Lipids are a class of biomolecules that play an essential role in numerous biochemical functions, including energy production and homeostasis, cellular communication, and plasma membrane structure. However, lipids are also linked to many diseases and pathological processes, the most common of which are heart disease, diabetes, and inflammation. In order to maintain homeostasis, the body has an intricate and complex transport system to deliver cholesterol and fatty acid from the diet to vital tissues and organs, as well as a method to transport excess cholesterol from tissues and cells back to the liver where it can be excreted or recycled. Disruption or dysregulation in any parts of this system results in life-threatening diseases such as coronary artery disease. Utilizing a variety of biochemical, cell biology, and imaging approaches, we describe several recent findings in two important aspects of lipid metabolism—intravascular triglyceride metabolism and macrophage reverse cholesterol transport.

In the first several sections, studies describe the protein GPIHBP1. GPIHBP1 is a protein of capillary endothelial cells that is responsible for capturing lipoprotein lipase (LPL) and transporting it to the capillary lumen where LPL functions in hydrolysis of triglyceride-rich lipoproteins in the bloodstream, releasing fatty acids for use by surrounding tissues. Without GPIHBP1, LPL never reaches the capillary lumen and triglyceride hydrolysis is deficient, resulting in severe hypertriglyceridemia. In the following studies, we first developed and characterized several monoclonal antibodies (mAbs) against human GPIHBP1, then utilized these mAbs to better understand GPIHBP1’s role in hypercholesterolemia and cancer lipid metabolism.

In the later sections, we investigated macrophages and their role in reverse cholesterol transport. Macrophage have been known to internalize cholesterol and offload excess cholesterol back to the bloodstream and the liver. Cholesterol efflux from macrophages have been studied extensively and has generally been thought to involve direct transport of cholesterol from ATP-binding cassette (ABC) transporters to acceptors in the plasma such as high density lipoproteins (HDL). In these studies, we demonstrate that macrophages release ~20 to 100-nm particles derived from the plasma membrane and that these particles are highly enriched in a pool of accessible cholesterol. This release cholesterol-rich particles would greatly augment macrophages’ ability to offload excess cholesterol in reverse cholesterol transport.

Nuclear lamins are intermediate filament proteins that form the nuclear lamina, a structural scaffolding for the nucleus. They interact with other nuclear proteins and the chromatin and are thought to participate in many fundamental cellular pathways. Most vertebrates have two-types of lamins: A-type (lamins A and C) and B-type (lamins B1 and B2). Nuclear lamins, particularly A-type lamins, have attracted considerable attention due to their association with many human genetic diseases. However, despite considerable interest in nuclear lamins, physiologic rationale for the different lamins is unclear. Also, the functional importance of the lamins in the brain has not been thoroughly investigated as the phenotypes of the associated genetic diseases are largely confined to mesenchymal tissues.

In this dissertation, we aimed to better understand distinctive functions and regulation of nuclear lamins in health and diseases, with an emphasis on the brain. In Chapter 2, we showed that prelamin A, but not lamin C, is down-regulated in the brain by a brain-specific microRNA, miR-9 using a series of in vitro studies in cultured cells. In Chapter 3, we generated two new lines of Lmna knock-in mice to investigate whether the regulation of prelamin A by miR-9 is relevant in vivo. These studies, taken together, provided a ready explanation why children with Hutchinson-Gilford progeria syndrome (a progeroid syndrome caused by a mutant form of prelamin A) are spared from neurodegenerative disease: miR-9 selectively eliminates the expression of the culprit molecule—the toxic prelamin A molecule that leads to disease. In Chapter 4, we addressed the physiologic importance of the farnesyl lipid anchor in B-type lamins using knock-in mice expressing nonfarnesylated versions of lamin B1 and lamin B2. In this study, we showed that the farnesyl lipid anchor on lamin B1 is crucial for retaining the nuclear chromatin within the bounds of nuclear lamina during neuronal migration. In Chapter 5, we investigated whether nuclear lamins are essential for proliferation and differentiation of non-neuronal cells in vivo using keratinocyte-specific Lmna/Lmnb1/Lmnb2 triple-knockout mice.

The nuclear lamina is an intermediate filament meshwork lining the inner nuclear membrane and is a key component of the nuclear envelope. The nuclear envelope separates the contents of the cell nucleus from the cytoplasm and consists of the inner and outer nuclear membranes, nuclear membrane proteins, nuclear pore complexes, and the nuclear lamina. The principal protein components of the nuclear lamina are lamins A and C (A-type lamins) and lamins B1 and B2 (B-type lamins). B-type lamins are expressed throughout development, whereas the A-type lamins are not expressed until late in development. Mutations in LMNA have been linked to a spectrum of human diseases, but only a single disease had been linked to mutations in LMNB1 or LMNB2. Several years ago, our group showed that both lamin B1 and lamin B2 are required for neuronal migration in the developing central nervous system (CNS), and for the survival of neurons. In the developing brain, the processes of neurogenesis and neuronal migration impart considerable mechanical stress on the cell nucleus. Neurogenesis in the ventricular zone of the developing cortex depends on interkinetic nuclear migration (INM), an oscillatory movement of nuclei between the apical and basal sides of the cell. Once neuronal fate is specified, the migration of neurons from the ventricular zone to the cortical plate depends on nuclear translocation (nucleokinesis) and passage through confined spaces. In cell culture studies, cell nuclei subjected to mechanical stresses (i.e., by physically deforming nuclei or forcing cells to traverse tight constrictions) had been shown to cause nuclear membrane (NM) ruptures. The frequency of NM ruptures was known to be increased in the setting of nuclear lamin deficiencies but the relevance of NM ruptures in cultured cells to pathology in tissues was unknown. In this dissertation, my goal was to investigate whether deficiencies in nuclear lamins predispose to nuclear membrane ruptures (particularly when the nucleus is subjected to mechanical stress during normal physiological processes, such as cell migration) and whether these ruptures may result in disease phenotypes. In Chapter 2, we showed that fibroblasts lacking all nuclear lamins exhibit spontaneous NM ruptures in vitro that are exacerbated by mechanical stress. In Chapter 3, we generated a mouse model with forebrain-specific loss of lamin B1 and expressing a nuclear fluorescent reporter, and showed that forebrain-specific loss of lamin B1 results in NM rupture and cell death in vivo. We also showed that both lamin B1– and lamin B2–deficient neuronal progenitor cells exhibit spontaneous NM ruptures, although the frequency and duration of NM ruptures was markedly different. In Chapter 4, we investigated whether overexpression of LAP2 (lamina-associated polypeptide 2, β isoform), a LEM-domain protein of the inner nuclear membrane, could prevent nuclear lamin–deficient fibroblasts and lamin B1–deficient neurons from exhibiting NM ruptures. In tandem, we discovered that reduced expression of LAP2 also rendered nuclear lamin–deficient cells more susceptible to NM ruptures.

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Lipoprotein lipase (LPL) is a key enzyme responsible for the lipolytic processing of triglyceride rich lipoproteins (TRLs), with deficiencies in LPL known to cause familial chylomicronemia and mutations associated with hypertriglyceridemia. Upon synthesis, LPL is secreted into the interstitial spaces where it binds to GPIHBP1, an endothelial cell (EC) GPI-anchored protein. GPIHBP1 facilitates the transport of LPL across the capillary lumen where it functions in the intravascular processing of TRLs. This study leverages single cell RNA sequencing (scRNA-seq) to investigate the cell-type-specific expression of Lpl and Gpihbp1 across tissues. In our study we confirm the presence of known LPL synthesizing cells in mouse cardiac and adipose tissues, validating previous findings, and providing further support for their role in lipid metabolism. Additionally, our investigation highlights a novel observation: the expression of Lpl in the endothelial cells of skeletal muscle tissue. Most notably, these analyses uncover new insights into Lpl and Gpihbp1 expression in the choroid plexus of embryonic mice, unveiling a possible role of LPL-mediated intravascular lipolysis in the central nervous system. Overall, the study provides a comprehensive overview of Lpl expression across tissues known for robust LPL-mediated lipoprotein processing. These findings underscore the importance of understanding tissue- and cell-type-specific gene expression patterns to elucidate the complex regulatory mechanisms underlying lipid metabolism and its implications for human health.