Cells Bearing Mutations Causing Leber's Hereditary Optic Neuropathy Are Sensitized to Fas-induced Apoptosis

Steven R Danielson,Alice Wong,Valerio Carelli,Andrea Martinuzzi,Anthony H.V Schapira,Gino A Cortopassi

doi:10.1074/jbc.m110119200

Abstract

Three prevalent mitochondrial DNA pathogenic mutations at positions 11778, 3460, and 14484, which affect different subunits of Complex I, cause retinal ganglion cell death and optic nerve atrophy in Leber's hereditary optic neuropathy (LHON). The cell death is painless and without inflammation, consistent with an apoptotic mechanism. We have investigated the possibility that the LHON mutation confers a pro-apoptotic stimulus and have tested the sensitivity of osteosarcoma-derived cybrid cells carrying the most common and severe mutations (11778 and 3460) to cell death induced by Fas. We observed that LHON cybrids were sensitized to Fas-dependent death. Control cells that bear the same mitochondrial genetic background (the J haplogroup) without the pathogenic 11778 mutation are no more sensitive than other controls, indicating that increased Fas-dependent death in LHON cybrids was induced by the LHON pathogenic mutations. The type of death was apoptotic by several criteria, including induction by Fas, inhibition by the caspase inhibitor zVAD-fmk (zVal-Ala-Asp-fluoro-methyl ketone), activation of DEVDase activity (Asp-Glu-Val-Asp protease), specific cleavage of caspase-3, DNA fragmentation, and increased Annexin-V labeling. These data indicate that the most common and severe LHON pathogenic mutations 11778 and 3460 predispose cells to apoptosis, which may be relevant for the pathophysiology of cell death in LHON, and potential therapy.

Highlights

Leber’s hereditary optic neuropathy (LHON)1 is a maternally inherited disorder characterized by a primary degeneration of the retinal ganglion cells (RGCs) and atrophy of the optic nerve [1, 2]
LHON is caused by three prevalent pathogenic mitochondrial DNA point mutations at positions 11778, 3460, and 14484, which alter coding genes for complex I subunits in the mitochondrial electron transport chain (4 – 6)
Increased Fas-dependent Death in LHON Cells—We investigated the hypothesis that the mitochondrial DNA (mtDNA) LHON pathogenic mutations may predispose cells to apoptosis

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Culture Conditions—Control and LHON cybrid cell lines were obtained as described elsewhere [19], by fusing 206 rhoo cells Attardi) with enucleated fibroblasts from controls or LHON patients previously characterized for their mtDNA haplogroup and presence of LHON pathogenic mutations. Three osteosarcoma-derived cybrid cell lines, constructed from control fibroblasts carrying a J haplogroup with no LHON pathogenic mutations, were used as haplogroup-matched controls. Five LHON cybrid cell lines were constructed from four individual patients and contained the 11778 pathogenic mutation. Two more LHON cybrid cell lines derived from two different patients contained the 3460 mtDNA point mutation. The cells were harvested by trypsinization and scored as alive or dead using the Trypan blue exclusion assay. DEVDase-specific Activity—DEVDase assay was performed following the manufacturer’s instructions (Biomol, Plymouth, PA), briefly, whole cells were treated with anti-Fas as before for 6 h, harvested by trypsinization, and lysed with lysis buffer provided in the kit (supplemented with 0.1% Triton X-100). All other p values were calculated using the paired two-tailed Student’s t test

RESULTS

LHON Cells Are Sensitized to Apoptosis

DISCUSSION