overexpression of genes using CRISPR activation (CRISPRa) has the potential for overcoming dependencies of gene essentiality. In this thesis, we created and utilized an in vivo genome-wide CRISPRa screening platform to identify novel genes, that when overexpressed, drive LBM. We have developed a patient-derived orthotopic murine xenograft model of LBM using a patient-derived NSCLC cell line (termed CRUK cells) from the Swanton Lab TRACERx study. We introduced a human genome-wide CRISPRa single guide RNA (sgRNA) library into non-metastatic and pro-metastatic lung cancer CRUK cells to achieve 500X representation of each sgRNA in the activation library. We then injected the cells into the lungs of immunocompromised mice and tracked lung tumor development and BM formation. Upon sequencing primary lung tumors and subsequent BM, we will identify enriched sgRNAs which may represent novel drivers of primary lung tumor formation and LBM. To the best of our knowledge, this study is the first in vivo genome-wide CRISPR activation screen using patient-derived NSCLC cells to help elucidate drivers of LBM. This work serves to provide a framework to gain a deeper understanding of the regulators of BM formation which will hopefully lead to targeted drug discovery that will ultimately be used in clinical trials to help eradicate brain metastasis in NSCLC patients. / Thesis / Master of Science (MSc) / Brain metastasis, or the spread of a primary cancer from another organ to the brain, is the most common adult brain tumor. Brain metastases can arise after the treatment of primary tumors and are only detected in the clinic at a highly malignant stage. Current treatments for brain metastasis consist of surgical removal and palliative chemoradiotherapy, which fail to fully eliminate the brain tumor. Over 20% of cancer patients develop brain metastases, with lung, breast, and skin cancers leading as the top three sources of metastasis. In particular, 40% of patients with non-small cell lung cancer develop brain metastasis, with survival of only 4-11 weeks once diagnosed without treatment, and 16 months with treatment. As systemic therapies for the treatment of non-small cell lung cancer are becoming increasingly effective at controlling primary disease, patients are ironically succumbing to their brain tumors. This highlights a large unmet need to develop novel targeted therapies for the treatment of lung-to-brain metastases (LBM). Functional genomic tools provide the opportunity to investigate the genetic underpinnings of LBM. With the advent of gene editing technologies, we are able to overexpress various genes and observe the impact genetic perturbations have on tumor initiation, growth, and metastasis. In this thesis, we devised a pre-clinical animal model of LBM that could be used to study genetic drivers of LBM using a gene overexpression tool such that one gene per tumor cell gets activated. We are then able to model the disease trajectory from a lung tumor to brain metastasis development using patient samples in our animal model and identify genes that, upon overexpression, drive LBM. This platform will lead to potential therapeutic targets to prevent the formation of LBM and prolong the survival of patients with non-small cell lung cancer.

No description available.

The retina is one of the most metabolically active tissues in the body and the high energetic demand is met by a well-organized vascular network. Aberrant vasculature is a prominent feature of many vision-threatening diseases, and although angiogenic pathways have been extensively studied the limited efficacy of therapies currently available for the treatment of these diseases suggests that there is more to be elucidated. The caspase family of proteases is best known for their roles in programmed cell death and inflammation, however members of this family have been found to have essential functions independent of cell death. Caspase-8, in particular, has been previously shown to be essential for embryonic vascular development, however, a requirement for caspase-8 in postnatal vascular development has not been established and it is unclear how caspase-8 exerts its function. In this study, we investigate the cell specific roles of caspase-8 in the development of the retinal vasculature using the postnatal mouse retina as our model and identified endothelial caspase-8 as a mediator of canonical Wnt signaling. Inducible endothelial cell-specific caspase-8 knockout (Casp8 iECKO) resulted in a delay in early angiogenesis and barrier establishment, and an increase in inflammation and premature vascular remodeling compared to littermate controls. Assessment of Lef1, a downstream effector of the Wnt pathway, confirmed that this phenotype was a result of inhibited Wnt signaling. We additionally show that caspase-8 mediates this pathway through degradation of its substrate HDAC7. HDAC7 has been shown previously to bind to β-catenin blocking its nuclear translocation. Caspase-8 mediated HDAC7 degradation restores β-catenin translocation and downstream Wnt signaling. We also explore the function of caspase-8 in myeloid cells – microglia and macrophages – during angiogenesis. We used an inducible myeloid-specific caspase-8 knockout (Casp8 imGKO) mouse and found that loss of caspase-8 in these cells did not affect angiogenesis. However, Casp8 imGKO resulted in a reduction in microglia number and a change in their morphology specifying a role for caspase-8 in mediating cell survival and activation in microglia. Altogether we show that caspase-8 exerts cell specific functions during retinal angiogenesis that are independent of cell death. We elucidate a novel role of caspase-8 in mediating Wnt/β-catenin signaling, and implicate caspase-8 as a potential therapeutic target in pathological angiogenesis.