Pathological ATAD3 variants and perturbed cholesterol metabolism in human and fly

183 p.Among the hundreds of nuclear-encoded components of mitochondria that have been linked to human disease, mutations in the ATAD3 gene cluster have emerged as one of the most frequent in recent years. The ATAD3 locus encodes three highly homologous mitochondrial transmembrane proteins that are members of the AAA+ protein family. ATAD3 has been implicated in mitochondrial DNA and cholesterol metabolism, as well as mitochondrial morphology and ultrastructure. In this thesis, several new ATAD3 mutants have been characterized and have been studied in cultured human cells and in a Drosophila model. These investigations have identified elevated free and membrane-embedded cholesterol as core features of pathological ATAD3 variants. Thus, this is a recurring cellular phenotype characteristic of mutant ATAD3, and as such, it represents a valuable new diagnostic tool, in combination with ATAD3 protein separation and immunodetection. Moreover, a model of the molecular mechanism of the disease has been proposed in which the high level of cholesterol results in cholesterol aggregates in membranes provoking an extraordinary expansion of lysosomes to digest the aberrant membranes. Multiple strategies aimed at modulating cholesterol and lipid metabolism increased cholesterol levels and none decreased it, which might exacerbate rather than mitigate the ATAD3 cellular phenotypes. However, sphingosine supplementation distinguished the ATAD3 mutants from control cells, which identifies an important future avenue for developing therapies

2-Deoxy-D-glucose couples mitochondrial DNA replication with mitochondrial fitness and promotes the selection of wild-type over mutant mitochondrial DNA

Pathological variants of human mitochondrial DNA (mtDNA) typically co-exist with wild-type molecules, but the factors driving the selection of each are not understood. Because mitochondrial fitness does not favour the propagation of functional mtDNAs in disease states, we sought to create conditions where it would be advantageous. Glucose and glutamine consumption are increased in mtDNA dysfunction, and so we targeted the use of both in cells carrying the pathogenic m.3243A>G variant with 2-Deoxy-D-glucose (2DG), or the related 5-thioglucose. Here, we show that both compounds selected wild-type over mutant mtDNA, restoring mtDNA expression and respiration. Mechanistically, 2DG selectively inhibits the replication of mutant mtDNA; and glutamine is the key target metabolite, as its withdrawal, too, suppresses mtDNA synthesis in mutant cells. Additionally, by restricting glucose utilization, 2DG supports functional mtDNAs, as glucose-fuelled respiration is critical for mtDNA replication in control cells, when glucose and glutamine are scarce. Hence, we demonstrate that mitochondrial fitness dictates metabolite preference for mtDNA replication; consequently, interventions that restrict metabolite availability can suppress pathological mtDNAs, by coupling mitochondrial fitness and replication.publishedVersio

Sequencing of snoRNAs from multiple sclerosis association regions and characterization of SNPs./Esklerosi anizkoitzaren asoziazio guneetako snoRNAen sekuentziazioa eta SNPen karakterizazioa.

[EN] Multiple sclerosis (MS) is a common inflammatory and neurodegenerative disease that causes neurological disability. Transcriptome regulation has been seen affected in MS patients’ blood cells. Recently, non-coding RNAs (ncRNAs) have emerged as prominent transcriptome regulators, and in turn, they could play an important role in MS pathogenesis. From this point of view, the multiple sclerosis group in IIS BioDonostia has already analyzed the expression of protein-coding RNAs and some ncRNAs, especially miRNAs, in search of putative MS-related genes. These studies highlighted some possible candidates, amongst others, the snoRNA SNORA40. Thus, the aim of this work is to analyze snoRNAs located in MS-associated regions of the genome and further investigate the possible MS relationship of SNORA40. For that purpose, after detecting the candidate snoRNAs and optimizing the conditions for amplifying them, we did the sequencing of 10 snoRNAs in 14 siblings from 7 Gipuzkoan families and the sequencing of SNORA40 in 47 samples previously included in a RNA expression study. That way, we could identify both already described and novel genetic variants in some of the snoRNAs, but could not relate those variants to the disease as to our samples, ruling out a possible Mendelian inheritance way of those genetic variants

2-Deoxy-D-glucose couples mitochondrial DNA replication with mitochondrial fitness and promotes the selection of wild-type over mutant mitochondrial DNA

Pathological variants of human mitochondrial DNA (mtDNA) typically co-exist with wild-type molecules, but the factors driving the selection of each are not understood. Because mitochondrial fitness does not favour the propagation of functional mtDNAs in disease states, we sought to create conditions where it would be advantageous. Glucose and glutamine consumption are increased in mtDNA dysfunction, and so we targeted the use of both in cells carrying the pathogenic m.3243A>G variant with 2-Deoxy-D-glucose (2DG), or the related 5-thioglucose. Here, we show that both compounds selected wild-type over mutant mtDNA, restoring mtDNA expression and respiration. Mechanistically, 2DG selectively inhibits the replication of mutant mtDNA; and glutamine is the key target metabolite, as its withdrawal, too, suppresses mtDNA synthesis in mutant cells. Additionally, by restricting glucose utilization, 2DG supports functional mtDNAs, as glucose-fuelled respiration is critical for mtDNA replication in control cells, when glucose and glutamine are scarce. Hence, we demonstrate that mitochondrial fitness dictates metabolite preference for mtDNA replication; consequently, interventions that restrict metabolite availability can suppress pathological mtDNAs, by coupling mitochondrial fitness and replication