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Severe Impairment of Complex I–Driven Adenosine Triphosphate Synthesis in Leber Hereditary Optic Neuropathy Cybrids
Alessandra Baracca, PhD;
Giancarlo Solaini, PhD;
Gianluca Sgarbi, PhD;
Giorgio Lenaz, MD;
Agostino Baruzzi, MD;
Anthony H. V. Schapira, MD;
Andrea Martinuzzi, MD, PhD;
Valerio Carelli, MD, PhD
Arch Neurol. 2005;62:730-736.
Background Leber hereditary optic neuropathy (LHON) is a maternally inherited form of central vision loss associated with mitochondrial DNA point mutations that affect the ND subunits of complex I.
Objective To elucidate the bioenergetic consequences of complex I dysfunction in LHON.
Design The biochemical phenotypes of LHON mutations have been investigated using the transmitochondrial cytoplasmic hybrid (cybrid) cell model derived from the osteocarcoma parental cell line 143B.TK–.
Setting Research laboratories at neuroscience and biochemistry departments at the University of Bologna, Scientific Institute "E. Medea," and University of College Medical School.
Participants Fibroblast cell lines were obtained from patients affected with LHON, as defined by the presence of 1 pathogenic mutation, and from healthy volunteers as controls to construct cybrid cell lines.
Main Outcome Measures Complex I (glutamate-malate)– and complex II (succinate)–dependent adenosine triphosphate (ATP) synthesis, their respective respiratory rates, and total cellular ATP content were investigated using digitonin permeabilized cybrid cells. Multiple cybrid cell lines were constructed, introducing into osteosarcoma-derived rho0 cells either wild-type or LHON mutant mitochondria carrying each of the 3 common mutations at positions 11778/ND4, 3460/ND1, and 14484/ND6.
Results All 3 LHON mutations impaired ATP synthesis and the respiratory control ratio driven by complex I substrates. In contrast, succinate-driven ATP synthesis, respiration rates, and respiratory control ratios were not affected. However, the defective ATP synthesis with complex I substrates did not result in reduced ATP cellular content, indicating a compensatory mechanism.
Conclusions The LHON pathogenic mutations profoundly impair complex I–dependent synthesis of ATP, providing a common biochemical feature that may play a major role in LHON pathogenesis. Stratification of the results by mutation suggests that the 11778/ND4 mutation may induce an uncoupling of cybrid respiration, whereas the other 2 mutations impair the oxygen consumption rate.
Author Affiliations: Dipartimento di Biochimica (Drs Baracca, Solaini, and Lenaz) and Dipartimento di Scienze Neurologiche (Drs Baruzzi and Carelli), University of Bologna, Bologna, Italy; Scuola Superiore di Studi Universitari e di Perfezionamento S Anna, Pisa, Italy (Dr Sgarbi); University Department of Clinical Neurosciences, Royal Free and University College Medical School, London, England (Dr Schapira); and Scientific Institute "E. Medea," Conegliano, Treviso, Italy (Dr Martinuzzi).
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