Cholesterol and Sphingolipid Perturbation of Membrane Traffic in Human Disease

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DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cAMP-dependent chloride channel CF transmembrane conductance regulator (CFTR). Some missense mutations, including deletion of phenylalanine 508 (?F508), the most common mutation in CF disease, cause retention of the protein in the ER and premature degradation. As a result there is a significant reduction in functional CFTR in the plasma membrane of airway epithelial cells leading to defective chloride secretion, hyper-absorption of sodium, and other changes that reduce the capacity of cilia to clear bacteria from the airways. Recently several groups, including our own, have shown that cholesterol and sphingolipids (SLs) accumulate in CF cells, similar to that seen in various sphingolipid storage diseases. These findings and key preliminary data led to our central hypothesis that increased levels of cholesterol and SLs exacerbate the symptoms of CF by interfering with normal membrane transport processes and CFTR function. This will be tested by pursuing three Specific Aims. Under Aim 1 we will focus on over-expression of Rab1 and Rab2, two GTPases involved in ER to Golgi transport, as a tool for enhancing the delivery of F508 to the plasma membrane. These studies will provide a new method for increasing ?F508-CFTR at the plasma membrane and allow us to carry out studies on the underlying mechanism for this enhanced delivery as well as the function of the mutant protein at the cell surface. Under Aim 2, we will study the effects of SLs and cholesterol on endocytosis, recycling, and function of WT- and ?F508-CFTR since these lipids can significantly affect membrane transport as well as the microenvironment of other membrane proteins. Based on preliminary data showing reduced cilia in ?F508-CFTR cells, we will also study the effect of alterations in cholesterol on (i) the distribution of cilia in cells expressing wild type or mutant CFTR, (ii) modulation of RabGTPases involved in ciliogenesis, and (iii) the distribution of cholesterol binding proteins that are normally associated with cilia. Under Aim 3, we will first study the mechanism underlying elevation of SLs and cholesterol in mutant CFTR cells to learn whether the absence of CFTR function or the presence of a misprocessed mutant CFTR in the distal secretory pathway is responsible for lipid storage. We will also investigate the causal relationships between mutant CFTR expression, elevated lysosomal pH, and lipid storage. Second, we will use methods developed in the previous grant period and in preliminary studies (e.g., Caveolin-1 knock-down) to deplete elevated cholesterol and SLs from cells, in an attempt to reverse various aspects of the ?F508 phenotype. Together these proposed experiments should increase our understanding of the connection between mutant CFTR and lipid storage and may also provide additional approaches for treatment of CF. PUBLIC HEALTH RELEVANCE Cystic fibrosis is an inherited disease caused by mutations in a protein that results in thick mucus secretions that are not properly cleared from the lung and can lead to chronic infection and respiratory failure. Recent studies have shown that cholesterol and other lipids accumulate in airway cells from Cystic fibrosis patients. The following studies will examine the role of these lipids in exacerbating the symptoms of Cystic fibrosis and will be used in developing new therapeutic approaches for this disease.