Aging trumps everything as a risk factor for sporadic AD, but after decades of study, researchers are still unclear why. One theory blames accumulating oxidative damage, a metabolic consequence of getting older. Three recent articles lend new support to that theory. Two cell culture studies point to mechanisms by which oxidative stress wreaks havoc on components of the γ-secretase complex and hence amyloid processing. Another reports that reducing oxidative damage in mice from birth alleviates amyloidβ accumulation. Together, the studies reveal that age-related oxidative stress is not just bad for neurons, it can specifically exacerbate AD pathology.
Presenilin (PS), the catalytic component of γ-secretase, cleaves APP to yield Aβ40 or Aβ42 (the species more prone to aggregation). Some familial AD PS mutations cause an increase in the Aβ42/Aβ40 ratio, a change that is pathogenic. Could age-related changes affect γ-secretase in similar ways? Bart De Strooper, K.U. Leuven in Belgium, and colleagues set out to determine just that, and published their findings April 10 in EMBO Molecular Medicine. First author Francesc Guix grew rat hippocampal neurons in culture and watched them age for four weeks, during which time they accumulated reactive oxygen species and underwent additional aging processes similar to what happens in vivo over much longer time frames. Between three and four weeks of age, the cells increased Aβ production and also raised the Aβ42:40 ratio. The group wondered if peroxynitrite, an oxidant that accumulates during aging and irreversibly modifies protein tyrosine residues by nitrating them (a process called nitrotyrosination), might somehow alter γ-secretase to change Aβ processing. Guix found that between three and four weeks of age (the same at which Aβ processing went awry), protein nitrotyrosination tripled relative to two-week-old neurons. Nitration is widespread in AD brains (see Smith et al., 1997 and Castegna et al., 2003) and nitrotyrosination has been implicated in AD pathogenesis (see Tran et al., 2003).
To see if peroxynitrite might be to blame for the modified Aβ levels observed in these aged cells, the team treated younger, two-week-old neurons with a peroxynitrite generator, SIN-1. Sure enough, Aβ42 and the Aβ42:40 ratio rose dramatically, just as it did in aged neurons. The same was true for human embryonic kidney (HEK) cells treated with SIN-1. Treatment with hydrogen peroxide however, did not produce the same results, suggesting the effect was specific to nitrosative stress. To determine if nitrotyrosination specifically modified γ-secretase, the research group isolated microsomes containing the protease from SIN-1 treated and untreated HEK cells and combined them with APP. The Aβ42:40 ratio nearly doubled in microsomes from treated HEK cells relative to that of the untreated ones.
How was γ-secretase being changed? On adding higher amounts of SIN-1, the C-terminal fragments and N-terminal fragments of presenilin 1 appeared to associate more strongly, based on immunoprecipitation reactions. Interaction between these two fragments is known to raise the Aβ42:40 ratio in familial AD (FAD) cases (see Berezovska et al., 2005). "We think that the nitration of the presenilin is mimicking, to some extent, this aspect of FAD," said Guix. Fluorescence-lifetime imaging microscopy, which estimates how close two protein partners are, confirmed that nitrosative stress brought the two PS ends closer together in HEK cells. After treating the cells with SIN-1, the C-terminal fragment of PS1 also bound an anti-nitrotyrosine antibody. Also, in postmortem AD patients' brains, high levels of presenilin nitrotyrosination turned up compared to age-matched controls. All this evidence points to nitrotyrosination of PS1 inducing a conformational change that leads to changes in Aβ.
Finally, Guix and colleagues wanted to know what causes the increase in peroxynitrite with age. Peroxynitrite is formed when superoxide anion, a product of the mitochondrial electron transport chain, reacts with nitric oxide. Superoxide dismutase 2 (SOD2) sops up superoxide, but its activity had dropped threefold in four-week-old neurons compared to three-week-old ones. SOD2 knockout neurons generated a higher Aβ42:40 ratio compared to wild type, and mice that produced only half the normal amount of SOD2 protein had widespread nitrotyrosination in their brains. The C- and N-terminal fragments of presenilin 1 were closer together in these mice and Aβ42:40 ratios were higher. The results indicate that a drop in SOD2 activity unfetters peroxynitrite, shifting γ-secretase processing toward Aβ42 production.
"It's a nice study with a provocative set of data that may end up suggesting some therapeutic approaches down the road," said Michael Wolfe, Brigham and Women's Hospital, Boston, Massachusetts. "The fact that a neuron undergoing this nitrosative stress has a change in its γ-secretase enzymatic properties so that you get more Aβ42 to Aβ40 is very novel and potentially important." However, he cautioned that cellular models do not necessarily provide an exact replica of what happens inside the brain, and that more work is needed to determine whether more Aβ is produced and a higher Aβ42:40 ratio exists in people with sporadic AD.
Additional work is needed to figure out why SOD2 activity drops with aging, said Gunnar Gouras, Lund University, Sweden, though overall, "it's a rigorous, logical paper that provides a mechanistic link between oxidative stress, aging and elevation of Aβ42—which is key to AD—via the nitration of presenilin," he said.
Antioxidants, which would counteract oxidative stress, have been tested before as AD therapeutics and most have shown little to no effect on people with the disease (for an overview, see the AlzRisk). But the current study supports early administration of such treatments—at mid-life or even before, Guix said. "I think it's important to treat the patient earlier so that the intervention is done before the damage occurs."
Nicastrin, another component of the γ-secretase complex, may also be modified by oxidative stress, suggests a study led by Mark Mattson, National Institute on Aging, Baltimore, Maryland and Dong-Gyu Jo, Sungkyunkwan University, Suwon, Korea and published online March 10 in Aging Cell. Co-first authors A-Ryeong Gwon, Jong-Sung Park and Thiruma Arumugam found that nicastrin, which acts as an APP receptor, had a higher binding affinity for the substrate after modification by 4-hydroxynonenal (HNE), a product of membrane lipid peroxidation. Higher HNE-nicastrin levels correlated with more γ-secretase activity as well as greater Aβ plaques in cultured neurons and in the brains of people with AD. Could blocking this nicastrin modification prove beneficial? Gwon and colleagues found that a histdine analog called AG/01, which scavenges HNE, diminished γ-secretase activity in cultured rat neurons and reduced Aβ42 production in human neuroblastoma-derived (SH-SY5Y) cells overexpressing the Swedish APP mutant. Treating triple transgenic mice (3xTg-AD) every other day for a month with AG/01 suppressed γ-secretase activity, Aβ42, HNE-modifed nicastrin and lowered the Aβ42:40 ratio in the brain compared to untreated mice, suggesting HNE-targeted treatments could be possible AD therapies, the authors wrote.
"The combination of both studies gives very strong support to the idea that oxidative stress links aging with γ-secretase and provides some mechanisms by which aging increases the risk for Alzheimer's disease," said Guix. Further, the two papers "are the first to identify specific oxidative stress-induced molecular modifications of proteins involved in APP processing that result in increased neurotoxic Aβ42," Mattson told Alzforum in an email. He and his co-authors also pointed out that Aβ reportedly enhances oxidative stress on cells, meaning a vicious, self-perpetuating cycle could be in play whereby lipid peroxidation leads to Aβ production, which in turn leads to further lipid peroxidation.
"There are almost too many smoking guns to decide that a single one is dominant," said Douglas Galasko, University of California, San Diego. "We need to understand biochemical mechanisms and pathways that predispose towards sporadic Alzheimer's disease to replace ' aging'—which is a black box—with a series of specific events, to study how they can effect pathways that are relevant to Alzheimer's disease." Galasko recently completed an unsuccessful trial of antioxidants aimed at treating people with mild to moderate AD (see ARF related news story). Studies such as the one from De Strooper and colleagues do make antioxidants attractive AD therapies, he said, but before undertaking any more large preventative studies, researchers should identify antioxidants most likely to enter the brain and protect from relevant damage, he added.
One potential treatment, suggests a Human Molecular Genetics paper published April 5 by Hemachandra Reddy, Oregon Health and Science University, Beaverton, and colleagues, aims to enhance mitochondrial catalase (MCAT), one of the body's own antioxidants. This enzyme quenches hydrogen peroxide, which is readily converted into damaging radicals that cause lipid peroxidation, mitochondrial dysfunction and neuron damage. First author Peizhong Mao crossed mice that overexpress human MCAT (see ARF related news story) with those that overproduce Aβ and show cognitive deficits (Tg2576) and found that a lifelong boost in MCAT expression lessens Aβ pathology. Compared to control Tg2576 mice, the double mutants had reduced evidence of oxidative damage, less BACE1, and fewer Aβ monomers, oligomers and plaques. They also processed APP to a greater extent through the non-amyloidogenic alpha-secretase pathway. The double mutants also enjoyed a longer lifespan. Not only do these results implicate oxidative stress in AD pathology, but they also suggest a potential way to prevent the disease, by enhancing the cell's own mitochondrial anti-oxidants, wrote the authors.
"If we can somehow enhance brain mitochondrial catalase early on in life, we can possibly delay or prevent the disease process," said Reddy.
"It's a nice approach to blocking the oxidative stress at its main source very early on, upstream of altered APP processing and altered APP production," said Mattson. Even without drugs, it may be possible to give the cell's own antioxidants a boost with exercise and dietary restriction, which mildly stresses cells and enhances their ability to cope with more severe stress, he said.—Gwyneth Dickey Zakaib
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