Synapses go haywire long before people with Alzheimer’s disease suspect problems with their memory, but what primes neurons for synaptic dysfunction in the first place? Oxidative changes, particularly in mitochondria, suggest two preclinical studies in this week’s Journal of Neuroscience. Gregory Brewer and colleagues at Southern Illinois University School of Medicine, Springfield, examined neurons from AD transgenic and aging wild-type mice and saw that, even before the cells accumulate harmful free radicals, they had changes in their reduction-oxidation (redox) reactions. These were partially reversed with nicotinamide, a B3 vitamin. Researchers led by Won-Kyung Ho at Seoul National University College of Medicine, Korea, report in the second paper that oxidative stress fans mitochondrial dysfunction that triggers synaptic impairment in a different AD mouse model. Here again, antioxidants came to the rescue. Albeit done in cultured neurons and brain slices, the new work sustains hope that antioxidants may help prevent or delay AD if given early enough. Clinical trials, however, have called this theory into question.
Since growing old is the biggest risk factor for sporadic AD, Brewer figured that things that happen during aging activate the pathological cascade leading to disease. Prior aging research in people showed that the plasma redox state of two natural antioxidants—glutathione and cysteine—starts dropping after middle age (Jones, 2006). “I thought this could be a trigger,” Brewer told ARF (see Brewer, 2010).
To address that possibility, first author Debolina Ghosh and colleagues isolated neurons from the brains of old wild-type or 3xTg-AD mice, and compared their NAD(P)H levels to those of neurons from young and middle-aged wild-type and 3xTg-AD animals. NAD(P)H is the primary supplier of the electrons mitochondria need to churn out ATP. Conveniently, it has intrinsic fluorescence that allows it to be visualized under a microscope without probes. The team analyzed cultured cells to identify neuron-specific differences, minimizing hormonal, vascular, and inflammatory changes that could confound in-vivo experiments.
Wild-type and 3xTg-AD neurons were equally viable, and looked alike morphologically in culture. At two months of age, they started with comparable amounts of NAD(P)H. However, while NAD(P)H levels in normal neurons nearly doubled by 11 months before falling in old age, in 3xTg-AD cells NAD(P)H rose only slightly and dropped more precipitously with age. The researchers found a similar trend with glutathione, another reducing agent that guards against buildup of reactive oxygen species (ROS). ROS levels, by contrast, did not look higher in 3xTg-AD cells until after four months of age. “That says to us that the redox shift precedes the ROS changes,” Brewer said. In addition, neurons from AD mice, at all ages, were less efficient than wild-type cells at regenerating NAD(P)H.
Most exciting to Brewer was that all these changes—in redox, ROS, NAD(P)H recycling, and even glutamate excitotoxicity—improved by incubating neurons overnight with the antioxidant nicotinamide. A prior study had found that feeding nicotinamide to 3xTg-AD mice restored their memory deficits (see ARF related news story on Green et al., 2008). “We think we may have found an earlier upstream target that is more critically linked to metabolism,” Brewer said.
Nicotinamide functions in so many cellular events that it is hard to determine if its impact on the cell’s redox state is directly responsible for something as complex as memory, noted Kim Green of the University of California, Irvine, who led the earlier study on nicotinamide-treated 3xTg-AD mice. The cognitive recovery could come from a single upstream event or from the combined effect of many events that include the nicotinamide-induced changes, Green suggested in an e-mail to Alzforum.
Last year, UC Irvine researchers completed a six-month Phase 2 trial of oral nicotinamide in ~50 mild to moderate AD patients. Its results are inconclusive and further study is required, wrote lead investigator Steven Schreiber in an e-mail to Alzforum. The trial data has not been published. Preliminary analysis indicates the intervention was safe, meeting the trial’s primary endpoint, but did not help cognition—though with just ~25 participants per group, recruitment was “not sufficient to show cognitive improvement,” Schreiber noted. His team is trying to raise funds for a longer, larger, multicenter trial with enough power to detect a cognitive signal.
In the other Journal of Neuroscience paper, first author Sang Hun Lee and colleagues arrived at oxidative changes in their search for early events that precede the synaptic deficits of AD. Compared to wild-type cells, mature dentate granule cells from Tg2576 mice had trouble with calcium clearance, which the researchers traced to dysfunctional mitochondria (as opposed to problems with calcium trafficking elsewhere in the cell). A closer look at the Tg2576 mitochondria revealed they were depolarized, i.e., making less ATP, and produced excess ROS, compared to wild-type mitochondria. The team linked these mitochondrial deficits to dysfunctional synapses in the hippocampal CA3 region that receives dentate granule cell projections along the mossy fiber pathway. They also showed that pre-treating Tg2576 brain slices with a form of vitamin E (Hoffman-LaRoche’s Trolox) restored mitochondrial calcium uptake as well as synaptic function.
The study “suggests that the increased ROS production in mitochondria is initiating the other dysfunctions,” the authors write. The work also “unravels causal relationships between mitochondrial dysfunction and impaired short-term plasticity in early AD pathogenesis.” Other pathways can also contribute to oxidative mechanisms for AD (see ARF related news story) and, as reported in a study posted online April 18 in Acta Neuropathologica, mitochondrial DNA polymorphisms that drive up ATP production can reduce Aβ load in mice (Scheffler et al., 2012).
“With a number of cautions, especially extrapolating from mice to humans, the [present studies] add support to the potential for antioxidant interventions as a preventive strategy for AD, although they do not discount the possibility that antioxidant pathways or damage may be important throughout the course of the disease,” wrote Douglas Galasko of the University of California, San Diego, in an e-mail to Alzforum (see full comment below). Galasko led a recent antioxidant trial with sobering results. In the four-month randomized trial, the antioxidant supplements hit their intended target in the brain, yet failed to move cerebrospinal fluid AD biomarkers, and even appeared to hasten cognitive decline, when given to people with mild to moderate AD (ARF related news story on Galasko et al., 2010).—Esther Landhuis