Exploring Yeast as a Study Model of Pantothenate Kinase-Associated Neurodegeneration and for the Identification of Therapeutic Compounds

Mutations in the pantothenate kinase 2 gene (PANK2) are the cause of pantothenate kinase-associated neurodegeneration (PKAN), the most common form of neurodegeneration with brain iron accumulation. Although different disease models have been created to investigate the pathogenic mechanism of PKAN, the cascade of molecular events resulting from CoA synthesis impairment is not completely understood. Moreover, for PKAN disease, only symptomatic treatments are available. Despite the lack of a neural system, Saccharomyces cerevisiae has been successfully used to decipher molecular mechanisms of many human disorders including neurodegenerative diseases as well as iron-related disorders. To gain insights into the molecular basis of PKAN, a yeast model of this disease was developed: a yeast strain with the unique gene encoding pantothenate kinase CAB1 deleted, and expressing a pathological variant of this enzyme. A detailed functional characterization demonstrated that this model recapitulates the main phenotypes associated with human disease: mitochondrial dysfunction, altered lipid metabolism, iron overload, and oxidative damage suggesting that the yeast model could represent a tool to provide information on pathophysiology of PKAN. Taking advantage of the impaired oxidative growth of this mutant strain, a screening for molecules able to rescue this phenotype was performed. Two molecules in particular were able to restore the multiple defects associated with PKAN deficiency and the rescue was not allele-specific. Furthermore, the construction and characterization of a set of mutant alleles, allowing a quick evaluation of the biochemical consequences of pantothenate kinase (PANK) protein variants could be a tool to predict genotype/phenotype correlation

A novel de novo dominant mutation in ISCU associated with mitochondrial myopathy

BACKGROUND: Hereditary myopathy with lactic acidosis and myopathy with deficiency of succinate dehydrogenase and aconitase are variants of a recessive disorder characterised by childhood-onset early fatigue, dyspnoea and palpitations on trivial exercise. The disease is non-progressive, but life-threatening episodes of widespread weakness, metabolic acidosis and rhabdomyolysis may occur. So far, this disease has been molecularly defined only in Swedish patients, all homozygous for a deep intronic splicing affecting mutation in ISCU encoding a scaffold protein for the assembly of iron-sulfur (Fe-S) clusters. A single Scandinavian family was identified with a different mutation, a missense change in compound heterozygosity with the common intronic mutation. The aim of the study was to identify the genetic defect in our proband. METHODS: A next-generation sequencing (NGS) approach was carried out on an Italian male who presented in childhood with ptosis, severe muscle weakness and exercise intolerance. His disease was slowly progressive, with partial recovery between episodes. Patient's specimens and yeast models were investigated. RESULTS: Histochemical and biochemical analyses on muscle biopsy showed multiple defects affecting mitochondrial respiratory chain complexes. We identified a single heterozygous mutation p.Gly96Val in ISCU, which was absent in DNA from his parents indicating a possible de novo dominant effect in the patient. Patient fibroblasts showed normal levels of ISCU protein and a few variably affected Fe-S cluster-dependent enzymes. Yeast studies confirmed both pathogenicity and dominance of the identified missense mutation. CONCLUSION: We describe the first heterozygous dominant mutation in ISCU which results in a phenotype reminiscent of the recessive disease previously reported.This work was supported by the TelethonItaly [GrantGGP15041]; the Pierfranco and Luisa Mariani Foundation; the MRC7QQR [201572020] grant; the ERC advanced grant [FP77322424]; the NRJ Foundation7Institut de France; the E7Rare project GENOMIT. RL acknowledges generous financial support from Deutsche Forschungsgemeinschaft [SFB 987 and SPP 1927] and the LOEWE program of state Hessen

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

first_pagesettingsOrder Article Reprints This is an early access version, the complete PDF, HTML, and XML versions will be available soon. Open AccessReview Modopathies Caused by Mutations in Genes Encoding for Mitochondrial RNA Modifying Enzymes: Molecular Mechanisms and Yeast Disease Models by Martina Magistrati 1,†ORCID,Alexandru Ionut Gilea 1,†,Camilla Ceccatelli Berti 1,2ORCID,Enrico Baruffini 1,*ORCID andCristina Dallabona 1ORCID 1 Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area delle Scienze 11/A, 43124 Parma, Italy 2 Department of Medicine and Surgery, University of Parma, Via Gramsci 14, 43126 Parma, Italy * Author to whom correspondence should be addressed. † These authors contributed equally to this work. Int. J. Mol. Sci. 2023, 24(3), 2178; https://doi.org/10.3390/ijms24032178 Received: 28 December 2022 / Revised: 17 January 2023 / Accepted: 20 January 2023 / Published: 22 January 2023 (This article belongs to the Special Issue RNA Regulatory Networks 2.0) Download Review Reports Versions Notes Abstract 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. Most of these mutations are sporadic or private, thus it is fundamental that their pathogenicity is confirmed through the use of a model system. This review will focus on the activity of genes that, when mutated, are associated with modopathies, on the molecular mechanisms through which the enzymes introduce the nucleotide modifications, on the pathological phenotypes associated with mutations in these genes and on the contribution of the yeast Saccharomyces cerevisiae to confirming the pathogenicity of novel mutations and, in some cases, for defining the molecular defects

Yeast as a model for mitochondrial aminoacyl-tRNA synthetase disorders: validation of mutations in NARS2 and WARS2

Aminoacyl-transfer ribonucleic acid synthetases (ARSs) catalyze the attachment of each amino acids to their cognate tRNAs. Mitochondrial ARSs (mtARSs), that ensure protein synthesis within mitochondrial compartment, are encoded by nuclear genes and imported in the organelle after translation in the cytosol. To date, thanks to the extensive use of next generation sequencing (NGS), an increasing number of variants in mtARS genes have been identified and associated with mitochondrial disease. The similarities between yeast and human mitochondrial translation machineries makes yeast a good model to evaluate the effect of variants in mtARSs genes in a quick and efficient way. We identified compound heterozygous missense WARS2 variants in a child with spastic paraparesis, tremor and ataxia and in another one with infantile parkinsonism, while compound heterozygous missense NARS2 variants were found in a baby with developmental delay, epilepsy and complex I deficiency. We have recently constructed two new yeast models to assess the functional consequences of novel mutations found in NARS2 and WARS2, encoding mitochondrial asparaginyl-tRNA (AsnRS) and tryptophanyl‐tRNA synthetases (TrpRS), respectively. Mitochondrial phenotypes such as oxidative growth, oxygen consumption rate (OCR) and Cox2 protein level were analyzed in yeast strains deleted in SLM5 and MSW1, the yeast orthologues of NARS2 and WARS2, and expressing the wild type or the mutant alleles both individually and in combination, confirming the pathogenicity of most the identified variants. Moreover, the beneficial effects deriving from supplementation of asparagine in the growth medium was investigated in the NARS2 yeast model. The results obtained suggest asparagine supplementation as a potential therapeutic approach

Modeling in yeast of KARS pathogenic variants associated with a progressive and multi-systemic disease: impact on cytosolic and mitochondrial isoforms

Aminoacyl-transfer ribonucleic acid synthetases (ARSes) are enzymes involved in translation of mRNAs into proteins. Lysyl-transfer RNA synthetase (LysRS) encoded by KARS loads lysine to its cognate tRNA. In contrast to other ARSes, which are encoded by two genes, two LysRS isoforms generated by alternative splicing of KARS pre-mRNA localizes either in cytosol (cytKARS, NM_005548.2) or in mitochondria (mtKARS, NM_001130089.1). To date, different KARS pathogenic variants have been associated to wide spectrum of clinical manifestations including sensorineural hearing loss, visual loss, neuropathy, seizures, leukodystrophy with spinal and brainstem calcification. In the context of an international collaborative project, ten further cases with a severe neurodegenerative and multi-system disease due to KARS bi-allelic variants were collected. The mapping of twelve amino acids affected in KARS patients on the crystal structure of KARS-p38 complex (PDB: 4dpg) showed that the majority of them localize in the catalytic domain or in the anticodon binding domain, probably affecting the aminoacylation reaction, the binding affinity for lysine or compromising the protein structure and its interaction with other proteins. Moreover, since the exact contribution of mitochondrial and/or cytosolic LysRS deficiency to KARS-related phenotype is difficult to untangle, we investigated the impact of each mutation on the activity of both compartments, modelling each mutation in the yeast Saccharomyces cerevisiae. While in human KARS encodes for both cytoplasmic and mitochondrial isoforms of lysyl-tRNA synthetase, in yeast two different genes, KRS1 and MSK1, encode the cytoplasmic and mitochondrial lysyl-tRNA synthetase, respectively. Taking advantage of this, we studied the effects of mutated alleles mtKARS and cytKARS separately through heterologous complementation in strains deleted in MSK1 and KRS1, respectively. The results obtained showed that most mutant strains displayed a growth defect, though at very different extent, and none affected only a single isoform. However, for several mutations, a variable degree of impairment was observed when the same variant was expressed by the cytKARS or the mtKARS, suggesting that defects of both cytKARS or the mtKARS contribute to the phenotype but also that the impairment of either cytKARS or the mtKARS activity was predominant. Moreover the detrimental effects of two variants in cytKARS, were partially improved by lysine supplementation in the medium, suggesting that mutations of these two amino acids, which are in the catalytic domain, may decrease the binding affinity for lysine, and thus supporting the therapeutic potential of lysine supplementation in patients

Modeling human Coenzyme A synthase mutation in yeast reveals altered mitochondrial function, lipid content and iron metabolism

Mutations in nuclear genes associated with defective coenzyme A biosynthesis have been identified as responsible for some forms of neurodegeneration with brain iron accumulation (NBIA), namely PKAN and CoPAN. PKAN are defined by mutations in PANK2, encoding the pantothenate kinase 2 enzyme, that account for about 50% of cases of NBIA, whereas mutations in CoA synthase COASY have been recently reported as the second inborn error of CoA synthesis leading to CoPAN. As reported previously, yeast cells expressing the pathogenic mutation exhibited a temperature-sensitive growth defect in the absence of pantothenate and a reduced CoA content. Additional characterization revealed decreased oxygen consumption, reduced activities of mitochondrial respiratory complexes, higher iron content, increased sensitivity to oxidative stress and reduced amount of lipid droplets, thus partially recapitulating the phenotypes found in patients and establishing yeast as a potential model to clarify the pathogenesis underlying PKAN and CoPAN diseases