[Show abstract][Hide abstract]ABSTRACT: Next Generation Sequencing (NGS) technologies are revolutionizing the diagnostic screening for rare disease entities, including primary mitochondrial disorders, particularly those caused by nuclear gene defects. NGS approaches are able to identify the causative gene defects in small families and even single individuals, unsuitable for investigation by traditional linkage analysis. These technologies are contributing to fill the gap between mitochondrial disease cases defined on the basis of clinical, neuroimaging and biochemical readouts, which still outnumber by approximately 50% the cases for which a molecular-genetic diagnosis is attained. We have been using a combined, two-step strategy, based on targeted genes panel as a first NGS screening, followed by whole exome sequencing (WES) in still unsolved cases, to analyze a large cohort of subjects, that failed to show mutations in mtDNA and in ad hoc sets of specific nuclear genes, sequenced by the Sanger's method. Not only this approach has allowed us to reach molecular diagnosis in a significant fraction (20%) of these difficult cases, but it has also revealed unexpected and conceptually new findings. These include the possibility of marked variable penetrance of recessive mutations, the identification of large-scale DNA rearrangements, which explain spuriously heterozygous cases, and the association of mutations in known genes with unusual, previously unreported clinical phenotypes. Importantly, WES on selected cases has unraveled the presence of pathogenic mutations in genes encoding non-mitochondrial proteins (e.g. the transcription factor E4F1), an observation that further expands the intricate genetics of mitochondrial disease and suggests a new area of investigation in mitochondrial medicine. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
Full-text Article · Mar 2016 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract]ABSTRACT: Mitochondrial dysfunction and altered proteostasis are central features of neurodegenerative diseases. The pitrilysin metallopeptidase 1 (PITRM1) is a mitochondrial matrix enzyme, which digests oligopeptides, including the mitochondrial targeting sequences that are cleaved from proteins imported across the inner mitochondrial membrane and the mitochondrial fraction of amyloid beta (Aβ). We identified two siblings carrying a homozygous PITRM1 missense mutation (c.548G>A, p.Arg183Gln) associated with an autosomal recessive, slowly progressive syndrome characterised by mental retardation, spinocerebellar ataxia, cognitive decline and psychosis. The pathogenicity of the mutation was tested in vitro, in mutant fibroblasts and skeletal muscle, and in a yeast model. A Pitrm1(+/-) heterozygous mouse showed progressive ataxia associated with brain degenerative lesions, including accumulation of Aβ-positive amyloid deposits. Our results show that PITRM1 is responsible for significant Aβ degradation and that impairment of its activity results in Aβ accumulation, thus providing a mechanistic demonstration of the mitochondrial involvement in amyloidotic neurodegeneration.
Full-text Article · Dec 2015 · EMBO Molecular Medicine
[Show abstract][Hide abstract]ABSTRACT: Forkhead box g1 (Foxg1) is a nuclear-cytosolic transcription factor essential for the forebrain development and involved in neurodevelopmental and cancer pathologies. Despite the importance of this protein, little is known about the modalities by which it exerts such a large number of cellular functions. Here we show that a fraction of Foxg1 is localized within the mitochondria in cell lines, primary neuronal or glial cell cultures, and in the mouse cortex. Import of Foxg1 in isolated mitochondria appears to be membrane potential-dependent. Amino acids (aa) 277-302 were identified as critical for mitochondrial localization. Overexpression of full-length Foxg1 enhanced mitochondrial membrane potential (ΔΨm) and promoted mitochondrial fission and mitosis. Conversely, overexpression of the C-term Foxg1 (aa 272-481), which is selectively localized in the mitochondrial matrix, enhanced organelle fusion and promoted the early phase of neuronal differentiation. These findings suggest that the different subcellular localizations of Foxg1 control the machinery that brings about cell differentiation, replication, and bioenergetics, possibly linking mitochondrial functions to embryonic development and pathological conditions.
Article · Oct 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract]ABSTRACT: The helicase Twinkle is indispensable for mtDNA replication in nucleoids. Previously, we showed that Twinkle is tightly membrane-associated even in the absence of mtDNA, which suggests that Twinkle is part of a membrane-attached replication platform. Here we show that this platform is a cholesterol-rich membrane structure. We fractionated mitochondrial membrane preparations on flotation gradients and show that membrane-associated nucleoids accumulate at the top of the gradient. This fraction was shown to be highly enriched in cholesterol, a lipid that is otherwise low abundant in mitochondria. In contrast, more common mitochondrial lipids, and abundant innermembrane associated proteins concentrated in the bottom-half of these gradients. Gene silencing of ATAD3, a protein with proposed functions related to nucleoid and mitochondrial cholesterol homeostasis, modified the distribution of cholesterol and nucleoids in the gradient in an identical fashion. Both cholesterol and ATAD3 were previously shown to be enriched in ER-mitochondrial junctions, and we detect nucleoid components in biochemical isolates of these structures. Our data suggest an uncommon membrane composition that accommodates platforms for replicating mtDNA, and reconcile apparently disparate functions of ATAD3. We suggest that mtDNA replication platforms are organized in connection with ER-mitochondrial junctions, facilitated by a specialized membrane architecture involving mitochondrial cholesterol.
[Show abstract][Hide abstract]ABSTRACT: Mitochondria are the major source of ATP that is synthesized by the respi- ratory chain through the process of oxidative phosphorylation (OXPHOS), a complex biochemical process carried out through the dual control of physically separated, but functionally interrelated, genomes, nuclear and mi- tochondrial DNAs. The genetic and biochemical intricacy of mitochondrial bioenergetics explains the extreme heterogeneity of mitochondrial disorders, a group of highly invalidating human conditions, for which no effective treatment is nowadays available. In addition to bioenergetic failure, other mechanisms are probably predominant in the pathogenesis of specific syn- dromes, such as alterations of cellular redox status, the production of reactive oxygen species, compromised Ca2+ homeostasis, mitochondrial protein and organelle quality control, and mitochondrial pathways of apoptosis. By inves- tigating selected families and patients, we have identified several new disease genes, each responsible of distinct defects of the respiratory chain, mtDNA metabolism, or both. Recently published and still unpublished findings will be presented and discussed. Structural analysis and the creation of ad hoc recombinant lines in yeast, flies, and mice have allowed us to dissect out the molecular consequences of the ablation or defects of some of these proteins, and their physical status in normal and disease conditions. These models have also been exploited to implement experimental therapeutic strategies, based on gene and cell replacement, or pharmacological control of mitochondrial biogenesis.
[Show abstract][Hide abstract]ABSTRACT: MGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete pro-cessing of 5 ′ ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nucle-ase S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an add-itional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA. INTRODUCTION
Full-text Article · Jul 2014 · Human Molecular Genetics
[Show abstract][Hide abstract]ABSTRACT: MPV17 is a mitochondrial protein of unknown function, and mutations in MPV17 are associated with mitochondrial deoxyribonucleic acid (DNA) maintenance disorders. Here we investigated its most similar
relative, MPV17L2, which is also annotated as a mitochondrial protein. Mitochondrial fractionation analyses demonstrate MPV17L2
is an integral inner membrane protein, like MPV17. However, unlike MPV17, MPV17L2 is dependent on mitochondrial DNA, as it
is absent from ρ0 cells, and co-sediments on sucrose gradients with the large subunit of the mitochondrial ribosome and the monosome. Gene
silencing of MPV17L2 results in marked decreases in the monosome and both subunits of the mitochondrial ribosome, leading to impaired protein
synthesis in the mitochondria. Depletion of MPV17L2 also induces mitochondrial DNA aggregation. The DNA and ribosome phenotypes
are linked, as in the absence of MPV17L2 proteins of the small subunit of the mitochondrial ribosome are trapped in the enlarged
nucleoids, in contrast to a component of the large subunit. These findings suggest MPV17L2 contributes to the biogenesis of
the mitochondrial ribosome, uniting the two subunits to create the translationally competent monosome, and provide evidence
that assembly of the small subunit of the mitochondrial ribosome occurs at the nucleoid.
Full-text Article · Jun 2014 · Nucleic Acids Research
[Show abstract][Hide abstract]ABSTRACT: Amino acids are essential for cell growth and proliferation for they can serve as precursors of protein synthesis, be remodelled for nucleotide and fat biosynthesis, or be burnt as fuel. Mitochondria are energy producing organelles that additionally play a central role in amino acid homeostasis. One might expect mitochondrial metabolism to be geared towards the production and preservation of amino acids when cells are deprived of an exogenous supply. On the contrary, we find that human cells respond to amino acid starvation by upregulating the amino acid-consuming processes of respiration, protein synthesis, and amino acid catabolism in the mitochondria. The increased utilization of these nutrients in the organelle is not driven primarily by energy demand, as it occurs when glucose is plentiful. Instead it is proposed that the changes in the mitochondrial metabolism complement the repression of cytosolic protein synthesis to restrict cell growth and proliferation when amino acids are limiting. Therefore, stimulating mitochondrial function might offer a means of inhibiting nutrient-demanding anabolism that drives cellular proliferation.