Deep sequencing unearths nuclear mitochondrial sequences under Leber's hereditary optic neuropathy-associated false heteroplasmic mitochondrial DNA variants.

Leber's hereditary optic neuropathy (LHON) is associated with mitochondrial DNA (mtDNA) ND mutations that are mostly homoplasmic. However, these mutations are not sufficient to explain the peculiar features of penetrance and the tissue-specific expression of the disease and are believed to be causative in association with unknown environmental or other genetic factors. Discerning between clear-cut pathogenetic variants, such as those that appear to be heteroplasmic, and less penetrant variants, such as the homoplasmic, remains a challenging issue that we have addressed here using next-generation sequencing approach. We set up a protocol to quantify MTND5 heteroplasmy levels in a family in which the proband manifests a LHON phenotype. Furthermore, to study this mtDNA haplotype, we applied the cybridization protocol. The results demonstrate that the mutations are mostly homoplasmic, whereas the suspected heteroplasmic feature of the observed mutations is due to the co-amplification of Nuclear mitochondrial Sequences.

Dysfunction of mitochondrial respiratory chain complex I in neurological disorders: genetics and pathogenetic mechanisms.

This chapter covers genetic and biochemical aspects of mitochondrial bioenergetics dysfunction in neurological disorders associated with complex I defects. Complex I formation and functionality in mammalian cells depends on coordinated expression of nuclear and mitochondrial genes, post-translational subunit modifications, mitochondrial import/maturation of nuclear encoded subunits, subunits interaction and stepwise assembly, and on proteolytic processing. Examples of complex I dysfunction are herein presented: homozygous mutations in the nuclear NDUFS1 and NDUFS4 genes for structural components of complex I; an autosomic recessive form of encephalopathy associated with enhanced proteolytic degradation of complex I; familial cases of Parkinson associated to mutations in the PINK1 and Parkin genes, in particular, homoplasmic mutations in the ND5 and ND6 mitochondrial genes of the complex I, coexistent with mutation in the PINK1 gene. This knowledge, besides clarifying molecular aspects of the pathogenesis of hereditary diseases, can also provide hints for understanding the involvement of complex I in neurological disorders, as well as for developing therapeutical strategies.

cAMP-dependent protein kinase regulates post-translational processing and expression of complex I subunits in mammalian cells.

Work is presented on the role of cAMP-dependent protein phosphorylation in post-translational processing and biosynthesis of complex I subunits in mammalian cell cultures. PKA-mediated phosphorylation of the NDUFS4 subunit of complex I promotes in cell cultures in vivo import/maturation in mitochondria of the precursor of this protein. The import promotion appears to be associated with the observed cAMP-dependent stimulation of the catalytic activity of complex I. These effects of PKA are counteracted by activation of protein phosphatase(s). PKA and the transcription factor CREB play a critical role in the biosynthesis of complex I subunits. CREB phosphorylation, by PKA and/or CaMKs, activates at nuclear and mitochondrial level a transcriptional regulatory cascade which promotes the concerted expression of nuclear and mitochondrial encoded subunits of complex I and other respiratory chain proteins.

Pathogenetic mechanisms in hereditary dysfunctions of complex I of the respiratory chain in neurological diseases.

This paper covers genetic and biochemical aspects of mitochondrial bioenergetics dysfunction in hereditary neurological disorders associated with complex I defects. Three types of hereditary complex I dysfunction are dealt with: (i) homozygous mutations in the nuclear genes NDUFS1 and NDUFS4 of complex I, associated with mitochondrial encephalopathy; (ii) a recessive hereditary epileptic neurological disorder associated with enhanced proteolytic degradation of complex I; (iii) homoplasmic mutations in the ND5 and ND6 mitochondrial genes of the complex, coexistent with mutation in the nuclear PINK1 gene in familial Parkinsonism. The genetic and biochemical data examined highlight different mechanisms by which mitochondrial bioenergetics is altered in these hereditary defects of complex I. This knowledge, besides clarifying molecular aspects of the pathogenesis of hereditary diseases, can also provide hints for understanding the involvement of complex I in sporadic neurological disorders and aging, as well as for developing therapeutical strategies.

Mitochondrial proteome analysis reveals depression of the Ndufs3 subunit and activity of complex I in diabetic rat brain.

Type-1 diabetes resulting from defective insulin secretion and consequent hyperglycemia, is associated with "diabetic encephalopathy." This is characterized by brain neurophysiological and structural changes resulting in impairment of cognitive function. The present proteomic analysis of brain mitochondrial proteins from streptozotocin-induced type-1 diabetic rats, shows a large decrement of the Ndufs3 protein subunit of complex I, decreased level of the mRNA and impaired catalytic activity of the complex in the diabetic rats as compared to controls. The severe depression of the expression and enzymatic activity of complex I can represent a critical contributing factor to the onset of the diabetic encephalopathy in type-1 diabetes.

The hUPF1-NMD factor controls the cellular transcript levels of different genes of complex I of the respiratory chain.

In this study the impact of hUPF1 and hUPF2 knockdown on alternative splicing (AS) isoforms of different genes encoding subunits of respiratory chain complex I and complex IV is described. As expected, loss of both hUPF1 and hUPF2 led to impairment of nonsense-mediated mRNA decay (NMD) and accumulation of PTC-containing NMD substrates generated by both complex I and complex IV genes. The levels of some complex I splice variants, which did not contain PTC as well as the level of some complex I canonical transcripts were, however, affected only by hUPF1 knockdown. This finding confirms that NMD plays a role in the maintenance of the transcriptome integrity and reveals a specific impact of hUPF1 on the regulation of complex I genes.

Induction of mitochondrial dysfunction and oxidative stress in human fibroblast cultures exposed to serum from septic patients.

AIMS: Sepsis which is the leading cause of death in intensive care units is usually related to the number and the severity of organ failure, but the mechanisms remain to be fully established. Findings of microvascular flow abnormalities, decreased oxygen consumption and elevated tissue oxygen tensions suggest that problems may lay in cellular oxygen utilization rather than in oxygen delivery per se. Several serum factors, released during sepsis syndrome, might be involved in induction of cytopathic hypoxia and increase of cellular oxidative stress. MAIN METHODS: Human fibroblast cultures were incubated 12h with 10% v/v severe septic patients' sera and measurements were carried out on cellular oxygen consumption, mitochondrial respiratory enzymes activity, H(2)O(2) generation and serum levels of cytokines/chemokines by multiplex assay. KEY FINDINGS: In fibroblast cultures a significant depression of cellular respiration and activity of mitochondrial complexes and increased H(2)O(2) production was observed after incubation with septic sera showing increased levels of TNFα, IL-1ß and IL-6. SIGNIFICANCE: During sepsis syndrome some increased cytokines might target specific mitochondrial enzymes inducing an impairment of cellular energy metabolism leading to multiple organ failure.

Dysfunctions of cellular oxidative metabolism in patients with mutations in the NDUFS1 and NDUFS4 genes of complex I.

The pathogenic mechanism of a G44A nonsense mutation in the NDUFS4 gene and a C1564A mutation in the NDUFS1 gene of respiratory chain complex I was investigated in fibroblasts from human patients. As previously observed the NDUFS4 mutation prevented complete assembly of the complex and caused full suppression of the activity. The mutation (Q522K replacement) in NDUFS1 gene, coding for the 75-kDa Fe-S subunit of the complex, was associated with (a) reduced level of the mature complex, (b) marked, albeit not complete, inhibition of the activity, (c) accumulation of H(2)O(2) and O(2)(.-) in mitochondria, (d) decreased cellular content of glutathione, (e) enhanced expression and activity of glutathione peroxidase, and (f) decrease of the mitochondrial potential and enhanced mitochondrial susceptibility to reactive oxygen species (ROS) damage. No ROS increase was observed in the NDUFS4 mutation. Exposure of the NDUFS1 mutant fibroblasts to dibutyryl-cAMP stimulated the residual NADH-ubiquinone oxidoreductase activity, induced disappearance of ROS, and restored the mitochondrial potential. These are relevant observations for a possible therapeutical strategy in NDUFS1 mutant patients.