Sulforaphane Ameliorates Metabolic Changes Associated With Status Epilepticus in Immature Rats.

Status epilepticus (SE) is a common paediatric emergency with the highest incidence in the neonatal period and is a well-known epileptogenic insult. As previously established in various experimental and human studies, SE induces long-term alterations to brain metabolism, alterations that directly contribute to the development of epilepsy. To influence these changes, organic isothiocyanate compound sulforaphane (SFN) has been used in the present study for its known effect of enhancing antioxidative, cytoprotective, and metabolic cellular properties via the Nrf2 pathway. We have explored the effect of SFN in a model of acquired epilepsy induced by Li-Cl pilocarpine in immature rats (12 days old). Energy metabolites PCr, ATP, glucose, glycogen, and lactate were determined by enzymatic fluorimetric methods during the acute phase of SE. Protein expression was evaluated by Western blot (WB) analysis. Neuronal death was scored on the FluoroJadeB stained brain sections harvested 24 h after SE. To assess the effect of SFN on glucose metabolism we have performed a series of 18F-DG µCT/PET recordings 1 h, 1 day, and 3 weeks after the induction of SE. Responses of cerebral blood flow (CBF) to electrical stimulation and their influence by SFN were evaluated by laser Doppler flowmetry (LDF). We have demonstrated that the Nrf2 pathway is upregulated in the CNS of immature rats after SFN treatment. In the animals that had undergone SE, SFN was responsible for lowering glucose uptake in most regions 1 h after the induction of SE. Moreover, SFN partially reversed hypometabolism observed after 24 h and achieved full reversal at approximately 3 weeks after SE. Since no difference in cell death was observed in SFN treated group, these changes cannot be attributed to differences in neurodegeneration. SFN per se did not affect the glucose uptake at any given time point suggesting that SFN improves endogenous CNS ability to adapt to the epileptogenic insult. Furthermore, we had discovered that SFN improves blood flow and accelerates CBF response to electrical stimulation. Our findings suggest that SFN improves metabolic changes induced by SE which have been identified during epileptogenesis in various animal models of acquired epilepsy.

Genetic Complementation of ATP Synthase Deficiency Due to Dysfunction of TMEM70 Assembly Factor in Rat.

Mutations of the TMEM70 gene disrupt the biogenesis of the ATP synthase and represent the most frequent cause of autosomal recessive encephalo-cardio-myopathy with neonatal onset. Patient tissues show isolated defects in the ATP synthase, leading to the impaired mitochondrial synthesis of ATP and insufficient energy provision. In the current study, we tested the efficiency of gene complementation by using a transgenic rescue approach in spontaneously hypertensive rats with the targeted Tmem70 gene (SHR-Tmem70ko/ko), which leads to embryonic lethality. We generated SHR-Tmem70ko/ko knockout rats expressing the Tmem70 wild-type transgene (SHR-Tmem70ko/ko,tg/tg) under the control of the EF-1α universal promoter. Transgenic rescue resulted in viable animals that showed the variable expression of the Tmem70 transgene across the range of tissues and only minor differences in terms of the growth parameters. The TMEM70 protein was restored to 16-49% of the controls in the liver and heart, which was sufficient for the full biochemical complementation of ATP synthase biogenesis as well as for mitochondrial energetic function in the liver. In the heart, we observed partial biochemical complementation, especially in SHR-Tmem70ko/ko,tg/0 hemizygotes. As a result, this led to a minor impairment in left ventricle function. Overall, the transgenic rescue of Tmem70 in SHR-Tmem70ko/ko knockout rats resulted in the efficient complementation of ATP synthase deficiency and thus in the successful genetic treatment of an otherwise fatal mitochondrial disorder.

TMEM70 facilitates biogenesis of mammalian ATP synthase by promoting subunit c incorporation into the rotor structure of the enzyme.

Biogenesis of F1Fo ATP synthase, the key enzyme of mitochondrial energy provision, depends on transmembrane protein 70 (TMEM70), localized in the inner mitochondrial membrane of higher eukaryotes. TMEM70 absence causes severe ATP-synthase deficiency and leads to a neonatal mitochondrial encephalocardiomyopathy in humans. However, the exact biochemical function of TMEM70 remains unknown. Using TMEM70 conditional knockout in mice, we show that absence of TMEM70 impairs the early stage of enzyme biogenesis by preventing incorporation of hydrophobic subunit c into rotor structure of the enzyme. This results in the formation of an incomplete, pathologic enzyme complex consisting of F1 domain and peripheral stalk but lacking Fo proton channel composed of subunits c and a. We demonstrated direct interaction between TMEM70 and subunit c and showed that overexpression of subunit c in TMEM70-/- cells partially rescued TMEM70 defect. Accordingly, TMEM70 knockdown prevented subunit c accumulation otherwise observed in F1-deficient cells. Altogether, we identified TMEM70 as specific ancillary factor for subunit c. The biologic role of TMEM70 is to increase the low efficacy of spontaneous assembly of subunit c oligomer, the key and rate-limiting step of ATP-synthase biogenesis, and thus to reach an adequately high physiologic level of ATP synthase in mammalian tissues.-Kovalcíková, J., Vrbacký, M., Pecina, P., Tauchmannová, K., Nusková, H., Kaplanová, V., Brázdová, A., Alán, L., Eliás, J., Cunátová, K., Korínek, V., Sedlacek, R., Mrácek, T., Houstek, J. TMEM70 facilitates biogenesis of mammalian ATP synthase by promoting subunit c incorporation into the rotor structure of the enzyme.

Bi-allelic Mutations in NDUFA6 Establish Its Role in Early-Onset Isolated Mitochondrial Complex I Deficiency.

Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects' fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects' fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis.

Selective replacement of mitochondrial DNA increases the cardioprotective effect of chronic continuous hypoxia in spontaneously hypertensive rats.

Mitochondria play an essential role in improved cardiac ischaemic tolerance conferred by adaptation to chronic hypoxia. In the present study, we analysed the effects of continuous normobaric hypoxia (CNH) on mitochondrial functions, including the sensitivity of the mitochondrial permeability transition pore (MPTP) to opening, and infarct size (IS) in hearts of spontaneously hypertensive rats (SHR) and the conplastic SHR-mtBN strain, characterized by the selective replacement of the mitochondrial genome of SHR with that of the more ischaemia-resistant brown Norway (BN) strain. Rats were adapted to CNH (10% O2, 3 weeks) or kept at room air as normoxic controls. In the left ventricular mitochondria, respiration and cytochrome c oxidase (COX) activity were measured using an Oxygraph-2k and the sensitivity of MPTP opening was assessed spectrophotometrically as Ca2+-induced swelling. Myocardial infarction was analysed in anaesthetized open-chest rats subjected to 20 min of coronary artery occlusion and 3 h of reperfusion. The IS reached 68±3.0% and 65±5% of the area at risk in normoxic SHR and SHR-mtBN strains, respectively. CNH significantly decreased myocardial infarction to 46±3% in SHR. In hypoxic SHR-mtBN strain, IS reached 33±2% and was significantly smaller compared with hypoxic SHR. Mitochondria isolated from hypoxic hearts of both strains had increased detergent-stimulated COX activity and were less sensitive to MPTP opening. The maximum swelling rate was significantly lower in hypoxic SHR-mtBN strain compared with hypoxic SHR, and positively correlated with myocardial infarction in all experimental groups. In conclusion, the mitochondrial genome of SHR modulates the IS-limiting effect of adaptation to CNH by affecting mitochondrial energetics and MPTP sensitivity to opening.

Acadian variant of Fanconi syndrome is caused by mitochondrial respiratory chain complex I deficiency due to a non-coding mutation in complex I assembly factor NDUFAF6.

The Acadian variant of Fanconi Syndrome refers to a specific condition characterized by generalized proximal tubular dysfunction from birth, slowly progressive chronic kidney disease and pulmonary interstitial fibrosis. This condition occurs only in Acadians, a founder population in Nova Scotia, Canada. The genetic and molecular basis of this disease is unknown. We carried out whole exome and genome sequencing and found that nine affected individuals were homozygous for the ultra-rare non-coding variant chr8:96046914 T > C; rs575462405, whereas 13 healthy siblings were either heterozygotes or lacked the mutant allele. This variant is located in intron 2 of NDUFAF6 (NM_152416.3; c.298-768 T > C), 37 base pairs upstream from an alternative splicing variant in NDUFAF6 chr8:96046951 A > G; rs74395342 (c.298-731 A > G). NDUFAF6 encodes NADH:ubiquinone oxidoreductase complex assembly factor 6, also known as C8ORF38. We found that rs575462405-either alone or in combination with rs74395342-affects splicing and synthesis of NDUFAF6 isoforms. Affected kidney and lung showed specific loss of the mitochondria-located NDUFAF6 isoform and ultrastructural characteristics of mitochondrial dysfunction. Accordingly, affected tissues had defects in mitochondrial respiration and complex I biogenesis that were corrected with NDUFAF6 cDNA transfection. Our results demonstrate that the Acadian variant of Fanconi Syndrome results from mitochondrial respiratory chain complex I deficiency. This information may be used in the diagnosis and prevention of this disease in individuals and families of Acadian descent and broadens the spectrum of the clinical presentation of mitochondrial diseases, respiratory chain defects and defects of complex I specifically.

Sex difference in the sensitivity of cardiac mitochondrial permeability transition pore to calcium load.

Most of the experimental studies have revealed that female heart is more tolerant to ischemia/reperfusion (I/R) injury as compared with the male myocardium. It is widely accepted that mitochondrial dysfunction, and particularly mitochondrial permeability transition pore (MPTP) opening, plays a major role in determining the extent of cardiac I/R injury. The aim of the present study was, therefore, to analyze (i) whether calcium-induced swelling of cardiac mitochondria is sex-dependent and related to the degree of cardiac tolerance to I/R injury and (ii) whether changes in MPTP components-cyclophilin D (CypD) and ATP synthase-can be involved in this process. We have observed that in mitochondria isolated from rat male and female hearts the MPTP has different sensitivity to the calcium load. Female mitochondria are more resistant both in the extent and in the rate of the mitochondrial swelling at higher calcium concentration (200 µM). At low calcium concentration (50 µM) no differences were observed. Our data further suggest that sex-dependent specificity of the MPTP is not the result of different amounts of ATP synthase and CypD, or their respective ratio in mitochondria isolated from male and female hearts. Our results indicate that male and female rat hearts contain comparable content of MPTP and its regulatory protein CypD; parallel immunodetection revealed also the same contents of adenine nucleotide translocator or voltage-dependent anion channel. Increased resistance of female heart mitochondria thus cannot be explained by changes in putative components of MPTP, and rather reflects regulation of MPTP function.