Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by the accumulation of amyloid-beta (Aβ) plaques and neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein. The development of sensitive and specific blood-based biomarkers for AD is crucial for early diagnosis, disease monitoring, and evaluating therapeutic interventions. While cerebrospinal fluid (CSF) biomarkers have shown promise, their invasive nature limits their widespread use. Recent research has focused on blood-based biomarkers, particularly phosphorylated tau (p-tau) isoforms, which have demonstrated strong associations with AD pathology and clinical progression. Several p-tau assays targeting different phosphorylation sites (e.g., p-tau181, p-tau217, p-tau231) have shown high diagnostic accuracy. However, the contribution of specific p-tau phosphorylation sites, such as p-tau212, remains less well understood. This study aimed to investigate the diagnostic utility of plasma p-tau212 as a novel biomarker for AD, focusing on its performance in detecting both amyloid and tau pathology, comparing it with other established p-tau biomarkers and validating it through autopsy confirmation.

Numerous studies have highlighted the potential of blood-based biomarkers, particularly plasma p-tau, in the diagnosis and prognosis of AD. Plasma Aβ biomarkers, while showing high diagnostic accuracy, exhibit modest changes between Aβ-positive and Aβ-negative individuals, limiting their clinical robustness. In contrast, plasma p-tau biomarkers (p-tau181, p-tau217, p-tau231) have shown stronger associations with brain Aβ load, the severity of AD pathology, and the prediction of longitudinal changes in brain pathology. These plasma p-tau assays have demonstrated higher sensitivity (>100%) in distinguishing between Aβ-positive and Aβ-negative individuals, especially in advanced disease stages. Moreover, they accurately identify abnormal amyloid PET scans, positive neuropathological diagnoses after autopsy, and predict longitudinal cognitive decline. Plasma p-tau variants have also shown promise as screening tools to identify individuals at risk of future cognitive impairment or AD pathology. Given their high performance and analytical robustness, plasma p-tau biomarkers are being used as surrogates of brain Aβ and tau pathology in clinical trials of anti-amyloid therapies. Head-to-head comparisons have consistently shown p-tau217 to have higher accuracy for AD pathology than p-tau181 and p-tau231, particularly in cognitively unimpaired individuals. This study builds upon these findings by focusing on the p-tau212 isoform and its potential as a novel diagnostic biomarker.

This study involved the development and validation of a novel blood-based immunoassay specific for p-tau212, without cross-reactivity to p-tau217. Two sheep monoclonal antibodies (mAbs) were generated, one specific to p-tau212 and another to p-tau217. The specificity of these antibodies was rigorously assessed using ELISAs against various synthetic peptides and recombinant proteins with different phosphorylation patterns. Immunohistochemical staining was performed on brain tissue slices from autopsy-verified AD patients to evaluate the antibody reactivity with neurofibrillary tangles. The p-tau212 immunoassay was developed using the Simoa HD-X platform, pairing the p-tau212 antibody with the N-terminal-tau targeting antibody Tau12. The assay's performance characteristics (linearity, precision, recovery) were thoroughly evaluated. The assay was then used to measure p-tau212 levels in plasma and CSF samples from five independent cohorts (total n=388): the Baltimore Longitudinal Study of Aging (BLSA)-Neuropathology cohort (autopsy-verified AD, asymptomatic AD, non-AD controls), the University of California San Diego (UCSD)-Neuropathology cohort (autopsy-confirmed AD), a Gothenburg cohort (biomarker-positive AD vs. biomarker-negative controls), a Slovenian memory clinic cohort (AD continuum), and a Polish cohort (paired CSF and plasma samples). Diagnostic performance was evaluated using receiver operating characteristic (ROC) curves, area under the curve (AUC), and fold changes compared to control groups. Associations between plasma p-tau212 levels and amyloid plaque and tangle densities were analyzed. Statistical comparisons were made using appropriate parametric and non-parametric tests, accounting for relevant covariates. Mass spectrometry was also employed for independent validation of the assay.

The p-tau212 and p-tau217 antibodies showed high specificity for their target phosphorylation sites. Immunohistochemical staining revealed prominent p-tau212 reactivity in neurofibrillary tangles, co-localizing with p-tau217 and p-tau202/205. The developed p-tau212 Simoa assay demonstrated strong analytical performance characteristics. In the BLSA-Neuropathology cohort, plasma p-tau212 was significantly higher in autopsy-verified AD compared to asymptomatic AD and non-AD controls. Similarly, in the UCSD-Neuropathology cohort, CSF p-tau212 showed significant increases with increasing severity of amyloid pathology and Braak stage. The Gothenburg cohort demonstrated a 3.7-fold higher plasma p-tau212 in biomarker-positive AD compared to biomarker-negative controls, exceeding the fold changes observed for p-tau181 and p-tau231. Plasma p-tau212 showed comparable diagnostic accuracy to CSF p-tau212 in differentiating Aβ+ AD from Aβ- controls. In the Slovenian memory clinic cohort, plasma p-tau212 levels increased with disease severity, mirroring the pattern observed for p-tau217. The concordance between plasma p-tau212 and p-tau217 in identifying Aβ-positivity was high (83.5%). Plasma p-tau212 showed comparable correlations with CSF Aβ, p-tau181, total tau, and Braak stage as p-tau217.

This study demonstrates the strong potential of plasma p-tau212 as a readily accessible biomarker for AD. The high diagnostic performance of plasma p-tau212, comparable to p-tau217 and superior to p-tau181 and p-tau231, across multiple independent cohorts and with autopsy verification, validates its utility. The close correlation between p-tau212 and p-tau217 levels suggests similar roles in disease pathogenesis, possibly reflecting a common pathway of tau hyperphosphorylation. The significant associations between p-tau212 levels and both amyloid and tau pathology, both in post-mortem and in vivo settings, further solidify its relevance to AD. The study's findings suggest that p-tau212 could be utilized for early diagnosis, disease monitoring, patient stratification for clinical trials, and population screening. The ease of obtaining blood samples compared to CSF makes p-tau212 a highly attractive biomarker for routine clinical practice.

Plasma p-tau212 is a promising novel biomarker for Alzheimer's disease. This study demonstrates its strong diagnostic accuracy and association with AD pathology, comparable to established p-tau biomarkers. Further research should focus on validating p-tau212's performance in pre-symptomatic stages and investigating its utility in monitoring disease progression and treatment response. The development of simple, inexpensive, and widely accessible assays for p-tau212 is crucial for its successful implementation in clinical settings.

While this study involved multiple cohorts and autopsy verification, the relatively small sample sizes in some cohorts might limit the generalizability of the findings. Future studies with larger, more diverse populations are needed to fully characterize the performance of p-tau212. The study primarily focused on AD, and further investigation is needed to assess the specificity of p-tau212 for AD compared to other neurodegenerative disorders. The cross-sectional nature of some cohorts limits inferences regarding longitudinal changes.