Decreased AMPK Signaling In Alveolar Stem Cells Impairs Alveolar Regeneration In Aged Mouse

Aging directly affects the health of living organisms.With the onset of aging,the physiological structure and function of lung will undergo the irreversibly changes,which strongly correlated with the occurrence of multiple lung diseases.Alveolar epithelial type 2 cells function as the alveolar stem cells and play the key roles in maintaining the alveolar physiological function and pathological injury repair.The functions of alveolar epithelial type 2 cells are also significantly altered by aging.Previous studies have shown that the dysfunction of alveolar epithelial type 2 cells would lead to multiple aging related-lung diseases.However,little is known about the mechanisms of aging on alveolar epithelial type 2 cells during alveolar regeneration.In this thesis,we study the cellular and molecular mechanisms on how aging affects alveolar regeneration by combining mouse genetics and single-cell RNA sequencing technology.By comparing alveolar regeneration processes of young and aged mice,we found that the alveolar regeneration capacity of aged mice was significantly decreased.In addition,we observed alveolar epithelial type 2 cells of aged mouse show impaired differentiation and kept clustered in alveolar injury area.To futher study the cellular and molecular mechanisms,we performed single-cell RNA sequencing and then analyzed the transcriptomic changes of young and aged alveolar epithelial type 2 cells.Based on the features of transcriptome,we found alveolar epithelial type 2 cells contain three different cell subpopulations.A subpopulation that enriched the genes related with alveolar regeneration was significantly increased in aged mice after alveolar injury.By further comparing the differentially expressed genes of this subpopulation between young and aged mice,we found that genes related with AMPK signaling pathway were significantly decreased in the aged alveolar epithelial type 2 cells.To investigate whether decreased AMPK signaling results in impaired differentiation of aged mice,we firstly analyzed AMPK activities in young and aged mice after lung injury.The results show that AMPK was only activated in young alveolar epithelia type 2 cells,but not in aged alveolar epithelia type 2 cells.Next,we specifically knocked out Stkll from alveolar epithelial type 2 cells to validate AMPK functions in alveolar regeneration.The results show that knocking out Stkll would significantly decrease AMPK signaling activity,and impaired alveolar epithelial type 2 cell differentiation.Finally,we used AICAR treatment to activate the AMPK signaling in alveolar epithelial type 2 cells in aged mice to demonstrate AMPK functions in aged mice.After AICAR treatment,we found that the differentiation of alveolar epithelial type 2 cells in aged mice was significantly improved both in vitro alveolar organoid culture and in vivo experiments.In summary,we demonstrated that the Stk11-AMPK signaling axis is required for the differentiation of alveolar epithelial type 2 cells and impaired differentiation of alveolar epithelial type 2 cells in aged mice is caused by decreased AMPK signaling.These findings provide us new insights for understanding the cellular and molecular mechanisms in lung regeneration and diseases.