Deletions and duplications in mitochondrial DNA (mtDNA) cause mitochondrial disease and accumulate in conditions such as cancer and age-related disorders, but validated high-throughput methodology ...that can readily detect and discriminate between these two types of events is lacking. Here we establish a computational method, MitoSAlt, for accurate identification, quantification and visualization of mtDNA deletions and duplications from genomic sequencing data. Our method was tested on simulated sequencing reads and human patient samples with single deletions and duplications to verify its accuracy. Application to mouse models of mtDNA maintenance disease demonstrated the ability to detect deletions and duplications even at low levels of heteroplasmy.
Despite being one of the most studied proteases in bacteria, very little is known about the role of ClpXP in mitochondria. We now present evidence that mammalian CLPP has an essential role in ...determining the rate of mitochondrial protein synthesis by regulating the level of mitoribosome assembly. Through a proteomic approach and the use of a catalytically inactive CLPP, we produced the first comprehensive list of possible mammalian ClpXP substrates involved in the regulation of mitochondrial translation, oxidative phosphorylation, and a number of metabolic pathways. We further show that the defect in mitoribosomal assembly is a consequence of the accumulation of ERAL1, a putative 12S rRNA chaperone, and novel ClpXP substrate. The presented data suggest that the timely removal of ERAL1 from the small ribosomal subunit is essential for the efficient maturation of the mitoribosome and a normal rate of mitochondrial translation.
Synopsis
A combination of proteomics and mouse knockout approaches identifies substrates of the mammalian ClpXP protease, showing that it controls mitochondrial homeostasis by ensuring assembly and function of mitochondrial ribosomes.
Mammalian ClpXP substrates are involved in mitochondrial translation, oxidative phosphorylation, and metabolism.
ClpXP determines the rate of mitochondrial protein synthesis by regulating the level of mitoribosome assembly.
Timely removal of ERAL1 from the small ribosomal subunit is essential for the efficient maturation of the mitoribosome and a normal rate of mitochondrial translation.
A combination of proteomics and mouse knockout approaches identifies substrates of the mammalian ClpXP protease, showing that it controls mitochondrial homeostasis by ensuring assembly and function of mitochondrial ribosomes.
Mitochondrial dysfunction is critically involved in Parkinson's disease, characterized by loss of dopaminergic neurons (DaNs) in the substantia nigra (SNc), whereas DaNs in the neighboring ventral ...tegmental area (VTA) are much less affected. In contrast to VTA, SNc DaNs engage calcium channels to generate action potentials, which lead to oxidant stress by yet unknown pathways. To determine the molecular mechanisms linking calcium load with selective cell death in the presence of mitochondrial deficiency, we analyzed the mitochondrial redox state and the mitochondrial membrane potential in mice of both sexes with genetically induced, severe mitochondrial dysfunction in DaNs (MitoPark mice), at the same time expressing a redox-sensitive GFP targeted to the mitochondrial matrix. Despite mitochondrial insufficiency in all DaNs, exclusively SNc neurons showed an oxidized redox-system, i.e., a low reduced/oxidized glutathione (GSH-GSSG) ratio. This was mimicked by cyanide, but not by rotenone or antimycin A, making the involvement of reactive oxygen species rather unlikely. Surprisingly, a high mitochondrial inner membrane potential was maintained in MitoPark SNc DaNs. Antagonizing calcium influx into the cell and into mitochondria, respectively, rescued the disturbed redox ratio and induced further hyperpolarization of the inner mitochondrial membrane. Our data therefore show that the constant calcium load in SNc DaNs is counterbalanced by a high mitochondrial inner membrane potential, even under conditions of severe mitochondrial dysfunction, but triggers a detrimental imbalance in the mitochondrial redox system, which will lead to neuron death. Our findings thus reveal a new mechanism, redox imbalance, which underlies the differential vulnerability of DaNs to mitochondrial defects.
Parkinson's disease is characterized by the preferential degeneration of dopaminergic neurons (DaNs) of the substantia nigra pars compacta (SNc), resulting in the characteristic hypokinesia in patients. Ubiquitous pathological triggers cannot be responsible for the selective neuron loss. Here we show that mitochondrial impairment together with elevated calcium burden destabilize the mitochondrial antioxidant defense only in SNc DaNs, and thus promote the increased vulnerability of this neuron population.
About the Authors: David Pla-Martin Affiliations Center for Physiology and Pathophysiology, Institute of Vegetative Physiology, University of Köln, Köln, Germany, Cologne Excellence Cluster on ...Cellular Stress Responses in Aging-associated Diseases (CECAD), University of Köln, Köln, Germany Rudolf J. Wiesner * E-mail: rudolf.wiesner@uni-koeln.de Affiliations Center for Physiology and Pathophysiology, Institute of Vegetative Physiology, University of Köln, Köln, Germany, Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), University of Köln, Köln, Germany, Center for Molecular Medicine Cologne, University of Köln, Köln, Germany ORCID logo http://orcid.org/0000-0003-1677-4476 Citation: Pla-Martin D, Wiesner RJ (2019) Reshaping membranes to build mitochondrial DNA. Mitochondrial DNA (mtDNA) is a double-stranded, circular DNA that encodes 37 essential genes and is present in thousands of copies in cells and packaged into nucleoids together with proteins involved in protection (transcription factor A, mitochondrial TFAM; mitochondrial single-stranded binding protein mtSSBP1), transcription (RNA polymerase, mitochondrial POLRMT; TFAM; transcription factor B, mitochondrial TFBM) and replication (DNA polymerase gamma POLG; mitochondrial helicase TWINKLE), and many other roles (Fig 1) 6. IMM, inner mitochondrial membrane; IMS, intermembrane space; KO, knock-out; MFN, mitofusin; mtDNA, mitochondrial DNA; mtSSBP1, mitochondrial single-stranded binding protein; OMM, outer mitochondrial membrane; OPA1, optic atrophy 1; POLG, DNA polymerase gamma; TFAM, transcription factor A, mitochondrial; TWINKLE, mitochondrial helicase. https://doi.org/10.1371/journal.pgen.1008140.g001 The fusion machinery is governed by two mitofusins for the outer membrane and OPA1 for the inner membrane. Mitochondrial fission has been related to clearance of dysfunctional mitochondria 14. ...fission has been shown to be essential also for mtDNA distribution during mitochondrial division 9.
To validate new mitochondrial myopathy serum biomarkers for diagnostic use.
We analyzed serum FGF21 (S-FGF21) and GDF15 from patients with (1) mitochondrial diseases and (2) nonmitochondrial ...disorders partially overlapping with mitochondrial disorder phenotypes. We (3) did a meta-analysis of S-FGF21 in mitochondrial disease and (4) analyzed S-Fgf21 and skeletal muscle Fgf21 expression in 6 mouse models with different muscle-manifesting mitochondrial dysfunctions.
We report that S-FGF21 consistently increases in primary mitochondrial myopathy, especially in patients with mitochondrial translation defects or mitochondrial DNA (mtDNA) deletions (675 and 347 pg/mL, respectively; controls: 66 pg/mL, p < 0.0001 for both). This is corroborated in mice (mtDNA deletions 1,163 vs 379 pg/mL, p < 0.0001). However, patients and mice with structural respiratory chain subunit or assembly factor defects showed low induction (human 335 pg/mL, p < 0.05; mice 335 pg/mL, not significant). Overall specificities of FGF21 and GDF15 to find patients with mitochondrial myopathy were 89.3% vs 86.4%, and sensitivities 67.3% and 76.0%, respectively. However, GDF15 was increased also in a wide range of nonmitochondrial conditions.
S-FGF21 is a specific biomarker for muscle-manifesting defects of mitochondrial translation, including mitochondrial transfer-RNA mutations and primary and secondary mtDNA deletions, the most common causes of mitochondrial disease. However, normal S-FGF21 does not exclude structural respiratory chain complex or assembly factor defects, important to acknowledge in diagnostics.
This study provides Class III evidence that elevated S-FGF21 accurately distinguishes patients with mitochondrial myopathies from patients with other conditions, and FGF21 and GDF15 mitochondrial myopathy from other myopathies.
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.
Aging is a progressive decline of body function, during which many tissues accumulate few cells with high levels of deleted mitochondrial DNA (mtDNA), leading to a defect of mitochondrial functions. ...Whether this mosaic mitochondrial deficiency contributes to organ dysfunction is unknown. To investigate this, we generated mice with an accelerated accumulation of mtDNA deletions in the myocardium, by expressing a dominant-negative mutant mitochondrial helicase. These animals accumulated few randomly distributed cardiomyocytes with compromised mitochondrial function, which led to spontaneous ventricular premature contractions and AV blocks at 18 months. These symptoms were not caused by a general mitochondrial dysfunction in the entire myocardium, and were not observed in mice at 12 months with significantly lower numbers of dysfunctional cells. Therefore, our results suggest that the disposition to arrhythmia typically found in the aged human heart might be due to the random accumulation of mtDNA deletions and the subsequent mosaic respiratory chain deficiency.
Display omitted
•Cardiomyocytes steadily accumulate mitochondrial DNA deletions with aging•This leads to the development of a tissue mosaic of mitochondrial deficiency•Impaired mitochondrial function in few cells promotes cardiac arrhythmia
Baris et al. test the idea of mosaic respiratory chain deficiency contributing to aging-related human cardiac disease. Using a genetic mouse model of accelerated accumulation of mtDNA deletions in the heart, they show that a few cardiomyocytes with mitochondrial DNA deletions progressively accumulate during aging, leading to cardiac arrhythmias.
Keratin intermediate filaments (KIFs) protect the epidermis against mechanical force, support strong adhesion, help barrier formation, and regulate growth. The mechanisms by which type I and II ...keratins contribute to these functions remain incompletely understood. Here, we report that mice lacking all type I or type II keratins display severe barrier defects and fragile skin, leading to perinatal mortality with full penetrance. Comparative proteomics of cornified envelopes (CEs) from prenatal KtyI(-/-) and KtyII(-/-)(K8) mice demonstrates that absence of KIF causes dysregulation of many CE constituents, including downregulation of desmoglein 1. Despite persistence of loricrin expression and upregulation of many Nrf2 targets, including CE components Sprr2d and Sprr2h, extensive barrier defects persist, identifying keratins as essential CE scaffolds. Furthermore, we show that KIFs control mitochondrial lipid composition and activity in a cell-intrinsic manner. Therefore, our study explains the complexity of keratinopathies accompanied by barrier disorders by linking keratin scaffolds to mitochondria, adhesion, and CE formation.
Here, we studied how epithelial energy metabolism impacts overall skin development by selectively deleting intraepithelial mtDNA in mice by ablating a key maintenance factor (TfamEKO), which induces ...loss of function of the electron transport chain (ETC). Quantitative (immuno)histomorphometry demonstrated that TfamEKO mice showed significantly reduced hair follicle (HF) density and morphogenesis, fewer intrafollicular keratin15+ epithelial progenitor cells, increased apoptosis, and reduced proliferation. TfamEKO mice also displayed premature entry into (aborted) HF cycling by apoptosis-driven HF regression (catagen). Ultrastructurally, TfamEKO mice exhibited severe HF dystrophy, pigmentary abnormalities, and telogen-like condensed dermal papillae. Epithelial HF progenitor cell differentiation (Plet1, Lrig1 Lef1, and β-catenin), sebaceous gland development (adipophilin, Scd1, and oil red), and key mediators/markers of epithelial–mesenchymal interactions during skin morphogenesis (NCAM, versican, and alkaline phosphatase) were all severely altered in TfamEKO mice. Moreover, the number of mast cells, major histocompatibility complex class II+, or CD11b+ immunocytes in the skin mesenchyme was increased, and essentially no subcutis developed. Therefore, in contrast to their epidermal counterparts, pilosebaceous unit stem cells depend on a functional ETC. Most importantly, our findings point toward a frontier in skin biology: the coupling of HF keratinocyte mitochondrial function with the epithelial–mesenchymal interactions that drive overall development of the skin and its appendages.
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 (K320Eskm mice) and MuSCs (K320Emsc 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
K320Eskm 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). K320Eskm mice revealed a progressive increase in the proportion of cytochrome c oxidase deficient (COX−) 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 K320Emsc mice showed 30% of COX− 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 K320Eskm mice, freshly isolated MuSCs from aged K320Emsc 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.
PDF
