Christopher A. Powell

Mutations in the mitochondrial genome (mtDNA) often lead to clinical pathologies. Mitochondrially-targeted zinc finger nucleases (mtZFNs) have been successful in reducing the levels of mutation-bearing mtDNA both in vivo and in vitro, resulting in a shift in the genetic makeup of affected mitochondria and subsequently to phenotypic rescue. Given the uneven distribution in the mtDNA mutation load across tissues in patients, and a great diversity in pathogenic mutations, it is of interest to develop mutation-specific, selective gene therapies that could be delivered to particular tissues. This study demonstrates the effectiveness of in vivo mitochondrial gene therapy using a novel mtZFN architecture on skeletal muscle using adeno-associated viral (AAV) platforms in a murine model harboring a pathogenic mtDNA mutation. We observed effective reduction in mutation load of cardiac and skeletal muscle, which was accompanied by molecular phenotypic rescue. The gene therapy treatment was shown to be safe when markers of immunity and inflammation were assessed. These results highlight the potential of curative approaches for mitochondrial diseases, paving the way for targeted and effective treatments.

Pathogenic variants in either the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial function. Within this group, an increasing number of families have been identified, where Mendelian genetic disorders implicate defective mitochondrial RNA biology. The PDE12 gene encodes the poly(A)-specific exoribonuclease, involved in the quality control of mitochondrial non-coding RNAs. Here, we report that disease-causing PDE12 variants in three unrelated families are associated with mitochondrial respiratory chain deficiencies and wide-ranging clinical presentations in utero and within the neonatal period, with muscle and brain involvement leading to marked cytochrome c oxidase (COX) deficiency in muscle and severe lactic acidosis. Whole exome sequencing of affected probands revealed novel, segregating bi-allelic missense PDE12 variants affecting conserved residues. Patient-derived primary fibroblasts demonstrate diminished steady-state levels of PDE12 protein, whilst mitochondrial poly(A)-tail RNA sequencing (MPAT-Seq) revealed an accumulation of spuriously polyadenylated mitochondrial RNA, consistent with perturbed function of PDE12 protein. Our data suggest that PDE12 regulates mitochondrial RNA processing and its loss results in neurological and muscular phenotypes.

The Tricarboxylic Acid (TCA) Cycle is arguably the most critical metabolic cycle in physiology and exists as an essential interface coordinating cellular metabolism, bioenergetics, and redox homeostasis. Despite decades of research, a comprehensive investigation into the consequences of TCA cycle dysfunction remains elusive. Here, we targeted two TCA cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), and combined metabolomics, transcriptomics, and proteomics analyses to fully appraise the consequences of TCA cycle inhibition (TCAi) in murine kidney epithelial cells. Our comparative approach shows that TCAi elicits a convergent rewiring of redox and amino acid metabolism dependent on the activation of ATF4 and the integrated stress response (ISR). Furthermore, we also uncover a divergent metabolic response, whereby acute FHi, but not SDHi, can maintain asparagine levels via reductive carboxylation and maintenance of cytosolic aspartate synthesis. Our work highlights an important interplay between the TCA cycle, redox biology, and amino acid homeostasis.

Summary: The Tricarboxylic Acid Cycle (TCA) cycle is arguably the most critical metabolic cycle in physiology and exists as an essential interface coordinating cellular metabolism, bioenergetics, and redox homeostasis. Despite decades of research, a comprehensive investigation into the consequences of TCA cycle dysfunction remains elusive. Here, we targeted two TCA cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), and combined metabolomics, transcriptomics, and proteomics analyses to fully appraise the consequences of TCA cycle inhibition (TCAi) in kidney epithelial cells. Our comparative approach shows that TCAi elicits a convergent rewiring of redox and amino acid metabolism dependent on the activation of ATF4 and the integrated stress response (ISR). Furthermore, we also uncover a divergent metabolic response, whereby acute FHi, but not SDHi, can maintain asparagine levels via reductive carboxylation and maintenance of cytosolic aspartate synthesis. Our work highlights an important interplay between the TCA cycle, redox biology and amino acid homeostasis. Highlights: TCA cycle inhibition promotes GSH synthesis and impairs de novo aspartate and proline synthesis Disruption of mitochondrial thiol redox homeostasis phenocopies TCA cycle inhibition by promoting GSH synthesis and impairing proline and aspartate synthesis Acute FHi, but not SDHi, can maintain asparagine levels via reductive carboxylation and maintenance of cytosolic aspartate synthesis TCA cycle inhibition mimics an amino acid deprivation-type response and activates ATF4 via the integrated stress response to maintain redox and amino acid homeostasis

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6‐months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt‐tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease.

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6 months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation, however only ∼1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt-tRNA ^Glu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. The spontaneous recovery in infants with digenic mutations is modulated by changes in amino acid availability in a multi-step process. First, the integrated stress-response associated with increased FGF21 and GDF15 expression enhances catabolism via β-oxidation and the TCA cycle increasing the availability of amino acids. In the second phase mitochondrial biogenesis increases via mTOR activation, leading to improved mitochondrial translation and recovery. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease.

RNA species play host to a plethora of post-transcriptional modifications which together make up the epitranscriptome. 5-methyluridine (m⁵U) is one of the most common modifications made to cellular RNA, where it is found almost ubiquitously in bacterial and eukaryotic cytosolic tRNAs at position 54. Here, we demonstrate that m⁵U54 in human mitochondrial tRNAs is catalysed by the nuclear-encoded enzyme TRMT2B, and that its repertoire of substrates is expanded to ribosomal RNAs, catalysing m⁵U429 in 12S rRNA. We show that TRMT2B is not essential for viability in human cells and that knocking-out the gene shows no obvious phenotype with regards to RNA stability, mitochondrial translation, or cellular growth.

RNA species play host to a plethora of post-transcriptional modifications which together make up the epitranscriptome. 5-methyluridine (m ⁵ U) is one of the most common modifications made to cellular RNA, where it is found almost ubiquitously in bacterial and eukaryotic cytosolic tRNAs at position 54. Here, we demonstrate that m ⁵ U54 in human mitochondrial tRNAs is catalysed by the nuclear-encoded enzyme TRMT2B, and that its repertoire of substrates is expanded to ribosomal RNAs, catalysing m ⁵ U429 in 12S rRNA. We show that TRMT2B is not essential for viability in human cells and that knocking-out the gene shows no obvious phenotype with regards to RNA stability, mitochondrial translation, or cellular growth.

Expression of human mitochondrial DNA is indispensable for proper function of the oxidative phosphorylation machinery. The mitochondrial genome encodes 22 tRNAs, 2 rRNAs and 11 mRNAs and their post-transcriptional modification constitutes one of the key regulatory steps during mitochondrial gene expression. Cytosine-5 methylation (m5C) has been detected in mitochondrial transcriptome, however its biogenesis has not been investigated in details. Mammalian NOP2/Sun RNA Methyltransferase Family Member 2 (NSUN2) has been characterized as an RNA methyltransferase introducing m5C in nuclear-encoded tRNAs, mRNAs and microRNAs and associated with cell proliferation and differentiation, with pathogenic variants in NSUN2 being linked to neurodevelopmental disorders. Here we employ spatially restricted proximity labelling and immunodetection to demonstrate that NSUN2 is imported into the matrix of mammalian mitochondria. Using three genetic models for NSUN2 inactivation—knockout mice, patient-derived fibroblasts and CRISPR/Cas9 knockout in human cells—we show that NSUN2 is necessary for the generation of m5C at positions 48, 49 and 50 of several mammalian mitochondrial tRNAs. Finally, we show that inactivation of NSUN2 does not have a profound effect on mitochondrial tRNA stability and oxidative phosphorylation in differentiated cells. We discuss the importance of the newly discovered function of NSUN2 in the context of human disease.