ARTICLE https://doi.org/10.1038/s41467-021-24319-x OPEN Phosphorylated tau181 in plasma as a potential biomarker for Alzheimer’s disease in adults with Down syndrome Alberto Lleó 1,2,13 ✉, Henrik Zetterberg 3,4,5,6,13, Jordi Pegueroles1,2, Thomas K. Karikari 3, María Carmona-Iragui1,2,7, Nicholas J. Ashton3,8,9,10, Victor Montal1,2, Isabel Barroeta1,2, Juan Lantero-Rodríguez 3, Laura Videla1,2,7, Miren Altuna 1,2, Bessy Benejam1,2,7, Susana Fernandez7, 1234567890():,; Silvia Valldeneu1,2, Diana Garzón1,2, Alexandre Bejanin 1,2, Maria Florencia Iulita1,2, Valle Camacho 1, Santiago Medrano-Martorell 11, Olivia Belbin 1,2, Jordi Clarimon1,2, Sylvain Lehmann12, Daniel Alcolea 1,2, Rafael Blesa1,2, Kaj Blennow 3,4,14 & Juan Fortea1,2,7,14 ✉ Plasma tau phosphorylated at threonine 181 (p-tau181) predicts Alzheimer’s disease (AD) pathology with high accuracy in the general population. In this study, we investigated plasma p-tau181 as a biomarker of AD in individuals with Down syndrome (DS). We included 366 adults with DS (240 asymptomatic, 43 prodromal AD, 83 AD dementia) and 44 euploid cognitively normal controls. We measured plasma p-tau181 with a Single molecule array (Simoa) assay. We examined the diagnostic performance of p-tau181 for the detection of AD and the relationship with other fluid and imaging biomarkers. Plasma p-tau181 concentration showed an area under the curve of 0.80 [95% CI 0.73–0.87] and 0.92 [95% CI 0.89–0.95] for the discrimination between asymptomatic individuals versus those in the prodromal and dementia groups, respectively. Plasma p-tau181 correlated with atrophy and hypometabolism in temporoparietal regions. Our findings indicate that plasma p-tau181 concentration can be useful to detect AD in DS. 1 Memory Unit, Department of Neurology, Hospital de la Santa Creu i Sant Pau, Biomedical Research Institute Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain. 2 Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain. 3 Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, the Sahlgrenska Academy at the University of Gothenburg, Möndal, Sweden. 4 Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden. 5 UK Dementia Research Institute at UCL, London, UK. 6 Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK. 7 Barcelona Down Medical Center, Fundació Catalana Síndrome de Down, Barcelona, Spain. 8 Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden. 9 Department of Old Age Psychiatry, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, UK. 10 NIHR Biomedical Research Centre for Mental Health and Biomedical Research Unit for Dementia at South London and Maudsley NHS Foundation, London, UK. 11 Hospital del Mar-Universitat Autònoma Barcelona (UAB), Barcelona, Spain. 12 The Institute for Neurosciences of Montpellier, Université de Montpellier, Centre Hospitalier Universitaire de Montpellier, INSERM, Montpellier, France. 13These authors contributed equally: Alberto Lleó, Henrik Zetterberg. 14These authors jointly supervised this work: Kaj Blennow, Juan Fortea. ✉email: alleo@santpau.cat; jfortea@santpau.cat NATURE COMMUNICATIONS | (2021)12:4304 | https://doi.org/10.1038/s41467-021-24319-x | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24319-x D own syndrome (DS) is the most frequent form of devel- There were no differences in plasma p-tau181 concentration opmental intellectual disability of genetic origin, affecting between asymptomatic DS participants and controls (11.36 pg/ 5.8 million people worldwide1. Due to improved medical mL vs 9.31 pg/mL; p = 0.139; Fig. 2A, Table 1). Median plasma p- care, life expectancy has dramatically increased, currently tau181 concentration in participants with DS and prodromal AD exceeding 60 years of age2. However, age-related comorbidities and AD dementia were increased approximately two-fold have also emerged. In particular, the lifetime risk of Alzheimer’s compared to those of asymptomatic participants with consider- disease (AD) in people with DS is over 90%3,4. As a result, AD able overlap (Fig. 2A, Table 1; p < 0.0001). Plasma p-tau181 has become the main cause of death in this population5. In spite concentration in participants with DS and AD dementia were of the strong association between DS and AD, this comorbidity is higher compared to those of participants with prodromal AD still underrecognized and underdiagnosed1, which prevents or (32.58 pg/mL vs 21.72 pg/mL; p = 0.024, Fig. 2A, Table 1). Plasma delays access to appropriate medical care and clinical trials. p-tau181 concentration was higher in participants with moderate We and others have described the usefulness of cerebrospinal or severe/profound intellectual disability than in those with mild fluid (CSF) and amyloid positron emission tomography (PET) intellectual disability (Supplementary Fig. 1). We next analysed biomarkers to detect the core neuropathological hallmarks of AD the accuracy of plasma p-tau181 concentration for the diagnosis in this population5–8. All these studies support the notion that of AD in DS (Fig. 2B). The AUC was 0.80 [95% CI 0.73–0.87] for AD in DS recapitulates the pattern observed in both sporadic and the comparison between asymptomatic individuals versus those autosomal-dominant AD. However, despite the high accuracy of with prodromal AD and 0.92 [95% CI 0.89–0.95] for the core CSF and imaging biomarkers to detect AD, the costs and low comparison between asymptomatic individuals versus those with accessibility of these tests may limit the broad application in AD dementia. The AUC was 0.88 [95% CI 0.84–0.91] for the clinical routine, restricting their use to specialized centres. In comparison between asymptomatic individuals versus those with recent years, blood biomarkers have emerged as an easy and cost- symptomatic AD (prodromal and dementia groups combined). effective alternative for the screening of AD9. In particular, The diagnostic performance of p-tau181 increased in combina- plasma NfL and the ratio Aβ1–42/1–40 have been described as tion with age and APOEε4 status (Supplementary Fig. 2). For accurate peripheral markers to detect neurodegeneration and comparison purposes, diagnostic performance of CSF biomarkers brain amyloidosis, respectively10–12. However, changes in plasma is shown in Supplementary Fig. 3. NfL are not specific to AD10, and changes in plasma Aβ levels (and the ratio Aβ1–42/1–40) are small compared to the change in Comparisons of plasma p-tau181 and plasma NfL. We next CSF13, which confers high demands on between-assay precision compared the diagnostic accuracy of plasma p-tau181 levels with for clinical routine. A recent advance has been the description of the levels in plasma of NfL in the subset of participants with both plasma p-tau assays, mainly p-tau181 and p-tau217, that can measures (Fig. 2C, Supplementary Table 1, n = 289)7. The AUC differentiate AD from other neurodegenerative diseases with high for the comparison between asymptomatic individuals versus accuracy14–19. Currently, only one small study20 has investigated those with prodromal AD was 0.88 [95% CI 0.82–0.93] for p- plasma p-tau181 levels in 20 adults with DS. However, the tau181 and 0.86 [95% CI 0.81–0.92] for NfL. The AUC for the method lacked analytical sensitivity to measure plasma p-tau in comparison between asymptomatic individuals versus those with around half of DS cases, and that study did not assess the AD AD dementia was 0.94 (95% CI 0.91–0.97) for p-tau181 and 0.96 clinical status or the diagnostic performance of this biomarker. [95% CI 0.93–0.98] for NfL (Fig. 2C and D). The AUC was 0.92 In this cross-sectional study, we assessed the accuracy of p- [95% CI 0.89–0.95] for plasma p-tau181 and 0.93 [95% CI tau181 in plasma to detect AD in a large cohort of adults with DS 0.90–0.96] for plasma NfL for the comparison between asymp- and describe the association with other biochemical and neuroi- tomatic individuals versus those with symptomatic AD (pro- maging AD biomarkers. dromal and dementia). The differences in diagnostic accuracy between plasma p-tau181 and plasma NfL were not statistically significant. Results Participants. We included 366 adults with DS and 44 euploid controls. Table 1 summarizes the demographics, cognitive and Associations with other fluid biomarkers. We also analysed the biomarker data. 170 (46.4%) of the participants with DS and 23 correlation between log-transformed plasma p-tau181 levels and (52.3%) of the controls were female. Among the participants with other core AD fluid biomarkers (Supplementary Fig. 2). In sub- DS, 21.9% had mild intellectual disability, 51.1% moderate and jects with DS plasma p-tau181 concentration correlated with 27.0% severe or profound. Of the participants with DS, 240 plasma NfL concentration (rho = 0.70; p < 0.0001). In paired (65.6%) were asymptomatic, 43 (11.7%) had prodromal AD and plasma-CSF samples there was a correlation between plasma p- 83 (22.7%) had AD dementia at the time of blood draw. The tau181 concentration and the CSF ratio Aβ1-42/1-40 (rho = −0.52; median age of diagnosis was 50.3 [48.0–54.1] for prodromal AD p < 0.0001), CSF concentration of total tau (rho = 0.63; p < and 53.2 [49.2–57.1] for AD dementia. As previously reported4,7, 0.0001), CSF concentration of p-tau181 (rho = 0.68; p < 0.0001) participants with DS and prodromal AD and AD dementia and CSF concentration of NfL (rho = 0.58; p < 0.0001, Supple- showed a decrease in the CSF ratio of Aβ1-42/1-40 and an increase mentary Fig. 4). in CSF concentration of total tau, p-tau181 and NfL, as well as an increase in plasma concentration of NfL compared with asymp- Associations with imaging biomarkers. Next, we analysed the tomatic subjects and euploid controls (Table 1). correlation between plasma p-tau181 and imaging biomarkers in participants with DS. Plasma p-tau181 concentration correlated with atrophy measured by MRI in characteristic AD regions Group comparisons and diagnostic accuracy of p-tau181. including the temporal regions angular and supramarginal gyri While plasma p-tau181 concentration was relatively stable in the and precuneus of both hemispheres (n = 121, Fig. 3). These euploid controls across age, they increased in the early thirties in associations also extended into the lateral frontal and orbito- DS (Fig. 1), and by the age of 40.5 years, the confidence intervals frontal and some occipital structures. The stratified analyses by of adults with DS and euploid controls did not overlap (10 years clinical group showed that these results were primarily driven by before expected prodromal AD diagnosis). patients with symptomatic AD. Similarly, plasma p-tau181 2 NATURE COMMUNICATIONS | (2021)12:4304 | https://doi.org/10.1038/s41467-021-24319-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24319-x ARTICLE Table 1 Demographics, cognitive and biomarker data of participants with Down syndrome and controls. Control aDS pDS dDS n 44 240 43 83 Age (years) (median [IQR]) 55.75 [47.50, 62.02]* 37.83 [29.90, 45.58]*+& 50.27 [48.02, 54.10]& 53.21 [49.19, 57.14]+ Gender = Male (%) 21 (47.7) 132 (55.0) 22 (51.2) 42 (50.6) APOEε4 positivity (%) 10 (23.3) 47 (19.7) 10 (23.3) 19 (23.5) MMSE score (median [IQR]) 30.00 [29.00, 30.00] CAMCOG score (median [IQR]) 77.00 [62.00, 86.00]+& 61.00 [46.50, 73.50]&^ 41.00 [30.00, 55.00]+^ Degree of disability (%) Mild 68 (28.3) 7 (16.3) 5 (6.0) Moderate 120 (50.0) 18 (41.9) 49 (59.0) Severe/Profound 52 (21.7) 18 (41.9) 29 (34.9) plasma p-tau181 (pg/ml) 9.31 [7.46, 13.82]$# 11.36 [8.10, 16.01]+& 21.72 [16.55, 37.02]&^ 32.58 [23.67, 44.57]+$^ (median [IQR]) plasma NfL (pg/ml) (median [IQR]) 3.38 [2.89, 4.16]$#* 5.93 [4.43, 10.25]+&* 13.61 [11.50, 18.26]&^ 23.86 [17.33, 33.65]+$^ CSF Aβ42/Aβ40 (median [IQR]) 0.11 [0.10, 0.11]$#* 0.08 [0.06, 0.09]+&* 0.04 [0.04, 0.05]& 0.05 [0.04, 0.05]+$ CSF t-tau (pg/ml) (median [IQR]) 239.00 [181.75, 295.50]$# 309.00 [163.00, 446.00] 711.50 [467.25, 1112.50] 963.00 [653.00, +& & 1222.00]+$ CSF p-tau (pg/ml) (median [IQR]) 32.15 [24.55, 41.52]$# 29.80 [17.30, 51.20]+& 111.90 [69.30, 201.07]& 152.80 [93.40, 192.80]+ $ CSF NfL (pg/ml) (median [IQR]) 362.00 [260.10, 497.56]$# 365.00 [233.30, 507.60] 705.05 [645.00, 1201.00 [893.58, +& 1027.05] &^ 1625.00]+$^ SUVR 18F-FBP (median [IQR]) 1.15 [1.09, 1.20]+& 1.35 [1.17, 1.43]& 1.33 [1.30, 1.34]+ SUVR 18F-FDG (median [IQR]) 1.35 [1.26, 1.41] +& 1.11 [1.07, 1.30]&^ 0.82 [0.74, 0.93]+^ N sample, IQR interquartile range, ID intellectual disability, CSF cerebrospinal fluid, Aβ Amyloid-β, NfL neurofilament light protein, FDG 18-fluorodeoxyglucose, SUVR Standardized Uptake Value Ratio. aDS asymptomatic Down syndrome, pDS prodromal Alzheimer’s disease Down syndrome, dDS Alzheimer’s disease dementia Down syndrome, Controls euploid healthy controls. Symbols designate significant differences between groups: control-aDS (*), control-pDS (#), control-dDS ($), aDS-pDS (&), aDS-dDS (+) and pDS-dDS (^). concentration correlated with lower brain metabolism in tem- dominant AD14–17,21. In DS, changes in p-tau181 concentration poroparietal regions measured by FDG-PET (n = 68), and this in blood are already detectable in the fourth decade with a very association was mainly driven by patients with symptomatic AD similar temporality and magnitude of change to plasma NfL (Fig. 3B). The areas with hypometabolism overlapped with the concentration and CSF p-tau181, total tau and NfL levels4. The areas with atrophy. pattern of change of plasma p-tau181 in DS is also very similar to Finally, we compared concentration of plasma p-tau181 in that described in autosomal-dominant AD21. Similarly to CSF participants with amyloid PET available. Of the 45 DS cases with total tau and CSF NfL concentrations4, plasma p-tau181 con- amyloid PET, 10 were amyloid negative (SUVr < 1.11). The mean centration was similar in asymptomatic adults with DS and plasma p-tau181 concentration was higher in participants with controls, but showed a two-fold increase in median plasma p-tau DS that had a positive amyloid PET scan compared with those concentration in patients with prodromal AD and AD dementia with a negative amyloid status (Fig. 4A). The AUC for the compared with asymptomatic individuals. This result is in dis- comparison between both groups was 0.77 (95% CI 0.61–0.93; agreement with the only other study to have investigated plasma Fig. 4B). p-tau181 concentration in DS20, which found higher levels in the 20 subjects with DS with respect to the 22 controls. The group Discussion differences might have been driven by undiagnosed symptomatic This study describes that plasma p-tau181 can be reliably used for patients as the study did not asses dementia status and all adults the screening of AD in a well-characterised cohort of participants with DS and high plasma p-tau181 concentrations were older with DS. Plasma p-tau181 concentration start to increase in the than 40. In contrast, we show that young adults with DS have early thirties and correlate with core fluid biomarkers of AD as normal concentration of plasma p-tau181. well as with cortical thinning and brain hypometabolism in AD- A relevant finding of this study is that we show a strong cor- related brain regions. relation of plasma p-tau181 and plasma NfL in adults with DS. Recent biomarker studies14–17,19,20 have shown that blood p- We have shown in a previous study that plasma NfL has an AUC tau181 can identify central tau pathology with high accuracy in of 0.95 (95% CI 0.92–0.98) for the differentiation of asympto- sporadic AD. Concentration of p-tau181 in blood is increased in matic vs AD dementia in Down syndrome7. In the present study, individuals with AD with respect to cognitively healthy indivi- there were no statistical differences in accuracy between both duals, they predict amyloid β PET positivity and are not elevated markers. Despite the lack of disease specificity of NfL, these in non-AD conditions14–17,19. In addition, concentration of p- results can be explained by the fact that DS is a genetically tau181 in blood correlate with CSF p-tau181, tau pathology determined form of AD4,22 and that individuals with DS have a measured by PET and Braak stages post-mortem15. There is only 90% lifetime risk of developing AD. In other words, a non- one small study with 20 adults with DS that has investigated specific neurodegeneration marker (NfL) mirrors the diagnostic plasma p-tau181 concentration20. However, this study did not performance of a specific AD marker (p-tau181) because the assess the diagnostic performance, did not evaluate the clinical other conditions that elevate NfL levels in adults with DS are dementia status of the participants and the method had insuffi- exceedingly rare. In contrast, the general population is at risk of cient analytical sensitivity. neurodegenerative dementias other than AD, which explains the Our data in individuals with DS are in good agreement with different performance. Nonetheless, both in clinical practice and recent studies on plasma p-tau-181 in sporadic and autosomal- trials, these two biomarkers could provide complementary NATURE COMMUNICATIONS | (2021)12:4304 | https://doi.org/10.1038/s41467-021-24319-x | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24319-x CSF NfL, but not CSF p-tau181 concentration, increase in autosomal-dominant and sporadic AD23,24. Future studies should assess whether the longitudinal trajectories of plasma NfL and plasma p-tau181 differ in DS (and in the general population). One relevant difference between our study and previous studies in sporadic AD is that concentration of p-tau181 in plasma in DS predicted amyloid β PET positivity (AUC 0.77) with less accuracy than in sporadic AD (AUC 0.80–91)14–16. This is not entirely unexpected given the context of application in a population genetically determined to develop AD. Indeed, in the elderly euploid population only a proportion of subjects have AD pathological changes. In DS and autosomal-dominant AD, all subjects eventually develop AD pathological changes with time4,25. In these populations, AD biomarkers show some degree of co-linearity in the AD continuum, more so than in healthy controls26, although with different temporality4,25. Therefore, the greater variability of AD pathological changes in the general population facilitates the accuracy to detect amyloid positivity. Furthermore, despite the co-linearity, the different biomarkers have different temporalities and sensitivities in the DS population with respect to other forms of AD. Thus, amyloid-β PET has indeed limited sensitivity to detect the earliest stages of amyloid deposition in DS27, and changes in amyloid burden with PET are detected ten years later than with CSF Aβ1-42/1-40 levels4. Of note, a similar offset has been reported in autosomal-dominant AD25. Thus, the context of application in a population with genetically determined AD and this sequence of changes, with early amyloid deposition in most individuals, likely explain the limited perfor- mance of plasma p-tau181 in predicting amyloid-β PET positivity in DS with respect to the general population. Finally, we found that plasma p-tau181 concentration corre- lated with cortical thickness and brain metabolism, mainly in subjects with DS with symptomatic AD. These findings suggest that plasma p-tau181 is associated with AD-related neurode- generation, as it has been suggested in sporadic AD16. If this is the case, then plasma p-tau181 could serve as a predictor of disease progression14. Future studies should assess if plasma p-tau181 concentration can be used to identify asymptomatic individuals with DS most likely to progress to AD dementia. It will be also important to investigate the longitudinal change of plasma p-tau and its association with the rate of decline, as well as the corre- lation with tau PET imaging and with post-mortem neuro- pathological measures of tau pathology, both intraneuronal Fig. 1 Changes with age in plasma p-tau181 concentrations in Down neurofibrillary tangles and tau-positive neurites surrounding syndrome and euploid controls. a Age-related changes in p-tau181 levels in amyloid plaques. individuals with Down syndrome (asymptomatic, prodromal Alzheimer’s Our results have several important implications. First, they disease and Alzheimer’s disease dementia, all in red) and in euploid support the use of plasma p-tau181 as an easily accessible bio- controls (blue). The central lines indicate the fitted linear model for each marker of tau pathology in DS. Second, these results show that group and the shadowed ribbons show the 95% confidence level intervals. the changes in this biomarker are similar to those described in b Integrated model of the natural history of Alzheimer’ disease in Down sporadic and autosomal-dominant AD. Finally, our findings syndrome. Comparison of the evolution of the standardized differences suggest that plasma p-tau181 may be a useful biomarker in between participants with Down syndrome and controls fitted with a locally clinical trials in DS, either for screening or inclusion purposes, or estimated scatterplot smoothing curve. Plasma p-tau levels are represented to monitor drug effects on tau pathology in clinical trials in DS. in a solid red line and are compared with CSF p-tau levels and both plasma People with DS are a suitable population to conduct AD clinical and CSF NfL levels (modified from ref. 4). Positive standardized differences trials, and the prevalence is higher than other genetic-determined represent higher biomarker values in participants with Down syndrome forms of AD. Unfortunately, despite the remarkably high risk to compared to euploid controls and negative values represent lower develop symptomatic AD in this population, very few trials have biomarker values. Standardized differences were computed by the been performed in individuals with DS. We previously showed difference between the DS and the controls divided by the standard that a significant proportion of adults with DS are capable and deviation of both groups. willing to perform all the multimodal studies required in a trial4. The implementation of easily accessible biomarkers, such as information. Plasma p-tau181 could be prioritized as a first plasma p-tau181 and NfL, may accelerate the testing of therapies diagnostic screening, and a measure of target engagement in trials for AD in people with DS. with anti-tau compounds. In turn, NfL could be used for mon- The main strengths of this study are the large sample size with itoring disease progression and neurodegeneration during follow- a wide range of age and the comprehensive and multimodal up. In this sense, it is worth mentioning that longitudinally only nature of the evaluation that includes clinical, biochemical and 4 NATURE COMMUNICATIONS | (2021)12:4304 | https://doi.org/10.1038/s41467-021-24319-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24319-x ARTICLE Fig. 2 Plasma p-tau181 and NfL concentrations in Down syndrome clinical groups and controls. a, b Box and whisker plots of the median concentrations of plasma p-tau181 and plasma NfL. Plasma p-tau181 concentrations a for aDS (n = 240), pDS (n = 43), dDS (n = 83) and euploid controls (n = 44) and plasma NfL concentrations b for aDS (n = 193), pDS (n = 26), dDS (n = 56) and euploid controls (n = 14). The central black lines indicate the median values. The boxes above and below these lines show the upper and lower quartiles, respectively, and the whiskers illustrate upper and lower 1.5× IQR limits. c, d ROC curves for plasma p-tau181 (c) and NfL (d) comparing the asymptomatic group with the Alzheimer’s disease dementia (full curve) and with the prodromal Alzheimer’s disease group (dotted curve). ROC receiver operating characteristic, NfL neurofilament light protein, p-tau tau phosphorylated at threonine 181. Fig. 3 Association of plasma p-tau181 levels with imaging biomarkers in Down syndrome. a Association of p-tau181 levels with cortical thickness measured by MRI in Down syndrome subjects. Levels of plasma p-tau181 correlated with atrophy in Alzheimer’s disease typical regions. b Association of plasma p-tau181 levels with brain metabolism measured by 18FDG-PET in Down syndrome subjects. Levels of plasma p-tau181 correlated with lower brain metabolism, also driven by patients with symptomatic Alzheimer’s disease. aDS asymptomatic Down syndrome, sDS Down syndrome with symptomatic Alzheimer’s disease. NATURE COMMUNICATIONS | (2021)12:4304 | https://doi.org/10.1038/s41467-021-24319-x | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24319-x imaging markers. The main limitations are the cross-sectional design, the lack of tau PET imaging or pathological confirmation and that the sample sizes differ between biomarker modalities. In conclusion, our study shows that p-tau181 in plasma can be useful for the detection of AD in DS. This biomarker correlates with the characteristic hallmarks of AD observed in other bio- marker modalities. Determinations of plasma p-tau181 may be useful as a first screening tool to detect AD in DS, particularly in those with severe intellectual disability where a precise clinical diagnosis is more difficult, as well as in and clinical trials. Methods Study design and participants. We performed a single-centre cross-sectional study of adults with Down syndrome and euploid controls in Barcelona (Spain). Adults with DS were recruited from a population-based health plan designed to screen for AD dementia, which includes yearly neurological and neuropsycholo- gical assessments. Those individuals interested in research studies are included in the Down Alzheimer Barcelona Neuroimaging Initiative (DABNI) cohort4,7. We recruited the euploid cognitively normal controls from the Sant Pau Initiative on Neurodegeneration (SPIN) cohort28. The period of recruitment was February 2013 to December 2019. The study was approved by the Ethical Review Board of the Sant Pau Research Institute, following the standards for medical research in humans recommended by the Declaration of Helsinki. All participants or their legally authorized representatives gave written informed consent before enrolment. We included all adults with DS that had plasma available. Procedures. For the purpose of dementia diagnosis, we administered a semi- structured adapted health questionnaire to the caregivers, the Cambridge Exam- ination for Mental Disorders of Older People with Down Syndrome and others with intellectual disabilities (CAMDEX-DS) developed in Cambridge (UK), and also adapted to the Spanish population. The CAMDEX includes a comprehensive battery covering seven different cognitive domains, the Cambridge Cognitive Examination for Older Adults with Down Syndrome (CAMCOG-DS). Using these tools, we classified participants with Down syndrome into asymptomatic (no clinical or neuropsychological suspicion of AD), prodromal AD (suspicion of AD, but symptoms do not fulfill criteria for dementia) or AD dementia (subjects with DS with full-blown dementia) in a consensus meeting between the neurologist/ psychiatrist and the neuropsychologists who assessed them blind to the biomarker data4,7. We stratified the level of intellectual disability according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition as mild, moderate, severe or profound (which were grouped together). Classification was based on the individuals’ best-ever level of functioning. The information was obtained through family interview and review of medical or educational records for past assessment results. Euploid controls underwent a structured neurological assessment and a comprehensive neuropsychological battery. Inclusion criteria were normal neuropsychological results for their age and education, a clinical dementia rating scale score of 0 and normal levels of core AD biomarkers in CSF28. Genetic screening of trisomy 21 was assessed in 308 adults with DS (75.3%), and APOEε4 carrier status was obtained following previously published protocols4,7. A subset of participants had 3T-MRI (n = 121) and/or 18F-fluorodeoxyglucose PET (FDG-PET, n = 68) and/or 18F-Florbetapir PET acquisitions (n = 45) available for analysis4,7. Structural T1 MRI was processed using the cortical reconstruction pipeline of Freesurfer v6 (https://surfer.nmr.mgh.harvard.edu/). The estimated cortical thickness (CTh) individual surfaces were inspected in order to detect segmentation errors, and a smoothing kernel of 15 mm was applied to all the images. FDG-PET images were co-registered to the individual MRI and intensity scaled by the uptake of the pons-vermis region and the resulting images were projected to the middle point of the cortical ribbon. Finally, these images were visually inspected for possible errors and a smoothing kernel of 10 mm was then Fig. 4 Plasma p-tau181 levels to predict Amyloid-β PET positivity in applied4. For the 18F-Florbetapir scans, whole cerebellum was used as reference Down syndrome. a Plasma p-tau181 levels for Down syndrome subjects region29. The cut-off point for amyloid positivity was 1.1129. CSF and blood samples were acquired concurrently on the same day following stratified by amyloid-β PET status (n PET negative = 0; n PET positive = established procedures14. Briefly, CSF samples were collected in 10 mL 35). The central black lines indicate the median values. The boxes polypropylene tubes (Sarstedt, Ref#62.610.018) and transferred to the Sant Pau above and below these lines show the upper and lower quartiles, Memory Unit’s laboratory where they were processed and aliquoted within the first respectively, and the whiskers illustrate upper and lower 1.5× IQR 2 h after the lumbar puncture and stored at −80 °C until analysis. Plasma samples were collected in 10 ml ETDA tubes, and centrifuged at 4 °C for 10 min, and limits. b ROC curves for plasma p-tau181 comparing the individuals aliquoted within the first 2 h in 100 µl tubes. The median storage time was 3.5 years with Down syndrome with positive and negative amyloid-β PET. ROC (IQR 1.7–4.4) and all samples had a maximum of two freeze/thaw cycles before the receiver operating characteristic, p-tau tau phosphorylated at analysis. Plasma levels of p-tau181 were measured with a validated Single molecule threonine 181. array (Simoa) immunoassay, as described previously in detail13, by technicians blinded to the biomarker and clinical data at the Sahlgrenska Academy at the University of Gothenburg, Möndal, Sweden. Plasma levels of NfL were measured using the commercially available NF-light kit (Quanterix, Billerica, MA) by Simoa at Centre Hospitalier Universitaire Montpellier (n = 289, Montpellier, France). CSF was available in a subset of participants (n = 129). CSF levels of Aβ11-42, Aβ1-40, 6 NATURE COMMUNICATIONS | (2021)12:4304 | https://doi.org/10.1038/s41467-021-24319-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24319-x ARTICLE p-tau181 and total tau were quantified in all samples using a commercially 12. Nakamura, A. et al. High performance plasma amyloid-beta biomarkers for available immunoassay in the fully automated platform LUMIPULSE (Fujirebio- Alzheimer’s disease. Nature 554, 249–254 (2018). Europe, Ghent, Belgium)29. CSF NfL levels were measured with a commercially 13. Schindler, S. E. et al. High-precision plasma beta-amyloid 42/40 predicts available ELISA (UmanDiagnostics, Umeå, Sweden) following the manufacturer’s current and future brain amyloidosis. Neurology 93, e1647–e1659 (2019). recommendations. All CSF samples were analysed at Hospital Sant Pau. Methods 14. Karikari, T. K. et al. Blood phosphorylated tau 181 as a biomarker for and results from plasma NfL and CSF biomarkers other than plasma p-tau181 in Alzheimer’s disease: a diagnostic performance and prediction modelling study this cohort have already been published4,7. All measurements were obtained from using data from four prospective cohorts. Lancet Neurol. 19, 422–433 (2020). distinct samples. 15. Thijssen, E. H. et al. Diagnostic value of plasma phosphorylated tau181 in Alzheimer’s disease and frontotemporal lobar degeneration. Nat. Med. 26, Statistical analysis. All the statistical analyses were performed using R statistical 387–397 (2020). software version 3.6.3. Baseline characteristics were reported using standard 16. Janelidze, S. et al. Plasma p-tau181 in Alzheimer’s disease: relationship to descriptive statistics. Continuous variables were indicated as median [IQR] and other biomarkers, differential diagnosis, neuropathology and longitudinal categorical data were summarised as absolute frequencies and percentages. Dif- progression to Alzheimer’s dementia. Nat. Med. 26, 379–386 (2020). ferences in baseline characteristics between the diagnostic groups were assessed 17. Mielke, M. M. et al. Plasma phospho-tau181 increases with Alzheimer’s using Kruskal–Wallis and Dunn’s tests with Holm multiple comparisons correc- disease clinical severity and is associated with tau- and amyloid-positron tion. To determine the temporality of plasma p-tau181 changes in DS we fitted a emission tomography. Alzheimers Dement. 14, 989–997 (2018). 1st degree locally estimated scatterplot smoothing curve in controls and in adults 18. Palmqvist, S. et al. Discriminative accuracy of plasma phospho-tau217 for with DS independently4. We defined biomarker change as the age at which the Alzheimer disease vs other neurodegenerative disorders. JAMA 324, 772–781 groups appear to start diverging visually, but we also provide the lower age at which (2020). the confidence intervals (at 95%) between groups did not overlap. 19. Lantero Rodriguez, J. et al. Plasma p-tau181 accurately predicts Alzheimer’s The diagnostic performance of plasma p-tau181 and NfL was determined with disease pathology at least 8 years prior to post-mortem and improves the receiver operating characteristic (ROC) curve analyses. DeLong’s test was used to clinical characterisation of cognitive decline. Acta Neuropathol. 140, 267–278 compare the different areas under the curve (AUC). To assess the correlation (2020). between plasma p-tau and other fluid biomarkers, Spearman correlation was used. 20. Tatebe, H. et al. Quantification of plasma phosphorylated tau to use as a P-tau181 values were non-normally distributed. Therefore, we present the raw data biomarker for brain Alzheimer pathology: pilot case-control studies including in the figures and ROC analyses although we used log-transformed the plasma p- patients with Alzheimer’s disease and Down syndrome. Mol. Neurodegener. tau181 values for the neuroimaging analyses. 12, 63 (2017). All significance tests were two-sided with the statistical significance set at 5%. 21. O’Connor, A. et al. Plasma phospho-tau181 in presymptomatic and Finally, for the neuroimaging variables, we first performed vertex‐wise correlation symptomatic familial Alzheimer’s disease: a longitudinal cohort study. Mol. analyses between p-tau181 plasma and both CTh and FDG-PET uptake in the Psychiatry Jul 14. https://doi.org/10.1038/s41380-020-0838-x (2020). whole sample. We then conducted separate stratified analyses in asymptomatic and 22. Dubois, B. et al. Advancing research diagnostic criteria for Alzheimer’s symptomatic AD participants. To correct for multiple comparisons, the threshold disease: the IWG-2 criteria. Lancet Neurol. 13, 614–629 (2014). of significance was set at family‐wise error (FWE)-corrected P < 0.05 for all vertex‐ 23. Fagan, A. M. et al. Longitudinal change in CSF biomarkers in autosomal- wise analyses. dominant Alzheimer’s disease. Sci. Transl. Med. 6, 226ra230 (2014). 24. Lleó, A. et al. Longitudinal cerebrospinal fluid biomarker trajectories along the Reporting summary. Further information on research design is available in the Nature Alzheimer’s disease continuum in the BIOMARKAPD study. Alzheimers Research Reporting Summary linked to this article. Dement. 15, 742–753 (2019). 25. Bateman, R. J. et al. Clinical and biomarker changes in dominantly inherited Data availability Alzheimer’s disease. N. Engl. J. Med. 367, 795–804 (2012). 26. Illan-Gala, I. et al. Challenges associated with biomarker-based classification Anonymized data will be shared upon request from a qualified academic investigator for systems for Alzheimer’s disease. Alzheimers Dement. 10, 346–357 (2018). the sole purpose of replicating procedures and results of the article and as long as data 27. Jennings, D. et al. Age dependence of brain beta-amyloid deposition in Down transfer is in agreement with EU legislation on the general data protection and the transfer is approved by the Ethical Review Board of the Sant Pau Research Institute. syndrome: an [18F]florbetaben PET study. Neurology 84, 500–507 (2015). 28. Alcolea, D. et al. The Sant Pau Initiative on Neurodegeneration (SPIN) cohort: a data set for biomarker discovery and validation in neurodegenerative Received: 26 October 2020; Accepted: 9 June 2021; disorders. Alzheimers Dement. 5, 597–609 (2019). 29. Alcolea, D. et al. Agreement of amyloid PET and CSF biomarkers for Alzheimer’s disease on Lumipulse. Ann. Clin. Transl. Neurol. 6, 1815–1824 (2019). References Acknowledgements 1. Ballard, C., Mobley, W., Hardy, J., Williams, G. & Corbett, A. Dementia in This study was supported by the Fondo de Investigaciones Sanitario (FIS), Instituto de Down’s syndrome. Lancet Neurol. 15, 622–636 (2016). Salud Carlos III (PI14/01126 and PI17/01019 to J.F., PI13/01532 and PI16/01825 to R.B., 2. Bittles, A. H., Bower, C., Hussain, R. & Glasson, E. J. The four ages of Down PI18/00335 to M.C.I., PI18/00435 and INT19/00016 to D.A., PI15/01618 to R.R., PI14/ syndrome. Eur. J. Public Health 17, 221–225 (2007). 1561 and AC19/00103 to A.L.) and the CIBERNED program (Program 1, Alzheimer 3. McCarron, M. et al. A prospective 20-year longitudinal follow-up of dementia Disease to A.L. and SIGNAL study, www.signalstudy.es), partly jointly funded by Fondo in persons with Down syndrome. J. Intellect. Disabil. Res. 61, 843–852 (2017). Europeo de Desarrollo Regional, Unión Europea, Una manera de hacer Europa. This 4. Fortea, J. et al. Clinical and biomarker changes of Alzheimer’s disease in adults work was also supported by the National Institutes of Health (NIA grants with Down syndrome: a cross-sectional study. Lancet 395, 1988–1997 (2020). 1R01AG056850 - 01A1; R21AG056974 and R01AG061566 to J.F.), Departament de Salut 5. Hithersay, R. et al. Association of dementia with mortality among adults with de la Generalitat de Catalunya, Pla Estratègic de Recerca i Innovació en Salut (SLT002/ Down syndrome older than 35 years. JAMA Neurol. 76, 152–160 (2019). 16/00408 to A.L.), Fundació La Marató de TV3 (20141210 to J.F., 044412 to R.B. and 6. Startin, C. M. et al. Plasma biomarkers for amyloid, tau, and cytokines in 201437.10 to R.R.); I. Illán-Gala is supported by the Rio Hortega grant (CM17/00074) Down syndrome and sporadic Alzheimer’s disease. Alzheimers Res. Ther. 11, from “Acción Estratégica en Salud 2013-2016” and the Global Brain Health Institute 26 (2019). (https://www.gbhi.org/). Fundació Catalana Síndrome de Down and Fundació Víctor 7. Fortea, J. et al. Plasma and CSF biomarkers for the diagnosis of Alzheimer’s Grífols i Lucas partially supported this work. This work was also supported by Gen- disease in adults with Down syndrome: a cross-sectional study. Lancet Neurol. eralitat de Catalunya (SLT006/17/00119 to J.F., SLT006/17/95 to E.V. and SLT006/17/ 17, 860–869 (2018). 00125 to D.A.) and a grant from the Fundació Bancaria La Caixa to R.B. M.F.I. 8. Henson, R. L. et al. Cerebrospinal fluid biomarkers of Alzheimer’s disease in a acknowledges support from the Jérôme Lejeune and Sysley D’Ornano Foundations. A cohort of adults with Down syndrome. Alzheimers Dement. (Amst.) 12, e12057 Bejanin was the recipient of a Juan de la Cierva-Incorporación grant from the Spanish (2020). Ministry of Economy and Competitiveness (IJCI-2017-32609) and a Miguel Servet I 9. Zetterberg, H. & Burnham, S. C. Blood-based molecular biomarkers for grant (CP20/00038) from the Carlos III Health Institute. T.K.K. holds a research fel- Alzheimer’s disease. Mol. Brain 12, 26 (2019). lowship from the BrightFocus Foundation (#A2020812F) and is further supported by the 10. Khalil, M. et al. Neurofilaments as biomarkers in neurological disorders. Nat. Swedish Alzheimer Foundation (Alzheimerfonden; #AF-930627), the Swedish Brain Rev. Neurol. 14, 577–589 (2018). Foundation (Hjärnfonden; #FO2020-0240), the Swedish Dementia Foundation 11. Ovod, V. et al. Amyloid beta concentrations and stable isotope labeling (Demensförbundet), Gamla Tjänarinnor Foundation, the Aina (Ann) Wallströms and kinetics of human plasma specific to central nervous system amyloidosis. Mary-Ann Sjöbloms Foundation, the Agneta Prytz-Folkes & Gösta Folkes Foundation Alzheimers Dement. 13, 841–849 (2017). (#2020-00124), the Gun and Bertil Stohnes Foundation, and the Anna Lisa and Brother Björnsson’s Foundation. H.Z. is a Wallenberg Scholar supported by grants from the NATURE COMMUNICATIONS | (2021)12:4304 | https://doi.org/10.1038/s41467-021-24319-x | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24319-x Swedish Research Council (#2018-02532), the European Research Council (#681712), WO2019175379 A1 Markers of synaptopathy in neurodegenerative disease issued. Swedish State Support for Clinical Research (#ALFGBG-720931), the Alzheimer Drug Dis- Dr. Blennow has served as a consultant or at advisory boards for Abcam, Axon, Biogen, covery Foundation (ADDF), USA (#201809-2016862), the AD Strategic Fund and the Alz- JOMDD/Shimadzu. Julius Clinical, Lilly, MagQu, Novartis, Roche Diagnostics, and heimer’s Association (#ADSF-21-831376-C, #ADSF-21-831381-C and #ADSF-21-831377-C), Siemens Healthineers, and is a co-founder of Brain Biomarker Solutions in Gothenburg the Olav Thon Foundation, the Erling-Persson Family Foundation, Stiftelsen för Gamla AB (BBS), which is a part of the GU Ventures Incubator Program. Dr. Fortea has served Tjänarinnor, Hjärnfonden, Sweden (#FO2019-0228), the European Union’s Horizon 2020 as a consultant or at advisory boards for AC Immune, Novartis, and Merck. In addition, research and innovation programme under the Marie Skłodowska-Curie grant agreement No Dr. Fortea has a patent WO2019175379 A1 Markers of synaptopathy in neurodegen- 860197 (MIRIADE), and the UK Dementia Research Institute at UCL. K.B. is supported by erative disease issued. The other authors declare no competing interests. the Swedish Research Council (#2017-00915), the Alzheimer Drug Discovery Foundation (ADDF), USA (#RDAPB-201809-2016615), the Swedish Alzheimer Foundation (#AF- 742881), Hjärnfonden, Sweden (#FO2017-0243), the Swedish state under the agreement Additional information Supplementary information The online version contains supplementary material between the Swedish government and the County Councils, the ALF-agreement (#ALFGBG- available at https://doi.org/10.1038/s41467-021-24319-x. 715986), and European Union Joint Program for Neurodegenerative Disorders (JPND2019- 466-236). The authors would like to thank all the participants with Down’s syndrome, their Correspondence and requests for materials should be addressed to A.L. or J.F. families and their carers for their support of, and dedication to this research. We also acknowledge the Fundació Catalana Síndrome de Down for global support; Soraya Torres, Peer review information Nature Communications thanks Nicolas Barthélemy, Cynthia Shaimaa El Bounasri, Laia Muñoz and Raúl Núñez for laboratory and sample handling; Reyes Lemere and the other, anonymous, reviewer(s) for their contribution to the peer review Alcoverro, Marta Salinas and Tania Martínez for administrative support; Concepción Escola of this work. Peer reviewer reports are available. for nursing handling. We also thank the clinicians for their help in acquiring the data reported in this article. Reprints and permission information is available at http://www.nature.com/reprints Author contributions Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in A.L. and J.F. did the literature search. J.F., H.Z., K.B. and A.L. conceived and designed the published maps and institutional affiliations. study. J.F., H.Z., T.K., M.C.I., N.A., V.M., I.B., L.V., M.A., B.B., S.F., S.V., D.A., K.B. and A.L. acquired data. J.F., H.Z., M.C.I., D.A., J.P., V.M., R.B., K.B. and A.L. analysed and interpreted the data. J.P. did the statistical analyses. J.F. and A.L. drafted the manuscript. Open Access This article is licensed under a Creative Commons All authors revised the manuscript for important intellectual content and edited the Attribution 4.0 International License, which permits use, sharing, manuscript. adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party Competing interests material in this article are included in the article’s Creative Commons license, unless Dr. Lleó has served as a consultant or at advisory boards for Fujirebio-Europe, Roche, indicated otherwise in a credit line to the material. If material is not included in the Biogen and Nutricia. In addition, Dr. Lleó has a patent WO2019175379 A1 Markers of article’s Creative Commons license and your intended use is not permitted by statutory synaptopathy in neurodegenerative disease issued. Dr. Zetterberg has served as a con- regulation or exceeds the permitted use, you will need to obtain permission directly from sultant or at advisory boards for Denali, Roche Diagnostics, Wave, Samumed, Siemens the copyright holder. To view a copy of this license, visit http://creativecommons.org/ Healthineers, Pinteon Therapeutics and CogRx, has given lectures in symposia sponsored licenses/by/4.0/. by Fujirebio, Alzecure and Biogen, and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program. Dr. Alcolea has served as a consultant or at advisory boards for Krka Farmacéutica S.L., © The Author(s) 2021 Fujirebio-Europe, Roche Diagnostics and Nutricia. In addition, Dr. Alcolea has a patent 8 NATURE COMMUNICATIONS | (2021)12:4304 | https://doi.org/10.1038/s41467-021-24319-x | www.nature.com/naturecommunications