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Mitochondrial respiratory chain (MRC) dysfunction has been implicated in a wide variety of neurodegenerative diseases including Leber's hereditary optic neuropathy (LHON), Parkinson's disease (PD), Huntington's disease (HD) and dystonia. The underlying causes of the MRC dysfunction are heterogenous and may be a consequence of primary mtDNA or nuclear mutations, secondary to the underlying disease process or the effects of endogenous toxins. Cybrids have been used to investigate the interactions between the mitochondrial and nuclear genomes, and their effects on MRC dysfunction in LHON and dystonia. Although primary mutations in mitochondrial DNA complex I genes have been identified in LHON, other factors are thought to influence disease expression. While the complex I defect was maintained in the 3460 LHON/206 p° cybrids, it was absent in the 3460 LHON/A549 p° cybrids suggesting the nuclear environment influences the expression of the complex I defect in these 3460 LHON patients and that nuclear factors may contribute to the clinical phenotype. The complex I defect in focal dystonia was not transferred to either mixed or clonal focal dystonia platelet/ A549 p° cybrids lines suggesting it is caused by either a nuclear mutation or a toxin and excludes a mtDNA mutation. The previously reported severe deficiencies of mitochondrial complexes II/III and IV in HD caudate were also seen in HD putamen, and a decrease in aconitase activity was found to parallel the anatomic distribution of HD pathology. In the R6/2 transgenic mouse model of HD there were significant reductions in aconitase and complex IV activities in striatum. The relative sensitivity of complexes II/III and aconitase activity to nitric oxide and the increased immunostaining for inducible nitric oxide synthase and nitrotyrosine in the R6/2 brain supports the hypothesis that defective energy metabolism, excitotoxicity and NO contribute to pathogenesis in HD. Stable inducible cell models of HD and PD have been constructed using mutant and wild-type N-termini of huntingtin, and mutant G209A and wild-type α-synuclein respectively, to facilitate the investigation of the pathophysiology of MRC dysfunction in these disorders.

BACKGROUND: Mitochondrial complex I deficiency often leads to a devastating neurodegenerative disorder of childhood. In most cases, the underlying genetic defect is unknown. Recessive nuclear gene mutations, rather than mitochondrial DNA mutations, account for the majority of cases. AIM: Our aim was to identify the genetic basis of complex I deficiency in 34 patients with isolated complex I deficiency, by studying six of the 39 nuclear encoded complex I subunit genes (NDUFV1, NDUFS1, NDUFS2, NDUFS4, NDUFS7 and NDUFS8). These genes have been conserved throughout evolution and carry out essential aspects of complex I function. METHODS: RNA was extracted from patient fibroblasts and cDNA made by reverse transcription. Overlapping amplicons that together spanned the entire coding area of each gene were amplified by PCR. The genes were screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC). Patient samples with abnormal dHPLC profiles underwent direct DNA sequencing. RESULTS: Novel mutations were identified in six of 34 (18%) patients with isolated complex I deficiency. Five patients had two mutations identified and one patient had a single mutation in NDUFS4 identified. All patients with mutations had a progressive encephalopathy and five out of six had Leigh syndrome or Leigh like syndrome. Mutations were found in three nuclear encoded subunit genes, NDUFV1, NDUFS2 and NDUFS4. Three novel NDUFV1 mutations were identified (R386H, K111E and P252R). The R386H mutation was found in two apparently unrelated patients. Four novel NDUFS2 mutations were identified (R221X, M292T, R333Q and IVS9+4A&ltG). The novel NDUFS4 mutation c.221delC was found in two patients - one in homozygous form and the other heterozygous. Specific genotype and phenotype correlations were not identified. CONCLUSIONS: Nuclear encoded complex I subunit gene mutations are an important contributor to the aetiology of isolated complex I deficiency in childhood. Screening of these genes is an essential part of the investigation of complex I deficiency.