Individuals with homozygous laminA/C p.R527C mutations manifest a severe form of Mandibuloacral dysplasia‐(MAD) and exhibit overlapping progeroid symptoms, for which the underlying molecular pathology remains unknown. Herein, it is shown that MAD patients achieved inflammaging with different pro‐inflammatory cytokines compared to progeria‐(HGPS) patient. Characterization of MAD iPSC‐derived Mesenchymal stem cells (MAD‐iMSC) uncovers deregulated mitochondrial Ca⁺² as the primary cause of inflammaging, mediated through inflammasome formation rather than the cGAS‐STING pathway. Moreover, MAD‐iMSCs extracellular vesicles (EVs) can also upregulate mitochondrial Ca⁺² in healthy cells. This deregulated Ca⁺² homeostasis is indirectly mediated by mitochondrial calcium mediator, signal transducer, and activator of transcription‐3 (STAT3), situated on the mitochondrial associated membrane (MAM). Inflammaging is mitigated by various FDA‐approved MAM‐STAT3 upstream inhibitors, such as (Tocilizumab) or by correcting R527C mutation with CRISPR/CAS9. These results provide new insights into MAD disease and propose targeting defective mitochondrial Ca⁺² homeostasis as a promising therapy for reversing immunosenescence.

Homozygous lamina/c p.R527C mutations result in severe mandibuloacral dysplasia (MAD) and progeroid syndrome, but the underlying molecular pathology remains unknown. Here, we report on three patients with MAD, all displaying severe systemic inflammaging and characterized the major molecular pathways involved in the manifestation of this disease. Analysis of induced pluripotent stem cell (IPSC)-derived mesenchymal stem cells (MAD-iMSCs) obtained from the patients revealed that increased mitochondrial Ca ⁺² loading was the root cause of lost mitochondrial membrane potential, abnormal fission/fusion and fragmentation, which then participated in inflammaging by inducing the inflammasome. These alterations in Ca ⁺² homeostasis were mediated by signal transducer and activator of transcription 3 (STAT3), which is located on the mitochondrial associated membrane (MAM). STAT3 function could be rescued by treatment with clinically-approved IL-6 blockers, or by correction of R527C mutations. In addition, extracellular vesicles (EVs) obtained from MAD-iMSCs displayed reduced immunomodulatory function, being unable to rescue bleomycin-induced lung fibrosis and triggering mitochondrial dysfunction, senescence, and fibrosis in healthy cells. Our results provide new insights into the pathology of complex lamin-associated MAD with systemic immunosenescence, and suggest that targeting defective mitochondrial Ca ⁺² homeostasis may represent a promising novel therapy for this condition.

Antisense vivo-morpholino oligonucleotides (vivo-MOs) allow transient gene knockdown in adult organisms with high specificity and low toxicity. Vivo-MOs are used in cell culture and in many established model organisms, but a method for their use has not been described in threepsine stickleback ( Gasterosteus aculeatus (Linnaeus, 1758)). Stickleback are an emerging model system used in evolutionary and ecological genetic studies. While genomic techniques are commonly used in stickleback research, there are few studies and tools available to assess gene function in-vivo, especially for genes that may be difficult to knock out by CRISPR (e.g., lethal knock-outs). Here, we test the use of splice-blocking vivo-MOs for gene knockdown in stickleback using intraperitoneal injection of vivo-MOs targeting three candidate genes. Gene expression was assessed in the liver, spleen, and intestine. Successful knockdown of Spi1b was observed in the spleen, however, we observed no other significant knockdown at either timepoint tested. Injection of a fluorescently labeled control vivo-MO confirmed delivery to each target organ, validating this approach, but delivery was variable which may explain inconsistent effects. These results indicate that vivo-MOs have potential as a tool for in-vivo gene knockdown in stickleback. Optimizing delivery methods could improve reproducibility and knockdown efficiency in future studies.