Islam T, Hill E, Abrahamson EE, Servaes S, Smirnov DS, Zeng X, Sehrawat A, Chen Y, Kac PR, Kvartsberg H, Olsson M, Sjons E, Gonzalez-Ortiz F, Therriault J, Tissot C, Del Popolo I, Rahmouni N, Richardson A, Mitchell V, Zetterberg H, Pascoal TA, Lashley T, Wall MJ, Galasko D, Rosa-Neto P, Ikonomovic MD, Blennow K, Karikari TK. Phospho-tau serine-262 and serine-356 as biomarkers of pre-tangle soluble tau assemblies in Alzheimer's disease. Nat Med. 2025 Feb 10; Epub 2025 Feb 10 PubMed.
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Washington University School of Medicine
This study is impactful because soluble tau assemblies (STA) containing phospho-tau serine-262 and phospho-tau-serine-356 were identified as components of pre-tangles, which are precursors of insoluble neurofibrillary tangles.
From previous Cryo-EM experiments, it is known that the tau filament core in AD brain is composed of amino acids 302 to 378. In this study, an integrative biochemical approach combining immunoprecipitation/immunodepletion using antibodies with various epitopes, and molecular profiling techniques, was used to identify aa 258-368 as one of the STA species. In vitro experiments were used to show that these have high aggregation competency and neurotoxicity. However, it is important to note that STA is not limited to aa 258-368, and there are various possible forms, including some containing adjacent residues.
This finding is also consistent with the previous report of Lövestam et al. on the effects of protein length on tau filament assembly (Lövestam et al., 2022).They produced recombinant tau of various lengths and reported that microtubule-binding region (MTBR) fragments with cleavage sites before the first repeat (R1) have low aggregation competency or do not form the AD filament structure. On the other hand, the aa 258-378 MTBR fragment, with a cleavage site in the middle of R1, does exhibit both strong aggregation competency and the AD filament structure.
Interestingly, Lövestam also reported that when the cleavage extends to the region spanning R3, such as aa 306 and aa 310, aggregation competency or AD filament structure is lost. This is consistent with Islam et al.’s finding that short MTBR species, e.g., aa 302-368, have relatively low aggregation competency and neurotoxicity.
Islam et al. also used pathologically confirmed, or tau-PET cohorts to test whether STAs can be used as CSF biomarkers to identify AD tau pathology in the brain, particularly at an early, pretangle stage. Their CSF STA sandwich assay used an anti-N368 neo-epitope antibody and an antibody to aa 321–371 as an antigenic epitope. In this method, the CSF STA/t-tau ratio tended to decrease with tau pathology progression, e.g., Braak stages, due to the sequestering effect of STA, which is consistent with the previous report with a similar method using an anti-N368 antibody (Blennow et al., 2020) and our other report on the R4 region peptide MTBR-tau354 (aa 354-369) in CSF using LC/MS (Horie et al., 2021; Horie et al., 2023).
However, there were large individual variations in this CSF STA biomarker, which include the tau filament core residues, and there were also large overlaps between groups. If highly sensitive methods are established for specifically measuring CSF p-tau262 and p-tau356, which are extensively discussed in this paper, then it may be possible to develop highly accurate biomarkers for identifying early stage AD tau pathology.
References:
Lövestam S, Koh FA, van Knippenberg B, Kotecha A, Murzin AG, Goedert M, Scheres SH. Assembly of recombinant tau into filaments identical to those of Alzheimer's disease and chronic traumatic encephalopathy. Elife. 2022 Mar 4;11 PubMed.
Blennow K, Chen C, Cicognola C, Wildsmith KR, Manser PT, Bohorquez SM, Zhang Z, Xie B, Peng J, Hansson O, Kvartsberg H, Portelius E, Zetterberg H, Lashley T, Brinkmalm G, Kerchner GA, Weimer RM, Ye K, Höglund K. Cerebrospinal fluid tau fragment correlates with tau PET: a candidate biomarker for tangle pathology. Brain. 2020 Feb 1;143(2):650-660. PubMed.
Horie K, Barthélemy NR, Sato C, Bateman RJ. CSF tau microtubule binding region identifies tau tangle and clinical stages of Alzheimer's disease. Brain. 2021 Mar 3;144(2):515-527. PubMed. Correction.
Horie K, Li Y, Barthélemy NR, Gordon B, Hassenstab J, Benzinger TL, Fagan AM, Morris JC, Karch CM, Xiong C, Allegri R, Mendez PC, Ikeuchi T, Kasuga K, Noble J, Farlow M, Chhatwal J, Day G, Schofield PR, Masters CL, Levin J, Jucker M, Lee JH, Roh JH, Sato C, Sachdev P, Koyama A, Reyderman L, Bateman RJ, McDade E, and the Dominantly Inherited Alzheimer Network. Change in Cerebrospinal Fluid Tau Microtubule Binding Region Detects Symptom Onset, Cognitive Decline, Tangles, and Atrophy in Dominantly Inherited Alzheimer's Disease. Ann Neurol. 2023 Jun;93(6):1158-1172. Epub 2023 Mar 16 PubMed.
View all comments by Kanta HorieEmory University
This study represents a significant step forward in understanding early tau pathology and advancing biomarker development for AD. The identification of phosphorylation at serine-262 and serine-356 within the soluble tau assembly (STA) core (~tau258–368) provides new insight into prefibrillar tau species that precede neurofibrillary tangles (NFTs). By developing a CSF assay capable of detecting these STA-specific phospho-epitopes, the authors offer a novel approach to distinguishing AD from other tauopathies while also enabling the tracking of early disease progression.
Unlike p-tau181 and p-tau217, which are strongly linked to amyloid and the emergence of fibrillar tau in NFTs, the STA-specific biomarkers (p-tau262 and p-tau356) highlight an earlier prefibrillar tau state, potentially offering a distinct window into tau pathogenesis before NFT formation. This positions them as potential tools for detecting AD at a stage where therapeutic interventions may still prevent irreversible neurodegeneration.
Beyond early detection, this STA-specific CSF assay holds promise as a pharmacodynamic marker for drug development. Current therapeutic strategies, including anti-tau antibodies and aggregation inhibitors, could benefit from a biomarker that reflects soluble tau levels before NFTs become prominent. Since clinical trials targeting amyloid-b have shown the greatest benefits in individuals with lower NFT pathology, monitoring STA levels may provide a more dynamic measure of treatment efficacy. Furthermore, the STA assay complements tau-PET imaging, which primarily detects fibrillar tau in later Braak stages. By capturing early soluble aggregates, this assay could refine patient stratification in clinical trials, ensuring that those at risk of developing symptomatic AD are identified sooner.
Despite these promising advances, there are challenges to address. Longitudinal studies are necessary to confirm whether STA levels predict disease progression reliably, and efforts to translate this assay into a less-invasive blood-based biomarker would significantly enhance accessibility. Additionally, exploring the functional impact of STA core peptides on synaptic function and network hyperexcitability may provide further mechanistic insight into tau-driven neurotoxicity.
Ultimately, this work represents a shift in focus from targeting fibrillar tau to intercepting its soluble precursors, aligning biomarker strategies with the earliest molecular drivers of AD pathology and opening new avenues for precision medicine in neurodegenerative disease.
View all comments by Seong KangNew York Institute for Basic Research in Developmental Disabilities
Tau pathology in humans or animals, without fail, is made up of the hyperphosphorylated protein. In AD tau is hyperphosphorylated sub-stoichiometrically at multiple sites by several combinations of protein kinases. Six isoforms of tau in the human brain and hyperphosphorylation at multiple sites in AD generate numerous protein species.
It is for this reason that hyperphosphorylation of tau at any one particular site in CSF or plasma has been insufficient to serve as a diagnostic test of tau pathology. The hyperphosphorylation makes tau from microtubule assembly-promoting to -disrupting molecule and promotes its self-assembly into aggregates leading to neurofibrillary tangles (NFT).
We found that p-tau had the same characteristics in plasma as in the brain from AD patients and thus can serve as a useful screening test for AD. The VeraBIND Tau test, a novel plasma assay for active tau pathology, identifies individuals with positive F18MK6240 tau-PET signal regardless of amyloid status (Feb 2025 conference news).
Although previous studies showed that tau aggregates through the microtubule binding domain repeats (MTBR) and that the abnormal hyperphosphorylation at Ser 262 and Ser 356, the only two sites on the MTBR, promotes its aggregation, the presence of this pathological change in the CSF of AD patients was not previously reported. Thus, this study by Karikari et al on the presence of tau hyperphosphorylated at Ser-262 and Ser-356 both in the brain and the CSF in in Alzheimer’s disease (AD) is highly significant.
Contrary to a previous study which detected only the amino terminal half of tau in the extracellular space in the brain, this study demonstrated the extracellular presence of full length or almost full-length hyperphosphorylated tau in AD. This suggests that passive immunization with tau antibodies is a viable therapeutic target and that the CSF level of p-tau262/356 or of the microtubule binding domain (MTBD) can serve as a useful biomarker of tau pathology to monitor tau therapeutics in clinical trials.
Given that the authors of this study have access to several cohorts of well-characterized AD and control brains and biological fluids, hopefully they will also study and report plasma levels of p-tau 262/356 from these cases.
View all comments by Khalid IqbalCliniques Universitaires Saint-Luc and Massachusetts General Hospital
This beautiful work uses human brain samples, particularly the FRET assay showing that there are soluble tau oligomers, named “soluble tau assemblies.” It is puzzling to see that these STAs are AD specific!
For the CSF analyses, I am less convinced of the authors’ interpretation. They used SIMOA with a capture antibody directed against the truncated version of tau at position 368, and then a detection antibody recognizing the C-terminal portion of the microtubule-binding region (321-371). They also named the resulting analytes “STAs,” as in the FRET experiments, when in reality I see no strong evidence that the assay measures tau assemblies. The assay quantifies all tau fragments 321-368, whether monomeric or oligomeric.
Furthermore, the normalization by total tau is questionable. When we look at the non-normalized SIMOA data (Ext. Fig. 6) nothing is significant. In my opinion, this means that increase in CSF total tau is driving the association with tau-PET stages.
A remarkable finding, though, is that they are able to reliably quantify tau MTBR in CSF using Simoa, despite low concentrations and tau fragmentation. Future studies are needed to further characterize MTBR fragments in CSF, including truncated but also post-translationally modified forms. Developing fluid biomarkers accurately reflecting AD and non-AD brain changes in tau protein is critical for including the appropriate patients in clinical trials and monitor biological responses to drug exposure.
View all comments by Bernard HanseeuwBrown University
This contains encouraging findings about the identification of phosphotau species that may correlate with pretangles and be a marker of risk for progression of AD-related cognitive decline. If replicated and expanded, these findings could provide important insights for biomarker and drug development.
The key to AD treatment will require intervening early in the disease course to arrest or substantially slow disease progression. We will need diagnostic, prognostic and potentially theragnostic tools, like those proposed, to make that possible. The phosphotau biomarkers coming into clinical practice correlate most closely with amyloid plaque burden in the brain and to a lesser degree with aggregated forms of tau.
MBTR-243 is emerging as a good marker of tau PET accumulation but it is not clear how well MBTR-243 correlates with pretangle fragments. Ultimately, it will be most impactful to have plasma tests for early-tau biomarkers.
View all comments by Stephen SallowayMichigan State University
This work is a tour de force, with a series of well-planned studies examining soluble tau assemblies (STAs) using a FRET assay in which both components are the same antibody, although the exact antibody used is not clear from the manuscript.
The authors define a core domain in these assemblies that is somewhat longer than that for neurofibrillary tangles, but, like NFTs, also has variable degrees of the tau sequence N-terminal and C-terminal to the core domains. From this, they have developed a CSF biomarker assay for these early stage pretangle assemblies, using the ratio of the STA values divided by the total tau values, which declines significantly as the fibrillar tau pathology accumulates. They demonstrated this relationship with both pathological specimens and by tau PET, providing a new fluid biomarker measure that predicts NFTs.
Curiously, both the STAs and total tau increase in parallel with NFTs, but the total tau denominator increases more, leading the ratio to decline. The apparent advantage of the ratio is to reduce variance found in the STA measure alone. A final important observation is that p-tau 262 is a good marker histologically of pretangles, which should prove useful for further experimental model and neuropathological studies.
View all comments by Dave MorganMake a Comment
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