Tau Biomarkers—More Informative Fragments in Spinal Fluid?

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
 

Comments

  1. This study confirms the observation we made years ago that the majority of tau in cerebrospinal fluid (CSF) is not full-length, but consists of fragments that are detectable using N-terminal antibodies. This new study makes use of an extensive panel of well-characterized antibodies, as well as more comprehensive separation techniques, and has identified that there are several different tau fragments in CSF. The steps that result in cleavage of tau as it is released from cells, or after its release, have not been clarified, and it is possible that different enzymes are involved in tau release into CSF in patients with AD relative to controls. Therefore it is a worthwhile exercise to try to characterize and compare the fragments, as has been done in this new study. The researchers found that different antibody combinations gave slightly different results regarding the concentrations of tau that were measured in CSF, and that the degree of distinction between AD and controls varied slightly depending on which antibody combination was used. These findings could help develop the most AD-appropriate assay to measure CSF tau, and also evaluate if treatment interventions alter tau processing as it gets into CSF.

    View all comments by Douglas Galasko

  2. This study confirms what we (see Nov 2012 news story) and others (Johnson et al., 1997) have shown—that tau is secreted into the cerebrospinal fluid (CSF) in both healthy people and AD patients as fragments and not full-length. The study also confirms our findings that tau is enriched in AD patient cerebrospinal fluids over those of healthy controls.

    We also have disclosed that tau fragments are detected in the conditioned media from cortical neurons, in interstitial fluids, and CSF from mouse tauopathy models. Our findings are consistent with this new data.

    We’re gratified to see complementary data being published by others in the field. Subsequent to the Society for Neuroscience 2012 meeting, we selected our lead therapeutic tau antibody based on our secreted tau findings and on our hypothesis that secreted tau fragments drive tauopathy progression. As such, we developed assays to specifically measure secreted tau in in vivo tauopathy models and correlate secreted tau with efficacy endpoints. We are very pleased with the strong efficacy our lead antibody has demonstrated in these models. Given the program’s progress and advanced preclinical stage, we will be presenting this at SfN this year. I believe this presents a significant advance in the field by providing evidence that blocking secreted tau fragments reduces the development of tauopathy.

    View all comments by Irene Griswold-Prenner

  3. We did not study CSF-tau in our lab, but our thinking was always primed by the early findings of Gail Johnson, Doug Galasko, and colleagues that CSF tau consists essentially of N-terminal fragments in the range of 25 kD (Johnson et al., 1997). In the new publication, that paper is quoted as reference 33, but Doug's name does not appear because of the method of citation used by PlosOne and many other journals, which often leaves key authors unmentioned. It must be pleasing to Galasko and colleagues that their result was confirmed, now using much more refined methods. Time will tell whether the new knowledge will improve tau's value as an early biomarker. But it is interesting to compare this study with the recent paper by Dave Holtzman and colleagues (Yamada et al., 2011), who found full-length tau in the nanomolar range (1,000 times lower than the estimated intraneuronal levels) in the interstitial fluid, as measured by in-vivo microdialysis of mouse brains. The question therefore arises, where and how does the tau that winds up in the CSF loose its C-terminal half? This could be due to various extracellular proteases, difficult to judge as long as the cleavage sites are not known in detail. However, the question is relevant in the context of the current discussion on tau migration across cell barriers and the possibility that certain fragments and aggregates thereof transmit pathological conformations (see e.g. recent papers by the groups of Marc Diamond, Brad Hyman, or Karen Duff). This is in particular true for fragments derived from the C-terminal half of tau containing the repeat domain because this contains the aggregation signal in the form of short hexapeptide motifs (von Bergen et al., 2000). I therefore suspect that the interesting part of the story is yet to come—where are the missing C-terminal peptides, and what might they be doing?

    References:

    Johnson GV, Seubert P, Cox TM, Motter R, Brown JP, Galasko D. The tau protein in human cerebrospinal fluid in Alzheimer's disease consists of proteolytically derived fragments. J Neurochem. 1997 Jan;68(1):430-3. PubMed.

    Yamada K, Cirrito JR, Stewart FR, Jiang H, Finn MB, Holmes BB, Binder LI, Mandelkow EM, Diamond MI, Lee VM, Holtzman DM. In vivo microdialysis reveals age-dependent decrease of brain interstitial fluid tau levels in P301S human tau transgenic mice. J Neurosci. 2011 Sep 14;31(37):13110-7. PubMed.

    von Bergen M, Friedhoff P, Biernat J, Heberle J, Mandelkow EM, Mandelkow E. Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5129-34. PubMed.

    View all comments by Eckhard Mandelkow

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References

News Citations

  1. Multi-Paper Alert: More Data That Brain Amyloid Is Bad for You
  2. SfN: Tau Toxicity in the Limelight
  3. Brain Microdialysis Reveals Tau, Synuclein Outside of Cells
  4. Truncated Tau Triggers Tangles, Transmits Pathology
  5. Antibodies Stop Toxic Tau in Its Extracellular Tracks

Paper Citations

  2. Hansson O, Zetterberg H, Buchhave P, Londos E, Blennow K, Minthon L. Association between CSF biomarkers and incipient Alzheimer's disease in patients with mild cognitive impairment: a follow-up study. Lancet Neurol. 2006 Mar;5(3):228-34. PubMed.
  3. Mattsson N, Zetterberg H, Hansson O, Andreasen N, Parnetti L, Jonsson M, Herukka SK, van der Flier WM, Blankenstein MA, Ewers M, Rich K, Kaiser E, Verbeek M, Tsolaki M, Mulugeta E, Rosén E, Aarsland D, Visser PJ, Schröder J, Marcusson J, de Leon M, Hampel H, Scheltens P, Pirttilä T, Wallin A, Jönhagen ME, Minthon L, Winblad B, Blennow K. CSF biomarkers and incipient Alzheimer disease in patients with mild cognitive impairment. JAMA. 2009 Jul 22;302(4):385-93. PubMed.
  4. Johnson GV, Seubert P, Cox TM, Motter R, Brown JP, Galasko D. The tau protein in human cerebrospinal fluid in Alzheimer's disease consists of proteolytically derived fragments. J Neurochem. 1997 Jan;68(1):430-3. PubMed.
  5. Sjögren M, Davidsson P, Tullberg M, Minthon L, Wallin A, Wikkelso C, Granérus AK, Vanderstichele H, Vanmechelen E, Blennow K. Both total and phosphorylated tau are increased in Alzheimer's disease. J Neurol Neurosurg Psychiatry. 2001 May;70(5):624-30. PubMed.
  6. Portelius E, Hansson SF, Tran AJ, Zetterberg H, Grognet P, Vanmechelen E, Höglund K, Brinkmalm G, Westman-Brinkmalm A, Nordhoff E, Blennow K, Gobom J. Characterization of tau in cerebrospinal fluid using mass spectrometry. J Proteome Res. 2008 May;7(5):2114-20. PubMed.
  7. von Bergen M, Friedhoff P, Biernat J, Heberle J, Mandelkow EM, Mandelkow E. Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5129-34. PubMed.

Further Reading

Primary Papers

  1. Meredith JE, Sankaranarayanan S, Guss V, Lanzetti AJ, Berisha F, Neely RJ, Slemmon JR, Portelius E, Zetterberg H, Blennow K, Soares H, Ahlijanian M, Albright CF. Characterization of Novel CSF Tau and ptau Biomarkers for Alzheimer's Disease. PLoS One. 2013;8(10):e76523. PubMed.

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