MAPT R406W increases tau T217 phosphorylation in absence of amyloid pathology.

OBJECTIVE: Tau hyperphosphorylation at threonine 217 (pT217) in cerebrospinal fluid (CSF) has recently been linked to early amyloidosis and could serve as a highly sensitive biomarker for Alzheimer's disease (AD). However, it remains unclear whether other tauopathies induce pT217 modifications. To determine if pT217 modification is specific to AD, CSF pT217 was measured in AD and other tauopathies. METHODS: Using immunoprecipitation and mass spectrometry methods, we compared CSF T217 phosphorylation occupancy (pT217/T217) and amyloid-beta (Aß) 42/40 ratio in cognitively normal individuals and those with symptomatic AD, progressive supranuclear palsy, corticobasal syndrome, and sporadic and familial frontotemporal dementia. RESULTS: Individuals with AD had high CSF pT217/T217 and low Aß42/40. In contrast, cognitively normal individuals and the majority of those with 4R tauopathies had low CSF pT217/T217 and normal Aß 42/40. We identified a subgroup of individuals with increased CSF pT217/T217 and normal Aß 42/40 ratio, most of whom were MAPT R406W mutation carriers. Diagnostic accuracies of CSF Aß 42/40 and CSF pT217/T217, alone and in combination were compared. We show that CSF pT217/T217 × CSF Aß 42/40 is a sensitive composite biomarker that can separate MAPT R406W carriers from cognitively normal individuals and those with other tauopathies. INTERPRETATION: MAPT R406W is a tau mutation that leads to 3R+4R tauopathy similar to AD, but without amyloid neuropathology. These findings suggest that change in CSF pT217/T217 ratio is not specific to AD and might reflect common downstream tau pathophysiology common to 3R+4R tauopathies.

Regional correlation of biochemical measures of amyloid and tau phosphorylation in the brain.

Alzheimer's disease (AD) neuropathologic change is characterized by amyloid plaques and neurofibrillary tangles (NFTs) that consist of aggregated amyloid beta (Abeta) and hyperphosphorylated tau proteins (p-tau), respectively. Although the global relationship between Abeta and p-tau has been studied for decades, it is still unclear whether a regional correlation exists between Abeta and p-tau in the human brain. Recent studies in cerebrospinal fluid (CSF) have suggested that tau phosphorylation at specific sites such as T217 is modified at an early stage of AD when amyloid plaques become detectable. We applied biochemical and mass spectrometry methods in human brain samples with and without Abeta plaque pathology to measure site-specific phosphorylation occupancies in soluble and insoluble tau. Our quantitative results identified multiple residues specifically hyper-phosphorylated in AD, including at sites T111, T153, S184 (or S185), T205, S208, T217, S262, and S285 in brain soluble tau. In contrast, the most enriched phosphorylated residues in brain insoluble tau were T111, S113, T153, T181, S199, S202, T205, T217, T231, S262, and S396. Tau phosphorylation occupancies in the insoluble fraction were relatively constant across brain regions, suggesting that tau has a consistent phosphorylation pattern once it has aggregated into NFTs. We did not find regional association between Abeta42 and insoluble tau. However, the phosphorylation profile of soluble tau in AD brain was highly correlated to that in AD CSF, which was analyzed in a previous study. We also found a higher regional association between total Abeta42 and soluble tau phosphorylation occupancy at residues T111, T153 and T217 in the brain. This study provides insights into regional interactions between amyloidosis and specific tau phosphorylated residues in the human brain and may explain the specific increases of tau species phosphorylation observed in AD CSF.

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.

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.