Regulation of Motile Cilia Assembly in Lung Disease

Washington University, Saint Louis MO

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Abstract

Project Summary/Abstract Motile cilia are essential for airway clearance and their dysfunction leads to lung diseases. Loss of motile cilia may be environmentally influenced in conditions like chronic obstructive lung disease, or due to genetic causes that are collectively known as primary ciliary dyskinesia (PCD). The goal of our studies is to determine the biologic basis of PCD lung disease phenotypes. Pathologic variants (mutations) occur in nearly 60 PCD genes. Variants result in abnormal cilia function due to defects in ciliary protein production, transport, or placement along the axoneme, which is the microtubule skeleton of cilia. While we know many components of the motile cilia machinery, we do not yet understand the assembly process, how it is interrupted and the consequences for disease in patients with PCD. Analysis of PCD patients reveals a wide spectrum of genotype-phenotype differences. Disease severity has been attributed primarily to impaired ciliary motility. Our studies reveal multiple cell pathologies that are independent of motility defects. The discoveries came as we sought to determine why PCD variants, CCDC39 and CCDC40, cause more severe lung disease than in other PCD variants. Using single particle cryo-electron microscopy, we found that CCDC39 and CCDC40 form a heterodimer attached to the axoneme. Proteomic analysis of cilia from CCDC39 variants identified 90 missing proteins from major ciliary structures. We deduced that a CCDC39/CCDC40 scaffold provides the addresses for a set of 14 key proteins that anchor an extensive connectome. In addition, the loss of CCDC39/CCDC40 leads to several downstream consequences on the airway cells that have not been previously described in PCD: the cilia are shortened, the axonemal microtubules lack structural integrity, the periciliary layer is disrupted, proteasomal activity is altered, and the multiciliated cells switch to a mucous cell fate. We hypothesize that loss of the CCDC39/40 scaffold, and its connectome, result in airway periciliary barrier failure and proteostatic stress leading to severe PCD. The mechanism and impact of these motility-independent phenotypes will be investigated in these Aims: (1) Determine how the CCDC39/CCDC40 scaffold provides addresses and anchors for ciliary structures that are affected in patients with PCD; (2) Identify and test the mechanistic role of ciliary structures that are required for maintaining the periciliary layer; (3) Determine how the large number of ciliary connectome proteins that remain in the cytoplasm are processed, which proteasomal machines are used, and how proteotoxic stress may influence cell fate. Completion of these aims will resolve questions related to lung disease severity in patients with PCD and identify new pathways for therapies.