Cristina Cerqua

Trichoplein/mitostatin regulates endoplasmic reticulum-mitochondria juxtaposition

Trichoplein/mitostatin (TpMs) is a keratin‐binding protein that partly colocalizes with mitochondria and is often downregulated in epithelial cancers, but its function remains unclear. In this study, we report that TpMs regulates the tethering between mitochondria and endoplasmic reticulum (ER) in a Mitofusin 2 (Mfn2)‐dependent manner. Subcellular fractionation and immunostaining show that TpMs is present at the interface between mitochondria and ER. The expression of TpMs leads to mitochondrial fragmentation and loosens tethering with ER, whereas its silencing has opposite effects. Functionally, the reduced tethering by TpMs inhibits apoptosis by Ca2+‐dependent stimuli that require ER–mitochondria juxtaposition. Biochemical and genetic evidence support a model in which TpMs requires Mfn2 to modulate mitochondrial shape and tethering. Thus, TpMs is a new regulator of mitochondria–ER juxtaposition.

Genetics of Coenzyme Q10 Deficiency

Coenzyme Q10 (CoQ10) is an essential component of eukaryotic cells and is involved in crucial biochemical reactions such as the production of ATP in the mitochondrial respiratory chain, the biosynthesis of pyrimidines, and the modulation of apoptosis. CoQ10 requires at least 13 genes for its biosynthesis. Mutations in these genes cause primary CoQ10 deficiency, a clinically and genetically heterogeneous disorder. To date mutations in 8 genes (PDSS1, PDSS2, COQ2, COQ4, COQ6, ADCK3, ADCK4, and COQ9) have been associated with CoQ10 deficiency presenting with a wide variety of clinical manifestations. Onset can be at virtually any age, although pediatric forms are more common. Symptoms include those typical of respiratory chain disorders (encephalomyopathy, ataxia, lactic acidosis, deafness, retinitis pigmentosa, hypertrophic cardiomyopathy), but some (such as steroid-resistant nephrotic syndrome) are peculiar to this condition. The molecular bases of the clinical diversity of this condition are still unknown. It is of critical importance that physicians promptly recognize these disorders because most patients respond to oral administration of CoQ10.

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

Summary 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.

Mutations of cytochrome c identified in patients with thrombocytopenia THC4 affect both apoptosis and cellular bioenergetics

Inherited thrombocytopenias are heterogeneous diseases caused by at least 20 genes playing different role in the processes of megakaryopoiesis and platelet production. Some forms, such as thrombocytopenia 4 (THC4), are very rare and not well characterized. THC4 is an autosomal dominant mild thrombocytopenia described in only one large family from New Zealand and due to a mutation (G41S) of the somatic isoform of the cytochrome c (CYCS) gene. We report a novel CYCS mutation (Y48H) in patients from an Italian family. Similar to individuals carrying G41S, they have platelets of normal size and morphology, which are only partially reduced in number, but no prolonged bleeding episodes. In order to determine the pathogenetic consequences of Y48H, we studied the effects of the two CYCS mutations in yeast and mouse cellular models. In both cases, we found reduction of respiratory level and increased apoptotic rate, supporting the pathogenetic role of CYCS in thrombocytopenia.

Copper and bezafibrate cooperate to rescue cytochrome c oxidase deficiency in cells of patients with sco2 mutations

BackgroundMutations in SCO2 cause cytochrome c oxidase deficiency (COX) and a fatal infantile cardioencephalomyopathy. SCO2 encodes a protein involved in COX copper metabolism; supplementation with copper salts rescues the defect in patients’ cells. Bezafibrate (BZF), an approved hypolipidemic agent, ameliorates the COX deficiency in mice with mutations in COX10, another COX-assembly gene.MethodsWe have investigated the effect of BZF and copper in cells with SCO2 mutations using spectrophotometric methods to analyse respiratory chain activities and a luciferase assay to measure ATP production..ResultsIndividual mitochondrial enzymes displayed different responses to BZF. COX activity increased by about 40% above basal levels (both in controls and patients), with SCO2 cells reaching 75-80% COX activity compared to untreated controls. The increase in COX was paralleled by an increase in ATP production. The effect was dose-dependent: it was negligible with 100 μM BZF, and peaked at 400 μM BZF. Higher BZF concentrations were associated with a relative decline of COX activity, indicating that the therapeutic range of this drug is very narrow. Combined treatment with 100 μM CuCl2 and 200 μM BZF (which are only marginally effective when administered individually) achieved complete rescue of COX activity in SCO2 cells.ConclusionsThese data are crucial to design therapeutic trials for this otherwise fatal disorder. The additive effect of copper and BZF will allow to employ lower doses of each drug and to reduce their potential toxic effects. The exact mechanism of action of BZF remains to be determined.

MCM5: a new actor in the link between DNA replication and Meier-Gorlin syndrome

Meier-Gorlin syndrome (MGORS) is a rare disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recessive mutations in ORC1, ORC4, ORC6, CDT1, CDC6, and CDC45, encoding members of the pre-replication (pre-RC) and pre-initiation (pre-IC) complexes, and heterozygous mutations in GMNN, a regulator of cell-cycle progression and DNA replication, have already been associated with this condition. We performed whole-exome sequencing (WES) in a patient with a clinical diagnosis of MGORS and identified biallelic variants in MCM5. This gene encodes a subunit of the replicative helicase complex, which represents a component of the pre-RC. Both variants, a missense substitution within a conserved domain critical for the helicase activity, and a single base deletion causing a frameshift and a premature stop codon, were predicted to be detrimental for the MCM5 function. Although variants of MCM5 have never been reported in specific human diseases, defect of this gene in zebrafish causes a phenotype of growth restriction overlapping the one associated with orc1 depletion. Complementation experiments in yeast showed that the plasmid carrying the missense variant was unable to rescue the lethal phenotype caused by mcm5 deletion. Moreover cell-cycle progression was delayed in patient’s cells, as already shown for mutations in the ORC1 gene. Altogether our findings support the role of MCM5 as a novel gene involved in MGORS, further emphasizing that this condition is caused by impaired DNA replication.

Mutations in COQ8B (ADCK4) found in patients with steroid-resistant nephrotic syndrome alter COQ8B function

Mutations in COQ8B cause steroid‐resistant nephrotic syndrome with variable neurological involvement. In yeast, COQ8 encodes a protein required for coenzyme Q (CoQ) biosynthesis, whose precise role is not clear. Humans harbor two paralog genes: COQ8A and COQ8B (previously termed ADCK3 and ADCK4). We have found that COQ8B is a mitochondrial matrix protein peripherally associated with the inner membrane. COQ8B can complement a ΔCOQ8 yeast strain when its mitochondrial targeting sequence (MTS) is replaced by a yeast MTS. This model was employed to validate COQ8B mutations, and to establish genotype–phenotype correlations. All mutations affected respiratory growth, but there was no correlation between mutation type and the severity of the phenotype. In fact, contrary to the case of COQ2, where residual CoQ biosynthesis correlates with clinical severity, patients harboring hypomorphic COQ8B alleles did not display a different phenotype compared with those with null mutations. These data also suggest that the system is redundant, and that other proteins (probably COQ8A) may partially compensate for the absence of COQ8B. Finally, a COQ8B polymorphism, present in 50% of the European population (NM_024876.3:c.521A > G, p.His174Arg), affects stability of the protein and could represent a risk factor for secondary CoQ deficiencies or for other complex traits.

A synonymous splicing mutation in the SF3B4 gene segregates in a family with highly variable Nager syndrome

Nager syndrome is a rare preaxial acrofacial dysostosis that is caused by heterozygous loss-of-function variants in SF3B4. This gene encodes for a protein required for the assembly of spliceosomal complexes, being a master gene for splicing regulation. The main clinical features of Nager syndrome include facial-mandibular and preaxial limb malformations, with normal cognitive functioning. Most Nager patients are sporadic, but few familial cases with a highly variable phenotype have been reported. In this work, we report a novel synonymous variant within exon 3 of the SF3B4 gene in a family with three members affected by Nager syndrome. No pathogenic variants have been detected in other 24 genes associated with syndromes characterized by mandibulo-facial anomalies. The pathogenicity of the mutation was demonstrated through a hybrid minigene assay, which confirmed an aberrant splicing with the creation of a cryptic splice site, and showed that this allele is hypomorphic. Our findings emphasize the importance to perform functional analyses to assess the possible consequences of synonymous variants and confirmed that hybrid minigenes represent an effective tool to evaluate the effects of variants on splicing, particularly when RNA is not available.

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.

In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy

ABSTRACT In yeast, Tom22, the central component of the TOMM (translocase of outer mitochondrial membrane) receptor complex, is responsible for the recognition and translocation of synthesized mitochondrial precursor proteins, and its protein kinase CK2-dependent phosphorylation is mandatory for TOMM complex biogenesis and proper mitochondrial protein import. In mammals, the biological function of protein kinase CSNK2/CK2 remains vastly elusive and it is unknown whether CSNK2-dependent phosphorylation of TOMM protein subunits has a similar role as that in yeast. To address this issue, we used a skeletal muscle-specific Csnk2b/Ck2β-conditional knockout (cKO) mouse model. Phenotypically, these skeletal muscle Csnk2b cKO mice showed reduced muscle strength and abnormal metabolic activity of mainly oxidative muscle fibers, which point towards mitochondrial dysfunction. Enzymatically, active muscle lysates from skeletal muscle Csnk2b cKO mice phosphorylate murine TOMM22, the mammalian ortholog of yeast Tom22, to a lower extent than lysates prepared from controls. Mechanistically, CSNK2-mediated phosphorylation of TOMM22 changes its binding affinity for mitochondrial precursor proteins. However, in contrast to yeast, mitochondrial protein import seems not to be affected in vitro using mitochondria isolated from muscles of skeletal muscle Csnk2b cKO mice. PINK1, a mitochondrial health sensor that undergoes constitutive import under physiological conditions, accumulates within skeletal muscle Csnk2b cKO fibers and labels abnormal mitochondria for removal by mitophagy as demonstrated by the appearance of mitochondria-containing autophagosomes through electron microscopy. Mitophagy can be normalized by either introduction of a phosphomimetic TOMM22 mutant in cultured myotubes, or by in vivo electroporation of phosphomimetic Tomm22 into muscles of mice. Importantly, transfection of the phosphomimetic Tomm22 mutant in muscle cells with ablated Csnk2b restored their oxygen consumption rate comparable to wild-type levels. In sum, our data show that mammalian CSNK2-dependent phosphorylation of TOMM22 is a critical switch for mitophagy and reveal CSNK2-dependent physiological implications on metabolism, muscle integrity and behavior.