Understanding the mechanisms governing selective turnover of mutation-bearing mtDNA is fundamental to design therapeutic strategies against mtDNA diseases. Here, we show that specific mtDNA damage leads to an exacerbated mtDNA turnover, independent of canonical macroautophagy, but relying on lysosomal function and ATG5. Using proximity labeling and Twinkle as a nucleoid marker, we demonstrate that mtDNA damage induces membrane remodeling and endosomal recruitment in close proximity to mitochondrial nucleoid sub-compartments. Targeting of mitochondrial nucleoids is controlled by the ATAD3-SAMM50 axis, which is disrupted upon mtDNA damage. SAMM50 acts as a gatekeeper, influencing BAK clustering, controlling nucleoid release and facilitating transfer to endosomes. Here, VPS35 mediates maturation of early endosomes to late autophagy vesicles where degradation occurs. In addition, using a mouse model where mtDNA alterations cause impairment of muscle regeneration, we show that stimulation of lysosomal activity by rapamycin, selectively removes mtDNA deletions without affecting mtDNA copy number, ameliorating mitochondrial dysfunction. Taken together, our data demonstrates that upon mtDNA damage, mitochondrial nucleoids are eliminated outside the mitochondrial network through an endosomal-mitophagy pathway. With these results, we unveil the molecular players of a complex mechanism with multiple potential benefits to understand mtDNA related diseases, inherited, acquired or due to normal ageing.

Background Mitochondrial dysfunction caused by mitochondrial (mtDNA) deletions have been associated with skeletal muscle atrophy and myofibre loss. However, whether such defects occurring in myofibres cause sarcopenia is unclear. Also, the contribution of mtDNA alterations in muscle stem cells (MuSCs) to sarcopenia remains to be investigated. Methods We expressed a dominant-negative variant of the mitochondrial helicase, which induces mtDNA alterations, specifically in differentiated myofibres (K320E skm mice) and MuSCs (K320E msc mice), respectively, and investigated their impact on muscle structure and function by immunohistochemistry, analysis of mtDNA and respiratory chain content, muscle transcriptome and functional tests. Results K320E skm mice at 24 months of age had higher levels of mtDNA deletions compared with controls in soleus (SOL, 0.07673% vs. 0.00015%, P = 0.0167), extensor digitorum longus (EDL, 0.0649 vs. 0.000925, P = 0.0015) and gastrocnemius (GAS, 0.09353 vs. 0.000425, P = 0.0004). K320E skm mice revealed a progressive increase in the proportion of cytochrome c oxidase deficient (COX À ) fibres in skeletal muscle cross sections, reaching a maximum of 3.03%, 4.36%, 13.58%, and 17.08% in EDL, SOL, tibialis anterior (TA) and GAS, respectively. However, mice did not show accelerated loss of muscle mass, muscle strength or physical performance. Histological analyses revealed ragged red fibres but also stimulated regeneration, indicating activation of MuSCs. RNAseq demonstrated enhanced expression of genes associated with protein synthesis, but also degradation, as well as muscle fibre differentiation and cell proliferation. In contrast, 7 days after destruction by cardiotoxin, regenerating TA of K320E msc mice showed 30% of COX À fibres. Notably, regenerated muscle showed dystrophic changes, increased fibrosis (2.5% vs. 1.6%, P = 0.0003), increased abundance of fat cells (2.76% vs. 0.23%, P = 0.0144) and reduced muscle mass (regenerated TA: 40.0 mg vs. 60.2 mg, P = 0.0171). In contrast to muscles from K320E skm mice, freshly isolated MuSCs from aged K320E msc mice were completely devoid of mtDNA alterations. However, after passaging, mtDNA copy number as well as respiratory chain subunits and p62 levels gradually decreased. Conclusions Taken together, accumulation of large-scale mtDNA alterations in myofibres alone is not sufficient to cause sarcopenia. Expression of K320E-Twinkle is tolerated in quiescent MuSCs, but progressively leads to mtDNA and respiratory chain depletion upon activation, in vivo and in vitro, possibly caused by an increased mitochondrial removal. Altogether, our results suggest that the accumulation of mtDNA alterations in myofibres activates regeneration during aging, which leads to sarcopenia if such alterations have expanded in MuSCs as well.

Integrity of mitochondrial DNA (mtDNA), encoding several subunits of the respiratory chain, is essential to maintain mitochondrial fitness. Mitochondria, as a central hub for metabolism, are affected in a wide variety of human diseases but also during normal ageing, where mtDNA integrity is compromised. Mitochondrial quality control mechanisms work at different levels, and mitophagy and its variants are critical to remove dysfunctional mitochondria together with mtDNA to maintain cellular homeostasis. Understanding the mechanisms governing a selective turnover of mutation-bearing mtDNA without affecting the entire mitochondrial pool is fundamental to design therapeutic strategies against mtDNA diseases and ageing. Here we show that mtDNA depletion after expressing a dominant negative version of the mitochondrial helicase Twinkle, or by chemical means, is due to an exacerbated mtDNA turnover. Targeting of nucleoids is controlled by Twinkle which, together with the mitochondrial transmembrane proteins ATAD3 and SAMM50, orchestrate mitochondrial membrane remodeling to form extrusions. mtDNA removal depends on autophagy and requires the vesicular trafficking protein VPS35 which binds to Twinkle-enriched mitochondrial subcompartments upon mtDNA damage. Stimulation of autophagy by rapamycin selectively removes mtDNA deletions which accumulated during muscle regeneration in vivo, but without affecting mtDNA copy number. With these results we unveil a new complex mechanism specifically targeting and removing mutant mtDNA which occurs outside the mitochondrial network. We reveal the molecular targets involved in a process with multiple potential benefits against human mtDNA related diseases, either inherited, acquired or due to normal ageing.

No abstract