Severe mitochondrial encephalomyopathy caused by variants in gene.

Background: Mitochondria adjust their shape in response to the different energetic and metabolic requirements of the cell, through extremely dynamic fusion and fission events. Several highly conserved dynamin-like GTPases are involved in these processes and, among those, the OPA1 protein is a key player in the fusion of inner mitochondrial membranes. Hundreds of monoallelic or biallelic pathogenic gene variants have been described in , all associated with a plethora of clinical phenotypes without a straightforward genotype-phenotype correlation.

Zebrafish polg2 knock-out recapitulates human POLG-disorders; implications for drug treatment.

The human mitochondrial DNA polymerase gamma is a holoenzyme, involved in mitochondrial DNA (mtDNA) replication and maintenance, composed of a catalytic subunit (POLG) and a dimeric accessory subunit (POLG2) conferring processivity. Mutations in POLG or POLG2 cause POLG-related diseases in humans, leading to a subset of Mendelian-inherited mitochondrial disorders characterized by mtDNA depletion (MDD) or accumulation of multiple deletions, presenting multi-organ defects and often leading to premature death at a young age. Considering the paucity of POLG2 models, we have generated a stable zebrafish polg2 mutant line (polg2) by CRISPR/Cas9 technology, carrying a 10-nucleotide deletion with frameshift mutation and premature stop codon.

Functional characterization of archaic-specific variants in mitonuclear genes: insights from comparative analysis in S. cerevisiae.

Neanderthal and Denisovan hybridisation with modern humans has generated a non-random genomic distribution of introgressed regions, the result of drift and selection dynamics. Cross-species genomic incompatibility and more efficient removal of slightly deleterious archaic variants have been proposed as selection-based processes involved in the post-hybridisation purge of archaic introgressed regions. Both scenarios require the presence of functionally different alleles across Homo species onto which selection operated differently according to which populations hosted them, but only a few of these variants have been pinpointed so far.

Recessive pathogenic variants cause an LHON-like optic neuropathy.

Background: Leber's hereditary optic neuropathy (LHON) is a mitochondrial disorder characterised by complex I defect leading to sudden degeneration of retinal ganglion cells. Although typically associated with pathogenic variants in mitochondrial DNA, LHON was recently described in patients carrying biallelic variants in nuclear genes , and . MCAT is part of mitochondrial fatty acid synthesis (mtFAS), as also MECR, the mitochondrial trans-2-enoyl-CoA reductase.

The Saccharomyces cerevisiae mitochondrial DNA polymerase and its contribution to the knowledge about human POLG-related disorders.

Most eukaryotes possess a mitochondrial genome, called mtDNA. In animals and fungi, the replication of mtDNA is entrusted by the DNA polymerase γ, or Pol γ. The yeast Pol γ is composed only of a catalytic subunit encoded by MIP1.

Drug Drop Test: How to Quickly Identify Potential Therapeutic Compounds for Mitochondrial Diseases Using Yeast .

Mitochondrial diseases (MDs) refer to a group of clinically and genetically heterogeneous pathologies characterized by defective mitochondrial function and energy production. Unfortunately, there is no effective treatment for most MDs, and current therapeutic management is limited to relieving symptoms. The yeast has been efficiently used as a model organism to study mitochondria-related disorders thanks to its easy manipulation and well-known mitochondrial biogenesis and metabolism.

Modopathies Caused by Mutations in Genes Encoding for Mitochondrial RNA Modifying Enzymes: Molecular Mechanisms and Yeast Disease Models.

In eukaryotes, mitochondrial RNAs (mt-tRNAs and mt-rRNAs) are subject to specific nucleotide modifications, which are critical for distinct functions linked to the synthesis of mitochondrial proteins encoded by mitochondrial genes, and thus for oxidative phosphorylation. In recent years, mutations in genes encoding for mt-RNAs modifying enzymes have been identified as being causative of primary mitochondrial diseases, which have been called modopathies. These latter pathologies can be caused by mutations in genes involved in the modification either of tRNAs or of rRNAs, resulting in the absence of/decrease in a specific nucleotide modification and thus on the impairment of the efficiency or the accuracy of the mitochondrial protein synthesis.

as a Tool for Studying Mutations in Nuclear Genes Involved in Diseases Caused by Mitochondrial DNA Instability.

Mitochondrial DNA (mtDNA) maintenance is critical for oxidative phosphorylation (OXPHOS) since some subunits of the respiratory chain complexes are mitochondrially encoded. Pathological mutations in nuclear genes involved in the mtDNA metabolism may result in a quantitative decrease in mtDNA levels, referred to as mtDNA depletion, or in qualitative defects in mtDNA, especially in multiple deletions. Since, in the last decade, most of the novel mutations have been identified through whole-exome sequencing, it is crucial to confirm the pathogenicity by functional analysis in the appropriate model systems.

A Yeast-Based Repurposing Approach for the Treatment of Mitochondrial DNA Depletion Syndromes Led to the Identification of Molecules Able to Modulate the dNTP Pool.

Mitochondrial DNA depletion syndromes (MDS) are clinically heterogenous and often severe diseases, characterized by a reduction of the number of copies of mitochondrial DNA (mtDNA) in affected tissues. In the context of MDS, yeast has proved to be both an excellent model for the study of the mechanisms underlying mitochondrial pathologies and for the discovery of new therapies via high-throughput assays. Among the several genes involved in MDS, it has been shown that recessive mutations in MPV17 cause a hepatocerebral form of MDS and Navajo neurohepatopathy.

Bi-allelic KARS1 pathogenic variants affecting functions of cytosolic and mitochondrial isoforms are associated with a progressive and multisystem disease.

KARS1 encodes a lysyl-transfer RNA synthetase (LysRS) that links lysine to its cognate transfer RNA. Two different KARS1 isoforms exert functional effects in cytosol and mitochondria. Bi-allelic pathogenic variants in KARS1 have been associated to sensorineural hearing and visual loss, neuropathy, seizures, and leukodystrophy.

Mitochondrial Aminoacyl-tRNA Synthetase and Disease: The Yeast Contribution for Functional Analysis of Novel Variants.

In most eukaryotes, mitochondrial protein synthesis is essential for oxidative phosphorylation (OXPHOS) as some subunits of the respiratory chain complexes are encoded by the mitochondrial DNA (mtDNA). Mutations affecting the mitochondrial translation apparatus have been identified as a major cause of mitochondrial diseases. These mutations include either heteroplasmic mtDNA mutations in genes encoding for the mitochondrial rRNA (mtrRNA) and tRNAs (mttRNAs) or mutations in nuclear genes encoding ribosomal proteins, initiation, elongation and termination factors, tRNA-modifying enzymes, and aminoacyl-tRNA synthetases (mtARSs).

In-frame deletion in canine PITRM1 is associated with a severe early-onset epilepsy, mitochondrial dysfunction and neurodegeneration.

We investigated the clinical, genetic, and pathological characteristics of a previously unknown severe juvenile brain disorder in several litters of Parson Russel Terriers. The disease started with epileptic seizures at 6-12 weeks of age and progressed rapidly to status epilepticus and death or euthanasia. Histopathological changes at autopsy were restricted to the brain.

The Power of Yeast in Modelling Human Nuclear Mutations Associated with Mitochondrial Diseases.

The increasing application of next generation sequencing approaches to the analysis of human exome and whole genome data has enabled the identification of novel variants and new genes involved in mitochondrial diseases. The ability of surviving in the absence of oxidative phosphorylation (OXPHOS) and mitochondrial genome makes the yeast an excellent model system for investigating the role of these new variants in mitochondrial-related conditions and dissecting the molecular mechanisms associated with these diseases. The aim of this review was to highlight the main advantages offered by this model for the study of mitochondrial diseases, from the validation and characterisation of novel mutations to the dissection of the role played by genes in mitochondrial functionality and the discovery of potential therapeutic molecules.

Efficient clofilium tosylate-mediated rescue of POLG-related disease phenotypes in zebrafish.

The DNA polymerase gamma (Polg) is a nuclear-encoded enzyme involved in DNA replication in animal mitochondria. In humans, mutations in the POLG gene underlie a set of mitochondrial diseases characterized by mitochondrial DNA (mtDNA) depletion or deletion and multiorgan defects, named POLG disorders, for which an effective therapy is still needed. By applying antisense strategies, ENU- and CRISPR/Cas9-based mutagenesis, we have generated embryonic, larval-lethal and adult-viable zebrafish Polg models.

Drug repositioning as a therapeutic strategy for neurodegenerations associated with OPA1 mutations.

OPA1 mutations are the major cause of dominant optic atrophy (DOA) and the syndromic form DOA plus, pathologies for which there is no established cure. We used a 'drug repurposing' approach to identify FDA-approved molecules able to rescue the mitochondrial dysfunctions induced by OPA1 mutations. We screened two different chemical libraries by using two yeast strains carrying the mgm1I322M and the chim3P646L mutations, identifying 26 drugs able to rescue their oxidative growth phenotype.

Mechanistic insights on the mode of action of an antiproliferative thiosemicarbazone-nickel complex revealed by an integrated chemogenomic profiling study.

Thiosemicarbazones (TSC) and their metal complexes display diverse biological activities and are active against multiple pathological conditions ranging from microbial infections to abnormal cell proliferation. Ribonucleotide reductase (RNR) is considered one of the main targets of TSCs, yet, the existence of additional targets, differently responsible for the multifaceted activities of TSCs and their metal complexes has been proposed. To set the basis for a more comprehensive delineation of their mode of action, we chemogenomically profiled the cellular effects of bis(citronellalthiosemicarbazonato)nickel(II) [Ni(S-tcitr)] using the unicellular eukaryote Saccharomyces cerevisiae as a model organism.

Mutation in the MICOS subunit gene (MIC26) associated with an X-linked recessive mitochondrial myopathy, lactic acidosis, cognitive impairment and autistic features.

Background: Mitochondria provide ATP through the process of oxidative phosphorylation, physically located in the inner mitochondrial membrane (IMM). The mitochondrial contact site and organising system (MICOS) complex is known as the 'mitoskeleton' due to its role in maintaining IMM architecture. encodes MIC26, a component of MICOS, whose exact function in its maintenance or assembly has still not been completely elucidated.

Modeling of pathogenic variants of mitochondrial DNA polymerase: insight into the replication defects and implication for human disease.

Background: Mutations in human gene encoding the mitochondrial DNA polymerase γ (HsPolγ) are associated with a broad range of mitochondrial diseases. Here we studied the impact on DNA replication by disease variants clustered around residue HsPolγ-K1191, a residue that in several family-A DNA polymerases interacts with the 3' end of the primer.

Methods: Specifically, we examined the effect of HsPolγ carrying pathogenic variants in residues D1184, I1185, C1188, K1191, D1196, and a stop codon at residue T1199, using as a model the yeast mitochondrial DNA polymerase protein, Mip1p.

A combination of two novel VARS2 variants causes a mitochondrial disorder associated with failure to thrive and pulmonary hypertension.

The VARS2 gene encodes a mitochondrial valyl-transfer RNA synthetase which is used in mitochondrial translation. To date, several patients with VARS2 pathogenic variants have been described in the literature. These patients have features of lactic acidosis with encephalomyopathy.

Amino and carboxy-terminal extensions of yeast mitochondrial DNA polymerase assemble both the polymerization and exonuclease active sites.

Human and yeast mitochondrial DNA polymerases (DNAPs), POLG and Mip1, are related by evolution to bacteriophage DNAPs. However, mitochondrial DNAPs contain unique amino and carboxyl-terminal extensions that physically interact. Here we describe that N-terminal deletions in Mip1 polymerases abolish polymerization and decrease exonucleolytic degradation, whereas moderate C-terminal deletions reduce polymerization.

Clinical-genetic features and peculiar muscle histopathology in infantile DNM1L-related mitochondrial epileptic encephalopathy.

Mitochondria are highly dynamic organelles, undergoing continuous fission and fusion. The DNM1L (dynamin-1 like) gene encodes for the DRP1 protein, an evolutionary conserved member of the dynamin family, responsible for fission of mitochondria, and having a role in the division of peroxisomes, as well. DRP1 impairment is implicated in several neurological disorders and associated with either de novo dominant or compound heterozygous mutations.

Deciphering OPA1 mutations pathogenicity by combined analysis of human, mouse and yeast cell models.

OPA1 is the major gene responsible for Dominant Optic Atrophy (DOA) and the syndromic form DOA "plus". Over 370 OPA1 mutations have been identified so far, although their pathogenicity is not always clear. We have analyzed one novel and a set of known OPA1 mutations to investigate their impact on protein functions in primary skin fibroblasts and in two "ad hoc" generated cell systems: the MGM1/OPA1 chimera yeast model and the Opa1-/- MEFs model expressing the mutated human OPA1 isoform 1.

Pathological alleles of MPV17 modeled in the yeast Saccharomyces cerevisiae orthologous gene SYM1 reveal their inability to take part in a high molecular weight complex.

Mitochondrial DNA depletion syndromes (MDDS) are a genetically and clinically heterogeneous group of human diseases caused by mutations in nuclear genes and characterized by a severe reduction in mitochondrial DNA (mtDNA) copy number leading to impaired energy production in affected tissues and organs. Mutations in the MPV17 gene, whose role is still elusive, were described as cause of the hepatocerebral form of MDDS and Navajo neuro-hepathopathy. The high degree of conservation observed between MPV17 and its yeast homolog SYM1 made the latter a good model for the study of the pathology.

Mitochondrial peptidase loss-of-function in childhood cerebellar atrophy.

Objective: To identify the genetic basis of a childhood-onset syndrome of variable severity characterised by progressive spinocerebellar ataxia, mental retardation, psychotic episodes and cerebellar atrophy.

Methods: Identification of the underlying mutations by whole exome and whole genome sequencing. Consequences were examined in patients' cells and in yeast.

Dominance of yeast aac2 and aac2 mutations, equivalent to pathological mutations in ant1, is due to gain of function.

The mitochondrial ADP/ATP carrier is a nuclear encoded protein, which catalyzes the exchange of ATP generated in mitochondria with ADP produced in the cytosol. In humans, mutations in the major ADP/ATP carrier gene, ANT1, are involved in several degenerative mitochondrial pathologies, leading to instability of mitochondrial DNA. Recessive mutations have been associated with mitochondrial myopathy and cardiomyopathy whereas dominant mutations have been associated with autosomal dominant Progressive External Ophtalmoplegia (adPEO).