Abstract
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.
Introduction
The accumulation over time of mutations in the mitochondrial genome (mtDNA) is a common process that has been shown to occur in many tissues as one of the hallmarks of ageing1. mtDNA is present in thousands of copies per cell, hence, impairment of mitochondrial function is observed only when the percentage of mutated mtDNA molecules surpasses a specific threshold2. Cells possess a plethora of quality control mechanisms to survey the intactness of DNA, RNA, and proteins, but also of entire organelles. In addition to bulk autophagy, which is responsible for the continuous and non-selective turnover of cellular material, specific mechanisms to remove malfunctioning organelles upon damage are known. The process of mitophagy has been investigated extensively in recent years as an important salvage pathway to remove dysfunctional mitochondria3. Upon mitochondrial damage, PINK1 is presented at the mitochondrial membrane signalizing the part of the network prompted to degradation. PINK1 stabilization induces recruitment of the cytosolic protein Parkin together with other autophagy proteins, and initiates the formation of an autophagosome which ends with the engulfment of damaged mitochondria3. Mitochondrial dynamics are important processes surveying mitochondrial quality. Mitochondrial fission facilitates removal of the mitochondrial parts with impaired function, while mitochondrial fusion is required for mtDNA replication. Thus, knockout of key players of mitochondrial fusion induce mtDNA instability by causing a rapid accumulation of mtDNA alterations over time4.
In addition to canonical forms of mitophagy, other special pathways have been described. A process with a high level of specificity involving mitochondrial-derived vesicles (MDVs) was shown to remove not the complete organelle, but rather mitochondrial fragments containing specific cargo5. This mechanism requires the coordination of mitochondrial dynamics, mitophagy, and also the vacuolar protein sorting (VPS) or retromer complex. Thus, changes in the mitochondrial membrane potential and the oxidation state of mitochondrial sub-compartments induce the curvature of the membrane, followed by recruitment of PINK1 and Parkin6. The retromer, formed by the proteins VPS26, VPS29 and VPS35, provides the force to generate a vesicle, which is delivered to lysosomes or peroxisomes, in a process independent of the autophagy proteins ATG5 or LC37,8,9. Other non-canonical pathways include direct transfer of mitochondrial parts to endosomes10. In these cases, mitochondrial damage triggers recruitment of Parkin, which is recognized by RAB5 endosomes engulfing mitochondrial parts guided towards the lysosomal compartment, where degradation occurs. Despite recent advances, little is known about how mitochondria contact the endo-lysosome system. In yeast, endo-lysosome-mitochondria tethering is regulated by physical interaction between the mitochondrial protein Tom40 and the endosomal proteins Vps39 and Vps1311. In mammalian cells, MFN2 and GDAP1 have been shown to mediate endo-lysosome-mitochondria contacts through RAB7 and LAMP1, respectively12,13.
Many inherited forms of neurodegenerative diseases are examples for insufficient mitochondrial quality control. Several forms of Charcot-Marie-Tooth neuropathy have been linked to mutations in genes encoding for GDAP1, MFN2, and RAB714. However, whether mitophagy impairment is the primary cause of the disease or a secondary effect is still not clear. The strongest link between neurodegeneration and mitochondrial quality control defects relies on Parkinson’s disease (PD). PD is caused by the specific degeneration of dopaminergic neurons, which have been comprehensively shown to be a hotspot for the accumulation of large-scale mtDNA deletions during normal ageing15,16. Thus, not only mutations of specific mitophagy receptors like PINK1 and Parkin, but also malfunction of the lysosomal proteins ATP13A2 and LAMP3, along with mutations in the retromer component VPS3517 cause familial forms of PD. Comparably, mutations in essential genes for mtDNA replication and maintenance, such as Twinkle and POLγ, lead to several forms of severe diseases including Parkinsonism, Progressive External Ophthalmoplegia, and Spinocerebellar Ataxia18. Therefore, therapeutic approaches to increase mitochondrial quality control and counteract the progression of mitochondrial-related diseases have been attempted19,20. Nevertheless, understanding the molecular mechanisms guiding the specificity of the different forms for the mitochondrial quality control systems is essential to design precise strategies and avoiding the activation of undesirable effects.
Expressing the mitochondrial helicase Twinkle, bearing disease-related dominant negative mutations, have been used to induce mtDNA instability and study mtDNA-related diseases21. In mouse models, expression of the Twinkle mutation p.K320E (from now on K320E), accelerates the accumulation of mtDNA deletions in postmitotic tissues22,23,24 and induces mtDNA depletion in proliferating cells25,26. Using a combination of genetic and chemical tools, we have now identified the proteins involved in a mechanism for the targeting and degradation of mutated mtDNA through mitochondria-endosome transfer without affecting the mitochondrial pool. Expression of K320E or chemical damage triggers the elimination of altered mtDNA molecules through endosomes, a process controlled by the interaction between mitochondrial nucleoids with the inner membrane protein ATAD3 and the outer membrane and translocase protein SAMM50. On the mitochondrial surface, SAMM50 regulates the distribution of BAK foci and balances nucleoid release, which are later captured by VPS35 endosomes. SAMM50 and VPS35 are essential to provide the required selectivity and specificity for mtDNA elimination. While VPS35 is necessary to avoid the activation of an exacerbated mitophagy response, SAMM50 acts as a sentinel, surveillant proper mtDNA transfer to VPS35 endosomes. Finally, we demonstrate that stimulation of lysosomal function by rapamycin in vivo is sufficient to specifically remove deleted mtDNA, without affecting the total mtDNA copy number, strengthening modulation of autophagy as an approach to counteract the accumulation of mutations in mtDNA, as observed in several mitochondrial pathologies and during ageing.
Results
mtDNA alterations in skeletal muscle do not induce mitophagy
Expression of K320E in skeletal muscles drives differential accumulation of mitochondrial damage depending on fiber-type23. In extraocular muscles, mtDNA alterations preferentially accumulate in fast-twitch fibers in contrast to slow-oxidative fibers, where mitochondrial function is fundamental for muscle performance. We hypothesized that these differences reflected different mitochondrial quality control systems surveillant mitochondrial integrity in different muscle fiber types. To study the nature of these mechanisms, we first analyzed fast-twitch M. tibialis anterior (TA) and slow-oxidative M. soleus (SOL), muscles both rich in fibers with high mitochondrial content in mice, but with preferentially glycolytic (TA) vs. oxidative metabolism (SOL), respectively. As shown before, K320E mutant mice carry a wide variety of reorganized mtDNA molecules27, causing an inefficient PCR amplification reaction and leading to a smear of products, however there were no changes in total mtDNA copy number (Fig. 1a, b). TA from aged WT mice also showed many mtDNA alterations, while only few were found in SOL. By conventional PCR, we analyzed the presence of common mtDNA deletions in aged mice23 and selected a deletion covering about 4000 b.p (Mus musculus mtDNA-Δ983-4977), which was present in both mutant muscles (Supplementary Fig. 1a, ca. 500 bp product). Considering that mtDNA copy number is 20% higher in SOL than in TA (Supplementary Fig. 1b), we performed qPCR quantification using the D-Loop region as a reference and found that indeed, this deletion was on average 20 times more abundant in TA compared to SOL (Fig. 1c)….
