Dysfunctions of Cellular Oxidative Metabolism in Patients with Mutations in the NDUFS1 and NDUFS4 Genes of Complex I

Abstract

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.Deficiency of complex I (NADH ubiquinone oxidoreductase, EC 1.6.5.3) of the respiratory chain is a major cause of inborn mitochondrial disease (1-6). Leigh syndrome (early onset fatal neurodegenerative disorder) and Leigh syndrome-like disease are the most common clinical phenotypes associated with complex I deficiency. Impairment of complex I has also been reported in Parkinson (7), Alzheimer (8, 9), and Huntington (10) diseases.Complex I is the largest of the respiratory chain enzymes, being composed of seven mitochondrial DNA and at least 39 nuclear DNA-encoded subunits (11). Mutations in structural subunits have been found in ϳ40% of the patients with inborn deficiency of complex I. Reported mutations include all of the mitochondrial DNA-encoded subunits (12) and twelve nuclear-encoded subunits (3, 6, 13-15).The nuclear NDUFS4 gene codes for an 18-kDa subunit of the complex (11), which in high eukaryotes contains potential phosphorylation sites for cAMP-dependent protein kinase in both the presequence and the carboxyl-terminal region (EMBL Data Bank). In mammalian (16 -18) and human (19) cell cultures cAMP promotes the phosphorylation of the NDUFS4 protein and enhances the functional capacity of complex I. Three recessive mutations in the nuclear NDUFS4 gene have been identified in three unrelated children affected by Leigh syndromelike syndrome with deficiency of complex I, including an AAGTC duplication at position 466 -470 in exon 5 (20), a single base deletion at position 289/290 in exon 3 (21), and a G44A nonsense mutation in the first exon of the gene, introducing a premature termination codon in the sequence coding for the mitochondrial leader peptide (13). All three mutations resulted in the disappearance of the 18-kDa subunit and defect in both the activity and assembly of the complex (22). In the 289/290 deletion in exon 3, whic...

Mitochondrial respiratory chain dysfunction: Implications in neurodegeneration

“…In primary mitochondrial diseases, as a consequence of the RC dysfunction, disturbances in the electron transport and proton pumping across the system occur, leading to decreased mitochondrial membrane potential and OXPHOS-derived ATP (Iuso et al, 2006;Distelmaier et al, 2009;Moran et al, 2010b). Besides the energetic defect, the altered electron transport can induce increased rates of superoxide production; probably due to a high reduction state of the components of the RC upstream the mutated point.…”

“…Another group reported increased superoxide and hydrogen peroxide levels in cells from a patient harboring the c.C1564A mutation in the NDUFS1 complex I subunit gene, which prevented complex I complete assembly and led to a marked complex I enzyme activity decrease (Iuso et al, 2006); however, the relatively more deleterious c.G44A nonsense mutation in the NDUFS4 structural gene displayed normal ROS levels (Iuso et al, 2006). Likewise, our group analyzed the hydrogen peroxide levels in complex I-deficient fibroblasts with mutations in the NDUFA1 and NDUFV1 structural genes, without finding differences in the H 2 O 2 levels between the mutant and control cells (Moran et al, 2010b).…”

cAMP controls oxygen metabolism in mammalian cells

“…2; c 0 refers to NDUFS1 mutant fibroblasts grown in the exponential phase and exposed to 100 lM dibutyryl-cAMP for 60 min. [18]). In the case of the NDUFS1-mutation, the inhibition of the NADH-ubiquinone oxidoreductase can be attributed to altered function of the 75 kDa Fe-S protein encoded by this gene.…”

The mitochondrial impairment, oxidative stress and neurodegeneration connection: reality or just an attractive hypothesis?

“…The notion that a complex I defect leads to a major increase in ROS production derives in part from the fact that rotenone, the most commonly used complex I inhibitor, increases ROS production in isolated mitochondria in the presence of excess NADH-linked substrates [30][31][32]. However, not all mutations in complex I subunits lead to an increase in ROS production [33]. Future detailed studies unraveling the molecular mechanisms whereby complex I is inhibited in sporadic PD patients will help us understand the contribution of complex I defects to oxidative stress and neurodegeneration in PD.…”