Mitochondrial membrane proteins and VPS35 orchestrate selective removal of mtDNA

Abstract

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.

A novel genetic fluorescent reporter to visualize mitochondrial nucleoids

“…Nonetheless, we propose that the novel mt-HI-NESS fluorescent proteins developed and described here will be valuable tools to study the dynamics of mtDNA nucleoids in live cell imaging experiments. These reporters will allow researchers to address questions about how nucleoids are distributed throughout the mitochondrial network, how mtDNA replication is coordinated with mitochondrial fission and fusion events, and to study release of mtDNA into the cytosol or its endosomal/vesicle-mediated transport to autophagosomes for degradation [56][57][58] .…”

Endosomal removal and disposal of dysfunctional, immunostimulatory mitochondrial DNA

“…Very recent evidence indicates that the endolysosomal pathway also mediates mitochondrial quality control, either by enhancing mitophagy 34,40 , or by mediating mitochondrial clearance in the absence of autophagy 41 . We demonstrate a role for the endosomal system in the disposal of nucleoids that fail to finish replication and/or segregation, elucidating another pathway of selective disposal of nucleoids, separate from mitophagy 37 . Finally, we propose that endolysosomal extraction of nucleoids represents a third mtDNA release pathway separate from that mediated by BAK/BAX 27,28 or VDAC pores 29 .…”

“…There is growing evidence for additional pathways beyond mitophagy to maintain mitochondrial quality control, including MDVs 31 as well as piecemeal autophagy of mitochondrial components such as nucleoids 37 and MICOS complexes 38 . Importantly, it was recently demonstrated that mitophagy suppresses mtDNA-driven inflammation 39 .…”