With continued focus on fluid biomarkers for Alzheimer’s disease and emerging evidence that pathological tau travels between neurons, scientists are coming up with better ways to measure tau in cerebrospinal fluid (CSF). In the October 7 PLoS ONE, researchers led by Charles Albright, Bristol-Myers Squibb, Wallingford, Connecticut, propose that N-terminal fragments of tau may be a more sensitive AD biomarker than the measures of total and phosphorylated tau that are now widely used in clinical research. The new assays also could give scientists insight into which tau fragments drive pathology in AD and other brain disorders.
CSF tau and phospho-tau levels climb two- to threefold in people with AD (Shaw et al., 2009; Hansson et al., 2006; Dec 2009 news story on Mattsson et al., 2009). Prior research suggests that tau populates CSF primarily as fragments rather than as a full-length protein (see Johnson et al., 1997; Sjögren et al., 2001; Portelius et al., 2008; Nov 2012 conference story). However, scientists have had trouble determining the molecular nature of CSF tau, partly because concentrations of the various fragments are low and limited fluid can be drawn from human volunteers. Concentrating the fluid by immunoprecipitation pulls down only a subset of the peptides recognized by available antibodies, thus restricting the number of fragments seen by Western blotting. Likewise, mass spectrometry may fail to capture the fragments’ full breadth, because it relies on proteolytic digestion prior to analysis, making it hard to determine the size of fragments in their native state.
To get a better picture of the different tau fragments in human CSF, co-first authors Jere Meredith and Sethu Sankaranarayanan used reverse-phase, high-performance liquid chromatography (HPLC) to enrich tau prior to Western blotting. Tau traverses the reverse-phase matrix faster than do other CSF proteins, and emerges at least 50 times more concentrated than when it goes in, Albright said. Immunoblotting such preparations with various tau antibodies revealed a wide range of N-terminal and mid-domain fragments in both control and AD CSF. At this point, the BMS researchers have no sequence information on the fragments. However, Meredith noted, the number of bands detected is “more than previously reported.” Curiously, but consistent with prior studies, antibodies to C-terminal tau regions picked up nothing.
Next, the researchers turned to the more quantitative ELISA to determine if the CSF tau fragments could be more informative than “total tau” measures from existing immunoassays. They developed five tau ELISAs and three phospho-tau ELISAs to capture different overlapping parts of the protein. They then determined how well the immunoassays could distinguish 20 people with AD from the same number of healthy volunteers. The two ELISAs that measured N-terminal sequences differentiated disease from control slightly better than currently used methods, which use antibodies that recognize the mid-domain of the protein. The ELISAs specific for the C-terminal region of tau came up empty. The p-tau ELISAs also discriminated between AD and control, but did so less robustly than immunoassays that captured N-terminal tau fragments. Taken together, the data support the idea that differentiating AD from controls “is dependent on the subset of CSF tau species measured,” the authors write.
“This is a very comprehensive effort. It creates a higher-resolution picture of the tau isoforms present in the CSF,” John Trojanowski, University of Pennsylvania School of Medicine, Philadelphia, told Alzforum. Douglas Galasko of the University of California, San Diego, agreed, and added that with further study in larger cohorts, the findings could help researchers develop better assays for measuring CSF tau in AD (see full comment below). The results suggest “that an assay more specific to tau’s N-terminal region [than those in current use] may be a more robust AD biomarker,” Meredith said. “We would like to test this idea.” Most CSF tau and p-tau data reported thus far come from the commercially available INNO-BIA AlzBio3 and INNOTEST plate ELISAs, which measure tau and p-tau with antibodies specific for the mid-domain region of the protein.
The authors are trying to develop additional antibodies for the C-terminal end of tau. The failure to pick up these fragments in the current analysis could be because they are below the level of detection, Albright told Alzforum. “There may be some C-terminal pieces in the CSF, but we need to improve our sensitivity.” He and others also raised the possibility that the C-terminal fragments are not making it into the CSF. The C-terminal end of tau includes the microtubule-binding domain, which contains short hexapeptide motifs needed for tau to self-assemble into pathological filaments (see von Bergen et al., 2000). “If fragments containing this region are generated, they may not get to CSF because they may aggregate,” Albright said. Eckhard Mandelkow of the German Center for Neurodegenerative Diseases (DZNE) in Bonn agreed. “The interesting part of the story is yet to come—where are the missing C-terminal peptides, and what might they be doing?” he asked in an email to Alzforum (see full comment below)
Another mystery is where and how the fragmentation occurs. In-vivo microdialysis finds full-length tau in the interstitial fluid (ISF) of mouse brains (see Sep 2011 news story), suggesting that neurons release full-length tau. Whether they release fragments as well is unclear. It is also possible that tau gets cleaved by intracellular proteases prior to secretion, researchers agreed. These issues are important in light of recent work suggesting that truncated tau may be more damaging than the full-length protein, and that antibodies curbing tau’s extracellular spread can relieve pathology in tauopathy mice (see Feb 2013 news story; Sep 2013 news story). Irene Griswold-Prenner of iPierian, a biotech company in South San Francisco, California, told Alzforum her company will report similar preclinical data on its therapeutic tau antibodies at the upcoming Society for Neuroscience meeting in San Diego (see also Nov 2012 conference story).
The authors plan to test the new ELISAs in people with other tauopathies to see if their CSF tau profiles differ from those in AD. The commercially available CSF tau assays do return different, and puzzling, results, where Alzheimer’s shows a CSF tau increase but taupathies such as frontotemporal dementia and supranuclear palsy do not. The scientists would also like to investigate the mechanisms for generation of the N-terminal fragments and their potential functions.—Esther Landhuis
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