Novel insights into surfactant protein C trafficking revealed through the study of a pathogenic mutant

Abstract

Alveolar epithelial cell dysfunction plays an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF) but remains incompletely understood. Some monogenic forms of pulmonary fibrosis are associated with expression of mutant surfactant protein C (SFTPC). The commonest pathogenic mutant, I73T, mislocalises to the alveolar epithelial cell plasma membrane and displays a toxic-gain-of-function. Because the mechanisms explaining the link between this mutant and IPF are incompletely understood, we sought to interrogate SFTPC trafficking in health and disease to understand the functional significance of SFTPC-I73T relocalisation.We performed mechanistic analysis of SFTPC trafficking in a cell model that reproduces the in vivo phenotype and validated findings in human primary alveolar organoids.We show that wild-type SFTPC takes an unexpected indirect trafficking route via the plasma membrane and undergoes the first of multiple cleavage events before reaching the multivesicular body (MVB) for further processing. SFTPC-I73T takes this same route, but its progress is retarded both at the cell surface and due to failure of trafficking into the MVB. Unable to undergo onward trafficking, it is recycled to the plasma membrane as a partially cleaved intermediate.These data show for the first time that all SFTPC transits the cell surface during normal trafficking, and the I73T mutation accumulates at the cell surface through both retarded trafficking and active recycling. This understanding of normal SFTPC trafficking and how the I73T mutant disturbs it provides novel insight into SFTPC biology in health and disease, and in the contribution of the SFTPC mutant to IPF development.

Regeneration or Repair? The Role of Alveolar Epithelial Cells in the Pathogenesis of Idiopathic Pulmonary Fibrosis (IPF)

“…The order of events occurring during the pathogenesis of IPF has not been elucidated yet, but it is known that the collapse of the alveolus occurs due to the alteration of its surfactant system. Autosomal dominant mutations in the surfactant protein C, such as the variant SFTPC-I73T, are expressed exclusively in ATII cells and are known to F-IPF [61]. A previous study conducted with a rat model of bleomycin-induced fibrosis confirmed downregulation of SP-B and SP-C gene expression [62].…”

The effect ofDnaaf5gene dosage on primary ciliary dyskinesia phenotypes

“…The gRNA/Cas9 complex with both ssODNs were electroporated into single cell C57BL/6 strain embryos. Embryos (20)(21)(22)(23)(24)(25) were transferred to each pseudo-pregnant C57BL/6 mother (59). Live born mice were genotyped using NGS, as during validation.…”

“…We leveraged our patients' clinical features and associated variants in DNAAF5 to develop mouse models harboring these mutations. Modeling specific human mutations in mice has been a powerful tool for understanding disease pathobiology while controlling for genetic background and environment (23)(24)(25). Models of human PCD variants in mice have not been reported.…”

A novel human fetal lung-derived alveolar organoid model reveals mechanisms of surfactant protein C maturation relevant to interstitial lung disease

“…Clonal GFP-SFTPC-expressing HeLa cell lines were derived as previously described 3 . A pool stably expressing Cas9 was generated by lentiviral transduction of the pHRSIN-Cas9 vector and selection of transduced cells using hygromycin (2 μg/ml; Invitrogen 10687010).…”