The Australian Genomics Mitochondrial Flagship: A national program delivering mitochondrial diagnoses.

Purpose: Families living with mitochondrial diseases (MD) often endure prolonged diagnostic journeys and invasive testing, yet many remain without a molecular diagnosis. The Australian Genomics Mitochondrial Flagship, comprising clinicians, diagnostic, and research scientists, conducted a prospective national study to identify the diagnostic utility of singleton genomic sequencing using blood samples.

Methods: A total of 140 children and adults living with suspected MD were recruited using modified Nijmegen criteria (MNC) and randomized to either exome + mitochondrial DNA (mtDNA) sequencing or genome sequencing.

Reduced Protein Import via TIM23 SORT Drives Disease Pathology in TIMM50-Associated Mitochondrial Disease.

TIMM50 is a core subunit of the TIM23 complex, the mitochondrial inner membrane translocase responsible for the import of pre-sequence-containing precursors into the mitochondrial matrix and inner membrane. Here we describe a mitochondrial disease patient who is homozygous for a novel variant in and establish the first proteomic map of mitochondrial disease associated with TIMM50 dysfunction. We demonstrate that TIMM50 pathogenic variants reduce the levels and activity of endogenous TIM23 complex, which significantly impacts the mitochondrial proteome, resulting in a combined oxidative phosphorylation (OXPHOS) defect and changes to mitochondrial ultrastructure.

CLPB disaggregase dysfunction impacts the functional integrity of the proteolytic SPY complex.

CLPB is a mitochondrial intermembrane space AAA+ domain-containing disaggregase. CLPB mutations are associated with 3-methylglutaconic aciduria and neutropenia; however, the molecular mechanism underscoring disease and the contribution of CLPB substrates to disease pathology remains unknown. Interactions between CLPB and mitochondrial quality control (QC) factors, including PARL and OPA1, have been reported, hinting at dysregulation of organelle QC in disease.

Mitochondrial biology and dysfunction in secondary mitochondrial disease.

Mitochondrial diseases are a broad, genetically heterogeneous class of metabolic disorders characterized by deficits in oxidative phosphorylation (OXPHOS). Primary mitochondrial disease (PMD) defines pathologies resulting from mutation of mitochondrial DNA (mtDNA) or nuclear genes affecting either mtDNA expression or the biogenesis and function of the respiratory chain. Secondary mitochondrial disease (SMD) arises due to mutation of nuclear-encoded genes independent of, or indirectly influencing OXPHOS assembly and operation.

Sideroflexin 4 is a complex I assembly factor that interacts with the MCIA complex and is required for the assembly of the ND2 module.

SignificanceMitochondria are double-membraned eukaryotic organelles that house the proteins required for generation of ATP, the energy currency of cells. ATP generation within mitochondria is performed by five multisubunit complexes (complexes I to V), the assembly of which is an intricate process. Mutations in subunits of these complexes, or the suite of proteins that help them assemble, lead to a severe multisystem condition called mitochondrial disease.

High-intensity training induces non-stoichiometric changes in the mitochondrial proteome of human skeletal muscle without reorganisation of respiratory chain content.

Mitochondrial defects are implicated in multiple diseases and aging. Exercise training is an accessible, inexpensive therapeutic intervention that can improve mitochondrial bioenergetics and quality of life. By combining multiple omics techniques with biochemical and in silico normalisation, we removed the bias arising from the training-induced increase in mitochondrial content to unearth an intricate and previously undemonstrated network of differentially prioritised mitochondrial adaptations.

Modelling Mitochondrial Disease in Human Pluripotent Stem Cells: What Have We Learned?

Mitochondrial diseases disrupt cellular energy production and are among the most complex group of inherited genetic disorders. Affecting approximately 1 in 5000 live births, they are both clinically and genetically heterogeneous, and can be highly tissue specific, but most often affect cell types with high energy demands in the brain, heart, and kidneys. There are currently no clinically validated treatment options available, despite several agents showing therapeutic promise.

The TIM22 complex mediates the import of sideroflexins and is required for efficient mitochondrial one-carbon metabolism.

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that contributes to protein biogenesis as a subunit of the TIM22 complex at the inner mitochondrial membrane. Mutations in cause Sengers syndrome, an autosomal recessive condition characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, and lactic acidosis. We mapped the proteomic changes in Sengers patient fibroblasts and AGK cell lines to understand the effects of AGK dysfunction on mitochondria.

Rothmund-Thomson Syndrome-Like RECQL4 Truncating Mutations Cause a Haploinsufficient Low-Bone-Mass Phenotype in Mice.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder characterized by defects in the skeletal system, such as bone hypoplasia, short stature, low bone mass, and an increased incidence of osteosarcoma. RTS type 2 patients have germ line compound biallelic protein-truncating mutations of . As existing murine models employ null alleles, we have attempted to more accurately model RTS by generating mice with patient-mimicking truncating mutations.

Assessment of mitochondrial respiratory chain enzymes in cells and tissues.

Measurement of the individual enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) forms a key part of diagnostic investigations in patients with suspected mitochondrial disease, and can provide crucial information on mitochondrial OXPHOS function in a variety of cells and tissues that are applicable to many research investigations. In this chapter, we present methods for analysis of mitochondrial respiratory chain enzymes in cells and tissues based on assays performed in two geographically separate diagnostic referral centers, as part of clinical diagnostic investigations. Techniques for sample preparation from cells and tissues, and spectrophotometric assays for measurement of the activities of OXPHOS complexes I-V, the combined activity of complexes II+III, and the mitochondrial matrix enzyme citrate synthase, are provided.

Function of hTim8a in complex IV assembly in neuronal cells provides insight into pathomechanism underlying Mohr-Tranebjærg syndrome.

Human Tim8a and Tim8b are members of an intermembrane space chaperone network, known as the small TIM family. Mutations in cause a neurodegenerative disease, Mohr-Tranebjærg syndrome (MTS), which is characterised by sensorineural hearing loss, dystonia and blindness. Nothing is known about the function of hTim8a in neuronal cells or how mutation of this protein leads to a neurodegenerative disease.

Mitochondrial energy generation disorders: genes, mechanisms, and clues to pathology.

Inherited disorders of oxidative phosphorylation cause the clinically and genetically heterogeneous diseases known as mitochondrial energy generation disorders, or mitochondrial diseases. Over the last three decades, mutations causing these disorders have been identified in almost 290 genes, but many patients still remain without a molecular diagnosis. Moreover, while our knowledge of the genetic causes is continually expanding, our understanding into how these defects lead to cellular dysfunction and organ pathology is still incomplete.

Loss of BIM increases mitochondrial oxygen consumption and lipid oxidation, reduces adiposity and improves insulin sensitivity in mice.

BCL-2 proteins are known to engage each other to determine the fate of a cell after a death stimulus. However, their evolutionary conservation and the many other reported binding partners suggest an additional function not directly linked to apoptosis regulation. To identify such a function, we studied mice lacking the BH3-only protein BIM.

Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a Subunit of the Human TIM22 Protein Import Complex.

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex.

ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism.

Although mitochondrial disorders are clinically heterogeneous, they frequently involve the central nervous system and are among the most common neurogenetic disorders. Identifying the causal genes has benefited enormously from advances in high-throughput sequencing technologies; however, once the defect is known, researchers face the challenge of deciphering the underlying disease mechanism. Here we characterize large biallelic deletions in the region encoding the ATAD3C, ATAD3B and ATAD3A genes.

Accessory subunits are integral for assembly and function of human mitochondrial complex I.

Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit membrane protein complexes. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson's disease and ageing.

Mitochondrial OXA Translocase Plays a Major Role in Biogenesis of Inner-Membrane Proteins.

The mitochondrial inner membrane harbors three protein translocases. Presequence translocase and carrier translocase are essential for importing nuclear-encoded proteins. The oxidase assembly (OXA) translocase is required for exporting mitochondrial-encoded proteins; however, different views exist about its relevance for nuclear-encoded proteins.

Deletion of the Complex I Subunit NDUFS4 Adversely Modulates Cellular Differentiation.

The vast majority of cellular ATP is produced by the oxidative phosphorylation (OXPHOS) system, which comprises the four complexes of the electron transfer chain plus the ATP synthase. Complex I is the largest of the OXPHOS complexes, and mutation of the genes encoding either the subunits or assembly factors of Complex I can result in Complex I deficiency, which is the most common OXPHOS disorder. Mutations in the Complex I gene NDUFS4 lead to Leigh syndrome, which is the most frequent presentation of Complex I deficiency in children presenting with progressive encephalopathy shortly after birth.

COA6 is a mitochondrial complex IV assembly factor critical for biogenesis of mtDNA-encoded COX2.

Biogenesis of complex IV of the mitochondrial respiratory chain requires assembly factors for subunit maturation, co-factor attachment and stabilization of intermediate assemblies. A pathogenic mutation in COA6, leading to substitution of a conserved tryptophan for a cysteine residue, results in a loss of complex IV activity and cardiomyopathy. Here, we demonstrate that the complex IV defect correlates with a severe loss in complex IV assembly in patient heart but not fibroblasts.

Characterization of mitochondrial FOXRED1 in the assembly of respiratory chain complex I.

Human mitochondrial complex I is the largest enzyme of the respiratory chain and is composed of 44 different subunits. Complex I subunits are encoded by both nuclear and mitochondrial (mt) DNA and their assembly requires a number of additional proteins. FAD-dependent oxidoreductase domain-containing protein 1 (FOXRED1) was recently identified as a putative assembly factor and FOXRED1 mutations in patients cause complex I deficiency; however, its role in assembly is unknown.

Neuronal and astrocyte dysfunction diverges from embryonic fibroblasts in the Ndufs4fky/fky mouse.

Mitochondrial dysfunction causes a range of early-onset neurological diseases and contributes to neurodegenerative conditions. The mechanisms of neurological damage however are poorly understood, as accessing relevant tissue from patients is difficult, and appropriate models are limited. Hence, we assessed mitochondrial function in neurologically relevant primary cell lines from a CI (complex I) deficient Ndufs4 KO (knockout) mouse (Ndufs4fky/fky) modelling aspects of the mitochondrial disease LS (Leigh syndrome), as well as MEFs (mouse embryonic fibroblasts).

Functional characterization of Friedreich ataxia iPS-derived neuronal progenitors and their integration in the adult brain.

Friedreich ataxia (FRDA) is an autosomal recessive disease characterised by neurodegeneration and cardiomyopathy that is caused by an insufficiency of the mitochondrial protein, frataxin. Our previous studies described the generation of FRDA induced pluripotent stem cell lines (FA3 and FA4 iPS) that retained genetic characteristics of this disease. Here we extend these studies, showing that neural derivatives of FA iPS cells are able to differentiate into functional neurons, which don't show altered susceptibility to cell death, and have normal mitochondrial function.