Novel tau biomarkers phosphorylated at T181, T217 or T231 rise in the initial stages of the preclinical Alzheimer's continuum when only subtle changes in Aβ pathology are detected

Abstract

In Alzheimer's disease (AD), tau phosphorylation in the brain and its subsequent release into cerebrospinal fluid (CSF) and blood is a dynamic process that changes during disease evolution. The main aim of our study was to characterize the pattern of changes in phosphorylated tau (p-tau) in the preclinical stage of the Alzheimer's continuum. We measured three novel CSF p-tau biomarkers, phosphorylated at threonine-181 and threonine-217 with an N-terminal partner antibody and at threonine-231 with a mid-region partner antibody. These were compared with an automated mid-region p-tau181 assay (Elecsys) as the gold standard p-tau measure. We demonstrate that these novel p-tau biomarkers increase more prominently in preclinical Alzheimer, when only subtle changes of amyloid-β (Aβ) pathology are detected, and can accurately differentiate Aβ-positive from Aβ-negative cognitively unimpaired individuals. Moreover, we show that the novel plasma N-terminal p-tau181 biomarker is mildly but significantly increased in the preclinical stage. Our results support the idea that early changes in neuronal tau metabolism in preclinical Alzheimer, likely in response to Aβ exposure, can be detected with these novel p-tau assays.

Keywords: Alzheimer’s disease; biomarker; cerebrospinal fluid; plasma; tau.

Figure EV1. Diagram of tau and the antibodies used in the assays

Tau protein domains and the fragments recognized by the combination of antibodies of the assays used in this study. We show the longest form of tau (2N4R), which comprises 441 amino acids. The two N‐terminal domains (N) are depicted in green, the two proline‐rich domains (P) in yellow and the four microtubule‐binding domains (R) in blue. The phosphorylation sites identified by the assays are also shown. Details of the assays can be found in the methods section. Abbreviations: Mid, mid‐region; N, N‐terminal; p‐tau, phosphorylated tau

Figure 1. P‐tau biomarkers changes with age

1. A–E

   Scatter plots showing the association of each of the p‐tau biomarkers with age in the Aβ‐negative (A−; blue; n = 250) and the Aβ‐positive (A+; red; n = 131) groups. The solid lines indicate the regression line and the 95% confidence intervals for each of the groups. For each group, the standardized regression coefficients (β) and the P‐values were computed using a linear model adjusting for sex. Additionally, we computed the “Age × Aβ status” interaction term. Abbreviations: CSF, cerebrospinal fluid; Mid, mid‐region; N, N‐terminal; p‐tau, phosphorylated tau.

Figure 2. Effect of Aβ pathology (CSF Aβ42/40) on p‐tau biomarkers

1. A–E

   Dot and box‐plot comparing each of the p‐tau biomarker between the Aβ‐negative (A−; blue; n = 250) and the Aβ‐positive (A+; red; n = 131) groups. Aβ positivity was defined as CSF Aβ42/40 ratio < 0.071. The box‐plots depict the median (horizontal bar), interquartile range (IQR, hinges) and 1.5 × IQR (whiskers). P‐values were assessed by a one‐way ANCOVA adjusted for age and sex.

2. F–J

   Scatter plots depicting the changes between each p‐tau biomarker as a function of CSF Aβ42/40. The horizontal axes directions were inverted; lower CSF Aβ42/40 ratio reflects higher Aβ pathology. For each Aβ status group, we computed the standardized regression coefficients (β) and the P‐values, adjusted for age and sex. The solid lines indicate the regression line and the 95% confidence intervals for each of the Aβ status groups. The dashed green lines indicate the CSF Aβ42/40 cut‐off. Participants were also colour‐coded based on the Aβ PET CL scale (≤ 12CL, black; > 12CL, turquoise; Aβ PET non‐available, grey).

Data information: Abbreviations: CSF, cerebrospinal fluid; Mid, mid‐region; N, N‐terminal; n.a. non‐available; p‐tau, phosphorylated tau.

Figure 3. Effect of Aβ pathology (Aβ PET) on p‐tau biomarkers

1. A–E

   Dot and box‐plot comparing each of the p‐tau biomarker between the Aβ‐negative (A−; blue; n = 287) and the Aβ‐positive (A+; red; n = 42) groups. Aβ positivity was defined with Aβ PET visual read. The box‐plots depict the median (horizontal bar), interquartile range (IQR, hinges) and 1.5 × IQR (whiskers). P‐values were assessed by a one‐way ANCOVA adjusted by age and sex.

2. F–J

   Scatter plots depicting the changes between each p‐tau biomarker as a function of Aβ PET Centiloids (CL). The standardized regression coefficients (β) and the P‐values were computed using a linear model adjusting for age and sex. The solid lines indicate the regression line and the 95% confidence intervals. The dashed green lines indicate the CL12 and CL30 cut‐offs. Participants were also colour‐coded based on the CSF Aβ42/40 ratio (A−, black; A+, turquoise).

3. K–O

   Dot and box‐plots depicting comparison between each of the p‐tau biomarker between Centiloid scale groups: (i) ≤ 12CL (blue; n = 278), (ii) 12–30CL (subthreshold Aβ pathology group; grey; n = 28), (iii) >30CL (red; n = 25). The box‐plots depict the median (horizontal bar), interquartile range (IQR, hinges) and 1.5 × IQR (whiskers). P‐values were assessed by a one‐way ANCOVA adjusted by age and sex, followed by a Bonferroni‐corrected post hoc pairwise comparison.

Data information: Abbreviations: CSF, cerebrospinal fluid; Mid, mid‐region; N, N‐terminal; p‐tau, phosphorylated tau.

Figure 4. Discrimination of cognitively unimpaired Aβ‐positive from Aβ‐negative individuals by p‐tau biomarkers

1. A–C

   ROC analysis was performed to test the accuracy to discriminate between Aβ‐positive (A+) from Aβ‐negative (A−) individuals. Aβ positivity was defined as CSF Aβ42/40 < 0.071 (A), Aβ PET‐positive visual read (B) or Aβ PET Centiloid (CL) > 12 (C). Abbreviations: CSF, cerebrospinal fluid; Mid, mid‐region; NfL, neurofilament light; N, N‐terminal; p‐tau, phosphorylated tau; t‐tau, total tau.

Figure EV2. Correlations between CSF p‐tau biomarkers

1. A–F

   Scatter plots depicting the correlations between the CSF p‐tau biomarkers. We computed the Spearman's correlation coefficient (r _s) and the P‐value for the whole cohort (n = 381) and for the A− (n = 250; blue dots) and A+ (n = 131; red dots) groups. The solid lines indicate the regression line and the 95% confidence intervals. Abbreviations: CSF, cerebrospinal fluid; Mid, mid‐region; N, N‐terminal; p‐tau, phosphorylated tau.

Figure EV3. Correlation between CSF and plasma N‐p‐tau181

N‐p‐tau181 was measured with the same assay in matched CSF and plasma samples. Scatter plots depicting the correlations between the CSF. We computed the Spearman's correlation coefficient (r _s) and the P‐value for the whole cohort (n = 381) and for the A− (n = 250; blue dots) and A+ (n = 131; red dots) groups. The solid lines indicate the regression line and the 95% confidence intervals. Abbreviations: CSF, cerebrospinal fluid; N, N‐terminal; p‐tau, phosphorylated tau.

Figure 5. Comparison of the p‐tau biomarkers between AT groups

1. A–D

   Dot and box‐plot showing the levels of each p‐tau biomarker in each of the AT groups. Aβ‐positive (A+) was defined by a CSF Aβ42/40 < 0.071 and Tau‐positive (T+) by an Elecsys CSF Mid‐p‐tau181 > 24 pg/ml. The box‐plots depict the median (horizontal bar), interquartile range (IQR, hinges) and 1.5 × IQR (whiskers). The horizontal dashed line indicates the median of the p‐tau biomarker in the A−T− group. P‐values were assessed by a one‐way ANCOVA adjusted by age and sex followed by Bonferroni‐corrected post hoc pairwise comparisons. Abbreviations: CSF, cerebrospinal fluid; Mid, mid‐region; N, N‐terminal; p‐tau, phosphorylated tau.

Figure EV4. Associations of p‐tau biomarkers and CSF NfL

1. A–E

   Scatter plots showing the association of each of the p‐tau biomarkers with CSF Neurofilament light (NfL) in the Aβ‐negative (A−; blue) and the Aβ‐positive (A+; red) groups. The solid lines indicate the regression line and the 95% confidence intervals for each of the groups. For each group, the standardized regression coefficients (β) and the P‐values were computed using a linear model adjusting for age and sex. We also computed the “CSF NfL × Aβ status” interaction term. Abbreviations: CSF, cerebrospinal fluid; Mid, mid‐region; N, N‐terminal; p‐tau, phosphorylated tau.

Figure 6. Trajectories of the p‐tau biomarkers in preclinical Alzheimer

The graphs represent the z‐scores changes of each CSF biomarker as a function of CSF Aβ42/40 ratio (as proxy of disease progression) using a robust local weighted regression method. The z‐scores were calculated using the mean and the SD of each CSF biomarker in the A−T− group as a reference. The solid lines depict the trajectory of each CSF biomarker. The dashed lines depict the trajectories of the plasma biomarkers. The vertical black dashed line indicates the CSF Aβ42/40 cut‐off for A+. Note that the CSF p‐tau biomarkers reach the 2 z‐scores (depicted with an horizontal dashed line) with the following sequence: Mid‐p‐tau231, N‐p‐tau181, N‐p‐tau217, Mid‐p‐tau181 and t‐tau. Abbreviations: CSF, cerebrospinal fluid; Mid, mid‐region; NfL, Neurofilament light; N, N‐terminal; p‐tau, phosphorylated tau; t‐tau, total tau.

Figure EV5. Trajectories of the p‐tau biomarkers as a function of Aβ PET

The graphs represent the z‐scores changes of each CSF biomarker as a function of Aβ PET Centiloid scale (instead of CSF Aβ42/40 as proxy of disease progression) using a robust local weighted regression method. The z‐scores were calculated using the mean and the SD of each CSF biomarker in the A−T− group as a reference. The solid lines depict the trajectory of each CSF biomarker. The dashed lines depict the trajectories of the plasma biomarkers. The vertical black dashed lines indicate the CL = 12 and CL = 30 cut‐offs. Consistent with the findings using CSF Aβ42/40 as a proxy of disease progression, the CSF p‐tau biomarkers reach the 2 z‐scores (depicted with an horizontal line) with the following sequence: Mid‐p‐tau231, N‐p‐tau181, N‐p‐tau217, Mid‐p‐tau181 and T‐tau. Abbreviations: CSF, cerebrospinal fluid; Mid, mid‐region; NfL, Neurofilament light; N, N‐terminal; p‐tau, phosphorylated tau; t‐tau, total tau.