Characterization of Novel CSF Tau and ptau Biomarkers for Alzheimer’s Disease

Abstract

Cerebral spinal fluid (CSF) Aβ42, tau and p181tau are widely accepted biomarkers of Alzheimer’s disease (AD). Numerous studies show that CSF tau and p181tau levels are elevated in mild-to-moderate AD compared to age-matched controls. In addition, these increases might predict preclinical AD in cognitively normal elderly. Despite their importance as biomarkers, the molecular nature of CSF tau and ptau is not known. In the current study, reverse-phase high performance liquid chromatography was used to enrich and concentrate tau prior to western-blot analysis. Multiple N-terminal and mid-domain fragments of tau were detected in pooled CSF with apparent sizes ranging from <20 kDa to ~40 kDa. The pattern of tau fragments in AD and control samples were similar. In contrast, full-length tau and C-terminal-containing fragments were not detected. To quantify levels, five tau ELISAs and three ptau ELISAs were developed to detect different overlapping regions of the protein. The discriminatory potential of each assay was determined using 20 AD and 20 age-matched control CSF samples. Of the tau ELISAs, the two assays specific for tau containing N-terminal sequences, amino acids 9-198 (numbering based on tau 441) and 9-163, exhibited the most significant differences between AD and control samples. In contrast, CSF tau was not detected with an ELISA specific for a more C-terminal region (amino acids 159-335). Significant discrimination was also observed with ptau assays measuring amino acids 159-p181 and 159-p231. Interestingly, the discriminatory potential of p181 was reduced when measured in the context of tau species containing amino acids 9-p181. Taken together, these results demonstrate that tau in CSF occurs as a series of fragments and that discrimination of AD from control is dependent on the subset of tau species measured. These assays provide novel tools to investigate CSF tau and ptau as biomarkers for other neurodegenerative diseases.

Differential Mass Spectrometry Profiles of Tau Protein in the Cerebrospinal Fluid of Patients with Alzheimer’s Disease, Progressive Supranuclear Palsy, and Dementia with Lewy Bodies

“…Because in our study the Tau protein was trypsin digested before analysis, we did not have a direct access to the exact fragments present in CSF. However, our results are coherent with the study of Meredith et al [30] which described N-and C-terminal fragments associated with a 17 kDa central core fragment. Differences in output observed between the present study and those by Portelius et al [28] and McAvoy et al [27] can be accounted for in various ways different origins.…”

Tau Monoclonal Antibody Generation Based on Humanized Yeast Models

“…This is in agreement with previously published studies (35)(36)(37). Recently, improved characterization of CSF Tau using reverse-phase high performance liquid chromatography led to the evaluation of five total Tau assays side by side, and the two assays containing N-terminal sequences exhibited the most significant differences between AD and control CSF (66). This suggests that the use of mAbs such as ADx215, recognizing the N terminus of Tau, may indeed improve diagnostic accuracy.…”

Tau Diagnostics and Clinical Studies

“…Importantly, despite the observation that primary tauopathies present severe neurodegeneration and tau pathology, CSF T-tau and Ptau show no clear change (Masters et al 2015).This may be due to the fact that current tau immunoassays are based on antibodies binding to the mid region of tau and may miss pathogenic tau due to truncation or masking during oligomer/aggregate formation. Recent studies suggest that CSF tau consists of several protein fragments (Meredith et al 2013, Barthelemy et al 2016, and metabolic processing of tau into N-, mid and C-terminal fragments may also be key for secretion, aggregation and spreading pathology of tau, and may differ between the tauopathies (see below). Furthermore, the potential of tau oligomers or cis-tau variants (Kondo et al 2015) as specific biomarkers for tauopathies has not been explored.…”

“…Recent data have shown that fragments of tau are able to cross the blood-brain barrier with different rates, depending on the precise amino acid sequence (Banks et al 2016). The CSF tau profile is composed entirely of tau fragments rather than full-length protein, with different fragmentation patterns dependent on disease state and progression (Johnson et al 1997;Meredith et al 2013). CSF tau CTF and NTF levels are decreased in PSP, while increases were observed in AD (Wagshal et al 2015).…”