Mitochondrial Lon Peptidase 1 Controls Diaphragm and Lung Development in a Context-Dependent Manner

Abstract

Congenital Diaphragmatic Hernia (CDH) is a rare neonatal disorder causing diaphragmatic defects and cardiopulmonary hypoplasia, traditionally attributed to mechanical compression from organ herniation. However, emerging evidence suggests genetic mutations may independently impair lung development, prompting debate over CDH etiology. Here, we investigated the requirement of mitochondrial function guarded by LON peptidase 1 (Lonp1), a CDH risk gene, in either diaphragm or lung development. Lonp1 loss in skeletal muscles of the diaphragm led to its thinning and membranization, recapitulating the pathology of sac-type CDH. On the other hand, lung-specific inactivation caused severe hypoplasia with defective branching morphogenesis, independent of diaphragm anomalies. Molecularly, Lonp1 disruption dysregulated key transcription factors and signaling pathways known to be critical for early lung development. Our findings here revealed that mitochondrial defects contribute to the pathogenesis of CDH in an organ and cell type specific manner, opening new avenues for drug and therapeutic development.

Keywords

CDH / Diaphragm / Lung / Mitochondria / LONP1 / SHH / FGF10

Acknowledgments

The authors thank Sun lab members for discussions. UCSD Microscopy Core supported by NINDS-P30NS047101, and IGM core with NovaSeq 6000 purchased using NIH S10-OD026929.

Author Contributions

L.X., C.T. and X.S. designed experiments. L.X., C.T., N.T. and A.S. performed experiments. L.X. and C.T. analyzed data. D.J.M., Y.S. and W.K.C. provided advice. L.X. and X.S. wrote the manuscript.

Ethics Statement

The animal experiments performed in this study were approved by The Institutional Animal Care and Use Committee (IACUC) in University of California San Diego (the protocol # is S16187 and approval date is 31 March 2025).

Informed Consent Statement

Not applicable.

Data Availability Statement

Raw and processed files from bulk RNA-seq have been uploaded to the GEO database under accession number GSE230334.

Funding

This work was supported by NIH R01HL160019 (to X.S.), AHA postdoctoral fellowship 827787 and NIH/NHLBI Pathway to Independence K99HL171830 (to L.X.).

Declaration of Competing Interest

The authors declare no competing interests.

References

[1] Zani A, Chung WK, Deprest J, Harting MT, Jancelewicz T, Kunisaki SM, et al.Congenital diaphragmatic hernia. Nat. Rev. Dis. Primers 2022, 8, 37.

[2] Olutoye Ii OO, Short WD, Gilley J, Hammond Ii JD, Belfort MA, Lee TC, et al. The Cellular and Molecular Effects of Fetoscopic Endoluminal Tracheal Occlusion in Congenital Diaphragmatic Hernia. Front. Pediatr. 2022, 10, 925106.

[3] Carmona R, Canete A, Cano E, Ariza L, Rojas A, Munoz-Chapuli R. Conditional deletion of WT 1 in the septum transversum mesenchyme causes congenital diaphragmatic hernia in mice. Elife 2016, 5, e16009.

[4] Jay PY, Bielinska M, Erlich JM, Mannisto S, Pu WT, Heikinheimo M, et al. Impaired mesenchymal cell function in Gata4 mutant mice leads to diaphragmatic hernias and primary lung defects. Dev. Biol. 2007, 301, 602-614.

[5] Ackerman KG, Herron BJ, Vargas SO, Huang HL, Tevosian SG, Kochilas L, et al. is required for normal diaphragm and lung development in mice and humans. PLoS Genet. 2005, 1, 58-65.

[6] You LR, Takamoto N, Yu CT, Tanaka T, Kodama T, DeMayo FJ, et al. Mouse lacking as an animal model of Bochdalek-type congenital diaphragmatic hernia. Prac. Natl. Acad. Sci. USA 2005, 102, 16351-16356.

[7] Rivas JFG, Applin NHM, Albrechtsen JFP, Ghazanfari A, Doschak M, Clugston RD. Mesenchymal retinoic acid signaling is required for normal diaphragm development in mice. Faseb J. 2025, 39, e70381.

[8] Guilbert TW, Gebb SA, Shannon JM. Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development. Am. J. Physiol. Lung Cell Mol. Physiol. 2000, 279, L1159-L1171.

[9] Mendelsohn C, Lohnes D, Decimo D, Lufkin T, LeMeur M, Chambon P, et al. Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities at various stages of organogenesis in RAR double mutants. Development 1994, 120, 2749-2771.

[10] Luna G, Verheyden J, Tan C, Kim E, Hwa M, Sahi J, et al. MYRF is Essential in Mesothelial Cells to Promote Lung Development and Maturation. bioRxiv 2025, doi:10.1101/2025.02.13.635155.

[11] Stokes G, Li ZW, Talaba N, Brix MB, Pham B, et al. Genthe W, Rescuing lung development through embryonic inhibition of histone acetylation. Sci. Transl. Med. 2024, 16, eadc8930.

[12] Han S, Lee M, Shin Y, Giovanni R, Chakrabarty RP, Herrerias MM, et al. Mitochondrial integrated stress response controls lung epithelial cell fate. Nature 2023, 620, 890-897.

[13] Islam MN, Gusarova GA, Das SR, Li L, Monma E, Anjaneyulu M, et al. The mitochondrial calcium uniporter of pulmonary type 2 cells determines severity of acute lung injury. Nat. Commun. 2022, 13, 5837.

[14] Ali M, Zhang X, LaCanna R, Tomar D, Elrod JW, Tian Y. MICU1-dependent mitochondrial calcium uptake regulates lung alveolar type 2 cell plasticity and lung regeneration. JCI Insight 2022, 7, e154447.

[15] Zhang K, Yao E, Chen B, Chuang E, Wong J, Seed RI, et al. Acquisition of cellular properties during alveolar formation requires differential activity and distribution of mitochondria. Elife 2022, 11, e68598.

[16] Rochard P, Rodier A, Casas F, Cassar-Malek I, Marchal-Victorion S, Daury L, et al. Mitochondrial activity is involved in the regulation of myoblast differentiation through myogenin expression and activity of myogenic factors. J. Biol. Chem. 2000, 275, 2733-2744.

[17] Qiao L, Xu L, Yu L, Wynn J, Hernan R, Zhou X, et al. Rare and de novo variants in 827 congenital diaphragmatic hernia probands implicate LONP1 as candidate risk gene. Am. J. Hum. Genet. 2021, 108, 1964-1980.

[18] Xu L, Tan C, Barr J, Talaba N, Verheyden J, Chin JS, et al. Context-dependent roles of mitochondrial LONP1 in orchestrating the balance between airway progenitor versus progeny cells. Cell Stem Cell 2024, 31, 1465-1483 e6.

[19] Harfe BD, Scherz PJ, Nissim S, Tian H, McMahon AP, Tabin CJ. Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell 2004, 118, 517-528.

[20] Zhang W, Menke DB, Jiang M, Chen H, Warburton D, Turcatel G, et al. Spatial-temporal targeting of lung-specific mesenchyme by a Tbx4 enhancer. BMC Biol. 2013, 11, 111.

[21] Engleka KA, Gitler AD, Zhang MZ, Zhou DD, High FA, Epstein JA. Insertion of Cre into the locus creates a new allele of and identifies unexpected derivatives. Dev. Biol. 2005, 280, 396-406.

[22] Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat. Methods 2012, 9, 357-359.

[23] Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 2010, 28, 511-515.

[24] Oliver ER, DeBari SE, Adams SE, Didier RA, Horii SC, Victoria T, et al. Congenital diaphragmatic hernia sacs: prenatal imaging and associated postnatal outcomes. Pediatr. Radiol. 2018, 49, 593-599.

[25] Guo M, Morley MP, Jiang C, Wu Y, Li G, Du Y, et al. Guided construction of single cell reference for human and mouse lung. Nat. Commun. 2023, 14, 4566.

[26] Abler LL, Mansour SL, Sun X. Conditional gene inactivation reveals roles for Fgf10 and Fgfr2 in establishing a normal pattern of epithelial branching in the mouse lung. Dev. Dyn. 2009, 238, 1999-2013.

[27] Herriges JC, Verheyden JM, Zhang Z, Sui P, Zhang Y, Anderson MJ, et al. FGF-Regulated ETV Transcription Factors Control FGF-SHH Feedback Loop in Lung Branching. Dev. Cell 2015, 35, 322-332.

[28] Wan H, Dingle S, Xu Y, Besnard V, Kaestner KH, Ang SL, et al. Compensatory roles of Foxa1 and Foxa2 during lung morphogenesis. J. Biol. Chem. 2005, 280, 13809-13816.

[29] Raitio A, Salim A, Losty PD. Congenital diaphragmatic hernia-does the presence of a hernia sac improve outcome? A systematic review of published studies. Eur. J. Pediatr. 2021, 180, 333-337.

[30] Metzakopian E, Lin W, Salmon-Divon M, Dvinge H, Andersson E, Ericson J, et al. Genome-wide characterization of Foxa2 targets reveals upregulation of floor plate genes and repression of ventrolateral genes in midbrain dopaminergic progenitors. Development 2012, 139, 2625-2634.