15 March 2013. Growing evidence suggests that neurons begin faltering decades in advance of cognitive symptoms in Alzheimer’s disease, but identifying the molecular culprits at play during this “invisible” phase has proven challenging. This is especially true of oligomeric forms of amyloid-β. Research reported in the March 11 JAMA Neurology proposes a step forward in the quest. Karen Ashe and colleagues at the University of Minnesota in Minneapolis, in collaboration with researchers at the University of Gothenburg in Sweden, have measured Aβ*56 in human cerebrospinal fluid (CSF). Aβ*56—convincingly to some, controversially to others, as much of the data on Aβ oligomers—is a species that reportedly wipes out memory in AD mice (see ARF related news story on Lesné et al., 2006).
In the new CSF analysis, Aβ*56 levels correlated with known CSF markers of neurodegeneration. This was the case only in cognitively normal seniors—not in people with AD or mild cognitive impairment (MCI). The findings imply that other factors elevate tau in symptomatic individuals. “This may help explain why anti-Aβ therapies have not been working in people who are symptomatic,” Ashe told Alzforum. Other scientists raised questions about the procedures used to measure the oligomers, which have been difficult to detect in human material, especially CSF.
In prior analyses with Tg2576 transgenic mice, an AD model generated in the Ashe lab, she and colleagues identified Aβ*56 as a putative 56 kDa dodecamer that correlates with cognitive impairment in this mouse strain. Aβ*56 shows up in their brains just as memory problems emerge, and levels of the oligomer track with the animal’s cognitive performance (ARF related news story on Lesné et al., 2006). Smaller Aβ species, including monomers, trimers, and hexamers, appear in Tg2576 mice prior to cognitive impairment. In this field, labs tend to stick to their own methods and protocols rather than adopt and compare each other’s for the purpose of multiple independent replication. This has so far prevented most reported oligomer species, including Aβ*56, from developing broad-based momentum.
The current paper, however, reports the first detection of Aβ*56 in human tissue. Lead author Maureen Handoko and colleagues report measuring this oligomer, as well as Aβ trimers, in the CSF of 48 cognitively impaired seniors (26 AD, 22 MCI), 49 age-matched controls with normal cognition, and 10 younger controls. The specimens came from a large, cross-sectional MCI study headed by coauthor Anders Wallin at the University of Gothenburg. One-fourth of each 1 ml CSF sample remained in Sweden, where researchers in the lab of coauthor Kaj Blennow measured CSF Aβ1-42, total tau, and phospho-tau 181 by using enzyme-linked immunosorbent assays (ELISA). The remaining 750 microliters went to the University of Minnesota, where Ashe’s group used immunoprecipitation (IP) and Western blotting to measure Aβ trimers and Aβ*56 in triplicate using 240-microliter aliquots. The Minnesota researchers were blinded to the participants’ clinical status—which the Gothenburg scientists revealed after the IP/Western data were quantitated and tabulated, Ashe said.
Among the cognitively normal volunteers, older individuals tended to have more Aβ trimers and Aβ*56 in their CSF. Moreover, Aβ*56 levels correlated with CSF tau and CSF phospho-tau levels, whereas Aβ1-42 did not. “This argues against fibrillar Aβ being coupled with tau, at least in the asymptomatic phase of disease,” Ashe said. About 10 percent of the cognitively normal older adults had an elevated CSF tau/Aβ1-42 ratio. The association was weaker in people with MCI or AD.
On a methodological level, the detection of Aβ oligomers in human CSF can be considered a feat in and of itself. Recent advances in sandwich ELISAs have enabled scientists to measure oligomers in human brain samples with high specificity, but these techniques did not detect oligomers in CSF (see ARF related news story). Ashe said her lab spent two years developing “a highly sensitive and specific immunoblot assay that can detect as little as 25-50 picograms of Aβ.” The Minneapolis group uses standard antibodies but has optimized transfer time, blocking conditions, and reagent concentrations. “All these things can drastically affect how clean your blots are,” Ashe said, noting that the detection limit for most Western blots is around 500 picograms—10 times less sensitive than her protocol. The detailed methods will appear in another manuscript, Ashe said.
The current study did not measure Aβ dimers, which some consider the most neurotoxic form of Aβ (see ARF related news story on Shankar et al., 2008; ARF news story on McDonald et al., 2010, and Villemagne et al., 2010). In unpublished work she is writing up for publication, Ashe found that Aβ dimers occur in human CSF at a ~10-fold lower concentration than Aβ trimers and Aβ*56; this ratio reverses in the brain. On her immunoblot, “you would need at least 1-2 milliliters of CSF to measure dimers,” Ashe said. Moreover, she said reliable visualization of Aβ dimers would require separate protein gels with different acrylamide concentration than those used in this study.
Some scientists were concerned about the biochemical evidence for Aβ oligomers in the present study. “It is not clear from the paper whether the detected species are Aβ rather than an APP fragment,” wrote David Brody of Washington University School of Medicine, St. Louis, Missouri, in an e-mail to Alzforum (see full comment below). He noted that levels of soluble APP in the CSF have been shown to be ~100-fold higher than Aβ (Nitsch et al., 1995). The paper reports the IP/Western blot data as densitometry light units; it does not show original blots. Some of the blots will appear in another paper currently in press in Brain.
The study does not control for the possibility that the IP/Western procedure itself could induce artifactual aggregation of Aβ, Brody noted. In a recent study (Esparza et al., 2013), he and colleagues found that monomeric Aβ can aggregate at high local concentrations, such as those occurring after immunoprecipitation. SDS, a detergent the researchers used to prepare samples for Westerns, can also induce Aβ aggregation (see Watt et al., 2013). Ashe acknowledges the potential for detergents such as SDS to trigger anomalous formation of oligomers. However, “if you do an immunoprecipitation from CSF, that eliminates this possibility because there is no detergent in CSF,” she said. On the concern about mistakenly detecting APP fragments, she said her lab has several lines of unpublished evidence indicating that they are, in fact, measuring Aβ. For example, when the researchers ran undiluted CSF on a size exclusion chromatography (SEC) column, Aβ*56 and Aβ monomers showed up in separate fractions on SDS-PAGE. Furthermore, they see no signal on Western blots probed with APP antibodies that recognize epitopes outside the Aβ region. “I’m very convinced that what we’re looking at is Aβ,” Ashe said.—Esther Landhuis.
Handoko M, Grant M, Kuskowski M, Zahs KR, Wallin A, Blennow K, Ashe KH. Correlation of Specific Amyloid-β Oligomers With Tau in Cerebrospinal Fluid From Cognitively Normal Older Adults. JAMA Neurol. 11 March 2013. Abstract