Alveolar macrophages (AMs) play critical roles in metabolizing surfactant and protecting the lung against inhaled pathogens. AMs mature following the newborn period and their immaturity at birth may contribute to the variety of infectious and inflammatory lung diseases specifically affecting infants. However, the specific molecular features differentiating neonatal and adult AMs remain poorly understood. Here we identify the unique transcriptomes and enhancer landscapes of neonatal and adult AMs that establish the molecular phenotypes specific to each developmental age. Adult AMs expressed higher levels of genes involved in lipid transport and metabolism (i.e. Fabp1, Pnpla5), consistent with their role in surfactant recycling. Neonatal AMs expressed higher levels of proinflammatory genes (Il1b, Il6, Tnf, and S100a8). ATAC-seq data detected adult AM peaks enriched with motifs recognizing KLF, GR, PPAR-gamma, and STAT. Accessible chromatin regions in neonatal AMs were highly enriched with binding motifs for the innate immunity transcription factors AP1, NF-kappa B, and IRF. To test how these baseline differences might impact AM innate immune function, we exposed neonatal and adult mice in vivo with inhaled LPS. AMs from both neonatal and adult mice demonstrated robust induction of Toll-Like Receptor 4 (TLR4) signaling pathways and similar patterns of chromatin accessibility. While neonatal and adult AMs did exhibit divergent expression patterns for some genes, the overall core innate response was similar. Intriguingly, neonatal AMs expressed higher basal levels of many LPS-induced genes, suggesting constitutive innate immune priming or activation in the neonatal lung. The lung microenvironment was a major factor regulating the unique molecular features of neonatal and adult AMs. Culturing isolated AMs for only 20h minimized the differences in gene expression between neonatal and adult cells. Interesting, while neonatal AMs in culture more closely resembled adult AMs, a core inflammatory signature of gene expression was retained. These data suggest that the proinflammatory phenotype of neonatal AMs results from both inherent properties of the cell and the lung microenvironment. Collectively, these studies provide new insights into the molecular mechanisms of lung innate immune development.

Successful development of effective therapeutics for Alzheimer’s disease (AD) requires a deep understanding of the mechanisms of disease pathogenesis. Recent human induced pluripotent stem cell (hiPSC)-derived models have shown it is possible to recapitulate the complex genetic diversity of a patient and more completely model AD pathology. While most work has focused on neuronal AD phenotypes, there is mounting evidence that failure to regulate cholesterol homeostasis by neighboring nonneuronal support cells may be involved in AD pathogenesis. Amyloid precursor protein (APP) or multiple fragments generated by APP proteolytic processing have previously been implicated in the regulation of cholesterol metabolism. However, the physiological function of APP in regulating lipoprotein homeostasis in astrocytes, which are responsible for the brain’s de novo cholesterol biosynthesis and regulation, remains unclear. To address this, we independently validated and characterized a novel in vitro differentiation protocol to generate human astrocytes from hiPSC. We found that cells derived by this method recapitulated native astrocytes in their form and function. We also utilized CRISPR/Cas9 genome editing to generate isogenic APP knockout (KO) hiPSCs. We found that APP KO astrocytes have reduced cholesterol, elevated levels of sterol regulatory element-binding protein (SREBP)-target gene transcripts, and increased low-density lipoprotein (LDL) receptor protein, all downstream consequences of reduced lipoprotein endocytosis. To elucidate which APP fragments regulate cholesterol homeostasis and examine whether familial AD mutations in APP affect lipoprotein metabolism, we analyzed an isogenic allelic series using the APP Swedish and APP V717F mutations. Only astrocytes homozygous for the APP Swedish (APPSwe/Swe) mutation, which had reduced full-length APP (FL APP) due to aggressive beta-secretase cleavage, recapitulated the APP KO phenotypes. Further, astrocytic internalization of amyloid-beta (A-beta), another ligand of LDL receptors, was impaired in APP KO and APPSwe/Swe astrocytes. Given that FL APP is known to bind to multiple LDL receptors, we propose that FL APP acts as a co-receptor for known LDL receptor ligands and is required for proper cholesterol homeostasis and A-beta clearance in human astrocytes.

A fundamental question in the study of motor control is how the nervous system accomplishes the complex task of integrating commands from multiple movement systems in order to perform a motor behavior. Even the simplest tasks, such as withdrawing the limb from a painful stimulus or taking a single step, involve multiple areas of the brain and spinal cord. Examples of movement parameters that are encoded within the nervous system include the selection and timing of muscle contraction in order to move a body region to a desired position, and the coordination of multiple body joints and body regions. Significant progress has been made in the field to understand the contribution of individual cell types to motor control, as well as the genetic mechanisms that regulate the development of individual cell types. However, the circuit components that link multiple motor control populations, as well as the mechanisms that regulate the formation of integrative motor networks, are poorly understood.

This dissertation describes a series of original work that aims to elucidate the cellular players and developmental mechanisms that assemble circuit components into functional neural networks. The first chapter provides a framework for understanding how different motor control pathways are organized within the central nervous system. The second chapter describes a group of premotor neurons that are located in the deep dorsal horn of the spinal cord, receive input from multiple motor control pathways, and bind together the activity of multiple motor pools. Importantly, genetic markers (Satb1, Satb2, and tcfAP2β) were identified that, to date, comprise the most significant fraction of molecularly defined premotor neurons in the spinal cord.

The third chapter describes a novel population of cells that express the genetic marker Satb2, spinal interneurons that are located at the intersection of incoming sensory and outgoing motor information. This work examines the role of the Satb2 gene in spinal interneuron development, as well as sensory-motor circuit assembly and function. This work provides an important contribution to our understanding of the organizational logic of integrative spinal networks and the types of movements they control.

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