Alpha-1 Antitrypsin (AAT) Polymers Induce Mitochondrial Dysfunction and Altered Metabolism in AAT Deficient Lungs and Precision-Cut Lung Slices (PCLS)

Abstract Rationale: In Alpha-1 Antitrypsin Deficiency (AATD), a genetic disease leading to early-onset emphysema, the Pi[asterisk]Z-SERPINA1 mutation results in polymerized Z-AAT protein. Z-AAT polymers within lung epithelial cells account for ER stress, increased mitochondrial reactive oxygen species (ROS) production, autophagy, and apoptosis. Emphysematous lungs of smokers demonstrate high Dynamin-Related Protein-1 (DRP-1) expression, a mediator of mitochondrial fission. Mitochondria are the primary site for ATP production through glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation, which depend on mitochondrial structure and function. Disruptions in these pathways can lead to inefficient ATP production, increased oxidative stress, and impaired mitochondrial respiration. Whether altered mitochondrial metabolism is present in AATD lungs remains unstudied. We hypothesize that Z-AAT polymers induce mitochondrial dysfunction and altered metabolism in our novel humanized Z-AAT transgenic mouse model on murine Serpina1Null background (Z-AAT Serpina1Null). Methods: Z-AAT polymers and DRP-1 expression were assessed using immunofluorescence and electron microscopy in lungs of Z-AAT Serpina1Null mice. Lung tissue from control and Z-AAT Serpina1Null mice was extracted using a methanol-water-chloroform solvent system and analyzed by LC-MS on a Bruker Impact II QTOF. Agarose-inflated lungs were used for airspace enlargement assessment and generation of viable PCLS. DRP-1 expression was analyzed via Western blot and qPCR in AATD PCLS and controls, and mitochondrial oxygen consumption rate (OCR) was assessed using the Seahorse XF analyzer. Results: Airspace enlargement, Z-AAT polymers, and increased DRP-1 expression were observed in Z-AAT Serpina1Null(p<0.05, t-test vs. wild-type) lungs. Metabolomic and lipidomic analysis of Z-AAT Serpina1Null lungs revealed significantly increased levels of acetoacetate, 6-hydroxydopamine, 6-Dimethylaminopurine, ADP, and citrulline, reflecting changes in energy metabolism, purine metabolism, and pathways involving fructose, mannose, arginine, and proline. Pathway enrichment analysis showed that Z-AAT lungs were enriched in metabolites related to phenylalanine and purine metabolism, arginine biosynthesis, and fructose and mannose metabolism, while controls displayed higher activity in glycolysis, pyrimidine metabolism, and the urea cycle. These metabolic shifts suggest a reprogramming in Z-AAT lungs, potentially driven by mitochondrial dysfunction and inflammation. Viable Serpina1Null PCLS sections showed increased levels of DRP-1 (p<0.05, t-test vs. wild-type), and decreased OCR and ATP production at baseline (p<0.05, t-test vs. wild-type). Conclusions: Our findings demonstrate that Z-AAT polymer accumulation in the lungs of Z-AAT Serpina1Null mice is linked to DRP-1 signaling and disrupted ATP production pathways, contributing to mitochondrial dysfunction and emphysema-like airspace enlargement. These results highlight potential targetable mechanisms for preventing lung injury in AATD, offering new avenues for therapeutic intervention.