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Functional mitochondria are required for amyloid ß-mediated neurotoxicity¹

SANDRA MORAIS CARDOSO^*, SANCHA SANTOS^*, RUSSELL H. SWERDLOW and CATARINA R. OLIVEIRA^*,2

^* Center for Neuroscience of Coimbra;
Department of Neurology, University of Virginia, Charlottesville, Virginia 22908, USA; and
Laboratory of Biochemistry, Faculty of Medicine of Coimbra, University of Coimbra, Coimbra, Portugal

²Correspondence: Center for Neuroscience of Coimbra, Laboratory of Biochemistry, Faculty of Medicine, University of Coimbra, 3004–504 Coimbra, Portugal. E-mail: catarina{at}cnc.cj.uc.pt

SPECIFIC AIMS

The objective of this work was to explore the involvement of mitochondria in the cellular toxicity induced by Aß protein and to analyze how mitochondrial respiratory chain injury induced by this protein occurs. For this purpose, we investigated the effect of the neurotoxic Aß peptides (25–35 and 1–42) on NT2 + and 0 mitochondrial DNA depleted cells.

PRINCIPAL FINDINGS

1. The differential toxic effect of Aß 25–35 on NT2 + and NT2 0 cells
Incubation with Aß 25–35 peptide (10 µM) or Aß 1–42 (1 µM) for 24 h induced a decrease in cell viability in NT2 + cells, but the viability of NT2 0 cells was maintained (Fig. 1 ). H₂O₂ (100 µM) induced a decrease in cell viability in both cell lines. Data in Fig. 1 also show that the percentage of MTT reduction determined in 0 cells is lower than that observed in + cells.

View larger version (25K): [in this window] [in a new window]   Figure 1. Effect of Aß peptide or H2O2 on cells lacking functional mitochondria. NT2 + and 0 cells were incubated in the presence of 10 µM Aß 25–35 peptide, 1 µM Aß 1–42, and 100 µM H2O2. Cell toxicity was evaluated by the reduction of the tetrazolium salt MTT, as described. Data are expressed as a percentage of control values, with the mean ± SE derived from 5 to 13 independent determinations. ***P < 0.001, significantly different as compared with untreated + cells, ++P < 001, significantly different as compared with untreated 0 cells.

2. Mitochondrial membrane potential and ATP levels in untreated and Aß-treated 0 cells
As shown in the inset of Fig. 2 , NT2 0 cells were able to retain rhodamine 123, known to be taken by the cell and to reflect the maintenance of the mitochondrial membrane potential, even when incubated with Aß 25–35. In 0 cells, ATP levels were similar to those measured in NT2 + cells, and Aß 25–35 was also not able to induce a statistically significant decrease in ATP levels in 0 cells.

View larger version (38K): [in this window] [in a new window]   Figure 2. Mitochondrial membrane potential in cells exposed to Aß peptide. mt was determined in NT2 + and 0. NT2 + cells were incubated with Aß 25–35 in the absence or presence of idebenone (1 µM), vitamin E (13 µM), melatonin (83 µM), nicotine (5 µM), and GSH (2 mM). Data was expressed as the percentage of control values as the mean ± SE for four to five independent determinations. *P < 0.05, significantly different when compared with NT2 + control cells.

Effect of Aß 25–35 on mitochondrial function in NT2 + and 0 cells
As shown in Fig. 2 , treatment of NT2 + cells with a cytotoxic dose of Aß 25–35 led to a significant decrease in the mitochondrial membrane potential. Nicotine, melatonin, and vitamin E prevented the decline of mitochondria membrane potential in NT2 + cells exposed to Aß 25–35. After incubation of NT2 + cells with the amyloid ß-peptide, enzymatic activity of the mitochondrial respiratory chain was decreased, namely, NADH-ubiquinone oxidoreductase (complex I), succinate cytochrome c oxidoreductase (complex II/III) and cytochrome c oxidase (complex IV) activities. Melatonin completely prevented Aß-induced inhibition of complex I activity, whereas vitamin E, idebenone, GSH, and nicotine only partially prevented the inhibition of this enzymatic complex activity. The inhibition of complexes II/III activity in + cells preincubated with melatonin, GSH, and vitamin E was prevented, whereas idebenone and nicotine did not show any protective effect. The preincubation with idebenone and vitamin E partially prevented complex IV activity, whereas melatonin, nicotine, and GSH were ineffective. The ATP levels in NT2 + cells submitted to Aß 25–35 (10 µM) for 24 h decreased compared with cells incubated under the same conditions, but in the absence of the peptide. In the presence of neuroprotectors, the ATP levels were maintained, although vitamin E, melatonin, and nicotine were shown to be more effective than idebenone and GSH.

CONCLUSIONS

Although it is widely assumed that MTT is reduced by active mitochondria in living cells, it has been demonstrated that cellular MTT reduction can also occur in cellular compartments other than mitochondria. Using NT2 0 cells we clearly proved that a functional mitochondria is required for Aß peptides to be toxic to the cells. After the incubation of NT2 + and 0 cells with Aß 25–35 (10 µM) or Aß 1–42 (1 µM), cell viability was measured by the MTT reduction assay (Fig. 1) . According to previous results, we also observed that MTT reduction capacity of 0 cells is lower than that of + cells, suggesting that it is due mainly to extramitochondrial dehydrogenases (Fig. 1) . When 0 cells were exposed to Aß 25–35 or Aß 1–42 peptides, no further decrease in MTT reduction was observed. The loss of toxicity of the Aß peptides in 0 cells was also confirmed using the LDH leakage test. These results strongly suggest that Aß-mediated cell toxicity requires a functional mitochondria.

In NT2 + cells treated with Aß 25–35 peptide, a significant decrease in mitochondrial membrane potential was demonstrated (Fig. 2) . We also observed that the exposure of + cells to Aß 25–35 induced a significant decrease in mitochondrial respiratory chain enzymatic complexes I, II/III, and IV activities, complex IV being the most affected. This inhibition of the mitochondrial function by the Aß 25–35 led to an impairment of the cellular energy metabolism, observed as a decrease in cell ATP levels. Accordingly, it was previously shown in our laboratory that in PC12 cells, Aß 25–35 induces a decrease in the activities of the complexes I, II/III and IV, complexes II and V not being inhibited by this peptide.

NT2 0 cells depleted of their mtDNA cannot support a normal electron transport, since 13 catalytic subunits of mitochondrial respiratory chain enzymatic complexes are encoded by mtDNA (7 subunits of NADH-Q reductase, 1 subunit of cytochrome c reductase, 3 subunits of cytochrome c oxidase, and 2 of ATP synthase). Mitochondrial DNA also encodes 2 rRNAs and 22 tRNAs, which are necessary for the expression of oxphos genes. Therefore 0 cells, lacking mtDNA, are not able to carry out normal mitochondrial electron transport and ATP synthesis and are dependent on ATP from anaerobic glycolysis. The lack of mitochondrial electron transport leads to a high NADH/NAD⁺ ratio; consequently, cell survival requires added pyruvate to the culture medium in order to prevent an extreme reductive state. Our results show that in NT2 0 cells, ATP levels are maintained despite an almost complete absence of active mitochondrial respiratory chain enzymatic complexes. The maintenance of mitochondrial membrane potential, also observed in 0 cells, can be explained by the transport of protons out of the mitochondrial matrix due to the reversal of ATP synthase, as was shown to occur in 143B-87 0 cells. In 0 cells, the ATP synthase subunits, which are encoded by nuclear DNA, were found to be assembled and able to maintain the proton gradient through the mitochondrial membrane. The Aß 25–35 peptide was not able to reduce ATP levels and mitochondrial membrane potential in 0 cells, in contrast to what was observed in + cells. These results strongly support the hypothesis that Aß peptide requires a functional mitochondria to exert its toxic effect.

In this study, the neuroprotective effect of the antioxidants vitamin E, idebenone, and GSH, of the hormone melatonin, and of nicotine, the agonist of the cholinergic nicotinic receptors, was tested in order to gain a better insight into the involvement of oxidative stress in the Aß 25–35 peptide-mediated cell toxicity. These results show that in the presence of Aß 25–35 (10 µM), a significant decrease in NT2 + cells mitochondrial function occurs. This effect can be less dramatic if the cells are preincubated with melatonin and idebenone, which are shown to protect the major proton pumps in mitochondria.

The results suggest that Aß, which is present in Alzheimer’s disease brains, can be acting at the mitochondrial level by inhibiting complex IV activity and promoting the increase on ROS production because idebenone, a free radical scavenger, protected this complex activity. The inhibition of complex IV activity leading to a bioenergetic failure, as observed in NT2 + cells treated with Aß 25–35, probably plays a key role in the neurodegenerative process occurring in Alzheimer’s disease. Evidence exists that Aß peptides do not influence on complex II and V activities, raising the question for the involvement of mtDNA in the etiopathogenesis of the disease. Since complex II subunits are codified by nDNA and 2 subunits of ATP synthase are codified by mtDNA, in 0 cells this complex can be assembled and as active as in ATPase. According to these results, we propose a possible mechanism for Alzheimer’s disease neurodegeneration (Fig. 3 ).

View larger version (28K): [in this window] [in a new window]   Figure 3. Schematic diagram of a probable mechanism for neurodegeneration in Alzheimer’s disease.

In conclusion, in this work we clearly demonstrate that Aß peptide requires a functional mitochondria to induce cell toxicity, mitochondria being simultaneously the source and the target for reactive oxygen species-induced neuronal injury. Reactive oxygen species generation by the mitochondria and injury of mitochondrial respiratory chain enzymatic complexes due to protein and/or lipid oxidation is probably occurring. However, the oxidative damage of mtDNA cannot be ruled out, since in 0 cells lacking mtDNA, no toxic effect of Aß was observed. Furthermore, 0 cells have proved to be helpful in understanding the role of mitochondria in the neurodegenerative process occurring in Alzheimer’s disease.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0561fje ; to cite this article, use FASEB J. (April 18, 2001) 10.1096/fj.00-0561fje

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