Studies on DNA interaction of organotin(IV) complexes of meso-tetra(4-sulfonatophenyl)porphine that show cellular activity

The interaction of the diorgano- and triorganotin(IV) derivatives of meso-tetra-(4-sulfonatophenyl)porphine (Me2Sn)2TPPS, (Bu2Sn)2TPPS, (Me3Sn)4TPPS and (Bu3Sn)4TPPS to natural DNA was analysed (together with free meso-tetra-(4-sulfonatophenyl)porphine (TPPS4-) for comparison purposes). Particular attention was paid to (Bu3Sn)4TPPS, a species that shows significant cellular action. Preliminary tests were done on the solution properties of the organotin(IV) compounds (pKA and possible self-aggregation). Spectrophotometric and spectrofluorometric experiments showed that all the investigated organotin(IV) derivatives strongly interact with DNA, the binding energy depending on the dye steric hindrance. In all cases experimental data concur in indicating that external binding mode prevails. Interestingly, fluorescence quenching and viscosity experiments show that the Bu-containing species, and in particular (Bu3Sn)4TPPS, are able to noticeably alter the DNA conformation

Case Series and DARS2 Variant Analysis in Early Severe Forms With Unexpected Presentations

Objective: Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is regarded a relatively mild leukodystrophy, diagnosed by characteristic long tract abnormalities on MRI and biallelic variants in DARS2, encoding mitochondrial aspartyl-tRNA synthetase (mtAspRS). DARS2 variants in LBSL are almost invariably compound heterozygous; in 95% of cases, 1 is a leaky splice site variant in intron 2. A few severely affected patients, still fulfilling the MRI criteria, have been described. We noticed highly unusual MRI presentations in 15 cases diagnosed by WES. We examined these cases to determine whether they represent consistent novel LBSL phenotypes. Methods: We reviewed clinical features, MRI abnormalities, and gene variants and investigated the variants' impact on mtAspRS structure and mitochondrial function. Results: We found 2 MRI phenotypes: early severe cerebral hypoplasia/atrophy (9 patients, group 1) and white matter abnormalities without long tract involvement (6 patients, group 2). With antenatal onset, microcephaly, and arrested development, group 1 patients were most severely affected. DARS2 variants were severer than for classic LBSL and severer for group 1 than group 2. All missense variants hit mtAspRS regions involved in tRNAAsp binding, aspartyl-adenosine-5'-monophosphate binding, and/or homodimerization. Missense variants expressed in the yeast DARS2 ortholog showed severely affected mitochondrial function. Conclusions: DARS2 variants are associated with highly heterogeneous phenotypes. New MRI presentations are profound cerebral hypoplasia/atrophy and white matter abnormalities without long tract involvement. Our findings have implications for diagnosis and understanding disease mechanisms, pointing at dominant neuronal/axonal involvement in severe cases. In line with this conclusion, activation of biallelic DARS2 null alleles in conditional transgenic mice leads to massive neuronal apoptosis

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

Evidence for a Conserved Function of Eukaryotic Pantothenate Kinases in the Regulation of Mitochondrial Homeostasis and Oxidative Stress

Human PANK1, PANK2, and PANK3 genes encode several pantothenate kinase isoforms that catalyze the phosphorylation of vitamin B5 (pantothenic acid) to phosphopantothenate, a critical step in the biosynthesis of the major cellular cofactor, Coenzyme A (CoA). Mutations in the PANK2 gene, which encodes the mitochondrial pantothenate kinase (PanK) isoform, have been linked to pantothenate-kinase associated neurodegeneration (PKAN), a debilitating and often fatal progressive neurodegeneration of children and young adults. While the biochemical properties of these enzymes have been well-characterized in vitro, their expression in a model organism such as yeast in order to probe their function under cellular conditions have never been achieved. Here we used three yeast mutants carrying missense mutations in the yeast PanK gene, CAB1, which are associated with defective growth at high temperature and iron, mitochondrial dysfunction, increased iron content, and oxidative stress, to assess the cellular function of human PANK genes and functional conservation of the CoA-controlled processes between humans and yeast. Overexpression of human PANK1 and PANK3 in these mutants restored normal cellular activity whereas complementation with PANK2 was partial and could only be achieved with an isoform, PanK2mtmΔ, lacking the mitochondrial transit peptide. These data, which demonstrate functional conservation of PanK activity between humans and yeast, set the stage for the use of yeast as a model system to investigate the impact of PKAN-associated mutations on the metabolic pathways altered in this disease

LBSL case series and DARS2 variant analysis in early severe forms with unexpected presentations

Abstract Objective: Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is regarded a relatively mild leukodystrophy, diagnosed by characteristic long tract abnormalities on MRI and biallelic variants in DARS2, encoding mitochondrial aspartyl-tRNA synthetase (mtAspRS). DARS2 variants in LBSL are almost invariably compound heterozygous; in 95% of cases, 1 is a leaky splice site variant in intron 2. A few severely affected patients, still fulfilling the MRI criteria, have been described. We noticed highly unusual MRI presentations in 15 cases diagnosed by WES. We examined these cases to determine whether they represent consistent novel LBSL phenotypes. Methods: We reviewed clinical features, MRI abnormalities, and gene variants and investigated the variants’ impact on mtAspRS structure and mitochondrial function. Results: We found 2 MRI phenotypes: early severe cerebral hypoplasia/atrophy (9 patients, group 1) and white matter abnormalities without long tract involvement (6 patients, group 2). With antenatal onset, microcephaly, and arrested development, group 1 patients were most severely affected. DARS2 variants were severer than for classic LBSL and severer for group 1 than group 2. All missense variants hit mtAspRS regions involved in tRNAAsp binding, aspartyl-adenosine-59-monophosphate binding, and/or homodimerization. Missense variants expressed in the yeast DARS2 ortholog showed severely affected mitochondrial function. Conclusions: DARS2 variants are associated with highly heterogeneous phenotypes. New MRI presentations are profound cerebral hypoplasia/atrophy and white matter abnormalities without long tract involvement. Our findings have implications for diagnosis and understanding disease mechanisms, pointing at dominant neuronal/axonal involvement in severe cases. In line with this conclusion, activation of biallelic DARS2 null alleles in conditional transgenic mice leads to massive neuronal apoptosis

Expanded phenotype of AARS1-related white matter disease

Purpose: Recent reports of individuals with cytoplasmic transfer RNA (tRNA) synthetase-related disorders have identified cases with phenotypic variability from the index presentations. We sought to assess phenotypic variability in individuals with AARS1-related disease. Methods: A cross-sectional survey was performed on individuals with biallelic variants in AARS1. Clinical data, neuroimaging, and genetic testing results were reviewed. Alanyl tRNA synthetase (AlaRS) activity was measured in available fibroblasts. Results: We identified 11 affected individuals. Two phenotypic presentations emerged, one with early infantile–onset disease resembling the index cases of AARS1-related epileptic encephalopathy with deficient myelination (n = 7). The second (n = 4) was a later-onset disorder, where disease onset occurred after the first year of life and was characterized on neuroimaging by a progressive posterior predominant leukoencephalopathy evolving to include the frontal white matter. AlaRS activity was significantly reduced in five affected individuals with both early infantile–onset and late-onset phenotypes. Conclusion: We suggest that variants in AARS1 result in a broader clinical spectrum than previously appreciated. The predominant form results in early infantile–onset disease with epileptic encephalopathy and deficient myelination. However, a subgroup of affected individuals manifests with late-onset disease and similarly rapid progressive clinical decline. Longitudinal imaging and clinical follow-up will be valuable in understanding factors affecting disease progression and outcome