Age-Associated Loss of OPA1 in Muscle Impacts Muscle Mass, Metabolic Homeostasis, Systemic Inflammation, and Epithelial Senescence

Mitochondrial dysfunction occurs during aging, but its impact on tissue senescence is unknown. Here, we find that sedentary but not active humans display an age-related decline in the mitochondrial protein, optic atrophy 1 (OPA1), that is associated with muscle loss. In adult mice, acute, muscle-specific deletion of Opa1 induces a precocious senescence phenotype and premature death. Conditional and inducible Opa1 deletion alters mitochondrial morphology and function but not DNA content. Mechanistically, the ablation of Opa1 leads to ER stress, which signals via the unfolded protein response (UPR) and FoxOs, inducing a catabolic program of muscle loss and systemic aging. Pharmacological inhibition of ER stress or muscle-specific deletion of FGF21 compensates for the loss of Opa1, restoring a normal metabolic state and preventing muscle atrophy and premature death. Thus, mitochondrial dysfunction in the muscle can trigger a cascade of signaling initiated at the ER that systemically affects general metabolism and aging

C. elegans as model to study neurometabolic conditions

The are several advantages in using Caenorhabditis elegans as organism model, among others its small size, the fast vital cycle and the easiness to maintain it in culture. Since between 60 and 80% of human genes have an orthologue in the worm genome, it has already been employed to investigate mitochondrial disorders, neurodegenerative diseases and also to search for neurotoxins and neuroprotective drugs. During my PhD program I used C. elegans as a model to study genes whose function is unknown or not fully understood and potentially involved in neurometabolic diseases. At this purpose I performed RNA interference (RNAi) experiments and CRISPR/Cas9 genome editing to generate knockdown and knockout (KO)/knock-in worm models, that were then extensively phenotypically characterized. Cytochrome c oxidase (COX) is the terminal enzymatic complex of the mitochondrial respiratory chain. Mutations in COX genes are responsible for COX deficiency, which is the most frequent cause of mitochondrial encephalomyopathies. COX16 is a COX assembly factor, with a homologue in the worm genome, that is cox-16. By a COX specific histochemical staining we found that cox-16 is required for COX biogenesis and function, since its knockdown in nematodes causes COX deficiency. These results confirm what was previously obtained in a COX16 KO cellular model, with the advantage of having a multicellular model that could be used for drug screening, since no cure is available so far for COX deficiencies. MYTHO is a recently identified FOXO-dependent gene which seems to be involved in autophagy and has an orthologue in the C. elegans genome. Since RNAi did not allow to detect a clearcut phenotype in nematodes, we therefore generated a KO model by CRISPR/Cas9 technology. KO animals did not show a significant reduction in survival after starvation, but manifested a precocious aging phenotype with locomotion impairment and reduced lifespan compared to controls. We are currently performing further experiments to analyze the autophagic flux in absence of MYTHO and characterize the pathway linking this gene to the IGF/Akt/FOXO signaling. A novel genetic variant has been identified in a gene that belongs to Crescerin1 family of proteins regulating microtubule dynamics, in patients with a Meckel-Gruber-like phenotype. The worm orthologue che-12 is expressed in the cilium of a subset of sensory neurons. We generated worm lines harboring the novel missense variant found in patients by CRISPR/Cas9 technology. These were then characterized to explore potential effects on behaviors controlled by sensory neurons expressing che-12. We did not observe an impairment in chemotaxis ability on a NaCl gradient, nor a strong reduction of lipophilic dye-uptake frequency, however preliminary results indicate that the cilium of sensory neurons is shortened in che-12 knock-in mutants. The demonstration of the pathogenic effect of the variant could establish an important link between mutations in this gene (that has not been so far associated with a human disease) and ciliopathies

Isolation and Structure Characterization of Two Novel Bioactive Sulphated Polyhydroxysteroids from the Antarctic OphiuroidOphioderma longicaudum

Two novel sulfated polyhydroxysteroids, I, possessing potent cytotoxic activity, and II, which proved to be cytoprotective against HIV-1, were isolated from the Antarctic ophiuroid Ophiosparte gigas, together with the known compd. III. The structures of I and II were detd. to be cholest-5-ene-2α,3α,4β, 21-tetraol 3,21-disulfate and cholest-5-ene-2β,3α, 21-triol 2,21-disulfate, resp

Cyclosporin A Promotes in vivo Myogenic Response in Collagen VI-Deficient Myopathic Mice.

Mutations of genes encoding for collagen VI cause various muscle diseases in humans, including Bethlem myopathy and Ullrich congenital muscular dystrophy. Collagen VI null (Col6a1 (-/-)) mice are affected by a myopathic phenotype with mitochondrial dysfunction, spontaneous apoptosis of muscle fibers, and defective autophagy. Moreover, Col6a1 (-/-) mice display impaired muscle regeneration and defective self-renewal of satellite cells after injury. Treatment with cyclosporin A (CsA) is effective in normalizing the mitochondrial, apoptotic, and autophagic defects of myofibers in Col6a1 (-/-) mice. A pilot clinical trial with CsA in Ullrich patients suggested that CsA may increase the number of regenerating myofibers. Here, we report the effects of CsA administration at 5\u2009mg/kg body weight every 12\u2009h in Col6a1 (-/-) mice on muscle regeneration under physiological conditions and after cardiotoxin (CdTx)-induced muscle injury. Our findings indicate that CsA influences satellite cell activity and triggers the formation of regenerating fibers in Col6a1 (-/-) mice. Data obtained on injured muscles show that under appropriate administration, regimens CsA is able to stimulate myogenesis in Col6a1 (-/-) mice by significantly increasing the number of myogenin (MyoG)-positive cells and of regenerating myofibers at the early stages of muscle regeneration. CsA is also able to ameliorate muscle regeneration of Col6a1 (-/-) mice subjected to multiple CdTx injuries, with a concurrent maintenance of the satellite cell pool. Our data show that CsA is beneficial for muscle regeneration in Col6a1 (-/-) mic

COX16 is required for assembly of cytochrome c oxidase in human cells and is involved in copper delivery to COX2

Cytochrome c oxidase (COX), complex IV of the mitochondrial respiratory chain, is comprised of 14 structural subunits, several prosthetic groups and metal cofactors, among which copper. Its biosynthesis involves a number of ancillary proteins, encoded by the COX-assembly genes that are required for the stabilization and membrane insertion of the nascent polypeptides, the synthesis of the prosthetic groups, and the delivery of the metal cofactors, in particular of copper. Recently, a modular model for COX assembly has been proposed, based on the sequential incorporation of different assembly modules formed by specific subunits. We have cloned and characterized the human homologue of yeast COX16. We show that human COX16 encodes a small mitochondrial transmembrane protein that faces the intermembrane space and is highly expressed in skeletal and cardiac muscle. Its knockdown in C. elegans produces COX deficiency, and its ablation in HEK293 cells impairs COX assembly. Interestingly, COX16 knockout cells retain significant COX activity, suggesting that the function of COX16 is partially redundant. Analysis of steady-state levels of COX subunits and of assembly intermediates by Blue-Native gels shows a pattern similar to that reported in cells lacking COX18, suggesting that COX16 is required for the formation of the COX2 subassembly module. Moreover, COX16 co-immunoprecipitates with COX2. Finally, we found that copper supplementation increases COX activity and restores normal steady state levels of COX subunits in COX16 knockout cells, indicating that, even in the absence of a canonical copper binding motif, COX16 could be involved in copper delivery to COX2

Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of NF1 Variants

Neurofibromatosis type 1 (NF1) is caused by heterozygous loss of function mutations in the NF1 gene. Although patients are diagnosed according to clinical criteria and few genotype-phenotype correlations are known, molecular analysis remains important. NF1 displays allelic heterogeneity, with a high proportion of variants affecting splicing, including deep intronic alleles and changes outside the canonical splice sites, making validation problematic. Next Generation Sequencing (NGS) technologies integrated with multiplex ligation-dependent probe amplification (MLPA) have largely overcome RNA-based techniques but do not detect splicing defects. A rapid minigene-based system was set up to test the effects of NF1 variants on splicing. We investigated 29 intronic and exonic NF1 variants identified in patients during the diagnostic process. The minigene assay showed the coexistence of multiple mechanisms of splicing alterations for seven variants. A leaky effect on splicing was documented in one de novo substitution detected in a sporadic patient with a specific phenotype without neurofibromas. Our splicing assay proved to be a reliable and fast method to validate novel NF1 variants potentially affecting splicing and to detect hypomorphic effects that might have phenotypic consequences, avoiding the requirement of patient’s RNA

Collagen VI regulates satellite cell self-renewal and muscle regeneration.

Adult muscle stem cells, or satellite cells play essential roles in homeostasis and regeneration of skeletal muscles. Satellite cells are located within a niche that includes myofibers and extracellular matrix. The function of specific extracellular matrix molecules in regulating SCs is poorly understood. Here we show that the extracellular matrix protein collagen VI is a key component of the satellite cell niche. Lack of collagen VI in Col6a1(–/–) mice causes impaired muscle regeneration and reduced satellite cell self-renewal capability after injury. Collagen VI null muscles display significant decrease of stiffness, which is able to compromise the in vitro and in vivo activity of wild-type satellite cells. When collagen VI is reinstated in vivo by grafting wild-type fibroblasts, the biomechanical properties of Col6a1(–/–) muscles are ameliorated and satellite cell defects rescued. Our findings establish a critical role for an extracellular matrix molecule in satellite cell self-renewal and open new venues for therapies of collagen VI-related muscle diseases

The COQ2 genotype predicts the severity of Coenzyme Q10 deficiency

COQ2 (p-hydroxybenzoate polyprenyl transferase) encodes the enzyme required for the second step of the final reaction sequence of Coenzyme Q10 (CoQ) biosynthesis. Its mutations represent a frequent cause of primary CoQ deficiency and have been associated to the widest clinical spectrum, ranging from fatal neonatal multisystemic disease to late-onset encephalopathy. However, the reasons of this variability are still unknown.We have characterized the structure of human COQ2, defined its subcellular localization and developed a yeast model to validate all the mutant alleles reported so far.Our findings show that the main functional transcript of COQ2 is shorter than what was previously reported and that its protein product localizes to mitochondria with the C-terminus facing the intermembrane space. Complementation experiments in yeast showed that the residual activity of the mutant proteins correlates with the clinical phenotypes observed in patients.We defined the structure of COQ2 with relevant implications for mutation screening in patients and demonstrated that, contrary to other COQ gene defects such as ADCK3, there is a correlation between COQ2 genotype and patient's phenotype