Tau Phosphorylation Rates Measured by Mass Spectrometry Differ in the Intracellular Brain vs. Extracellular Cerebrospinal Fluid Compartments and Are Differentially Affected by Alzheimer’s Disease

Abstract

Tau protein aggregation into neurofibrillary tangles in the central nervous system contributes to the etiology of certain neurodegenerative disorders, including Alzheimer’s disease (AD). Though the mechanism of tau destabilization is not fully understood yet, tau protein has been found to be hyperphosphorylated in tau aggregates. To investigate this further, we developed a highly sensitive and specific mass spectrometry (MS) method using parallel reaction monitoring (PRM) to identify tau phosphorylation sites. This method enables us to compare the abundance of phosphorylation sites in tau proteins in the brain and cerebrospinal fluid (CSF) in humans with and without AD. We detected 29 distinct phosphorylated tau (p-tau) sites in full-length tau from soluble human brain lysate and 12 sites on truncated tau in CSF, mainly in the mid-domain. Brain soluble tau phosphorylation sites are localized on three domains including a proline-rich mid-domain, the C-terminus, and a cluster on the N-terminal projection domain not previously characterized. Some phosphorylation sites increased in CSF, while others decreased compared to brain. Notably, phosphorylation on T205 and S208, recognized by AT8 antibody defining Braak stages of brain tau aggregation, were not detected in normal brain soluble tau but were found in the CSF. Comparison of the p-tau rates from the brain and the CSF indicated that the abundance of phosphorylated sites varied in a site-specific manner. CSF tau proteins from non-AD participants were significantly hyperphosphorylated on T111, T205, S208, T217 and T231. In AD CSF, hyperphosphorylation on these sites was exacerbated, and phosphorylation on T153 and T175 specifically were detected. This supports the hypothesis that tau hyperphosphorylation could be a physiological process amplified by AD pathology. Conversely, we found that S202 was hypophosphorylated in CSF and was not hyperphosphorylated in AD, demonstrating that p-tau isoforms could have different metabolisms depending on which sites are phosphorylated. These site-specific p-tau rates are independent of tau concentration and distinct of current CSF tau and p-tau assays measuring tau isoforms levels. Targeted MS multiplexing ability and high-throughput capacity lets us envision the use of these new p-tau measurements as promising biomarkers for AD diagnosis and tracking therapeutic responses.

Sleep Deprivation Affects Tau Phosphorylation in Human Cerebrospinal Fluid

“…For tau phosphorylated at T217 (pT217), there was a large increase of 60 to 80% in the sleep‐deprived group compared to the control, with no change in the drug group (see Fig D–F, Table ). To assess the phosphorylation occupancy on each site, the ratio of p‐tau to the unphosphorylated form was calculated . Each p‐tau species responded to sleep deprivation in opposite directions (see Fig G–I, Table ).…”

“…All samples were collected and processed as previously described . Because previous studies have shown a linear rise in serially sampled CSF Aβ concentrations in Aβ‐negative individuals that is dependent on sampling frequency and volume, 6ml of CSF was sampled every 2 hours in all groups.…”

“…Because previous studies have shown a linear rise in serially sampled CSF Aβ concentrations in Aβ‐negative individuals that is dependent on sampling frequency and volume, 6ml of CSF was sampled every 2 hours in all groups. After 1ml of CSF at each time point and media standards were thawed, tau was purified and processed for mass spectrometry (MS) by immunoprecipitation (IP) as reported . The IP mixture was comprised of 1ml of CSF, 5ng of 15N‐441 tau internal standard, 5mM guanidine, and 1% NP‐40 and 1X protease inhibitor cocktail (Roche, Basel, Switzerland).…”

“…After rinsing, immobilized samples were digested overnight at 37°C with 400ng of Trypsin Gold (Promega, Madison, WI) then extracted on C18 tips (TopTip; Glygen, Columbia, MD). Unphosphorylated tau and p‐tau peptides were separated using nano‐liquid chromatography (nanoACQUITY UPLC; Waters, Milford, MA) on a Waters HSS T3 column (75μm × 100mm, 1.8μm) operating at a flow rate of 400ng/μl and measured on mass spectrometer (Fusion Tribrid; Thermo Scientific, San Jose, CA) operating in parallel reaction monitoring using high‐energy collisional dissociation fragmentation as reported . Skyline software (MacCoss lab, University of Washington, Seattle, WA) was used to collect and quantify MS data.…”

Perspectives in fluid biomarkers in neurodegeneration from the 2019 biomarkers in neurodegenerative diseases course—a joint PhD student course at University College London and University of Gothenburg

“…AD pathology also significantly affects phosphorylation patterns, with hyperphosphorylation seen of a number CSF tau sites in comparison to healthy controls. In addition, a distinct phosphorylation site (T153) has been identified in AD CSF, which is absent in non-AD CSF [26].…”