Nicolas R. Barthélemy

Tau Protein Quantification in Human Cerebrospinal Fluid by Targeted Mass Spectrometry at High Sequence Coverage Provides Insights into Its Primary Structure Heterogeneity

Tau protein plays a major role in neurodegenerative disorders, appears to be a central biomarker of neuronal injury in cerebrospinal fluid (CSF), and is a promising target for Alzheimers disease immunotherapies. To quantify tau at high sensitivity and gain insights into its naturally occurring structural variations in human CSF, we coupled absolute quantification using protein standard with the multiplex detection capability of targeted high-resolution mass spectrometry (MS) on a Quadrupole-Orbitrap instrument. Using recombinant tau we developed a step-by-step workflow optimization including an extraction protocol that avoided affinity reagents and achieved the monitoring of 22 tau peptides uniformly distributed along the tau sequence. The lower limits of quantification ranged (LLOQ) from 150 to 1500 pg/mL depending on the peptide. Applied to endogenous CSF tau, up to 19 peptides were detected. Interestingly, there were significant differences in the abundance of peptides depending on their position in the sequence, with peptides from the tau mid-domain appearing significantly more abundant than peptides from the N- and C-terminus domains. This MS-based strategy provided results complementary to those of previous ELISA or Western Blot studies of CSF tau and could be applied to tau monitoring in human CSF cohorts.

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.

Microtubule-associated Tau proteins are major actors in neurological disorders, the so-called tauopathies. In some of them, and specifically in Alzheimers disease (AD), hyperphosphorylated forms of Tau aggregate into neurofibrillary tangles. Following and understanding the complexity of Taus molecular profile with its multiple isoforms and post-translational modifications represent an important issue, and a major analytical challenge. Immunodetection methods are, in fact, limited by the number, specificity, sensitivity, and capturing property of the available antibodies. Mass spectrometry (MS) has recently allowed protein quantification in complex biological fluids using isotope-labeled recombinant standard for absolute quantification (PSAQ). To study Tau proteins, which are found at very low concentrations within the cerebrospinal fluid (CSF), we relied on an innovative two-step pre-fractionation strategy, which was not dependent on immuno-enrichment. We then developed a sensitive multiplex peptide detection capability using targeted high-resolution MS to quantify Tau-specific peptides covering its entire sequence. This approach was used on a clinical cohort of patients with AD, progressive supranuclear palsy (PSP), and dementia with Lewy body (DLB) and with control non-neurodegenerative disorders. We uncovered a common CSF Tau molecular profile characterized by a predominance of central core expression and 1N/3R isoform detection. While PSP and DLB tau profiles showed minimal changes, AD was characterized by a unique pattern with specific modifications of peptide distribution. Taken together these results provide important information on Tau biology for future therapeutic interventions, and improved molecular diagnosis of tauopathies.

Quantitative detection of amyloid-β peptides by mass spectrometry: state of the art and clinical applications

Abstract Alzheimer’s disease (AD) is the most common form of dementia in humans, and a major public health concern with 35 million of patients worldwide. Cerebrospinal fluid (CSF) biomarkers being early diagnostic indicators of AD, it is essential to use the most efficient analytical methods to detect and quantify them accurately. These biomarkers, and more specifically amyloid-β (Aβ) peptides, are measured in routine clinical practice using immunoassays. However, there are several limits to this immunodetection in terms of specificity and multiplexing of the multiple isoforms of the Aβ peptides. To overcome these issues, the quantification of these analytes by mass spectrometry (MS) represents an interesting alternative, and several assays have been described over the past years. This article reviews the different Aβ peptides quantitative MS-based approaches published so far, compares their pre-analytical phase, and the different quantitative strategies implemented that might be suitable for clinical applications.

Antibody-free quantification of seven tau peptides in human CSF using targeted mass spectrometry

Tau protein concentration in cerebrospinal fluid (CSF) is currently used as a sensitive and specific biomarker for Alzheimers disease. Its detection currently relies on ELISA but the perspective of using mass spectrometry (MS) to detect its different proteoforms represents an interesting alternative. This is however an analytical challenge because of its low concentration in the CSF, a biological fluid collected in small volume by lumbar puncture, and with a high structural heterogeneity. To overcome these issues, instead of using immunocapture as previously done, we rather relied on an original two steps pre-fractionation technique of CSF: perchloric acid (PCA) followed by micro solid phase extraction (μSPE). We could then measure seven tau trypsic peptides by Multiple Reaction Monitoring (MRM) on a triple quadrupole mass spectrometer. Quantification was performed using isotopically labeled 15N- recombinant tau protein as internal standard and validated using CSF pools with low, medium, or high tau concentrations (HTCs). Repeatability, intermediate precision, linearity, limit of quantification (LOQ), and recovery were calculated for the different peptides. This new MRM assay, which allowed for the first time CSF tau protein quantification without immunocapture, has important potential application to follow tau metabolism in both diagnostic and therapeutic research.

Quantitative mass spectrometry (SRM/MRM) for amyloid peptides, tau protein and apolipoprotein E in human cerebrospinal fluid for Alzheimer's disease diagnosis

RR intervals(SDNN) and the square root of the mean of the squares of successive RR intervals differences(rMSSD) mean values were significantly lower in patients with AD than in those with MCI or in controls. There were no significant differences between MCI and control groups in mean R-R interval, 5-min total power, VLF, LF and HF or LF/HF ratio. MMSE was negatively correlation with age and duration with illness. HF power emerged as the parameters better related to MMSE. Conclusions: These results suggest that the presence of autonomic cardiac dysfunction in AD patients might be due to a cholinergic deficit in the peripheral autonomic nervous system. The ongoing follow-up of this study population will verify the potential role of the HRV parameters in predicting the degeneration of cognitive functions and mortality rates.

Tau hyperphosphorylation on T217 in cerebrospinal fluid is specifically associated to amyloid-β pathology

Introduction Modification of CSF tau phosphorylation in AD remains controversial since total-tau and phospho-tau levels measured by immunoassays are highly correlated. Methods Stoichiometry of phosphorylation of CSF tau was monitored on five sites by mass spectrometry. We compared 50 participants with AD at mild to moderate stages, others tauopathies and controls then confirmed our results in a cohort of 86 participants cognitively normal or with mild cognitive impairment and stratified according to amyloid-β status. Results Changes in tau phosphorylation rates were observed in AD participants but not in other tauopathies. T181 appeared slightly hyperphosphorylated in AD. In comparison, T217 phosphorylation, was considerably modified. We demonstrated T217 hyperphosphorylation occurred systematically in amyloid positive participants even at preclinical stage (AUC 0.999). T217 phosphorylation change specificity overpasses other phosphorylated tau sites investigated in this study, but also CSF total-tau and p-tau levels. Discussion CSF T217 hyperphosphorylation defines a specific tauopathy status concomitant with ß-amyloidosis.

Erratum: Tau Kinetics in Neurons and the Human Central Nervous System (Neuron (2018) 97(6) (1284–1298.e7)(S0896627318301363)(10.1016/j.neuron.2018.02.015))

Chihiro Sato,* Nicolas R. Barthélemy, Kwasi G. Mawuenyega, Bruce W. Patterson, Brian A. Gordon, Jennifer Jockel-Balsarotti, Melissa Sullivan, Matthew J. Crisp, Tom Kasten, Kristopher M. Kirmess, Nicholas M. Kanaan, Kevin E. Yarasheski, Alaina Baker-Nigh, Tammie L.S. Benzinger, Timothy M. Miller, Celeste M. Karch,* and Randall J. Bateman* *Correspondence: satochihiro@wustl.edu (C.S.), karchc@wustl.edu (C.M.K.), batemanr@wustl.edu (R.J.B.) https://doi.org/10.1016/j.neuron.2018.04.035

MASS SPECTROMETRY–BASED MEASUREMENT OF LONGITUDINAL CSF TAU IDENTIFIES DIFFERENT PHOSPHORYLATED SITES THAT TRACK DISTINCT STAGES OF PRESYMPTOMATIC DOMINANTLY INHERITED AD

exosomes isolated from injected mice contained human tau species as measured by ELISA, suggesting exosome trafficking from the CNS to the periphery. Conclusions: Here, we are the first to demonstrate that secreted exosomes isolated from hiPSCs can propagate human tau and oAb species to multiple brain regions and traffic into the periphery. Our model system provides a means to study transcellular propagation of exosome cargo, and related neurodegenerative mechanisms. Together, our data suggests that exosomes are mediators of AD pathogenesis and can serve as potential targets for AD therapeutics.

Tau kinetics in the human cns

new datasets), increasing our discovery sample to 21,433 cases and 44,340 controls. Methods: All datasets were imputed to a 1000 Genomes reference panel (Phase 1 v3, March 2012) of over 37 million variants, many of which are low-frequency single nucleotide variants (SNV) and indels. Single-variant-based association analysis was conducted adjusting for age, sex and population substructure. Individual datasets were analyzed with the score test for case-control datasets and general estimating equations (with generalized linear mixed model for rare variants) for family-based analyses. Within-study results were meta-analyzed in METAL. Gene-based testing was conducted on summary statistics using VEGAS. Results: Imputation produced approximately nine million high-quality low-frequency variants for analyses. Twenty-five loci were genomewide significant at P 5310-8, including five novel loci. Three of these novel loci are driven by significant low-frequency variants, while two are associations of common intergenic variants between the genes USP6NL and ECHDC3 at Chr10: 10:11720308 (P1⁄42.91x10) and the genes CYYR1 and ADAMTS1 at Chr21: 28,156,856 (P1⁄41.44x10). Previously reported rare and low-frequency variants in TREM2 and SORL1 were also significantly associated, while low-frequency SNVs in the common loci BIN1 (MAF1⁄40.026) and CLU (MAF1⁄40.029) show suggestive significance (P 5310-7). Twelve additional loci produced signals with suggestive significance, seven driven by low-frequency or rare variants and five driven by common variants. Genotyping to confirm imputation quality, and replication genotyping using the Sequenom MassArray are underway. Gene-based analyses identified 13 significantly associated genes (Bonferroni P 2.83x10-6), four of which are novel loci driven by nominally significant low-frequency variants. Conclusions: Using an imputation set with a large number of rare variants we identified several novel candidate loci for LOAD, giving support to the hypothesis that rare and low-frequency variant imputation can identify novel associations with disease.

Mass spectrometry follow-up of t181, s199, s202, t205, and T217 tau phosphorylation in cerebrospinal fluid from patients revealed a specific Alzheimer’s disease pattern

P4-234 MASS SPECTROMETRY FOLLOW-UP OF T181, S199, S202, T205, AND T217 TAU PHOSPHORYLATION IN CEREBROSPINAL FLUID FROM PATIENTS REVEALED A SPECIFIC ALZHEIMER’S DISEASE PATTERN Nicolas Barthelemy, Christophe Hirtz, Susanna Schraen, Martial Seveno, Randall Bateman, Philippe Marin, Francois Becher, Audrey Gabelle, Sylvain Lehmann, Washington University School of Medicine, St. Louis, MO, USA; CHU de Montpellier, Montpellier, France; Inserm, UMR 837, Alzheimer & Tauopathies, Facult e de M edecine, Lille, France; PPF, IGF, CNRS-UMR 5203, Inserm U661, Montpellier, France; CEA, iBiTec-S, SPI, LEMM, Gif-sur-Yvette, France; CHRU Gui de Chauliac Hospital, Montpellier, France. Contact e-mail: barthelemyn@ neuro.wustl.edu