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Keywords:

  • Alzheimer's disease;
  • beta amyloid;
  • complex IV;
  • mitochondrial permeability transition;
  • neurotoxicity;
  • oxidative stress

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Beta amyloid (Aβ) peptides accumulate in Alzheimer's disease and are neurotoxic possibly through the production of oxygen free radicals. Using brain microdialysis we characterized the ability of Aβ to increase oxygen radical production in vivo. The 1–40 Aβ fragment increased 2,3-dehydroxybenzoic acid efflux more than the 1–28 fragment, in a manner dependent on nitric oxide synthase and NMDA receptor channels. We then examined the effects of Aβ peptides on mitochondrial function in vitro. Induction of the mitochondrial permeability transition in isolated rat liver mitochondria by Aβ(25–35) and Aβ(35–25) exhibited dose dependency and required calcium and phosphate. Cyclosporin A prevented the transition as did ruthenium red, chlorpromazine, or N-ethylmaleimide. ADP and magnesium delayed the onset of mitochondrial permeability transition. Electron microscopy confirmed the presence of Aβ aggregates and swollen mitochondria and preservation of mitochondrial structure by inhibitors of mitochondrial permeability transition. Cytochrome c oxidase (COX) activity was selectively inhibited by Aβ(25–35) but not by Aβ(35–25). Neurotoxic Aβ peptide can increase oxidative stress in vivo through mechanisms involving NMDA receptors and nitric oxide sythase. Increased intracellular Aβ levels can further exacerbate the genetically driven complex IV defect in sporadic Alzheimer's disease and may precipitate mitochondrial permeability transition opening. In combination, our results provide potential mechanisms to support the feed-forward hypothesis of Aβ neurotoxicity.

Abbreviations used
Ab

beta amyloidprotein

APP

amyloid precursor protein

COX

cytochrome c oxidase

DHB

dihydroxybenzoic acid

ETC

electron transport chain

MPT

mitochondrial permeability transition

NOS

nitric oxide synthase

ROS

reactive oxygen species

Alzheimer's disease (AD), the most commonly occurring form of late-onset dementia in adults, occurs in ≈ 90% of cases in a sporadic form, and in the remaining 10% of cases has an autosomal dominant pattern of inheritance. Known causes of autosomal dominant forms are mutations in either the amyloid precursor protein (APP) or one of the two known presenilin genes (PS1, PS2) (Goate et al. 1991; Alzheimer's Disease Collaborative Group 1995; Levy-Lahad et al. 1995; Sherrington et al. 1995; Hardy 1997). Autosomal dominant forms of AD are characterized by excessive secretion of β amyloid (Aβ) (Scheuner et al. 1995), and both autosomal dominant and sporadic AD brains show deposition in brain parenchyma and blood vessels (Iwatsubo et al. 1994; Wang et al. 1999) of fragments of APP derived from processing by proteases known as secretases, several of which have recently been characterized (Hussain et al. 1999; Sinha et al. 1999; Vassar et al. 1999; Yan et al. 1999). These APP fragments, known as Aβ 1–40 and 1–42/43, are also deposited in the brains of mice carrying transgenes for human APP with or without PS mutations (Masliah et al. 1996; McGowan et al. 1999; Moechars et al. 1999).

Whether Aβ deposition in brain is a primary component of AD pathogenesis or an important marker of a separate underlying pathogenic process remains unclear. In vitro experiments have demonstrated that aggregated Aβ can be neurotoxic (Bruce et al. 1996; Puttfarcken et al. 1996; Klein et al. 1999; Diana et al. 2000; Wei et al. 2000) and can perturb intracellular calcium signaling (Sheehan et al. 1997). Aβ peptides can enhance metal-catalyzed oxidation reactions (Dikalov et al. 1999) and stimulate increases in intracellular reactive oxygen species (ROS) in several cell models (Yatin et al. 1999). Aβ deposition in the brains of mice carrying a mutant APP transgene causes the development of markers of oxidative stress (Smith et al. 1998), similar to that observed in human AD brain sections (Smith et al. 1997). These findings have led to the formulation of the ‘feed-forward hypothesis’ of Aβ neurotoxicity. The major tenet of this hypothesis is that, independent of whatever causes increased Aβ deposition in AD, once present, Aβ can accelerate cell damage through oxidative stress and other mechanisms. Thus, a given cell oversecreting Aβ can damage many of its neighbors. Examples include the ability of Aβ to activate microglia and cause the secretion of several neurotoxic chemicals (Combs et al. 1999).

We have explored potential mechanisms for Aβ neurotoxicity, particularly ways in which Aβ might further disrupt mitochondrial electron transport chain (ETC) activity, already defective in AD brain (Parker et al. 1994) and transmitted through mitochondrial genes (Swerdlow et al. 1997). We found that Aβ peptides infused into brain through microdialysis probes increased ROS production in an NMDA receptor- and nitric oxide-dependent manner. Furthermore, the toxic 25–35 Aβ analog was a potent inhibitor of mitochondrial complex IV ETC activity and was able to open the mitochondrial transition pore at submicromolar concentrations. These findings support multiple potential mechanisms of Aβ neurotoxicity interacting at the mitochondrial level.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Microdialysis infusion of Aβ peptides

All animal experiments were carried out under the auspices of a protocol approved by the University of Virginia Animal Research Committee according to NIH guidelines. Under deep surgical anesthesia 3 mm Carnegie-Medicin polycarbonate dialysis probes were implanted into the dorsolateral striatum of male Sprague–Dawley rats (250–300 g) and secured to the skull using dental acrylic and screws. The coordinates from Bregma were A + 0.5, L 2.5, and V −7.0 from dura. The animals were allowed to recover for 24 h before use and were studied awake. On the day of the experiment the dialysis probe was perfused with Tris acetate-buffered artificial CSF at 2 µL/min for 2 h before any drugs were added to the perfusate. The perfusate was then changed to one containing 5 mm salicylic acid for 1 h, collection of samples began after the perfusate had been changed to contain Aβ. Aβ(1–40) peptide (Bachem) was activated by incubation for 4 days at 37°C according to the manufacturer's instructions and then diluted in artificial CSF to 200 µg/mL, which is ≈ 50 µm for the monomer peptide. Other smaller amyloid peptide fragments were diluted directly into artificial CSF without pre-incubation and used at a final concentration of 50 µm. Amyloid peptides in CSF/salicylic acid were perfused at 2 µL/min for 4–5 h; 15-min dialysate fractions were collected into 5 µL of 0.5 m HCl and stored at −80°C until assayed. In experiments involving d- or l nitroarginine (each at 10 mm) or MK-801 (100 µm) infusion, these drugs were added to the dialysate for 2 h before amyloid peptide was added.

The salicylic acid free radical trapping method is based on the hydroxylation of salicylate by hydroxyl radical or peroxynitrite anion to yield two adducts, 2,5- and 2,3-dihydroxybenzoic acid (DHB; Kaur et al. 1997; Narayan et al. 1997). DHB levels in dialysate were measured by reverse-phase HPLC. They were separated on a 3-µ C18 column (Catecholamine, Alltech, Deerfield, IL, USA) perfused at 0.8 mL/min with 50 mm monobasic sodium phosphate (pH 2.8–3.0) containing 1 mm disodium EDTA and 10% methanol. DHBs were detected with an ESA Coulochem II detector, guard cell +400 mV, E1 = −100 mV, E2 = +250 mV. Data were analyzed as the hourly output of 2,3-DHB.

Preparation of rat liver mitochondria

Following sacrifice by decapitation the liver was rapidly removed, homogenized and liver mitochondria were isolated by differential centrifugation in a buffer containing 220 mm mannitol, 70 mm sucrose, 5 mm HEPES and 0.2 mm EGTA. The crude mitochondrial pellet was washed twice in the same buffer without EGTA and then resuspended in assay buffer containing 214 mm mannitol, 70 mm sucrose, 5 mm HEPES, 1 mm potassium phosphate, 5 mm glutamate and 0.5 mm malate to a protein concentration of ≈ 25 mg/mL and held on ice.

Mitochondrial assays

The MPT assay was determined by light scatter at 540 nm at 25°C in a Cary 4E spectrophotometer operating in single beam mode. Approximately 0.8–1.0 mg of mitochondria were added to buffer to yield a final volume of 1 mL which yielded an initial absorbance of ≈ 1.9 AU. In most experiments, recording was started after a 3-min pre-incubation period in the presence or absence of Aβ. Recording began and calcium was added 1 min later to induce the transition. Swelling was monitored until maximal swelling (decrease in absorbance) had occurred or for 10–12 min.

The mitochondrial suspension was then removed from the cuvette immediately after completion of recording and centrifuged for 1 min at 14 000 g in an Eppendorf centrifuge. Supernatants were frozen immediately and stored at −80°C until assay. The portions of the mitochondrial pellets intended for electron microscopy were immediately placed in 1 mL of 2% paraformaldehyde/2.5% glutaraldehyde fixative and processed for electron microscopy as described previously (Khan et al. 2000). The remainders of the pellets were resuspended in 1 mL of assay buffer frozen at −80°C until assay of COX activity or analysis of cytochrome spectra. Duplicate experiments were acrried out to generate pellets for assay of COX.

Room temperature reduced–oxidized difference spectra were measured with a Beckman DU70 spectrophotometer. Freshly thawed pellets were resuspended in 800 µL of 20 mm potassium phosphate, pH 7.0, containing 40 µg N-dodecyl-maltoside. Freshly thawed supernatants were assayed without further treatment. The oxidized background was subtracted electronically from the reduced spectrum which was obtained following the addition of a few grains of dithionite. The concentration of cytochrome c was calculated using the 551–538 nm wavelength pair and an extinction coefficient of 21.1 mm−1.

ETC complexes were assayed using previously published methods (Parker et al. 1994). Direct inhibition of individual complexes was determined following pre-incubation of 5–20 µg mitochondrial protein for 3 min at 30°C with either Aβ(25–35) or Aβ(35–25). Assay of succinate : cytochrome c oxidoreductase followed activation of the enzyme in the presence of 10 mm sodium succinate for 7 min prior to the 3-min incubation in the presence of peptides and substrate. Cytochrome c was used to initiate the reaction. Assay of resuspended pellets from pore experiments were assayed omitting the 3 min pre-incubation.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Microdialysis infusion of Aβ into awake rat striata increases ROS production

As shown in Fig. 1, infusion of neurotoxic, aggregated Aβ(1–40) or the shorter Aβ(25–35) fragment increased the outflow of 2,3-DHB, the reaction product of salicylate with hydroxyl radicals and/or peroxynitrite (Kaur et al. 1997; Narayan et al. 1997). 2,3-DHB output was increased by both the 25–35 fragment and aggregated 1–40 amyloid and was reduced stereoselectively by nitro-l-arginine, a nonselective inhibitor of NOS (Fig. 1). Blockade of NMDA receptor channels with MK-801 also reduced 2,3-DHB output to about the same degree as did NOS inhibition. The relatively inactive Aβ(1–28) fragment was much less potent in stimulating 2,3-DHB output.

image

Figure 1. Microdialysis infusion of 1–40 or 25–35 Aβ peptide increases the outflow of 2,3-DHB. Shown are results from microdialysis infusion into striata of awake rats of 1–40, 25–35 or 1–28 Aβ peptides, as described in Materials and methods. Data presented are mean ± SEM of dialysate (2,3-DHB) levels from 4–8 independent experiments at each condition. Dialysis samples represent 15-min collections of dialysate perfused at 2 µL/min. Elimination of Aβ peptide from dialysate yielded maximal 2,3-DHB levels < 20% of peak levels found when 1–40 Aβ was present. Statistical analysis by two-way anova and Tukey's test for multiple comparisons' revealed the following: 1–40 alone different from 1–28 (p < 0.001); 1–40 alone not different from 1–40 + nitro-d-arginine (p = 0.23); 1–40 alone different from 1–40 + nitro-l-arginine (p < 0.001); 1–40 alone different from 1–40 + MK-801 (p < 0.001).

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Neurotoxic Aβ inhibit mitochondrial complex IV activity

We next examined the ability of Aβ to inhibit the mitochondrial complex IV activity of the ETC. In isolated, purified rat liver, mitochondria Aβ(25–35) produced dose-dependent inhibition of complex IV activity (Fig. 2). No significant inhibition of complex IV activity was observed with 80 µm Aβ(35–25), whereas 80 µm of the neurotoxic Aβ(25–35) fragment produced ≈ 60% inhibition of complex IV activity, well above the concentration required to demonstrate transition of the pore. The effect of Aβ(25–35) was not due to random effects on enzyme activity, as 80 µm levels did not inhibit activities of NADH : ubiquinone reductase (complex I) or succinate : cytochrome c reductase (Fig. 2).

image

Figure 2. (a) Concentration-dependent inhibition of complex IV activity by 25–35 Aβ peptide. Complex IV activity was assayed in isolated rat liver mitochondria as described in Materials and methods. Shown are mean ± SEM values from three independent experiments. Data are expressed as percentage of control complex IV activity in each experiment. Values for 40 µm 25–35 Aβ and 80 µm 25–35 Aβ were significantly different from 10 µm 25–35 Aβ by paired t-test (p < 0.01). (b) Lack of inhibition of complex I or succinate cytochrome c reductase activity by 80 µm 25–35 Aβ. Shown are mean ± SEM values for absolute enzyme activities from three independent experiments.

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Neurotoxic Aβ opens the MPT pore

We next searched for additional mitochondrial actions of neurotoxic Aβ. We observed that the 25–35 fragment was capable of initiating a classical permeability transition in isolated rat liver mitochondria at nm concentrations (Fig. 3). The permeability transition caused by the 25–35 peptide was structurally specific and produced much more slowly by comparable concentrations of the nontoxic 35–25 peptide (Fig. 3a). Beta amyloid peptide-induced permeability transition could be induced by submicromolar levels of 25–35 peptide (Fig. 3b) and was inhibited completely by cyclosporin A (Fig. 3b), which binds to mitochondrial cyclophillin D, ruthenium red which blocks mitochondrial calcium ion entry through the uniporter (Figs 3b and 4), chlorpromazine (not shown) or N-ethylmaleimide (not shown). Aβ-induced permeability transition was also blocked by ADP and magnesium ions (Fig. 4), which inhibit opening of the MTP. Electron microscopic observations of isolated mitochondria incubated with aggregated Aβ showed swelling and membrane disruption (Fig. 5). This swelling could be prevented by pre-incubation with ruthenium red, chlorpromazine, or N-ethylmaleimide.

image

Figure 3. (a) 25–35 Aβ peptide causes a mitochondrial permeability transition (MPT). MPT in rat liver mitochondria was assayed by monitoring light scattering at 540 nm as described in Materials and methods. All Aβ peptides were added at time (0) and 50 µmCaCl2 was added at +1 min. Shown are traces typical of at least three independent experiments. (b) Ruthenium red (RR) or cyclosporin A (CsA) block the induction of MPT from 20 µm 25–35 Aβ peptide. Shown also is MPT induced by 0.2 µm 25–35 Aβ. Traces are typical of at least three independent experiments. CaCl2 (50µm) was added to all samples at +1 min.

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image

Figure 4. Ruthenium red (RuRed) and Mg-ADP slow rates of mitochondrial swelling induced by 25–35 Aβ peptide and calcium. MPT was induced with 20 µm 25–35 Aβ peptide at t = −3 min. CaCl2 (50µm) was added at t = +1 min, followed by 0.1 µm RuRed at t = +2 min where noted. In other experiments Mg+2/ADP was added at t = 0. MTP was monitored by light scattering at 540 nm as described in Materials and methods. Traces are typical of at least three independent experiments.

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image

Figure 5. 25–35 Aβ peptide causes mitochondrial swelling blocked by ruthenium red, chlorpromazine or N-ethylmaleimide. See Materials and methods for details. (a) Control mitochondria, (b) mitochondria incubated with 10 µm 25–35 Aβ peptide, (c) mitochondria incubated with ruthenium red and 10 µm 25–35 Aβ peptide, (d) mitochondria incubated with 2 µm chlorpromazine and 10 µm 25–35 Aβ peptide, (e) mitochondria incubated with 20 µmN-ethylmaleimide and 10 µm 25–35 Aβ peptide.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Our results have shown that local infusion of Aβ into the striata of awake rats can induce increased oxidative stress. The hydroxylation of salicylate to 2,3-DHB is believed to represent trapping of specific ROS, either nitric oxide/peroxynitrite anion or the very reactive hydroxyl radical (Kaur et al. 1997; Narayan et al. 1997). Our dialysis results in awake animals indicate that much, but not all, of the Aβ-induced increase in ROS is dependent on the activity of NMDA receptors and NOS. While these may represent independent mechanisms of increasing ROS production, they may also be linked, as appears to be the case for NMDA-induced increases in ROS production in vivo. How Aβ might increase activation of NMDA receptors in vivo is not clear, but it may derive from processes similar to the recently described Aβ-induced increase in glutamate release from microglia (Noda et al. 1999). Subsequent glutamate-induced increases in NOS activity through NMDA receptor activation and the constitutive NOS-1 pathway are well known. Because our microdialysis experiments were carried out in striatum, it is also conceivable that stimulation of dopamine release and subsequent oxidation could contribute to our observed increase in ROS generation following Aβ infusion. Future studies in dopamine-depleted striata could address this possibility.

Our microdialysis results may assist in understanding the origin of markers of oxidative stress that are increased in AD brain, including increased nitrosylated proteins in amyloid plaques (Smith et al. 1997). Aβ deposition also activates brain microglia to increase production of NO by inducible NOS-2 (Rossi and Bianchini 1996; Akama et al. 1998; Akama and Van Eldik 2000), and increased NOS-2 activity may account for some of our results. Because we did not utilize a selective NOS inhibitor and did not perform combined NOSi–MK-801 infusions, we cannot discern which NOS isoforms are responsible for our results and whether the dependencies we observed on NOS activity and NMDA receptor function are related or independent.

Our results also provide potential intracellular mechanisms for the neurotoxicity of Aβ. Interestingly, we observed in isolated mitochondria that opening of the MTP was brought about more potently by Aβ(25–35) than was inhibition of complex IV activity. This suggests that under appropriate conditions of intraneuronal stress (elevated cytoplasmic calcium, reduced pH), increased intracellular Aβ in AD neurons may directly bring about opening of the MTP, which can initiate cell death cascades. However, because we used liver mitochondria to examine the effects of Aβ, our results may not be directly applicable to brain mitochondria. The Aβ-induced complex IV inhibition which we and colleagues (Canevari et al. 1999) have observed would be predicted to increase ROS production, initially superoxide anion, from electrons lost from the ETC. If extracellular Aβ is simultaneously causing increased NO production, the NO produced may combine with the increased superoxide anion to yield peroxynitrite, a more damaging ROS than its two precursors. Also, because exogenous NO is capable of initiating MTP opening (Cassarino et al. 1999), one can envision a situation in which build-up of intracellular and extracellular Aβ could yield exponentially increasing neurotoxicity.

There are several important limitations of our study. First, it is not known what the active species are in our Aβ(1–40) infusion studies. Pre-aggregation of Aβ(1–40), which we performed, is necessary to increase cytotoxicity. Because our dialysis probes pass molecules up to ≈ 20 kDa, it is unknown whether Aβ monomers or polymers are responsible for producing ROS. Second, we do not know if Aβ had to cross membrane barriers to bring about the apparent increase in free radical production. Also, Aβ can generate radicals by reducing metal ions. Although this may have occurred to some degree in vivo, it is not likely to have contributed significantly to our observed increases in 2,3-DHB, which were dependent on NMDA receptors and NOS activity. However, we did not specifically test for any dependence of our dialysis results on the presence of metal ions. In addition, we could not detect significant levels of 2,3-DHB in Aβ-containing dialysates that had not entered the brain. Although aggregated Aβ can produce free radicals in solution (Dikalov et al. 1999), detection by ESR is necessary to demonstrate this phenomenon. It is likely that the salicylate trapping technique we employed is less sensitive than ESR.

The source of increased Aβ deposition in sporadic AD (≈ 90% of cases), is not known with certainty. Our recent work with the cybrid model of AD supports the hypothesis that defective mitochondrial genes in AD patients are responsible. In AD cybrids there is increased secretion of 1–40 and 1–42 Aβ and increased intracellular Aβ levels (Khan et al. 2000). These increases in Aβ production in AD cybrids are caspase 3 dependent and appear to derive from an oxidative stress-mediated loss of mitochondrial bioelectric potential (ΔΨM), with increased release of cytochrome c into cytoplasm and activation of caspase 3. Because the cybrid model also recapitulates the reduced complex IV activity found in AD (Parker et al. 1994), one can envision a situation in which cells with AD mitochondrial genes accelerate their demise by overproduction of toxic Aβ, ironically through activation of cell death pathways.

If abnormal mitochondrial genes are present in AD throughout life, this paradigm may also provide a mechanism for the age-related increase in neuronal death. Cells may be able to compensate for a baseline level of oxidative stress derived initially from reduced complex IV activity alone, but as Aβ accumulation increases, these protective mechanisms fail under the burden of further loss of complex IV activity and increased NO-mediated damage to intracellular proteins. Aβ-induced disruption of mitochondrial integrity through opening of MTPs would further compromise a cell's ability to restrain activation of cell death cascades.

In summary, we propose that our results provide mechanisms to support the ‘feed-forward’ hypothesis of Aβ neurotoxicity. Our studies in cybrids have defined abnormal mitochondrial genes as a potential source of the initial increase in Aβ production in AD. The capacities to increase NO-mediated oxidative stress combined with further impairment of mitochondrial function define Aβ as a uniquely paradoxical toxin, which can accelerate its own production in vulnerable cells. Blockade of the processes responsible for its initially increased production in AD, combined with therapies to ameliorate its presence in increased amounts, could represent rational approaches to neuroprotection in AD.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This research was supported by grants from NIH and from the Dana Foundation.

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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Footnotes
  1. J. K. Parks and T. S. Smith contributed equally to this study.