MTORC1 activation in lung mesenchyme drives sex- and age-dependent pulmonary structure and function decline

Kseniya Obraztsova,David B Frank,Susan M Lin,Jilly F Evans,Elena N Atochina‐Vasserman ,Michael F Beers,Shanru Li,Andrei I Gritsiuta,Edward E Morrisey,Meghan C Kopp ,Aravind Sivakumar,Justine Carl,Apoorva Babu,Edward Cantu,Alexander R Mukhitov,Jeremy Katzen,Maria C Basil,Ryan W Rue ,Annette Robichaud,Michael Morley ,Vera P Krymskaya

doi:10.1038/s41467-020-18979-4

Abstract

Lymphangioleiomyomatosis (LAM) is a rare fatal cystic lung disease due to bi-allelic inactivating mutations in tuberous sclerosis complex (TSC1/TSC2) genes coding for suppressors of the mechanistic target of rapamycin complex 1 (mTORC1). The origin of LAM cells is still unknown. Here, we profile a LAM lung compared to an age- and sex-matched healthy control lung as a hypothesis-generating approach to identify cell subtypes that are specific to LAM. Our single-cell RNA sequencing (scRNA-seq) analysis reveals novel mesenchymal and transitional alveolar epithelial states unique to LAM lung. This analysis identifies a mesenchymal cell hub coordinating the LAM disease phenotype. Mesenchymal-restricted deletion of Tsc2 in the mouse lung produces a mTORC1-driven pulmonary phenotype, with a progressive disruption of alveolar structure, a decline in pulmonary function, increase of rapamycin-sensitive expression of WNT ligands, and profound female-specific changes in mesenchymal and epithelial lung cell gene expression. Genetic inactivation of WNT signaling reverses age-dependent changes of mTORC1-driven lung phenotype, but WNT activation alone in lung mesenchyme is not sufficient for the development of mouse LAM-like phenotype. The alterations in gene expression are driven by distinctive crosstalk between mesenchymal and epithelial subsets of cells observed in mesenchymal Tsc2-deficient lungs. This study identifies sex- and age-specific gene changes in the mTORC1-activated lung mesenchyme and establishes the importance of the WNT signaling pathway in the mTORC1-driven lung phenotype.

Highlights

Lymphangioleiomyomatosis (LAM) is a rare fatal cystic lung disease due to bi-allelic inactivating mutations in tuberous sclerosis complex (TSC1/tuberous sclerosis complex 2 (TSC2)) genes coding for suppressors of the mechanistic target of rapamycin complex 1
Typical LAM lesions consist of a nest of cells formed by “immature”-looking mesenchymal smoothmuscle-like cells expressing smooth-muscle α-actin (SMA) and positive for phospho-ribosomal protein S6, a marker of mechanistic target of rapamycin complex 1 (mTORC1) activation[2,3,4] (Fig. 1b)
The alveolar epithelial cell (AEC) niche was divided into two canonical clusters: alveolar type 2 (AT2), marked by the high differential expression of SFTPC, SFTPD, and SFTPA genes, and alveolar type-1 (AT1) cells, characterized by HOPX, AGER, and AQP4 gene expression (Fig. 2b, d, Supplementary Fig. 1A, B)

Summary

Introduction

Lymphangioleiomyomatosis (LAM) is a rare fatal cystic lung disease due to bi-allelic inactivating mutations in tuberous sclerosis complex (TSC1/TSC2) genes coding for suppressors of the mechanistic target of rapamycin complex 1 (mTORC1). The rare genetic disease pulmonary lymphangioleiomyomatosis (LAM) occurs almost exclusively in women of childbearing age due to loss of function of the tumor suppressor gene tuberous sclerosis complex 2 (TSC2), a negative regulator of the mechanistic target of rapamycin complex 1 (mTORC1)[2,3]. The spontaneous occurrence of lung tumors in heterozygous Tsc1+/− and Tsc2+/− mice, or in Eker rats with a naturally occurring Tsc[2] mutation, is extremely rare and occurs without major changes of lung parenchyma and only in very aged animals[13] To address these challenges and to generate a hypothesis about the cell of origin of LAM, we perform single-cell RNA sequencing (scRNA-seq) of a LAM lung and compare this with an age-and sex-matched normal human lung. Our study demonstrates a critical role for Tsc2-dependent mTORC1 and WNT signaling pathways in the regulation of the lung structure and function

Methods

Results

Conclusion