Sub-lethal apoptotic stress enables mtDNA release during senescence and drives the SASP
Posted on: 19 July 2022
Preprint posted on 10 March 2022
Categories: biochemistry, cell biology
Background
External stressors and ageing push cells to undergo senescence. Senescent cells irreversibly stop their growth to limit transformation, but also start to secrete inflammatory mediators to help resolving the originating stressor by removing senescent cells from the body (Coppé et al., 2008). During aging or in pathological conditions, the body loses its capacity to clear senescent cell and as consequence the senescence-associated secretory phenotype (SASP) increases, leading to pathologic inflammation. Mitochondrial dysfunction is central in senescence and targeting damaged mitochondria in senescent cells could potentially inhibit the SASP, whilst keeping the tumor-suppressor roles of senescent cells (Correia‐Melo et al. 2016).
In this work, Chapman, Salmonowicz, Victorelli and colleagues found that a limited fraction of mitochondria in senescent cells undergo permeabilization of the outer membrane through the oligomerization of BAX-BAK and release of mtDNA. This process resembles what happens to mitochondria during apoptosis; but senescent-inducing stressors are not enough to trigger cell death, because only few mitochondria lose their integrity. Oligomerization of BAX-BAK proteins in the outer membrane of mitochondria induces the release of mtDNA, but in senescent cells, caspases activation is not enough to kept immunologically silent cytosolic mtDNA, as instead happens during apoptosis. Therefore, mtDNA activates the cGAS-STING cytosolic DNA sensing pathway, leading to the SASP.
Key findings of this preprint
- Peripheral mitochondria are more permeable in senescent cells
Using 3D SIM super-resolution microscopy and complementary biochemical experiments, the authors discover that the outer membrane protein TOM20 and the cytochrome c are not properly colocalized in a subset of peripheral mitochondria of senescent cells. Cytochrome c is generally released from mitochondria to the cytosol through BAX-BAK pores during apoptosis. Indeed, increased levels of cytosolic cytochrome c and BAX-BAK oligomers can be detected in senescent but not in proliferative fibroblasts by western blot.
- mtDNA is released in senescence
Airyscan confocal microscopy of dsDNA and TOM20 was used to quantify the number of DNA spots present into the cytoplasm of different fibroblastic cell lines during replicative conditions or in senescence. Senescent cells were observed to carry more cytosolic DNA and q-PCR with specific primers confirmed the source of this DNA as mitochondrial and not nuclear.
- miMOMP drives SASP
To probe whether mitochondrial outer membrane permeabilization (MOMP) occurring in a minority of mitochondria (miMOMP) could orchestrate by itself the SASP, the authors genetically delete by CRISPR-Cas9 both BAX and BAK and measured different hallmark of senescence, including cytokine secretion, proliferation, LMNB1 loss and DNA damage.
Of these, only cytosolic mtDNA and cytokine secretion were restored by BAX-BAK deletion, while proliferation markers (Ki-67), LMNB1 loss and DNA damage markers (γH2AX) were not rescued. To further confirm that BAX-BAK-mediated mtDNA leakage contributes to the SASP in senescent cell, the author also proved that other mechanisms of mtDNA leakage (i.e. formation of the mitochondrial permeability transition pore) were not concomitantly present in senescent cells; nor that BAX-BAK deletion generates confounding factors such as compromised oxidative phosphorylation and ROS production.
- Caspase activation following miMOMP is not able to block the SASP
During apoptosis, secretion of inflammatory mediators is blunted by concomitant activation of caspases that cleave cGAS (Ning et al., 2019). However, senescence-driven activation of caspases downstream miMOMP appeared to be not sufficient to suppress the SASP. Indeed, deleting a critical activator of caspases (APAF1), did not alter the SASP induced by irradiation. If caspases activity is not sufficient to prevent inflammatory activation, then cGAS and STING are likely to participate in the SASP, as previously suggested (Yang et al., 2017).
- Investigating cGAS-STING in senescence
As a tabula rasa to investigate involvement of cGAS-STING in the SASP, the authors wanted to generate cells completely lacking mitochondria, to study the effect of mtDNA sensing in senescent cells without any confounding factor. To this end, cells were stably transfected with a component of mitochondria disposal machinery (mitophagy) and mitochondria depolarization was triggered by the decoupler of the respiratory chain compound carbonyl cyanide m-chlorophenyl hydrazone (CCCP) to induce mitophagy and deplete cells from mitochondria. mtDNA reintroduction by transfection was sufficient to recapitulate the SASP phenotype. Moreover, TFAM deficient cells, that show increased cytosolic mtDNA, were confirmed to have an inflammatory phenotype which promotes senescence. Thus, mtDNA in the cytosol can accelerate senescence.
- BAX inhibition improves healthspan in aged mice
As a proof of concept, the authors treated aged mice with a BAX inhibitor, known to prevent apoptosis and block mtDNA release. Notably, musculoskeletal parameters and healthspan were improved in treated mice, without affecting lifespan.
In conclusion, this paper characterized miMOMP in senescence and linked mtDNA and cGAS-STING functions in aging. Moreover, it shows that cGAS-driven SASP is a major rate limiting factor to healthy aging in mice.
Why I chose this preprint
cGAS-STING is a hot topic, but its physiological involvement in senescence and aging is still debated. This paper, with cool microscopy and clever experiments, add an important piece of evidence to the cGAS-STING field of research and clarify the link between this cytosolic DNA sensing pathway and the SASP in senescence.
Questions to the authors
· Why miMOMP is more frequently detected in peripheral mitochondria of senescent cells
· Could the tested BAX inhibitor be used also in humans? Are there any trial with those kinds of small molecules to improve healthspan in humans?
References
· Coppé, J.-P. et al. Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor. PLOS Biology 6, e301, doi:10.1371/journal.pbio.0060301 (2008).
· Correia‐Melo, C. et al. Mitochondria are required for pro‐ageing features of the senescent phenotype. The EMBO Journal 35, 724, doi:10.15252/embj.201592862 (2016).
· X. Ning, Y. Wang, M. Jing, M. Sha, M. Lv, P. Gao, R. Zhang, X. Huang, J. M. Feng, Z. Jiang, Apoptotic Caspases Suppress Type I Interferon Production via the Cleavage of cGAS, MAVS, and IRF3. Mol. Cell. 74, 19-31.e7 (2019).
· H. Yang, H. Wang, J. Ren, Q. Chen, Z. J. Chen, cGAS is essential for cellular senescence. PNAS (2017), doi:10.1073/pnas.1705499114.
doi: https://doi.org/10.1242/prelights.32397
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