8 November 2011. One of the paradoxes of Alzheimer’s disease is that while the brain is stuffed with amyloid, levels of Aβ42 in the cerebrospinal fluid (CSF) drop off. Many scientists have proposed that this is because soluble brain Aβ gets mopped up by plaques, but direct evidence for this idea has been scarce. In the November 2 Journal of Neuroscience, researchers led by Dennis Selkoe at Brigham and Women’s Hospital, Boston, in collaboration with David Holtzman and John Cirrito at Washington University in St. Louis, Missouri, strengthen the case for this hypothesis. They used microdialysis to measure brain interstitial fluid (ISF) Aβ in young, middle-aged, and old mouse models of AD. In the old mice, Aβ42 levels were low in ISF and high in plaques, and newly generated Aβ quickly absorbed onto existing deposits, suggesting the peptide gets sequestered there. In addition, the authors report that most of the free-floating Aβ42 in old, plaque-ridden mice appears to come from plaques, not neurons. Their data provide perhaps the most direct evidence to date for processes that many researchers have speculated are occurring in AD brains, to which scientists have no direct access for study.
On the other hand, a paper in the October 26 Journal of Neuroscience puts forth a more novel explanation for falling extracellular Aβ in aging AD brains. Researchers led by Gunnar Gouras, previously at Weill Cornell Medical College in New York City and now at Lund University, Sweden, report that neurons from transgenic AD mice secrete less Aβ as they age in culture. Over time, these neurons also lose the ability to spit out Aβ in response to synaptic stimulation. The results imply that AD brains might accumulate more intraneuronal Aβ as they age than healthy brains do, Gouras said, suggesting that the failure to secrete Aβ may be central to pathology. “This turns the traditional amyloid hypothesis on its head,” he noted. If the findings are confirmed by other labs, and are found to apply to aging mice in vivo, they could have implications for therapeutic strategies.
“Both papers are interesting and support findings of decreased CSF amyloid-β dynamics in aging and Alzheimer's disease,” Randall Bateman at WashU wrote to ARF. Bateman was not involved in either study.
To examine Aβ dynamics in situ, first author Soyon Hong in Selkoe’s group performed microdialysis on more than 40 awake, active APP (J20) mice at three, 12, and 24 months of age. Placing the dialysis probe in the mice hippocampus, she found that ISF Aβ, particularly Aβ42, the most toxic and aggregation-prone species, fell steadily with age. Meanwhile, levels of less soluble forms of Aβ climbed, as seen in brain extracts obtained with saline, detergent, or formic acid. Scientists have always assumed that saline extracts represented soluble, free-floating Aβ, Selkoe noted. However, they also contained large aggregates of more than 500 kD, which may actually represent Aβ globs that were loosely bound to membranes or plaques, or were intracellular before homogenization, Hong said. Dave Morgan at the University of South Florida, Tampa, who was not involved in the research, suggested the data may cause scientists to rethink what various chemically extracted fractions actually represent.
Although the microdialysis probe cutoff size allowed Aβ monomers and, to a lesser extent, dimers to pass, Hong and colleagues recovered only monomers in ISF. The complete lack of dimers suggests that this species is scarce in the ISF, Selkoe told ARF. Dimers and other oligomeric Aβ forms have more exposed hydrophobic portions than monomeric Aβ does, he pointed out, meaning they may tend to bind to nearby hydrophobic surfaces such as cell membranes and plaques. In ongoing work, Hong said, they are injecting wild-type mice with human Aβ monomers and dimers, and seeing where the peptides go.
To find out what happens to newly produced Aβ in older mouse brains, the authors injected radiolabeled, synthetic Aβ40 monomers into the hippocampal ISF through a cannula attached to the microdialysis probe. In the oldest, plaque-ridden mice, microdialysis recovered less than half as much radiolabeled Aβ as the procedure did in younger mice within 90 minutes after injection. The missing Aβ turned up in saline brain extracts. This provides direct evidence that plaques act as a sink, rapidly pulling Aβ out of the ISF and into less soluble fractions, Hong said. Some previous ISF studies in mice (see ARF related news story on Cirrito et al., 2003), and amyloid imaging studies in people (see ARF related news story on Fagan et al., 2006), also support this idea.
Finally, to get at the question of where endogenous ISF Aβ comes from at different ages, Hong and colleagues inhibited γ-secretase, the enzyme that produces Aβ. In young AD mice, this treatment resulted in a rapid fall in levels of all Aβ monomers tested. In the older mice, by contrast, Aβ38 and Aβ40 levels fell, but Aβ42 levels barely budged. The authors suggest that most of the Aβ42 in the ISF in older AD mice diffuses off deposits, rather than being freshly synthesized. Some previous work has turned up support for this idea as well (see, e.g., ARF related news story on Koffie et al., 2009).
Oligomers are widely believed to be the most toxic form of Aβ. If oligomers are drifting off plaques, then, “You’re not going to be able to do something that selectively affects oligomers without also getting rid of the fibrillar deposits, because the fibrillar deposits may be the source of the oligomers,” Morgan noted.
Gouras’ group took a different approach to the mystery of falling Aβ levels, focusing on Aβ secretion from neurons. Numerous studies have shown that when synapses are stimulated, they disgorge more of the peptide (see ARF related news story on Kamenetz et al., 2003; ARF related news story on Cirrito et al., 2005; and ARF related news story on Tampellini et al., 2009). This raised the question of whether brain activity accelerates AD pathology by contributing to extracellular plaque formation.
Gouras and colleagues suggested, however, that synaptic activity is beneficial because it removes intraneuronal Aβ that would otherwise harm synapses (see Gouras et al., 2010; Capetillo-Zarate et al., 2011). Some researchers have questioned studies on intraneuronal Aβ, claiming that existing technology is not good enough to distinguish it from its precursor (see ARF Webinar). That controversy aside, when Gouras’ group inhibited synaptic activity in AD transgenic mice, the animals made fewer extracellular plaques, but had more synapse damage and worse memory (see Tampellini et al., 2010).
Since age is the primary risk factor for AD, Gouras wondered how it might affect Aβ secretion. To investigate, first author Davide Tampellini prepared hippocampal and cortical cultures from embryonic Tg2576 mice, which express human APP with the Swedish mutation, and compared the cells at 12 and 19 days after plating. Previous work has suggested that this culture model mirrors in-vivo aging on an accelerated timeframe, with one week in culture corresponding to changes seen in one year in animals, Gouras said (see, e.g., Takahashi et al., 2004; Almeida et al., 2005; ARF related news story on Almeida et al., 2006; ARF related news story on Snyder et al., 2005; Martin et al., 2008; and Sodero et al., 2011). Some researchers expressed doubts about this in-vitro model, however. “I'm just not sure what 12-day versus 19-day neuronal cultures equate to in an aging disease,” Cirrito wrote to ARF.
At 19 days in vitro, Tampellini and colleagues saw no changes in wild-type neurons, but found that the transgenic neurons secreted about one-third less Aβ, and had about 50 percent more intracellular Aβ42, than they did at 12 days. Moreover, when older neurons were stimulated with glycine, the wild-type neurons spat out more than twice as much Aβ as before, but transgenic neurons failed to respond. These findings were unexpected, Gouras told ARF. “People have always assumed secretion went up [with age] because you see plaques outside cells.” It is not clear if secretion of other proteins also changes as the cells age.
If neurons release less Aβ with age, then why does the peptide accumulate in AD? The authors looked at Aβ clearance. They report that when the 12-day-old transgenic neurons were stimulated with glycine, the Aβ-degrading protease neprilysin migrated to the cell surface and colocalized with Aβ. The 19-day-old transgenic neurons expressed about 20 percent less neprilysin than the younger cultures. This dovetails with work showing that neprilysin expression falls with age in wild-type mouse brain (see Iwata et al., 2002). The drop in neprilysin could “play a critical role in synaptic accumulation of Aβ with aging and AD,” the authors write.
These data may shed some light on why AD develops in older brains, but not young ones, Gouras said. He pointed out that people at risk for AD show reduced brain activity decades before symptoms develop (see Reiman et al., 2004). Therefore, “AD patients should be secreting less Aβ than controls,” Gouras said, and may accumulate more intraneuronal Aβ and more synapse damage. He emphasized that he does not discount a role for extracellular Aβ and plaques in promoting pathology as well. These cell culture data would suggest that trying to lower Aβ secretion may be the wrong approach to the disease, Gouras said. Would promoting Aβ secretion be good, bad, or neutral? That question remains to be answered.—Madolyn Bowman Rogers.
Hong S, Quintero-Monzon O, Ostaszewski BL, Podlisny DR, Cavanaugh WT, Yang T, Holtzman DM, Cirrito JR, Selkoe DJ. Dynamic analysis of amyloid β-protein in behaving mice reveals opposing changes in ISF versus parenchymal Aβ during age-related plaque formation. J Neurosci. 2011 Nov 2;31(44):15861-9. Abstract
Tampellini D, Rahman N, Lin MT, Capetillo-Zarate E, Gouras GK. Impaired beta-amyloid secretion in Alzheimer’s disease pathogenesis. J Neurosci. 2011 Oct 26;31(43):15384-90. Abstract