Motile cilia are essential for lung defense, as evidenced by the genetic syndrome primary ciliary dyskinesia (PCD). PCD is characterized by impaired motile cilia resulting in respiratory distress at birth, followed by chronic sinopulmonary infection and bronchiectasis, which can lead to respiratory failure. There are no specific therapies for PCD, in part because key pathways for motile cilia biogenesis and pathogenesis are not defined. PCD has been linked to mutations in nearly 40 genes. Those that encode dynein proteins, the motors necessary for cilia beating, comprise the largest class of PCD mutations. We have found that mutations in dynein motor proteins result in abnormal cytoplasmic aggregates in ciliated cells. Importantly, these aggregates sequester normal proteins of the machinery required to assemble the dynein motor complexes, suggesting a global disruption of cilia assembly. To further determine the impact of mutations, we biopsied and cultured airway cells from patients with PCD, revealing increased expression of genes related to cell stress, including IL-1? and IL6. We hypothesize that accumulation of mutant protein leads to failure of the cilia assembly machinery and to cellular stress. This will be tested through the following Specific Aims: (1) Determine how mutant dynein proteins interrupt the cilia motor assembly pathway and (2) Define the transcriptional and stress responses in cells containing PCD mutant proteins. We will leverage primary culture nasal cells from patients with mutations in dynein motor proteins seen at our PCD clinic. To determine how mutant proteins perturb cilia assembly, we will use proteomics and advanced microscopy to quantify the interaction of the mutant dynein proteins with the assembly machinery. To characterize the effect of mutant protein on cell stress, we will employ RNA sequencing to define the transcriptional profile of PCD cells and test known cell stress pathways. Data generated from this proposal will identify shared pathways in PCD, that can be exploited to develop future therapeutic strategies. This proposal comprises a plan to provide Dr. Horani with the mentored research, technical skill development, and tailored didactic training needed to achieve his goal of becoming an independent physician-scientist. The training will cover areas of genetics and genomics, sequencing data analysis, and advanced fluorescent microscopy imaging, which are key areas of this proposal. This project will be overseen by a scientific advisory committee with expertise in motile cilia biology, protein interactions, proteostatic pathways and imaging. The committee will ensure that career milestones are realized, formal course work is completed, and collaborations are developed locally and internationally. Findings generated through the proposed studies can be applied to other genetic airway disease and training will allow Dr. Horani to develop new approaches and therapies that may improve patients? health as an independent investigator.
Primary ciliary dyskinesia (PCD) is a genetic disease caused by the failure of cilia to beat, leading to recurrent infections and chronic lung disease, for which there is no treatment. Our proposed project will investigate the motor proteins that supply the force for cilia beating and are mutant in PCD. We will determine the how the cell handles mutant motor proteins to identify shared pathways that could be therapeutically manipulated to treat PCD in the early course of disease.