Introduction
Alzheimer’s disease (AD) is the most prevalent cause of dementia, accounting for 50–60% of the 50 million reported cases worldwide, which is expected to triple by 2050 [
25]. A definitive diagnosis of AD remains to be only possible via neuropathological examination that demonstrates the presence of the classical disease hallmarks, namely amyloid-β (Aβ) plaques together with tau neurofibrillary tangles (NFT) [
6,
14]. However, increasingly, clinical assessment of AD is now being aided by neuropathologically validated biomarkers that reflect Aβ and tau pathologies which have led to the improved accuracy in diagnosing AD during life [
9,
13,
20]. The importance of biological markers has been emphasised in the recent National Institute of Aging and Alzheimer Association (NIA-AA) Research Framework [
13]. In this framework, AD is defined as a biological construct, documented by post-mortem examination or in vivo by biomarkers, and not as a clinical syndrome. Therefore, the term AD is applied whenever there is biomarker evidence of Aβ and tau pathology. There are two main types of biomarkers for AD, that is, neuroimaging and fluid biomarkers. Neuroimaging biomarkers include in vivo positron emission tomography (PET) using ligands specific for fibrillar Aβ [
3] and paired-helical filament tau [
17,
29]. Regarding the fluid biomarkers, the triad of cerebrospinal fluid (CSF) biomarkers, broadly referred as “
core AD biomarkers”, are widely used in both clinical and research settings. They comprise Aβ42 (or the Aβ 42/40 ratio), phosphorylated tau181 (p-tau181) and total tau (t-tau), which reflect Aβ pathology, tau pathology and neuronal injury, respectively [
21].
Despite their high specificity and sensitivity in detecting AD pathophysiology, both CSF and imaging biomarkers present certain limitations, e.g. perceived invasiveness or complexity attached to a lumbar puncture or limited access to and high costs for molecular imaging, which restrict the use of these biomarkers to specialised centres [
21]. Therefore, a blood biomarker that reliably reflects cerebral Aβ and tau pathologies has huge potential as a scalable test for primary care and frequent disease monitoring in clinical and therapeutic settings. In recent years, numerous promising studies have explored the potential of blood biomarkers to provide information on cerebral pathology. Mass spectrometry [
22,
28] and automated immunoassays [
23] measuring Aβ species have proven highly accurate,however, the considerable peripheral expression of Aβ remains to be a significant cofounder for these assays, making the fold change in Aβ42/40 ratio in amyloid PET-positive individuals much less pronounced in plasma than in CSF [
28]. On the other hand, blood immunoassays targeting tau species, specifically tau fragments phosphorylated at threonine 181, have shown promising results, proving to be reliable tools for AD diagnosis and correlating well with in vivo assessments of Aβ and tau pathologies [
15,
16,
32].
To our knowledge, and despite the very promising results in blood p-tau181, all studies conducted so far have mainly focused on research cohorts accurately characterised by CSF or PET biomarkers. Some of these studies have also validated their results in a subset of pathologically confirmed cases [
32] but it is unclear if plasma p-tau181, determined years before death, can predict the eventual neuropathological diagnosis of AD. Therefore, the main aim of this study was to investigate (1) if plasma p-tau181 specifically reflects AD pathology in neuropathologically confirmed cases, (2) if plasma p-tau181 would inform on a more accurate diagnosis of AD and highlight dementia of a non-AD type at the time of clinical assessment, and (3) if the longitudinal trajectories of plasma p-tau181 are different between neuropathologically confirmed AD patients, non-AD patients and controls. For this purpose, we measured plasma p-tau181 in a longitudinal cohort comprising cognitively unimpaired controls and participants with the clinical diagnosis of mild cognitive impairment (MCI) and AD dementia. At post-mortem, each patient was re-classified into control, AD and non-AD dementia based on a detailed neuropathological assessment.
Discussion
In a longitudinal cohort with neuropathological characterisation, we demonstrated that plasma p-tau181 predicts AD pathology at least 8 years prior to death and neuropathological confirmation, and accurately discriminates between AD and non-AD pathologies. Our data suggest, an individual clinically diagnosed of AD dementia syndrome but with low concentrations of plasma p-tau181 is more indicative of non-AD than AD pathology. We have also demonstrated that plasma p-tau181 increases over time in cases with AD pathology, most likely in parallel to neurofibrillary tangle neurodegeneration (as shown by Braak stages). This increase, however, plateaus at the very advanced stages of the disease. Altogether, our results support the idea of using plasma p-tau181 as a biomarker of AD at the clinical setting or in clinical trials when CSF and/or PET biomarkers are not available. Alternatively, plasma p-tau181 could be used as a pre-screening tool to select those patients who would further undergo lumbar puncture or PET imaging.
As the field of fluid biomarkers in neurodegeneration moves towards targeted analysis in blood, several novel assays have been developed. Assays targeting Aβ [
22,
23,
28], t-tau [
8,
24] and NfL [
2,
5,
18] in blood have demonstrated promising results. However, they are compromised by either substantial peripheral expression of the targeted protein [
26], poor correlations with CSF measures of the same protein [
19] and large overlaps between the neurodegenerative disease groups [
1,
10]. These limitations may make the use of these biomarkers to diagnose and/or predict the development of AD pathology more difficult at the individual level. A major breakthrough has been the development of assays to sensitively measure plasma p-tau181 by us and other groups [
15,
16,
32]. In contrast to the aforementioned biomarkers, p-tau181 can be robustly measured in plasma, is highly specific for AD, provides high diagnostic accuracy for discriminating AD and non-AD dementia, and it also finely discriminates between Aβ-positive CU older adults from those that are Aβ-negative. Furthermore, plasma p-tau181 correlates with CSF p-tau181 and identifies tau PET uptake, suggesting that p-tau181 found in plasma is predominately derived from the central nervous system and not from a peripheral source.
To the best of our knowledge, this is the first study of longitudinal plasma p-tau181 with a confirmed neuropathological diagnosis. Although the clinical diagnosis of AD by an expert neurologist is very reliable, it is not rare to find discordances between the clinical and the final pathological diagnosis [
4]. In our study, we initially demonstrated a significant increase in plasma p-tau181 in the AD dementia syndrome group compared to the CU groups. However, there was a large overlap in plasma p-tau181 with the control group likely owing to the lack of CSF and PET characterisation in this cohort. Remarkably, when this comparison was performed between pathologically defined groups, the magnitude of the differences between the AD pathology and control group was considerably higher. These results indicate that plasma p-tau181 is specific for AD pathology, irrespective of whether the clinical presentation resembles a typical AD dementia or another type of dementia. We further confirmed this idea by the fact that plasma p-tau181 discriminates AD pathology from non-AD pathology with an AUC of 97.4% 8 years prior to post-mortem, which is of equivalent performance to the well-established CSF AD core biomarkers (Aβ42, p-tau and t-tau) or Aβ and Tau PET. Our study to some extent mimics a still common situation in several non-specialised clinics, where these CSF or PET biomarkers are not available. Our results open the possibility of routinely using plasma p-tau181 to improve the confidence in administering symptomatic treatment (e.g. acetylcholinesterase inhibitors or memantine), or better inform on patient management. Also, it may be used in both clinical practice and clinical trials as a first screening tool that may be followed, if needed, by CSF or PET biomarker confirmation.
In addition to comparing plasma p-tau181 in clinical and neuropathological classifications, we also examined the relationship of plasma p-tau181 with Braak staging. We observed that a significant increase of plasma p-tau181 occurred between Braak I–II (Transentorhinal) and V–VI (Isocortical) at all timepoints. We also observed, at 8 years prior to post-mortem, a significant increase of plasma p-tau181 between Braak stages III–IV (Limbic) and V–VI but this was not apparent at later timepoints. Instead, 4 years and 2 years antemortem, the significant differences occurred between Braak stages I–II and III–IV. Moreover, plasma p-tau181 concentrations in individuals classified as Braak stage III–IV followed an increasing mean trend across all three timepoints. In contrast, no mean change in Braak stages I–II was observed and individuals that reached a higher degree of tau pathology, that is Isocortical Braak V–VI, began to plateau after initial significant increase between 8 and 4 years antemortem. These trajectories are probably parallel to that of Aβ pathology, which also plateaus at more advanced stages. In fact, plasma p-tau181 highly correlates with Aβ PET [
15,
16,
32]. These results suggest that plasma p-tau181 is a good diagnostic marker in both early and late stages of the disease. However, plasma p-tau181 may only be useful as a biomarker of the burden of the disease at early stages, when its levels follow an increasing trajectory, rather than at late stages, when the levels plateau.
Another novel contribution of our study is that we demonstrate that the trajectories of plasma p-tau181 change over several years in patients with AD. The availability of repeated plasma samples over the course of a decade allowed us to define this trajectory. Interestingly, we found that the main increase in plasma p-tau181 in the AD pathology group occurred from timepoint 1 (8 years prior to post-mortem) and timepoint 2 (4 years prior to post-mortem). However, between timepoint 2 (4 years prior to post-mortem) and timepoint 3 (2 years prior to post-mortem) p-tau181 began to plateau. This suggests that the main increase in plasma p-tau181 occurs several years before there is overt AD pathology. In the case of AD with contaminate pathology, this increase was less steep but still significant and following the same trajectory as AD pathology. These findings are consistent with the longitudinal studies showing that CSF p-tau or t-tau does not increase, or it may even decrease, in advanced stages of the disease, which may indicate a deceleration in neurodegeneration due to substantial neuronal loss [
31]. This hypothesis has also gained support from recent stable isotope labelling kinetics (SILK) studies that track the turnover of tau in the human central nervous system (CNS) [
27]. In contrast to the AD pathology groups, we observed a slight but statistically significant increase of plasma p-tau181 between timepoints 2 and 3 for the non-AD dementia and control groups. This slight increase cannot be attributed to underlying Aβ pathology, due to the neuropathological report, but there may be other factors that have an effect on p-tau181, such as ageing or co-pathology (TDP-43, α-synuclein).
Our study has some limitations. First, the availability of CSF or PET biomarker data would have allowed us to compare the predictive value of plasma p-tau181 with accepted gold-standard biomarkers for AD. Second, the number of CU participants that eventually were pathologically diagnosed as AD was low and, therefore, we could not test whether plasma p-tau181 can also predict AD pathology at early preclinical stages. In a similar manner, the number of MCI patients in this study was low and, therefore, the progression from MCI to AD dementia could not be investigated. Furthermore, the AD dementia patients were quite impaired already at the first timepoint, with an average MMSE score of 12. Finally, detailed Aβ pathological data, such as Thal staging, were not available for all cases. However, recently, Thal staging has been shown to not substantially contribute to predicting antemortem cognition as compared to neuritic plaque scores and Braak NFT stages [
30]. The main strength is that we studied a very well-characterised cohort of participants, with longitudinal samples and neuropathological confirmation. Furthermore, we used a very sensitive and robust p-tau181 assay which, importantly, can be easily set up in other centres and hence replicate our results.
In conclusion, our study demonstrates that plasma p-tau181 predicts AD pathology, even if the blood sample was obtained several years before the post-mortem examination. This has obvious consequences in both the design of clinical trials and at the routine clinical practice. Plasma p-tau181 could be used as a rapid and cost-effective screening tool for participant selection for therapeutic trials of AD. Furthermore, the high accuracy of p-tau181 in predicting confirmed AD neuropathology may guide clinicians in an accurate diagnosis of the underlying mechanism causing cognitive decline (AD pathology, non-AD or mixed) and, therefore, symptomatic treatment and patient management can be governed at the earliest stage with a higher degree of confidence.
Acknowledgements
Open access funding provided by University of Gothenburg. This study represents independent research partly funded by the National Institute for Health Research (NIHR) Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King’s College London. Tissue samples were supplied by The London Neurodegenerative Diseases Brain Bank, which receives funding from the UK Medical Research Council and as part of the Brains for Dementia Research programme, jointly funded by Alzheimer’s Research UK and the Alzheimer’s Society. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. TKK holds a postdoctoral fellowship from the BrightFocus Foundation (#A2020812F), and was further supported by the Swedish Alzheimer Foundation (Alzheimerfonden; #AF-930627), the Swedish Brain Foundation (Hjärnfonden; #FO2020-0240), the Swedish Dementia Foundation (Demensförbundet), the Agneta Prytz-Folkes and Gösta Folkes Foundation, Gamla Tjänarinnor, the Aina (Ann) Wallströms and Mary-Ann Sjöbloms Foundation, the Gun and Bertil Stohnes Foundation, and the Anna Lisa and Brother Björnsson’s Foundation. MSC received funding from the European Union’s Horizon 2020 Research and Innovation Program under the Marie Sklodowska-Curie action grant agreement no. 752310, and currently receives funding from Instituto de Salud Carlos III (PI19/00155) and from the Spanish Ministry of Science, Innovation and Universities (Juan de la Cierva Programme grant IJC2018-037478-I). AH is funded by Research Centre for Mental Health and Biomedical Research Unit for Dementia. KB holds the Torsten Söderberg Professorship in Medicine and is supported by grants from the Swedish Research Council, the Swedish Alzheimer Foundation, and the Swedish Brain Foundation. AH is funded by Research Centre for Mental Health and Biomedical Research Unit for Dementia. KB holds the Torsten Söderberg Professorship in Medicine and is supported by grants from the Swedish Research Council, the Swedish Alzheimer Foundation, and the Swedish Brain Foundation. KB holds the Torsten Söderberg Professorship in Medicine at the Royal Swedish Academy of Sciences, and is supported by the Swedish Research Council (#2017-00915), the Swedish Alzheimer Foundation (#AF-742881), Hjärnfonden, Sweden (#FO2017-0243), and a grant (#ALFGBG-715986) from the Swedish state under the agreement between the Swedish government and the County Councils, the ALF-agreement. NJA is supported by the Wallenberg Centre for Molecular and Translational Medicine, the Swedish Alzheimer Foundation (Alzheimerfonden), the Swedish Dementia Foundation (Demensförbundet), and Hjärnfonden, Sweden.
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