A Role for PICALM in Macroautophagy and Cellular Cholesterol Homeostasis

<p>The dissertation will focus on deciphering novel roles for PICALM in cellular biology. PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid Protein) is a ubiquitously expressed protein that was initially identified as a partner for AF10 in a chromosomal translocation in a lymphoma cell line. Since its identification, PICALM has been shown to act as an accessory adaptor protein in clathrin-mediated endocytosis and to regulate the internalization of proteins involved in vesicular trafficking (SNARE proteins). In addition, mutations in the PICALM gene have been shown to be linked to the development of leukemia and Alzheimer’s Disease. As a result of our studies, we have determined that PICALM is involved in two previously unappreciated cellular processes: macroautophagy and cellular cholesterol metabolism. This dissertation will address each of these processes in turn.</p><p>The thesis begins with an introduction to PICALM, including a description of PICALM’s known cellular functions and its relationship to disease. In addition, general aspects of macroautophagy and cellular cholesterol metabolism will be introduced (Chapter 1). Chapter 2 will describe the materials and methods that were used in the experimental analysis.</p><p> Chapter 3 describes our observation of a novel role for PICALM in macroautophagy. PICALM regulates SNARE protein internalization and localization. Intriguingly, SNARE proteins (VAMP3 and VAMP8) are involved in vesicular trafficking and macroautophagy. Thus, we sought to determine a role for PICALM in regulating macroautophagy by experimentally reducing or overexpressing PICALM. Our studies show that both reduction and overexpression of PICALM can modulate macroautophagy. In addition, our work indicates that PICALM modulates macroautophagy by altering autophagosome breakdown, without having an effect on autophagosome formation. This section of the thesis concludes with a possible mechanism by which PICALM may modulate macroautophagy. A substantial portion of this Chapter appeared in Moreau et al, Nature Communications, 2014 (1).</p><p> Chapter 4 focuses on PICALM’s ability to modulate cellular cholesterol homeostasis. We initially performed a microarray experiment using picalm-deficient and PICALM-expressing cells in order to obtain biological insight into possible novel roles for PICALM. This study suggested that modulating the level of PICALM expression alters cellular cholesterol homeostasis. We went on to demonstrate that PICALM reduction and overexpression result in altered cholesterol metabolism gene expression. In addition, we examined the effect of PICALM deficiency on cholesterol flux, and unexpectedly showed that PICALM reduction results in elevated cholesterol internalization, and cellular cholesterol levels. The LDL receptor is the primary route by which cholesterol is internalized. Thus, we measured LDL receptor internalization by flow cytometry. We showed that internalization of the LDL receptor is elevated in the absence of PICALM. This portion of the thesis concludes with a possible mechanism by which PICALM alters cellular cholesterol metabolism. The majority of this Chapter appeared in Mercer et al, PLoS ONE, 2015 (2).</p><p> Finally, Chapter 5 summarizes our observations and discusses the relationship among PICALM, macroautophagy and cellular cholesterol metabolism. In addition, future directions of these projects and how these studies are relevant to disease will be discussed.</p>Dissertatio