A comparison between tau and amyloid-β cerebrospinal fluid biomarkers in chronic traumatic encephalopathy and Alzheimer disease

One hundred ninety-two participants with post-mortem CSF available were enrolled from three study groups. 100 donated their brains to the Veteran’s Affairs-Boston University-Concussion Legacy Foundation Brain Bank (VA-BU-CLF) as part of the Understanding Neurologic Injury and Traumatic Encephalopathy (UNITE) study, 38 were donated to the Framingham Heart Study (FHS), and 54 were donated to Boston University’s Alzheimer’s Disease Research Center (ADRC) as part of the Health Outreach Program for the Elderly study. The UNITE group consisted of participants with a history of exposure to contact sports such as football, ice hockey, boxing, soccer, rugby, and martial arts at either the professional or amateur level [14]. For most brain donations, the next of kin contacted the brain bank to donate tissue at or near the time of death. The participants from Boston University’s Alzheimer’s Disease Research Center (BU ADRC) with and without cognitive impairment underwent annual cognitive evaluations using the National Alzheimer’s Disease Coordinating Center (NACC) Uniform Data Set (UDS) protocol [1]. The third cohort consisted of participants from the Framingham Heart Study (FHS), a longitudinal, community-based study. Consents for brain donation and research participation were provided by donor next of kin.

For UNITE study participants, retrospective clinical evaluations were performed using semi-structured post-mortem interviews, through online surveys, and review of medical records as described previously [14]. Information was obtained regarding repetitive head impact (RHI) exposure, traumatic brain injury (TBI) exposure, military history, athletic history, and clinical symptoms prior to death. In addition, medical records were examined to provide a determination of clinical symptoms and course. For the FHS participants, an athletic history assessment identical to UNITE was performed with the donor’s next of kin [15]. Athletic history was not available for BU ADRC participants. All interviews were conducted independently and blinded to the results of neuropathological examination.

All brains were neuropathologically evaluated for changes consistent with CTE, AD, and other neurodegenerative disorders using previously described selection criteria and protocols. Specifically, participants were separated into pathologic groups: CTE, AD, both (CTE+AD), or neither no CTE/no AD (control). Pathologic diagnosis of CTE was based on consensus criteria [1] and CTE staging I–IV was determined using previously published staging criteria [12, 16]. Participants were stratified using NIA-Reagan criteria to high, intermediate, or low probability of dementia caused by AD, based on Braak Score and CERAD score.

There is growing recognition that with increasing age co-occurring neurodegenerative pathologies become more common [17, 18]; therefore, comorbid neurodegenerative pathologies were not excluded from any of the groups, including the control group. Other neurodegenerative diseases were diagnosed using well-established criteria for Lewy body disease (LBD) [19] and frontotemporal lobar degeneration (FTLD) [20, 21] (Table 1). Participants with Amyotrophic Lateral Sclerosis/motor neuron disease (ALS/MND) pathology were also not excluded and included two participants in the no CTE/no AD (control) group and one participant in the high CTE group. The inclusion of a wider group of participants with concurrent neurodegenerative pathologies increases the generalizability of the current findings as patients presenting for clinical evaluation of cognitive complaints often have multiplane neurodegenerative diagnoses and underlying pathologies.

Participants were divided into pathological groups based on NIA-Reagan Criteria and CTE stage as follows. Participants with no evidence of CTE and no elements of NIA-Reagan were labeled as “No CTE/no AD” and were used and referenced as the control group throughout. Participants with CTE Stage of I–II were determined to have early stage disease and termed “Low CTE,” while those with CTE Stage of III–IV were determined to have late stage disease and were termed “High CTE.” Those with no evidence of CTE and NIA-Reagan of high or intermediate probability were termed “Intermediate/High AD,” while those with no evidence of CTE and NIA-Reagan of low probability were classified as “Low AD.” Subjects with CTE and intermediate or high probability of AD were combined to “CTE+AD.”

CSF was obtained post-mortem from the foramen magnum by gently lifting the frontal lobes to access with a large bore needle. CSF was then mixed by gently inverting the tube 5 times. The tubes were centrifuged at 1500 g for 15 min at 4°C. The CSF supernatant was removed with a transfer pipet and aliquoted into 1.5mL microcentrifuge polypropylene tubes. CSF was stored at −80 C prior to use. CSF was then diluted 1:2 with 1% Blocker A (MSD, Rockville, MD, USA, #R93BA) in wash buffer. Immunoassay was performed for Aβ_1-42 and Aβ_1-40, using a multiplex plate from MSD (#K15200E), as well as for levels of p-tau₂₃₁ and total tau (MSD #K15121D) according to manufacturer’s protocol. To capture tau phosphorylated at Thr residue 181, antibody AT270 was used and the detecting antibody was the biotinylated HT7 that recognizes residue 159–163 of tau (Thermo Scientific, Rockford, IL). For hemoglobin quantification, CSF was diluted 1:3000 and applied to the RayBio Human Hemoglobin ELISA kit (# ELH-Hgb). All standards and samples were run in duplicate.

The buffer conditions, protease inhibitors, and centrifugation protocols have been reported previously [11]. A 4-mm-tissue punch was used to isolate and remove gray matter from the gyral crests and sulcal depths of the middle frontal gyrus and neighboring sulci and superior temporal gyrus and sulcus. The brain tissue was homogenized in five-fold volume of 5 M Guanidine Hydrocholride/50 mM Tris-HCL, pH 8.0, with protease inhibitors (Thermo Scientific, 78439) and phosphatase inhibitors (Sigma, P5726 and P0044). The tissue was homogenized using a mechanical homogenizer for 25 strokes followed by ultrasonic disruption on ice. The homogenates were shaken at room temperature overnight. The lysate was diluted 1:80 with 1% Blocker A (MSD, #R93BA) in wash buffer, and immunoassay was performed for Aβ_1-42 using a multiplex plate from MSD.

Statistical analysis was performed using SPSS 26.0 (IBM Corp, Armonk, NY) and Prism v8 (Graph-Pad Software, La Jolla, CA). A one-way analysis of variance (ANOVA) was used to compare age among groups. Levels of Aβ_1-40, Aβ_1-42, p-tau₁₈₁, p-tau₂₃₁, and total tau that were outside 3X the interquartile range were eliminated as outliers and included n=1 from no CTE/no AD (control) group, n=1 from low CTE group, n=1 high CTE group, n=1 from low AD group, and n=1 from intermediate/high AD group for ptau 181 analysis, n=3 from the control group, n=1 from low AD group, n= 1 for the high CTE group, n=6 from intermediate/high AD group, and n=1 from CTE+AD group for ptau 231 analysis, n=1 from no CTE/no AD (control) group, n=2 from high CTE group for total tau analysis, n=5 from control group, n=3 from low CTE group, n=4 from high CTE group, n=1 from intermediate/high AD group, n= 1 for the CTE+AD group for Aβ_1-42 analysis, n=2 from the control group, n=1 from low CTE group, n=3 from high CTE group, n=1 from low AD group, and n=2 from intermediate/high AD group for Aβ_1-40 analysis. A two-sample chi-square test weighted by sample size was used to compare the frequency of men in each pathologic group, as well as the frequency of FTLD and LBD pathologies between pathologic groups. CSF analyte levels were used in a Kruskal-Wallis test performed to compare levels of analytes between pathologic groups. CSF measures were also rank-normalized (supplementary Figures e-1 and e-2) and used in one-way ANCOVAs in order to correct for age as a covariate to compare relative amounts of biomarkers between groups (supplementary results).

Statistical significance was set to p<0.05 following adjustments for multiple comparisons for all planned analyses. Binary logistic regression analyses were used to determine association between p-tau₂₃₁ and Aβ_1-42 and CTE or AD pathologic diagnosis controlling for age, sex, PMI, and other variables where appropriate. Linear regressions were performed to determine the relationship between CSF and brain Aβ_1-42 levels in AD and CTE. Receiver operating characteristic (ROC) curve analysis was used to determine sensitivity and specificity of CSF analytes between diagnoses.

Participants were grouped based on the presence or absence of CTE and/or AD pathology. Group demographic differences for age at death, post-mortem interval (PMI), sex, RNA integrity number (RIN), pH, and presence/absence of FTLD and LBD pathologies are listed in Table 1. Pathologic groups differed in age at death (p <0.001) and PMI (p <0.001) (Table 1). The low CTE group (M= 64.6± 4.6) was younger than the low AD (M= 86.7±1.1, p <.05), intermediate/high AD (M= 81.1±1.3, p < 0.05), and CTE+AD groups (M= 79.6 ± 2.8, p <0.05). The low AD group was older than the high CTE and control groups. The low CTE group had significantly longer PMIs (M= 46.6±6.4) than the control (M= 23.2±3.15 p<0.001), low AD (M= 21.4±3.85, p <0.001), and intermediate/high AD group (M= 20.7±2.3, p < 0.05). The high CTE group (M= 33.88±3.02) had significantly longer PMIs than the intermediate/high AD group (M= 20.7±2.3, p < 0.05). Despite these differences in PMI, there was no difference in RIN, pH, or CSF hemoglobin between groups. A majority of men were present in each pathological group for all groups except for the intermediate/high AD group which had a majority of women. Years of contact sports differed between groups (p =0.013) for the subset of participants that had this history taken (n=95); post hoc pairwise comparisons revealed the control group (M= 9.46±2.97) had decreased years of contact sports compared to mild CTE group (M= 20.85±3.43, p=0.036 Bonferroni-corrected).

We performed a binary logistic regression analysis to determine the contributions of p-tau₂₃₁ and Aβ_1-42 in predicting pathological diagnosis of CTE (low and high CTE combined, excluding CTE +AD cases) vs. control. Variables included in the model were p-tau₂₃₁, Aβ_1-42, age at death, and sex (n= 77). Both p-tau₂₃₁ (OR 1.53, 95% CI 1.08–2.16) and Aβ_1-42 (OR 0.35, 95% CI 0.17–0.74) were significant predictors of CTE status while controlling for age and sex, neither of which were significant predictors (Table 2). Secondary analyses including PMI, pH, and presence of LBD pathology as additional variables did not substantially change the results and were not significant predictors of CTE diagnosis. Although RIN and FTLD were associated with CTE, their presence in the model did not change the associations between ptau₂₃₁, Aβ_1-42, and CTE status.

We also performed a binary logistic regression to determine the contributions of p-tau₂₃₁ and Aβ_1-42 in predicting pathological diagnosis of CTE (low and high CTE combined) vs. AD (low AD and intermediate/high AD combined) (Table 3). Variables included in the model were p-tau₂₃₁, Aβ_1-42, age at death, and sex (n= 111). Both p-tau₂₃₁ (OR 1.34, 95% CI 1.02–1.76, p = 0.036) and Aβ_1-42 (OR 0.51, 95% CI 0.28–0.91) were found to distinguish between CTE and AD diagnoses, controlling for age at death (OR 0.91, 95% CI 0.84–0.97) and sex which was not a significant predictor. A secondary analysis adjusting for PMI (n= 102), demonstrated that this trend continued but with a decrease in significance most likely due to decreased power, with p-tau₂₃₁ (OR 1.23, 95% CI 0.90–1.72), Aβ_1-42 (OR 0.60, 95% CI 0.31–1.15), adjusting for age (OR 0.88, 95% CI 0.81–0.97), PMI (OR 1.06, 95% CI 1.01–1.12), and sex, which was not a significant predictor. Additional secondary analyses included the addition of RIN and pH separately as well as presence/absence of FTLD pathology and LBD pathology, none of which were significant predictors of CTE diagnosis.

Given the surprisingly low levels of Aβ_1-42 in CTE, we performed a secondary analysis to test the hypothesis CSF beta-amyloid levels reflect brain tissue Aβ_1-42 levels in AD, but not in CTE. Using a linear regression it was found that a model including age, sex, and CSF Aβ_1-42 levels predicted a significant amount of the variance of brain Aβ_1-42 levels among the AD and control groups combined (F(3, 100)=5.42, p=0.002, R²= 0.14, adjusted R²= 0.114) and both CSF Aβ_1-42 (β = −0.21, p =0.032) and age (β = 0.21, p =0.036) predicted frontal cortex Aβ_1-42 while sex (β = 0.13, p =0.19) did not. In a separate linear regression among the combined CTE and control groups, the overall regression model including CSF Aβ_1-42 levels, age, and sex predicted a significant amount of the variance of brain Aβ_1-42 levels (F(3, 77)= 4.52, p=0.006, R²= 0.15, adjusted R²= .117), though CSF Aβ_1-42 levels (β = −0.17, p =0.11) and sex (β = −0.14, p =0.21) were not found to be a significant predictors of cortical Aβ_1-42 levels while age was found to be a significant predictor (β = 0.38, p =0.001).

To assess the diagnostic accuracy of CSF p-tau₂₃₁ and Aβ_1-42 levels, an ROC analysis was performed to determine if p-tau_231, Aβ_1-42, age at death, and sex were predictive of CTE vs. control diagnosis. Area under the curve (AUC) was 0.88 (SEM= 0.04, p <0.001) (Fig. 3A). A separate ROC analysis was performed for CTE vs. AD diagnosis to determine if p-tau₂₃₁, Aβ_1-42, age at death, and sex were predictive and demonstrated that AUC was 0.93 (SEM= 0.023, p <0.001) (Fig. 3B).

This study is limited in that age is a major risk factor for neurodegenerative conditions and the CTE groups were younger than the AD groups. As a result, age at death was included in the regression analysis but there is still the possibility that age could act as a potential confounder in the current study. Differences in years of play generally differ by diagnosis with CTE groups having increased years of play exposure; however, this variable was missing in a significant percentage of cases and as such contact sports play is a potential confounder that could not be accounted for in our models. Furthermore, the groups did not have individuals of each gender evenly distributed and the AD group had increased numbers of women compared to the CTE groups which did not have women. This is a concern as gender differences in neuropathology have previously been reported [34]. However, we have attempted to correct for this in part by running regression and sensitivity analyses among men only and have found similar results for both ptau₂₃₁ and Aβ_1-42 compared to analyses run with both men and women. In addition, this study was autopsy-based and thus potentially subject to selection bias as individuals whose brains are donated by family may not represent the population more broadly. However, grouping by known pathology allows for definitive associations not possible in a clinical sample where the pathology is unknown. The current study was also limited in that it is post-mortem, and CSF abeta and total tau levels are known to be altered from antemortem levels in post-mortem samples [35]. Both the low and high CTE groups had significantly longer PMIs than the no CTE/no AD (control), low AD, and intermediate/high AD groups which raises concern that a longer PMI could lead to increased tau levels in the CTE groups. We attempted to take this into account by controlling for PMI when possible. Furthermore, given its post-mortem nature, the current study serves not to set a definitive cut-off but to warrant ante-mortem comparison of these amyloid and tau levels in CSF among patients with suspected CTE and AD. Of note, t-tau, p-tau₁₈₁, and Aβ₄₂ have been previously evaluated during life in former professional American football players, at increased risk of CTE, and it was found that increased cumulative head impact exposure in former players predicted t-tau levels [36].

Several promising modalities have been investigated for their use as in vivo CTE biomarkers including PET imaging of p-tau [31], peripheral blood levels of total tau and exosomal tau [37, 38], and CSF biomarkers [39, 40]. CSF biomarkers are particularly promising as they closely reflect the dynamic relationship of solute clearance in the glymphatic space [41] and thus may provide a window to relatively early neuropathological changes. Future studies should investigate other tau isoforms that have recently shown promise in AD including p-tau₂₁₇ [42] as well as plasma biomarkers of tau isoforms [7, 43, 44].