Combined fibre atrophy and decreased muscle regeneration capacity driven by mitochondrial DNA alterations underlie the development of sarcopenia.

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.

CMT-linked loss-of-function mutations in GDAP1 impair store-operated Ca2+ entry-stimulated respiration.

GDAP1 is an outer mitochondrial membrane protein involved in Charcot-Marie-Tooth (CMT) disease. Lack of GDAP1 gives rise to altered mitochondrial networks and endoplasmic reticulum (ER)-mitochondrial interactions resulting in a decreased ER-Ca2+ levels along with a defect on store-operated calcium entry (SOCE) related to a misallocation of mitochondria to subplasmalemmal sites. The defect on SOCE is mimicked by MCU silencing or mitochondrial depolarization, which prevent mitochondrial calcium uptake. Ca2+ release from de ER and Ca2+ inflow through SOCE in neuroblastoma cells result in a Ca2+-dependent upregulation of respiration which is blunted in GDAP1 silenced cells. Reduced SOCE in cells with CMT recessive missense mutations in the α-loop of GDAP1, but not dominant mutations, was associated with smaller SOCE-stimulated respiration. These cases of GDAP1 deficiency also resulted in a decreased ER-Ca2+ levels which may have pathological implications. The results suggest that CMT neurons may be under energetic constraints upon stimulation by Ca2+ mobilization agonists and point to a potential role of perturbed mitochondria-ER interaction related to energy metabolism in forms of CMT caused by some of the recessive or null mutations of GDAP1.

CLUH regulates mitochondrial metabolism by controlling translation and decay of target mRNAs.

Mitochondria are essential organelles that host crucial metabolic pathways and produce adenosine triphosphate. The mitochondrial proteome is heterogeneous among tissues and can dynamically change in response to different metabolic conditions. Although the transcriptional programs that govern mitochondrial biogenesis and respiratory function are well known, posttranscriptional regulatory mechanisms remain unclear. In this study, we show that the cytosolic RNA-binding protein clustered mitochondria homologue (CLUH) regulates the expression of a mitochondrial protein network supporting key metabolic programs required under nutrient deprivation. CLUH exerts its function by controlling the stability and translation of target messenger RNAs. In the absence of Cluh, mitochondria are severely depleted of crucial enzymes involved in catabolic energy-converting pathways. CLUH preserves oxidative mitochondrial function and glucose homeostasis, thus preventing death at the fetal-neonatal transition. In the adult liver, CLUH ensures maximal respiration capacity and the metabolic response to starvation. Our results shed new light on the posttranscriptional mechanisms controlling the expression of mitochondrial proteins and suggest novel strategies to tailor mitochondrial function to physiological and pathological conditions.

Junctophilin-1 is a modifier gene of GDAP1-related Charcot-Marie-Tooth disease.

Mutations in the GDAP1 gene cause different forms of Charcot-Marie-Tooth (CMT) disease, and the primary clinical expression of this disease is markedly variable in the dominant inheritance form (CMT type 2K; CMT2K), in which carriers of the GDAP1 p.R120W mutation can display a wide range of clinical severity. We investigated the JPH1 gene as a genetic modifier of clinical expression variability because junctophilin-1 (JPH1) is a good positional and functional candidate. We demonstrated that the JPH1-GDAP1 cluster forms a paralogon and is conserved in vertebrates. Moreover, both proteins play a role in Ca(2+) homeostasis, and we demonstrated that JPH1 is able to restore the store-operated Ca(2+) entry (SOCE) activity in GDAP1-silenced cells. After the mutational screening of JPH1 in a series of 24 CMT2K subjects who harbour the GDAP1 p.R120W mutation, we characterized the JPH1 p.R213P mutation in one patient with a more severe clinical picture. JPH1(p.R213P) cannot rescue the SOCE response in GDAP1-silenced cells. We observed that JPH1 colocalizes with STIM1, which is the activator of SOCE, in endoplasmic reticulum-plasma membrane puncta structures during Ca(2+) release in a GDAP1-dependent manner. However, when GDAP1(p.R120W) is expressed, JPH1 seems to be retained in mitochondria. We also established that the combination of GDAP1(p.R120W) and JPH1(p.R213P) dramatically reduces SOCE activity, mimicking the effect observed in GDAP1 knock-down cells. In summary, we conclude that JPH1 and GDAP1 share a common pathway and depend on each other; therefore, JPH1 can contribute to the phenotypical consequences of GDAP1 mutations.

Mitochondrial dysfunction induced by frataxin deficiency is associated with cellular senescence and abnormal calcium metabolism.

Friedreich ataxia is considered a neurodegenerative disorder involving both the peripheral and central nervous systems. Dorsal root ganglia (DRG) are the major target tissue structures. This neuropathy is caused by mutations in the FXN gene that encodes frataxin. Here, we investigated the mitochondrial and cell consequences of frataxin depletion in a cellular model based on frataxin silencing in SH-SY5Y human neuroblastoma cells, a cell line that has been used widely as in vitro models for studies on neurological diseases. We showed that the reduction of frataxin induced mitochondrial dysfunction due to a bioenergetic deficit and abnormal Ca(2+) homeostasis in the mitochondria that were associated with oxidative and endoplasmic reticulum stresses. The depletion of frataxin did not cause cell death but increased autophagy, which may have a cytoprotective effect against cellular insults such as oxidative stress. Frataxin silencing provoked slow cell growth associated with cellular senescence, as demonstrated by increased SA-ßgal activity and cell cycle arrest at the G1 phase. We postulate that cellular senescence might be related to a hypoplastic defect in the DRG during neurodevelopment, as suggested by necropsy studies.

Silencing of the Charcot-Marie-Tooth disease-associated gene GDAP1 induces abnormal mitochondrial distribution and affects Ca2+ homeostasis by reducing store-operated Ca2+ entry.

GDAP1 is an outer mitochondrial membrane protein that acts as a regulator of mitochondrial dynamics. Mutations of the GDAP1 gene cause Charcot-Marie-Tooth (CMT) neuropathy. We show that GDAP1 interacts with the vesicle-organelle trafficking proteins RAB6B and caytaxin, which suggests that GDAP1 may participate in the mitochondrial movement within the cell. GDAP1 silencing in the SH-SY5Y cell line induces abnormal distribution of the mitochondrial network, reduces the contact between mitochondria and endoplasmic reticulum (ER) and alters the mobilization of mitochondria towards plasma membrane upon depletion of ER-Ca(2+) stores. GDAP1 silencing does not affect mitochondrial Ca(2+) uptake, ER-Ca(2+), or Ca(2+) flow from ER to mitochondria, but reduces Ca(2+) inflow through store-operated Ca(2+) entry (SOCE) following mobilization of ER-Ca(2+) and SOCE-driven Ca(2+) entry in mitochondria. Our studies suggest that the pathophysiology of GDAP1-related CMT neuropathies may be associated with abnormal distribution and movement of mitochondria throughout cytoskeleton towards the ER and subplasmalemmal microdomains, resulting in a decrease in SOCE activity and impaired SOCE-driven Ca(2+) uptake in mitochondria.