Intracellular vesicle trafficking plays an essential role in mitochondrial quality control

The Drosophila gene products Bet1, Slh, and CG10144, predicted to function in intracellular vesicle trafficking, were previously found to be essential for mitochondrial nucleoid maintenance. Here we show that Slh and Bet1 cooperate to maintain mitochondrial functions. In their absence, mitochondrial content, membrane potential, and respiration became abnormal, accompanied by mitochondrial proteotoxic stress, but without direct effects on mtDNA. Immunocytochemistry showed that both Slh and Bet1 are localized at the Golgi, together with a proportion of Rab5-positive vesicles. Some Bet1, as well as a tiny amount of Slh, cofractionated with highly purified mitochondria, while live-cell imaging showed coincidence of fluorescently tagged Bet1 with most Lysotracker-positive and a small proportion of Mitotracker-positive structures. This three-way association was disrupted in cells knocked down for Slh, although colocalized lysosomal and mitochondrial signals were still seen. Neither Slh nor Bet1 was required for global mitophagy or endocytosis, but prolonged Slh knockdown resulted in G2 growth arrest, with increased cell diameter. These effects were shared with knockdown of betaCOP but not of CG1044, Snap24, or Syntaxin6. Our findings implicate vesicle sorting at the cis-Golgi in mitochondrial quality control.Peer reviewe

RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

Mitochondrial DNA (mtDNA) replication uses a simple core machinery similar to those of bacterial viruses and plasmids, but its components are challenging to unravel. Here, we found that, as in mammals, the single Drosophila gene for RNase H1 (rnh1) has alternative translational start sites, resulting in two polypeptides, targeted to either mitochondria or the nucleus. RNAi-mediated rnh1 knockdown did not influence growth or viability of S2 cells, but compromised mtDNA integrity and copy number. rnh1 knockdown in intact flies also produced a phenotype of impaired mitochondrial function, characterized by respiratory chain deficiency, locomotor dysfunction, and decreased lifespan. Its overexpression in S2 cells resulted in cell lethality after 5-9 days, attributable to the nuclearly localized isoform. rnh1 knockdown and overexpression produced opposite effects on mtDNA replication intermediates. The most pronounced effects were seen in genome regions beyond the major replication pauses where the replication fork needs to progress through a gene cluster that is transcribed in the opposite direction. RNase H1 deficiency led to an accumulation of replication intermediates in these zones, abundant mtDNA molecules joined by four-way junctions, and species consistent with fork regression from the origin. These findings indicate replication stalling due to the presence of unprocessed RNA/DNA heteroduplexes, potentially leading to the degradation of collapsed forks or to replication restart by a mechanism involving strand invasion. Both mitochondrial RNA and DNA syntheses were affected by rnh1 knockdown, suggesting that RNase H1 also plays a role in integrating or coregulating these processes in Drosophila mitochondria.Peer reviewe

Specific MRI abnormalities reveal severe perrault syndrome due to CLPP defects

In establishing a genetic diagnosis in heterogeneous neurological disease, clinical characterization and whole exome sequencing (WES) go hand-in-hand. Clinical data are essential, not only to guide WES variant selection and define the clinical severity of a genetic defect but also to identify other patients with defects in the same gene. In an infant patient with sensorineural hearing loss, psychomotor retardation, and epilepsy, WES resulted in identification of a novel homozygous CLPP frameshift mutation (c.21delA). Based on the gene defect and clinical symptoms, the diagnosis Perrault syndrome type 3 (PRLTS3) was established. The patient's brain-MRI revealed specific abnormalities of the subcortical and deep cerebral white matter and the middle blade of the corpus callosum, which was used to identify similar patients in the Amsterdam brain-MRI database, containing over 3000 unclassified leukoencephalopathy cases. In three unrelated patients with similar MRI abnormalities the CLPP gene was sequenced, and in two of them novel missense mutations were identified together with a large deletion that covered part of the CLPP gene on the other allele. The severe neurological and MRI abnormalities in these young patients were due to the drastic impact of the CLPP mutations, correlating with the variation in clinical manifestations among previously reported patients. Our data show that similarity in brain-MRI patterns can be used to identify novel PRLTS3 patients, especially during early disease stages, when only part of the disease manifestations are present. This seems especially applicable to the severely affected cases in which CLPP function is drastically affected and MRI abnormalities are pronounced

Ciona intestinalis NADH dehydrogenase NDX confers stress-resistance and extended lifespan on Drosophila

Peer reviewe

A zebrafish model to study small-fiber neuropathy reveals a potential role for GDAP1

Mutations in genes involved in mitochondrial dynamics (fusion and fission) have been implicated in many peripheral neuropathies. We hypothesized that defects in these genes could result in a phenotype resembling features of small-fiber neuropathy (SFN). This was investigated in zebrafish by knocking down two genes involved in mitochondrial dynamics gdapl (possibly fission and motility) and opal (fusion) using established morpholinos. Our read-outs were nerve density in the caudal fin and a behavioral response to temperature changes, both based on comparable hallmarks of SFN in patients. Knockdown of gdapl resulted in zebrafish embryos with a reduced density of sensory neurites compared to control morpholino-injected embryos. Furthermore, these embryos demonstrated a decreased temperature-related activity. In contrast, a knockdown of opal did not affect the density of sensory neurites nor the temperature-related activity. However, only the opal morphants had an effect on mitochondrial network morphology. As we were not able to visualize the mitochondria in the neurons, it could well be that changes in the mitochondrial network remained undetected. Our data indicate that GDAP1 knockdown affects sensory neurite development, however, it is unclear if a problem in mitochondrial fission and network formation is the pathophysiological mechanism. Although we did not observe an effect of inhibiting mitochondrial fusion during development, we still propose that genes involved in mitochondrial dynamics should be screened for mutations in patients with SFN.</p

Manipulating mtDNA in vivo reprograms metabolism via novel response mechanisms

Author summary Mitochondria, subcellular compartments (organelles) found in virtually all eukaryotes, contain DNA which is believed to be a remnant of an ancestral bacterial genome. They are best known for the synthesis of the universal energy carrier ATP, but also serve as the hub of various metabolic and signalling pathways. We report here that mtDNA integrity is linked to a signaling system that influences metabolic fuel selection between fats and sugars. By disrupting mtDNA in the fruit fly we induced a strong shift towards lipid catabolism. This was caused both by a widespread decrease in post-translational acetylation of proteins, as well as specific inhibition of the machinery that transports glucose into cells across the plasma membrane. This phenomenon is very similar to the pathophysiology of diabetes, where the inability to transport glucose to cells is considered the main hallmark of the disease. Moreover, decreased protein acetylation was associated with lower levels of certain neurotransmitters, causing various effects on feeding and fertility. Our discovery reveals an unexpected role for mtDNA stability in regulating global metabolic balance and suggests that it could be instrumental in pandemic metabolic disorders such as diabetes and obesity.Peer reviewe

<i>Tfam</i>Knockdown Results in Reduction of mtDNA Copy Number, OXPHOS Deficiency and Abnormalities in Zebrafish Embryos

High mitochondrial DNA (mtDNA) copy numbers are essential for oogenesis and embryogenesis and correlate with fertility of oocytes and viability of embryos. To understand the pathology and mechanisms associated with low mtDNA copy numbers, we knocked down mitochondrial transcription factor A (tfam), a regulator of mtDNA replication, during early zebrafish development. Reduction oftfamusing a splice-modifying morpholino (MO) resulted in a 42 +/- 17% decrease in mtDNA copy number in embryos at 4 days post fertilization. Morphant embryos displayed abnormal development of the eye, brain, heart, and muscle, as well as a 50 +/- 22% decrease in ATP production. Transcriptome analysis revealed a decrease in protein-encoding transcripts from the heavy strand of the mtDNA, and down-regulation of genes involved in haem production and the metabolism of metabolites, which appear to trigger increased rRNA and tRNA synthesis in the nucleoli. However, this stress or compensatory response appears to fall short as pathology emerges and expression of genes related to eye development are severely down-regulated. Taken together, this study highlights the importance of sufficient mtDNA copies for early zebrafish development. Zebrafish is an excellent model to manipulate the mtDNA bottleneck and study its effect on embryogenesis rapidly and in large numbers of offspring

Defective complex I assembly due to C20orf7 mutations as a new cause of Leigh syndrome

Background Leigh syndrome is an early onset, progressive, neurodegenerative disorder with developmental and motor skills regression. Characteristic magnetic resonance imaging abnormalities consist of focal bilateral lesions in the basal ganglia and/or the brainstem. The main cause is a deficiency in oxidative phosphorylation due to mutations in an mtDNA or nuclear oxidative phosphorylation gene. Methods and results A consanguineous Moroccan family with Leigh syndrome comprise 11 children, three of which are affected. Marker analysis revealed a homozygous region of 11.5 Mb on chromosome 20, containing 111 genes. Eight possible mitochondrial candidate genes were sequenced. Patients were homozygous for an unclassified variant (p.P193L) in the cardiolipin synthase gene (CRLS1). As this variant was present in 20% of a Moroccan control population and enzyme activity was only reduced to 50%, this could not explain the rare clinical phenotype in our family. Patients were also homozygous for an amino acid substitution (p.L159F) in C20orf7, a new complex I assembly factor. Parents were heterozygous and unaffected sibs heterozygous or homozygous wild type. The mutation affects the predicted S-adenosylmethionine (SAM) dependent methyltransferase domain of C20orf7, possibly involved in methylation of NDUFB3 during the assembly process. Blue native gel electrophoresis showed an altered complex I assembly with only 30-40% of mature complex I present in patients and 70-90% in carriers. Conclusions A new cause of Leigh syndrome can be a defect in early complex I assembly due to C20orf7 mutations