Combined enzymatic complex I and III deficiency associated with mutations in the nuclear encoded NDUFS4 gene

Combined OXPHOS-system enzyme deficiencies are observed in approximately 25% of all OXPHOS-system disturbances. Of these, combined complex I and III deficiency is relatively scarce. So far, only mtDNA and thymidine phosphorylase (TP) mutations have been associated with combined OXPHOS-system disturbances. In this report we show, for the first time, that a nuclear gene mutation in a structural, nuclear encoded complex I gene is associated with combined complex I and III deficiency. After our initial report we describe mutations in the NDUFS4 gene of complex I in two additional patients. The first mutation is a deletion of G at position 289 or 290. Amino acid 96 changes from a tryptophan to a stop codon. The mutation was found homozygous in the patient; both parents are heterozygous for the mutation. The second mutation is a transition from C to T at cDNA position 316. Codon is changed from CGA (arginine) to TGA (stop). The patient is homozygous for the mutation; both parents are heterozygous. Both mutations in the NDUFS4 gene led to a premature stop in Leigh-like patients with an early lethal phenotype. We hypothesise that the structural integrity of the OXPHOS system, in mammal supermolecular structures, may be responsible for the observed biochemical features.     

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.     

Mutations in human nuclear genes encoding for subunits of mitochondrial respiratory complex I: the NDUFS4 gene

Among the mitochondrial disorders, complex I deficiencies are encountered frequently. Although some complex I deficiencies have been associated with mitochondrial DNA mutations, in the majority of the complex I-deficient patients mutations of nuclear genes are expected. This review attempts to summarize genetic defects affecting nuclear encoded subunits of complex I reported to date focusing on those found in the NDUFS4 gene. NDUFS4 product is 18 kDa protein which appears to have a dual role in complex I, at least: cAMP-dependent phosphorylation activates the complex; non-sense mutation of NDUFS4 prevents normal assembly of a functional complex in the inner mitochondrial membrane.     

NDUFV1 mutations in complex I deficiency: Case reports and review of symptoms

Mitochondrial complex I (CI) deficiency is the most common oxidative phosphorylation disorder described. It shows a wide range of phenotypes with poor correlation within genotypes. Herein we expand the clinics and genetics of CI deficiency in the brazilian population by reporting three patients with pathogenic (c.640G>A, c.1268C>T, c.1207dupG) and likely pathogenic (c.766C>T) variants in the NDUFV1 gene. We show the mutation c.766C>T associated with a childhood onset phenotype of hypotonia, muscle weakness, psychomotor regression, lethargy, dysphagia, and strabismus. Additionally, this mutation was found to be associated with headaches and exercise intolerance in adulthood. We also review reported pathogenic variants in NDUFV1 highlighting the wide phenotypic heterogeneity in CI deficiency.     

Identification of mitochondrial complex I assembly intermediates by tracing tagged NDUFS3 demonstrates the entry point of mitochondrial subunits

Biogenesis of human mitochondrial complex I (CI) requires the coordinated assembly of 45 subunits derived from both the mitochondrial and nuclear genome. The presence of CI subcomplexes in CI-deficient cells suggests that assembly occurs in distinct steps. However, discriminating between products of assembly or instability is problematic. Using an inducible NDUFS3-green fluorescent protein (GFP) expression system in HEK293 cells, we here provide direct evidence for the stepwise assembly of CI. Upon induction, six distinct NDUFS3-GFP-containing subcomplexes gradually appeared on a blue native Western blot also observed in wild type HEK293 mitochondria. Their stability was demonstrated by differential solubilization and heat incubation, which additionally allowed their distinction from specific products of CI instability and breakdown. Inhibition of mitochondrial translation under conditions of steady state labeling resulted in an accumulation of two of the NDUFS3-GFP-containing subcomplexes (100 and 150 kDa) and concomitant disappearance of the fully assembled complex. Lifting inhibition reversed this effect, demonstrating that these two subcomplexes are true assembly intermediates. Composition analysis showed that this event was accompanied by the incorporation of at least one mitochondrial DNA-encoded subunit, thereby revealing the first entry point of these subunits.     

cAMP-dependent protein kinase regulates the mitochondrial import of the nuclear encoded NDUFS4 subunit of complex I

The subunits of complex I encoded by the mammalian nuclear genes NDUFS4 (AQDQ protein) and NDUFB11 (ESSS protein) contain serine/threonine consensus phosphorylation sequences (CPS) in their presequence, the first also in the C-terminus. We have studied the impact of PKA mediated phosphorylation on the mitochondrial import of in vitro and in vivo synthesized NDUFS4 protein. The intramitochondrial accumulation of the mature form of in vitro synthesized NDUFS4 protein, but not that of ESSS protein, was promoted by PKA and depressed by alkaline phosphatase (AP). In HeLa cells, control or transfected with the NDUFS4 cDNA construct, the mitochondrial level of mature NDUFS4 protein was promoted by 8-Br-cAMP and depressed by H89. Ser173Ala mutagenesis in the C-terminus CPS abolished the appearance in mitochondria of the mature form of NDUFS4 protein. The promoting effect of PKA on the mitochondrial accumulation of mature NDUFS4 protein appears to be due to inhibition of its retrograde diffusion into the cytosol.     

The structure of the human NDUFV1 gene encoding the 51-kDa subunit of mitochondrial complex I

The genomic organization of the human 51-kDa subunit gene (NDUFV1) on human Chromosome (Chr) 11q13 was determined. The NDUFV1 gene consists of 10 exons. Exon 1 encodes for the 20-amino-acids-long import sequence, and exon 1 through 10 codes for the 444-amino-acids-long mature protein. The protein sequence is highly conserved between human and bovine. Northern blotting analysis showed that the NDUFV1 gene expression varies widely among tissues and that in testis a unique mRNA species is present. In comparison with the other complex I flavoproteins, the expression of the 51-kDa gene in pancreatic tissue is high. Received: 5 May 1998 / Accepted: 28 August 1998     

The NDUFS4 nuclear gene of complex I of mitochondria and the cAMP cascade

Results of studies on the role of the 18 kDa (IP) polypeptide subunit of complex I, encoded by the nuclear NDUFS4 gene, in isolated bovine heart mitochondria and human and murine cell cultures are presented.The mammalian 18 kDa subunit has in the carboxy-terminal sequence a conserved consensus site (RVS), which in isolated mitochondria is phosphorylated by cAMP-dependent protein kinase (PKA). The catalytic and regulatory subunits of PKA have been directly immunodetected in the inner membrane/matrix fraction of mammalian mitochondria. In the mitochondrial inner membrane a PP2Cgamma-type phosphatase has also been immunodetected, which dephosphorylates the 18 kDa subunit, phosphorylated by PKA. This phosphatase is Mg(2+)-dependent and inhibited by Ca(2+). In human and murine fibroblast and myoblast cultures "in vivo", elevation of intracellular cAMP level promotes phosphorylation of the 18 kDa subunit and stimulates the activity of complex I and NAD-linked mitochondrial respiration.Four families have been found with different mutations in the cDNA of the NDUFS4 gene. These mutations, transmitted by autosomal recessive inheritance, were associated in homozygous children with fatal neurological syndrome. All these mutations destroyed the phosphorylation consensus site in the C terminus of the 18 kDa subunit, abolished cAMP activation of complex I and impaired its normal assembly.     

Pathogenic mutations of nuclear genes associated with mitochondrial disorders

Mitochondrial disorders are clinical phenotypes associated with mitochondrial dysfunction, which can be caused by mutations in mitochondrial DNA （mtDNA） or nuclear genes. In this review, we summarized the pathogenic mutations of nuclear genes associated with mitochondrial disorders. These nuclear genes encode, components of mitochondrial translational machinery and structural subunits and assembly factors of the oxidative phosphorylation, that complex. The molecular mechanisms, that nuclear modifier genes modulate the phenotypic expression of mtDNA mutations, are discussed in detail.     

A constant and similar assembly defect of mitochondrial respiratory chain complex I allows rapid identification of NDUFS4 mutations in patients with Leigh syndrome

Isolated complex I deficiency is a frequent cause of respiratory chain defects in childhood. In this study, we report our systematic approach with blue native PAGE (BN-PAGE) to study mitochondrial respiratory chain assembly in skin fibroblasts from patients with Leigh syndrome and CI deficiency. We describe five new NDUFS4 patients with a similar and constant abnormal BN-PAGE profile and present a meta-analysis of the literature. All NDUFS4 mutations that have been tested with BN-PAGE result in a constant and similar abnormal assembly profile with a complete loss of the fully assembled complex I usually due to a truncated protein and the loss of its canonical cAMP dependent protein kinase phosphorylation consensus site. We also report the association of abnormal brain MRI images with this characteristic BN-PAGE profile as the hallmarks of NDUFS4 mutations and the first founder NDUFS4 mutations in the North-African population.     

Assembly defects induce oxidative stress in inherited mitochondrial complex I deficiency

Complex I (CI) deficiency is the most common respiratory chain defect representing more than 30% of mitochondrial diseases. CI is an L-shaped multi-subunit complex with a peripheral arm protruding into the mitochondrial matrix and a membrane arm. CI sequentially assembled into main assembly intermediates: the P (pumping), Q (Quinone) and N (NADH dehydrogenase) modules. In this study, we analyzed 11 fibroblast cell lines derived from patients with inherited CI deficiency resulting from mutations in the nuclear or mitochondrial DNA and impacting these different modules. In patient cells carrying a mutation located in the matrix arm of CI, blue native-polyacrylamide gel electrophoresis (BN-PAGE) revealed a significant reduction of fully assembled CI enzyme and an accumulation of intermediates of the N module. In these cell lines with an assembly defect, NADH dehydrogenase activity was partly functional, even though CI was not fully assembled. We further demonstrated that this functional N module was responsible for ROS production through the reduced flavin mononucleotide. Due to the assembly defect, the FMN site was not re-oxidized leading to a significant oxidative stress in cell lines with an assembly defect. These findings not only highlight the relationship between CI assembly and oxidative stress, but also show the suitability of BN-PAGE analysis in evaluating the consequences of CI dysfunction. Moreover, these data suggest that the use of antioxidants may be particularly relevant for patients displaying a CI assembly defect.     

Pharmacological targeting of mitochondrial complex I deficiency: the cellular level and beyond

Complex I (CI) represents a major entry point of electrons in the mitochondrial electron transport chain (ETC). It consists of 45 different subunits, encoded by the mitochondrial (mtDNA) and nuclear DNA (nDNA). In humans, mutations in nDNA-encoded subunits cause severe neurodegenerative disorders like Leigh Syndrome with onset in early childhood. The pathophysiological mechanism of these disorders is still poorly understood. Here we summarize the current knowledge concerning the consequences of nDNA-encoded CI mutations in patient-derived cells, present mouse models for human CI deficiency, and discuss potential treatment strategies for CI deficiency.     

Neurospora strains harboring mitochondrial disease-associated mutations in iron-sulfur subunits of complex I

We subjected the genes encoding the 19.3-, 21.3c-, and 51-kDa iron-sulfur subunits of respiratory chain complex I from Neurospora crassa to site-directed mutagenesis to mimic mutations in human complex I subunits associated with mitochondrial diseases. The V135M substitution was introduced into the 19.3-kDa cDNA, the P88L and R111H substitutions were separately introduced into the 21.3c-kDa cDNA, and the A353V and T435M alterations were separately introduced into the 51-kDa cDNA. The altered cDNAs were expressed in the corresponding null-mutants under the control of a heterologous promoter. With the exception of the A353V polypeptide, all mutated subunits were able to promote assembly of a functional complex I, rescuing the phenotypes of the respective null-mutants. Complex I from these strains displays spectroscopic and enzymatic properties similar to those observed in the wild-type strain. A decrease in total complex I amounts may be the major impact of the mutations, although expression levels of mutant genes from the heterologous promoter were sometimes lower and may also account for complex I levels. We discuss these findings in relation to the involvement of complex I deficiencies in mitochondrial disease.     

Leigh syndrome associated with mitochondrial complex I deficiency due to novel mutations In NDUFV1 and NDUFS2

Leigh syndrome (LS) is a progressive neurodegenerative disease caused by either mitochondrial or nuclear DNA mutations resulting in dysfunctional mitochondrial energy metabolism. Mutations in genes encoding for subunits of the respiratory chain or assembly factors of respiratory chain complexes are often documented in LS cases. Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) enzyme deficiencies account for a significant proportion of mitochondrial disorders, including LS. In an attempt to expand the repertoire of known mutations accounting for LS, we describe the clinical, radiological, biochemical and molecular data of six patients with LS found to have novel mutations in two complex I subunits (NDUFV1 and NDUFS2). Two siblings were homozygous for the previously undescribed R386C mutation in NDUFV1, one patient was a compound heterozygote for the R386C mutation in NDUFV1 and a frameshift mutation in the same gene, one patient was a compound heterozygote for the R88G and R199P mutations in NDUFV1, and two siblings were compound heterozygotes for an undescribed E104A mutation in NDUFS2. After the novel mutations were identified, we employed prediction models using protein conservation analysis (SIFT, PolyPhen and UCSC genome browser) to determine pathogenicity. The R386C, R88G, R199P, and E104A mutations were found to be likely pathogenic, and thus presumably account for the LS phenotype. This case series broadens our understanding of the etiology of LS by identifying new molecular defects that can result in complex I deficiency and may assist in targeted diagnostics and/or prenatal diagnosis of LS in the future.     

Region-specific expression of mitochondrial complex I genes during murine brain development

Mutations in the nuclear encoded subunits of mitochondrial complex I (NADH:ubiquinone oxidoreductase) may cause circumscribed cerebral lesions ranging from degeneration of the striatal and brainstem gray matter (Leigh syndrome) to leukodystrophy. We hypothesized that such pattern of regional pathology might be due to local differences in the dependence on complex I function. Using in situ hybridization we investigated the relative expression of 33 nuclear encoded complex I subunits in different brain regions of the mouse at E11.5, E17.5, P1, P11, P28 and adult (12 weeks). With respect to timing and relative intensity of complex I gene expression we found a highly variant pattern in different regions during development. High average expression levels were detected in periods of intense neurogenesis. In cerebellar Purkinje and in hippocampal CA1/CA3 pyramidal neurons we found a second even higher peak during the period of synaptogenesis and maturation. The extraordinary dependence of these structures on complex I gene expression during synaptogenesis is in accord with our recent findings that gamma oscillations--known to be associated with higher cognitive functions of the mammalian brain--strongly depend on the complex I activity. However, with the exception of the mesencephalon, we detected only average complex I expression levels in the striatum and basal ganglia, which does not explain the exquisite vulnerability of these structures in mitochondrial disorders.