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- Materials and methods
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.
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.
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- Materials and methods
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.