Leber's Hereditary Optic Neuropathy (LHON) Pathogenic Mutations Induce Mitochondrial-dependent Apoptotic Death in Transmitochondrial Cells Incubated with Galactose Medium

Abstract

Leber's hereditary optic neuropathy (LHON), a maternally inherited form of central vision loss, is associated with mitochondrial DNA pathogenic point mutations affecting different subunits of complex I. We here report that osteosarcoma-derived cytoplasmic hybrids (cybrid) cell lines harboring one of the three most frequent LHON pathogenic mutations, at positions 11778/ND4, 3460/ND1, and 14484/ND6, undergo cell death when galactose replaces glucose in the medium, contrary to control cybrids that maintain some growth capabilities. This is a well known way to produce a metabolic stress, forcing the cells to rely on the mitochondrial respiratory chain to produce ATP. We demonstrate that LHON cybrid cell death is apoptotic, showing chromatin condensation and nuclear DNA laddering. Moreover, we also document the mitochondrial involvement in the activation of the apoptotic cascade, as shown by the increased release of cytochromec into the cytosol in LHON cybrid cells as compared with controls. Cybrids bearing the 3460/ND1 and 14484/ND6 mutations seemed more readily prone to undergo apoptosis as compared with the 11778/ND4 mutation. In conclusion, LHON cybrid cells forced by the reduced rate of glycolytic flux to utilize oxidative metabolism are sensitized to an apoptotic death through a mechanism involving mitochondria. Leber's hereditary optic neuropathy (LHON), a maternally inherited form of central vision loss, is associated with mitochondrial DNA pathogenic point mutations affecting different subunits of complex I. We here report that osteosarcoma-derived cytoplasmic hybrids (cybrid) cell lines harboring one of the three most frequent LHON pathogenic mutations, at positions 11778/ND4, 3460/ND1, and 14484/ND6, undergo cell death when galactose replaces glucose in the medium, contrary to control cybrids that maintain some growth capabilities. This is a well known way to produce a metabolic stress, forcing the cells to rely on the mitochondrial respiratory chain to produce ATP. We demonstrate that LHON cybrid cell death is apoptotic, showing chromatin condensation and nuclear DNA laddering. Moreover, we also document the mitochondrial involvement in the activation of the apoptotic cascade, as shown by the increased release of cytochromec into the cytosol in LHON cybrid cells as compared with controls. Cybrids bearing the 3460/ND1 and 14484/ND6 mutations seemed more readily prone to undergo apoptosis as compared with the 11778/ND4 mutation. In conclusion, LHON cybrid cells forced by the reduced rate of glycolytic flux to utilize oxidative metabolism are sensitized to an apoptotic death through a mechanism involving mitochondria. Leber's hereditary optic neuropathy mitochondrial DNA cytoplasmic hybrids 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Dulbecco's modified Eagle's medium thymidine kinase In 1988, Leber's hereditary optic neuropathy (LHON)1 has been the first human pathology to be associated with a mitochondrial DNA (mtDNA) point mutation (1Wallace D.C. Singh G. Lott M.T. Hodge J. Schurr T.G. Lezza A.M. Elsas L.J.D. Nikoskelainen E.K. Science. 1988; 242: 1427-1430Crossref PubMed Scopus (1912) Google Scholar). However, its pathogenesis remains poorly understood (2Howell N. Int. Rev. Cytol. 1999; 186: 49-116Crossref PubMed Google Scholar, 3Brown M.D. J. Neurol. Sci. 1999; 165: 1-5Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 4Carelli V. Ross-Cisneros F.N. Sadun A.A. Neurochem. Int. 2002; 40: 573-584Crossref PubMed Scopus (210) Google Scholar). Currently, three most frequent (11778/ND4, 3460/ND1, 14484/ND6) (2Howell N. Int. Rev. Cytol. 1999; 186: 49-116Crossref PubMed Google Scholar, 3Brown M.D. J. Neurol. Sci. 1999; 165: 1-5Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 4Carelli V. Ross-Cisneros F.N. Sadun A.A. Neurochem. Int. 2002; 40: 573-584Crossref PubMed Scopus (210) Google Scholar) and other rare (14459/ND6, 10663/ND4L, 4171/ND1, 14482/ND6) (5Chinnery P.F. Brown D.T. Andrews R.M. Singh-Kler R. Riordan-Eva P. Lindley J. Applegarth D.A. Turnbull D.M. Howell N. Brain. 2001; 124: 209-218Crossref PubMed Scopus (145) Google Scholar, 6Brown M.D. Starikovskaya E. Derbeneva O. Hosseini S. Allen J.C. Mikhailovskaya I.E. Sukernik R.I. Wallace D.C. Hum. Genet. 2002; 110: 130-138Crossref PubMed Scopus (188) Google Scholar, 7Kim J.Y. Hwang J-M. Park S.S. Ann. Neurol. 2002; 51: 630-634Crossref PubMed Scopus (46) Google Scholar, 8Valentino M.L. Avoni P. Barboni P. Pallotti F. Rengo C. Torroni A. Bellan M. Baruzzi A. Carelli V. Ann. Neurol. 2002; 51: 774-778Crossref PubMed Scopus (41) Google Scholar) pathogenic mutations are found in the majority of patients affected with this maternally inherited form of optic neuropathy. LHON is due to a massive acute or subacute retinal ganglion cell death, characteristically leading to central vision loss (2Howell N. Int. Rev. Cytol. 1999; 186: 49-116Crossref PubMed Google Scholar, 3Brown M.D. J. Neurol. Sci. 1999; 165: 1-5Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 4Carelli V. Ross-Cisneros F.N. Sadun A.A. Neurochem. Int. 2002; 40: 573-584Crossref PubMed Scopus (210) Google Scholar). These pathogenic mutations invariably affect complex I subunits, which possibly interact with the quinone substrate (9Carelli V. Ghelli A. Ratta M. Bacchilega E. Sangiorgi R. Mancini R. Leuzzi V. Cortelli P. Montagna P. Lugaresi E. Degli Esposti M. Neurology. 1997; 48: 1623-1632Crossref PubMed Scopus (119) Google Scholar, 10Carelli V. Ghelli A. Bucchi L. Montagna P., De Negri A. Leuzzi V. Carducci C. Lenaz G. Lugaresi E. Degli Esposti M. Ann. Neurol. 1999; 45: 320-328Crossref PubMed Scopus (118) Google Scholar), and a combination of partial respiratory deficiency and increased oxidative stress is documented to be the pathological consequence in transmitochondrial cell systems (11Vergani L. Martinuzzi A. Carelli V. Cortelli P. Montagna P. Schievano G. Carrozzo R. Angelini C. Lugaresi E. Biochem. Biophys. Res. Commun. 1995; 210: 880-888Crossref PubMed Scopus (112) Google Scholar, 12Brown M.D. Trounce I.A. Jun A.S. Allen J.C. Wallace D.C. J. Biol. Chem. 2000; 275: 39831-39836Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 13Wong A. Cortopassi G. Biochem. Biophys. Res. Commun. 1997; 239: 139-145Crossref PubMed Scopus (74) Google Scholar, 14Wong A. Cavelier L. Collins-Schram H.E. Selding M.F. McGrogan M. Savontaus M.L. Cortopassi A.G. Hum. Mol. Genet. 2002; 11: 431-438Crossref PubMed Scopus (157) Google Scholar, 15Carelli V. Napoli E. Valente L. Valentino M.L. Martinuzzi A. Neurology. 2002; 58: A507Crossref PubMed Scopus (1) Google Scholar). However, the mtDNA pathogenic mutations are a necessary, but not sufficient, condition to actually develop LHON, as suggested by the variable penetrance (2Howell N. Int. Rev. Cytol. 1999; 186: 49-116Crossref PubMed Google Scholar, 16Carelli V. Schapira A.H.V. DiMauro S. Mitochondrial Disorders in Neurology 2, Blue Books of Practical Neurology. Butterworth-Heinemann Woburn, MA2002: 115-142Google Scholar). Thus, other mitochondrial or nuclear genetic factors, as well as environmental factors, are believed to be necessary for triggering the optic neuropathy. At the present time, most of these factors are not well defined. One potential modulator is possibly represented by the mtDNA background, as indicated by the association of some of the pathogenic mutations (11778/ND4, 14484/ND6, 10663/ND4L) with a specific mtDNA haplogroup characteristic of the European population, classified as J (2Howell N. Int. Rev. Cytol. 1999; 186: 49-116Crossref PubMed Google Scholar, 6Brown M.D. Starikovskaya E. Derbeneva O. Hosseini S. Allen J.C. Mikhailovskaya I.E. Sukernik R.I. Wallace D.C. Hum. Genet. 2002; 110: 130-138Crossref PubMed Scopus (188) Google Scholar, 16Carelli V. Schapira A.H.V. DiMauro S. Mitochondrial Disorders in Neurology 2, Blue Books of Practical Neurology. Butterworth-Heinemann Woburn, MA2002: 115-142Google Scholar). LHON has many other peculiar and a yet unexplained features. Among these, we emphasize the age-dependent, highly tissue-specific, mostly apoplectic, and wave of retinal ganglion cell death in the absence of classical signs of inflammation (16Carelli V. Schapira A.H.V. DiMauro S. Mitochondrial Disorders in Neurology 2, Blue Books of Practical Neurology. Butterworth-Heinemann Woburn, MA2002: 115-142Google Scholar). Vascular signs, such as microangiopathy and small vessel tortuosity at the optic nerve head, usually precede the onset of the disease (16Carelli V. Schapira A.H.V. DiMauro S. Mitochondrial Disorders in Neurology 2, Blue Books of Practical Neurology. Butterworth-Heinemann Woburn, MA2002: 115-142Google Scholar). Some of these features are thought to suggest a prevalent apoptotic mode of cell death (17Howell N. J. Bioenerg. Biomembr. 1997; 29: 165-173Crossref PubMed Scopus (55) Google Scholar), and by analogy with other pathological conditions, such as glaucoma, in which apoptosis has been directly documented (17Howell N. J. Bioenerg. Biomembr. 1997; 29: 165-173Crossref PubMed Scopus (55) Google Scholar,18Kerrigan L.A. Zack D.J. Quigley H.A. Smith S.D. Pease M.E. Arch. Ophthalmol. 1997; 115: 1031-1035Crossref PubMed Scopus (418) Google Scholar). Growth impairment of transmitochondrial cytoplasmic hybrids (cybrids) carrying the most frequent LHON pathogenic mutation at position 11778/ND4 has been first reported by Hofhaus et al. (19Hofhaus G. Johns D.R. Hurko O. Attardi G. Chomyn A. J. Biol. Chem. 1996; 271: 13155-13161Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) when cells were incubated in a medium containing galactose in place of glucose (19Hofhaus G. Johns D.R. Hurko O. Attardi G. Chomyn A. J. Biol. Chem. 1996; 271: 13155-13161Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). This finding was in agreement with previous studies showing that cultured cells with defective mitochondrial metabolism often die in glucose-free galactose medium (20Robinson B.H. Petrova-Benedict R. Buncic J.R. Wallace D.C. Biochem. Med. Metab. Biol. 1992; 48: 122-126Crossref PubMed Scopus (181) Google Scholar, 21Robinson B.H. Methods Enzymol. 1996; 264: 454-464Crossref PubMed Google Scholar). Indeed, in glucose medium, most of the total ATP yield of proliferating cells is produced by glycolytic glucose breakdown to lactate, and only a minimal amount of pyruvate is oxidized to CO2 and water. Galactose can also enter the glycolytic pathway; however, the restricted flow of galactose to glucose-6-phosphate determines the formation of very little lactate since pyruvate, which is formed at a much slower rate, is further oxidized within the mitochondria (21Robinson B.H. Methods Enzymol. 1996; 264: 454-464Crossref PubMed Google Scholar). In the present study, we have characterized the loss of cell viability of osteosarcoma-derived cybrids containing the three main LHON pathogenic mutations (11778/ND4, 3460/ND1, 14484/ND6) grown in a medium in which glucose was replaced by galactose. We show that LHON cybrids, but not control cybrids or the parental osteosarcoma cell line (143B.TK−), died of apoptosis. It is widely accepted that the apoptotic process is triggered through two major mechanisms, one involving the engagement of plasma membrane-associated death receptors and another involving the of mitochondria in C. Biochem. Sci. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). that the only control and LHON cybrids is the of mtDNA mutations, on the involvement of these in the apoptotic death triggered by a metabolic and were was and were cell lines were two and LHON as mitochondria and the osteosarcoma cell line as cell line and were by Attardi and I the cybrid cell lines in this and (11Vergani L. Martinuzzi A. Carelli V. Cortelli P. Montagna P. Schievano G. Carrozzo R. Angelini C. Lugaresi E. Biochem. Biophys. Res. Commun. 1995; 210: 880-888Crossref PubMed Scopus (112) Google Scholar, 15Carelli V. Napoli E. Valente L. Valentino M.L. Martinuzzi A. Neurology. 2002; 58: A507Crossref PubMed Scopus (1) Google Scholar, Schapira A.H.V. Ann. Neurol. PubMed Scopus Google Scholar, A. Carelli V. Martinuzzi A. Schapira A.H.V. Cortopassi A.G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, V. L. C. Bucchi L. Valentino M.L. Rengo C. Torroni A. Martinuzzi A. Biophys. 2002; PubMed Scopus Google Scholar). of the mtDNA haplogroup and of the LHON pathogenic mutations were by as reported A. Carelli V. Martinuzzi A. Schapira A.H.V. Cortopassi A.G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, V. L. C. Bucchi L. Valentino M.L. Rengo C. Torroni A. Martinuzzi A. Biophys. 2002; PubMed Scopus Google Scholar). and cybrid cell lines were grown in medium with and the cells were and incubated in glucose-free medium with pyruvate, and galactose at in an with a of mutations and mtDNA of the cell lines in the present pathogenic parental cell line cell cell line cell cell J J J J in a was to the cell as in A.S. R. 1996; were into and incubated in galactose medium for different and was to the of and were to the and were in the of was with a at the of a was by the cells at to and the of was in cells the at the with for at and with were with a of an with a and A.M. P. E.K. A. M. R. Biol. 2001; PubMed Google Scholar). of apoptotic and cells was by with and the into however, only can the plasma and the bromide is and apoptotic which have an plasma A. J. T.G. in Scholar). cells were in and incubated with galactose medium for different and incubated with and bromide and with an or apoptotic cells or with cells with of DNA cells were into in galactose medium for the indicated and and DNA was as in M. P. P. R. F. J. Biol. Chem. 1992; Full Text PDF PubMed Google DNA was by in and with in galactose medium, cells were in of and for with a This and the were at were for at and the was for at was at of was as in Biochem. PubMed Scopus Google of of were by and was with in for and incubated with the for at were by in with and incubated for at were an and with the growth impairment has been reported in cells carrying the 11778/ND4 LHON mutation incubated in a medium containing galactose in place of glucose (19Hofhaus G. Johns D.R. Hurko O. Attardi G. Chomyn A. J. Biol. Chem. 1996; 271: 13155-13161Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). this has been to the other LHON pathogenic 3460/ND1 and 14484/ND6 parental cell two control cybrid cell and two different cybrid cell lines for of the three LHON mutations were incubated in galactose medium, and in viability were are shown in that cells and control cybrids a very of a small in the of cells was and the of cells or increased to of and at the of the shown in the growth medium was that the metabolic stress a different in cybrid cell lines bearing LHON In a of cells was in the cybrids bearing the and 14484/ND6 mutations, which were in an of and of of with galactose medium, the of the 11778/ND4 mutation was found in an of cells that were and of cells that were It that cybrids carrying LHON pathogenic mutations are more to metabolic stress by growth in galactose medium control cybrids, some the three mutations are also control and LHON cybrid cell lines were characterized for mtDNA background, as shown in I. in the mtDNA haplogroup not in growth of control cybrids and the parental cell line in galactose medium, as reported V. L. C. Bucchi L. Valentino M.L. Rengo C. Torroni A. Martinuzzi A. Biophys. 2002; PubMed Scopus Google Scholar). Moreover, the of different mtDNA in the LHON cybrids, in haplogroup which has been associated with the 11778/ND4 and 14484/ND6 mutations (16Carelli V. Schapira A.H.V. DiMauro S. Mitochondrial Disorders in Neurology 2, Blue Books of Practical Neurology. Butterworth-Heinemann Woburn, MA2002: 115-142Google Scholar), not show on cell We in to the of cybrids carrying the 11778/ND4 mutation but in J and and cybrids carrying the 14484/ND6 mutation and haplogroup J seemed to present the to cell death in galactose However, in this we the with a further cell line carrying the pathogenic mutation with a different of cell death, apoptosis in LHON cybrids by metabolic stress was first by of nuclear of cells with In cells apoptosis are characterized by the that in the chromatin condensation and nuclear DNA A. J. T.G. in Scholar). that of in galactose medium, the of a in cybrids bearing the pathological LHON mutations, the nuclear chromatin of most cells was and highly of on the of apoptotic and cells were by the bromide with or with chromatin were as or apoptotic in of in galactose medium, the of apoptotic cells was for parental cells and for control cybrids, was in cybrids carrying the 11778/ND4 mutation and and in cybrids carrying the 3460/ND1 and 14484/ND6, of cells with with was not of apoptotic cells in cybrids incubated in galactose and LHON cybrids were in galactose medium for the indicated and incubated with and bromide and with an are S.D. of at three of apoptotic cells in LHON cybrids was different control or parental cells and and the nuclear characterized by chromatin condensation is by specific of leading to the or of DNA into small which can be of nuclear DNA and with In the three show of DNA the parental cell line and two control cybrid cell lines incubated for the indicated in galactose DNA was to of a DNA was of with galactose in the two cybrid cell lines bearing the 11778/ND4 mutation and in the cybrids with the 3460/ND1 and 14484/ND6 mitochondria are in the apoptotic process in LHON cybrids by metabolic stress, the release of mitochondria to cytosol has been as a of in galactose release of this was in the of the parental cells a cytochromec release was in cybrids with the 11778/ND4 mutation and In cybrids with the 3460/ND1 and 14484/ND6 mutations, a release of was of in galactose medium first of this was the growth impairment documented in cybrids carrying the LHON pathogenic mutations when incubated in a this the reduced rate of the glycolytic the forced of pyruvate through the mitochondrial respiratory This condition has been to an respiratory (20Robinson B.H. Petrova-Benedict R. Buncic J.R. Wallace D.C. Biochem. Med. Metab. Biol. 1992; 48: 122-126Crossref PubMed Scopus (181) Google Scholar, 21Robinson B.H. Methods Enzymol. 1996; 264: 454-464Crossref PubMed Google Scholar), and Hofhaus et al. (19Hofhaus G. Johns D.R. Hurko O. Attardi G. Chomyn A. J. Biol. Chem. 1996; 271: 13155-13161Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) report a reduced of cybrids to in galactose medium (19Hofhaus G. Johns D.R. Hurko O. Attardi G. Chomyn A. J. Biol. Chem. 1996; 271: 13155-13161Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). this to the of the main LHON pathogenic mutations, also showing that growth impairment was in cybrids as compared with carrying the and and reported by al. (19Hofhaus G. Johns D.R. Hurko O. Attardi G. Chomyn A. J. Biol. Chem. 1996; 271: 13155-13161Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) the of the parental cell line and the other control In control cells were reported to have a growth rate in galactose medium, reduced as compared with growth in glucose medium (19Hofhaus G. Johns D.R. Hurko O. Attardi G. Chomyn A. J. Biol. Chem. 1996; 271: 13155-13161Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). we report here that the viability of these cells of and increased to to further at This loss of viability is due to since was the growth medium not We that the rate at the be due to the of cell we a growth of control cells in galactose medium when at a of not growth impairment of LHON cybrids with a different mtDNA haplogroup not show that be to the mitochondrial However, we that some the of cell and different within the in this is that the of cell death in LHON cybrids incubated in galactose medium is In we for the first time, that LHON cybrids, but not control cybrids and the parental cell some of such as the in nuclear chromatin and DNA laddering. these is that cybrids with the 11778/ND4 mutation to the metabolic stress in with the 3460/ND1 and 14484/ND6 is an and its or with the glucose can to in cells Sci. S. A. PubMed Scopus (145) Google Scholar) or to apoptosis in cells Sci. S. A. PubMed Scopus Google Scholar). has been shown that of the glycolytic rate by the glucose in the medium or glucose by of or the apoptosis by growth D.R. J.C. Mol. Biol. 2001; PubMed Scopus Google Scholar). in the metabolism be to the to cell death D.R. Metab. 2002; Full Text Full Text PDF Google Scholar). However, in cell only LHON cybrids undergo apoptosis in galactose We that the glycolytic flux in is with an of for mitochondrial and for cell and of growth capabilities. that the only control and LHON cybrids is the of the 11778/ND4, 3460/ND1, and 14484/ND6 pathogenic mutations, known to affect complex I M.D. J. Neurol. Sci. 1999; 165: 1-5Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, V. Ghelli A. Ratta M. Bacchilega E. Sangiorgi R. Mancini R. Leuzzi V. Cortelli P. Montagna P. Lugaresi E. Degli Esposti M. Neurology. 1997; 48: 1623-1632Crossref PubMed Scopus (119) Google Scholar, 10Carelli V. Ghelli A. Bucchi L. Montagna P., De Negri A. Leuzzi V. Carducci C. Lenaz G. Lugaresi E. Degli Esposti M. Ann. Neurol. 1999; 45: 320-328Crossref PubMed Scopus (118) Google Scholar, 12Brown M.D. Trounce I.A. Jun A.S. Allen J.C. Wallace D.C. J. Biol. Chem. 2000; 275: 39831-39836Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), we that this genetic is for the of the LHON cybrids to with the metabolic finding this is the of mitochondrial involvement in the apoptotic in the death of the LHON This has been by the release of mitochondria to cytosol documented in LHON of stress to have been reported to a for apoptosis by these In this the engagement of of the of to mitochondria and the release of such as and C. Biochem. Sci. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). This is by the of which are to the of mitochondria and which at in by the release of C. Biochem. Sci. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Science. PubMed Scopus Google Scholar, J.C. 1997; PubMed Scopus Google Scholar). It is of that glucose of cultured cells has been reported to in the of the cytosol to mitochondria P. PubMed Scopus Google Scholar). We to of in cell cell line and LHON cybrids Ghelli and C. studies are in to the in the process of release galactose cell of LHON pathogenic mutations have been characterized M.D. J. Neurol. Sci. 1999; 165: 1-5Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). However, we have that a variable respiratory is associated with these mutations with the of a partial of ATP This be due to a reduced release of or due to a at complex I which in be in of potential However, is not mechanisms, such as complex actually maintain the ATP or the of ATP the other the complex I in LHON characterized by the of affecting the with the quinone and an of has been as a consequence (9Carelli V. Ghelli A. Ratta M. Bacchilega E. Sangiorgi R. Mancini R. Leuzzi V. Cortelli P. Montagna P. Lugaresi E. Degli Esposti M. Neurology. 1997; 48: 1623-1632Crossref PubMed Scopus (119) Google Scholar, 10Carelli V. Ghelli A. Bucchi L. Montagna P., De Negri A. Leuzzi V. Carducci C. Lenaz G. Lugaresi E. Degli Esposti M. Ann. Neurol. 1999; 45: 320-328Crossref PubMed Scopus (118) Google Scholar). Some studies are this by and A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), in which a partial complex I impairment is or by that apoptotic cell death was with with a in respiratory chain Moreover, a of was in a cybrid cell carrying the 11778/ND4 and 3460/ND1 LHON mutations only A. Cavelier L. Collins-Schram H.E. Selding M.F. McGrogan M. Savontaus M.L. Cortopassi A.G. Hum. Mol. Genet. 2002; 11: 431-438Crossref PubMed Scopus (157) Google Scholar). these in the osteosarcoma cybrids with LHON mutations V. Napoli E. Valente L. Valentino M.L. Martinuzzi A. Neurology. 2002; 58: A507Crossref PubMed Scopus (1) Google Scholar). Thus, conditions, the of of be in triggering a apoptotic cell death in to the of different line of possibly be for the LHON mutations, affecting different subunits, with apoptosis. for complex I and has been in the of the mitochondrial E. O. F. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. F. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). It is to that the LHON pathogenic mutations, which affect the of complex I with the also the with the release of cytochromec and activation of the apoptotic of the are in agreement with and reported the very LHON cybrid cell lines but to a different of apoptotic activation A. Carelli V. Martinuzzi A. Schapira A.H.V. Cortopassi A.G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In et al. A. Carelli V. Martinuzzi A. Schapira A.H.V. Cortopassi A.G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) that these LHON cybrids were more to apoptosis A. Carelli V. Martinuzzi A. Schapira A.H.V. Cortopassi A.G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In this apoptotic the death activation of can directly and the such as and M. G. Sci. S. A. 1996; PubMed Scopus Google Scholar). In also these by release as a consequence of of the of and its into the mitochondrial J. Full Text Full Text PDF PubMed Scopus Google Scholar, I C. Full Text Full Text PDF PubMed Scopus Google Scholar). the of these two to apoptosis in LHON cybrids has not been the increased of these cells to and metabolic apoptosis that the complex I mutations can cell further of the of the apoptotic in LHON more on the of this disease and more in on the mitochondrial by complex I mutations and cell This is of for a of and for to or the apoptotic This a for LHON patients the acute of the when the retinal ganglion cell loss is thought to the retinal ganglion cell death and the We are to Cortopassi and of for some cybrids cell lines and to of for the