Rapid advances in fluid and neuroimaging biomarkers have facilitated the understanding of the dynamic associations between Alzheimer's disease (AD)-related pathophysiological processes in the living human brain. These biomarker studies suggest that brain accumulation of amyloid-β (Aβ) precedes tau pathology in cognitively unimpaired (CU) individuals1-3, which is closely related to the development of cognitive symptoms4-6. However, the reasons why Aβ pathology is not associated with AD-related progression in some CU individuals is one of the most pressing questions in the field7,8. In addition to revealing key biological players associated with disease progression, finding predictive markers of early Aβ-related tau pathology would allow for the identification of CU individuals who are more likely to develop AD even before the first signs of pathological tau, facilitating enrollment in early prevention clinical trials.
The fact that Aβ leads to tau pathology in some individuals, but not in others, suggests the presence of other biological processes capable of triggering the deleterious effects of Aβ in the early disease stages. Postmortem studies show that astrocyte reactivity is a common neuropathological finding and, like cortical Aβ plaques, one of the earliest abnormalities in the AD brain9-11. The extent to which astrocyte reactivity contributes to Aβ and tau pathology is not clear. Experimental literature suggests that astrocyte reactivity is critical for triggering Aβ-induced tau phosphorylation12 and that the attenuation of astrocyte reactivity mitigates tau pathology13,14. Additionally, glial fibrillary acidic protein (GFAP)-positive astrocytes can internalize tau and might contribute to its propagation15,16. Furthermore, the release of signaling mediators, including cytokines and caspases, has also been postulated as a mechanism linking reactive astrocytes with tau phosphorylation12,17-19.
Clinical studies support that plasma measures of GFAP correlate with its CSF levels, and are increased in CU individuals with AD pathophysiology, representing a robust proxy of astrocyte reactivity in the brains of living individuals20-22. Based on this previous literature, we designed a multi-site biomarker study including three cohorts to test the hypothesis that the presence of astrocyte reactivity biomarker abnormality is a key element determining the association of Aβ with early tau phosphorylation and aggregation biomarkers in preclinical AD.
To this end, we investigated 1,016 CU individuals (mean age = 69.6 ± 8.9, CDR = 0) from two research (TRIAD, McGill University, Canada and Pittsburgh, University of Pittsburgh, USA) and one community-based (MYHAT, Pittsburgh, USA) cohort with in vivo biomarkers. Individuals were classified as negative (Ast-) or positive (Ast+) for astrocyte reactivity biomarker according to their plasma GFAP levels (see Methods). Demographic and clinical characteristics of participants are summarized in Table 1. Overall, participants classified as Aβ+/Ast+ presented increased plasma p-tau181, p-tau231, and p-tau217 compared to other groups. No differences in Aβ levels were observed between CU Aβ+/Ast- and Aβ+/Ast+ in any cohort. Demographic characteristics of individuals segregated by cohort are presented in Supplemental Tables 1-3.
First, we z-scored biomarker levels inside each cohort and applied a robust local weighted regression to model the trajectory of plasma p-tau181, the only p-tau biomarker available in all cohorts, as a function of Aβ burden [plasma or positron emission tomography (PET)] in CU individuals classified as Ast- (n = 743) or Ast+ (n = 273). Notably, we observed that plasma p-tau181 levels increased as a function of Aβ only in CU Ast+ individuals (Fig.1a). Similarly, linear regression showed a significant association between Aβ burden and plasma p-tau181 in CU Ast+ (β = 0.34, t = 5.37, p < 0.0001; Fig.1b, Supplemental Table 4) but not in CU Ast- (β = 0.04, t = 1.06, p = 0.29; Fig.1b) individuals. A significant interaction between Aβ burden and astrocyte reactivity status on plasma p-tau181 (β = 0.31, t = 4.62, p < 0.0001; Fig.1b) further supported the presence of astrocyte reactivity was key to determining Aβ effects on tau phosphorylation. Local weighted regression using only continuous values for Aβ, p-tau181, and GFAP levels confirmed that these results were not influenced by biomarker thresholds (β = 0.10, t = 3.22, p = 0.0013, Fig.1c). Cohen’s d analysis revealed that the presence of Aβ+ and Ast+ has a large magnitude of effect on tau phosphorylation (Cohen’s d = 0.80), whereas Aβ+ in the absence of Ast+ presented a negligible effect size (Fig.1d). Voxel-wise analysis confirmed that Aβ levels in brain regions known to present early Aβ plaque accumulation in AD, including the posterior cingulate, precuneus, and insula23 associated with plasma p-tau181 only in the presence of astrocyte reactivity (Fig.1e).
Consistently, the stratified analysis within cohorts showed the same results. In the three enrolment sites, plasma p-tau181 levels increased as a function of Aβ burden only in CU Ast+ [Pittsburgh: β= -0.35, t = 3.10, p = 0.003 (Fig.1f), MYHAT: β = -0.20, t = 2.26, p = 0.026 (Fig.1h) and TRIAD: β = 0.57, t = 4.36, p < 0.0001 (Fig.1j)]. A steeper increase in plasma p-tau181 was observed in the research cohorts (TRIAD and Pittsburgh) compared to the community-based cohort (MYHAT). Similarly, we observed a significant interaction between Aβ burden and astrocyte reactivity status on plasma p-tau181 levels in the Pittsburgh (β = -0.29, t = 2.30, p = 0.022; Fig.1g), MYHAT (β = -0.19, t = 2.07, p = 0.039; Fig.1i) and TRIAD (β = 0.46, t = 2.92, p = 0.004; Fig.1k) cohorts. In a subset of participants from the Pittsburgh and MYHAT cohorts with available Aβ-PET (n = 150), we found the same results with increased plasma p-tau181 as a function of Aβ only in Ast+ (Supplemental Fig.1).
We also explored the impact of Ast+ in the associations of Aβ burden with plasma p-tau231 (available for Pittsburgh and TRIAD cohorts, n = 502) and p-tau217 (available for the TRIAD cohort, n = 136) levels in subsets of individuals that had these markers available. Plasma p-tau231 increased as a function of Aβ only in CU Ast+ individuals (Fig.1l). Additionally, we found a significant association between Aβ and plasma p-tau231 in CU Ast+ (β = 0.36, t = 4.62, p < 0.0001; Fig.1m, Supplemental Table 5) but not in CU Ast- individuals (β = 0.10, t = 1.87, p = 0.06). We also observed a significant interaction between Aβ and astrocyte reactivity status on plasma p-tau231 (β = 0.26, t = 2.84, p = 0.004; Fig.1m). Cohen’s d analysis suggests that the presence of both Aβ+ and Ast+ also had a strong effect on the levels of p-tau231 (Cohen’s d=0.91), whereas pathologies independently did not have a significant effect (Fig.1n). Similarly, plasma p-tau217 presented a steeper increase as a function of Aβ burden in Ast+ compared to Ast- (Fig.1o, Supplemental Table 5). An association between Aβ burden and plasma p-tau217 was observed in Ast- (β = 0.19, t = 3.48, p = 0.0008, Fig.1p), but with a much larger magnitude in Ast+ (β = 0.74, t = 5.62, p < 0.0001, Fig.1p) individuals. The stronger association in CU Ast+ individuals was further evidenced by a significant interaction between Aβ burden and astrocyte reactivity status on plasma p-tau217 (β = 0.52, t = 3.61, p = 0.0004, Fig.1p). The presence of both Aβ+ and Ast+ had the largest effect size on plasma p-tau217 increase (Cohen’s d = 1.41; Fig.1q) compared to p-tau181 and p-tau231. Importantly, a sex effect was observed in the association between Aβ and plasma p-tau epitopes in CU Ast+ individuals in all cohorts, with the association being stronger in men than women (Fig.1r, Supplemental Fig.2). The greater effect of Aβ burden on tau phosphorylation in the presence of Ast+ in men than in women may prove to play a role in the larger magnitude of effect of anti-Aβ therapies in man24. Finally, the presence of astrocyte reactivity did not impact the association between Aβ burden and NfL levels in any of the three cohorts (Supplemental Table 6), supporting that astrocyte reactivity unleashes Aβ effects on early tau pathology.
We used PET imaging available in the TRIAD cohort to determine the topographic localization of p-tau protein aggregates in the form of tangles (n = 147). Tau-PET deposition occurred as a function of Aβ burden only in CU Ast+ and in regions expected to present the earliest tau deposition (Fig.2a), affecting 100% and 62% of the extension of the Braak I and II regions, respectively (Fig.2b). As expected, in later Braak regions tau-PET did not increase as a function of Aβ in either group (Fig.2b). Finally, we investigated the link of baseline Aβ and astrocyte reactivity status with future tau-PET burden (n = 71; mean follow-up = 2.3 years; Supplemental Table 7). We observed that the annual rate of tau-PET accumulation was higher in the CU Ast+ group (Fig.2c) and predicted by baseline Aβ burden only in CU Ast+ (Fig.2d). Interestingly, while the baseline association was confined to the mesial temporal cortex, the longitudinal tau-PET accumulation as a function of Aβ/Ast presented initial tau spread over the neocortex in Braak III-IV regions (Fig.2e), further supporting the notion that these individuals are following a tau accumulation pathway consistent with AD progression25.
In summary, we provide biomarker evidence across multiple cohorts that increased astrocyte reactivity, measured by a plasma GFAP assay, plays a key role in the association of Aβ with early tau pathology in preclinical AD. The fact that the presence of abnormal astrocyte reactivity potentiates Aβ-triggered tau pathology may prove to favor the inclusion of astrocyte reactivity biomarkers in the biomarker modeling1 and biological definitions26 of AD. The strengths of our study include large sample size and the use of well-characterized research and population-based cohorts. While our cohort represents significant socioeconomic diversity, the main limitation is that our cohorts are composed mainly of White participants, which limits the generalizability of our findings to a more diverse world population. As biomarkers are naturally continuous, dichotomizing thresholds are invariably subject to conceptual and analytical idiosyncrasies and may change depending on the method used.
Furthermore, our findings support recent observations suggesting that plasma p-tau is a state marker of Aβ in preclinical AD3, but also add that this occurs mainly with the concomitant presence of astrocyte reactivity biomarker abnormality. As preventive clinical trials have increasingly focused on individuals in the earliest preclinical phases of AD, our results highlight that the selection of CU individuals Aβ+/Ast+ without overt p-tau abnormality may offer a time window very early in the disease process but with increased risk of AD-related progression. Finally, based on our results, we may speculate that a combination of drugs targeting both Aβ and astrocyte activation can potentiate the prevention of early tau pathology in preclinical AD.