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

Abstract

Background: Microtubule-associated protein tau and its phosphory-lation in cerebrospinal fluid (CSF) are currently used as biomarkers for the diagnostic of Alzheimer Disease (AD) by ELISA. In AD, tau phosphorylation in neurons is known to be significantly affected and aggregated hyperphosphorylated tau in neurofibrillary tangles (NFTs) is a feature of the disease physiopathology. However, AD tau phosphorylation modification is difficult to establish in CSF since immuno-detection are unable to provide site stoichiometry quantitation. Mass spectrometry (MS) appears as a promising technique to simultaneously monitor CSF tau and its numerous phos-phorylation sites. Nevertheless, CSF tau concentrations are particularly low and to date, no detection of CSF tau phosphopep-tides was reported in vivo by MS. Methods: We developed a high sensitive MS strategy to detect 5 sites of phosphorylation in CSF tau (T181, S199, S202, T205 and T217). Corresponding tau phos-phopeptides and their unmodified counterparts were simultaneously quantified by quantitative mass spectrometry with labeled standards as reference material. The method was therefore applied to the CSF analysis of a cohort of 50 memory clinics patients affected by various neurological disorders including AD, DLFT or LBD. Results: Results highlighted the strong correlation between the level of phosphopeptides and unmodified one confirming results obtained by ELISA in numerous previous studies. However , the amount of phosphorylation was not similar depending on the position of the residues. This allowed us to distinguish specific patterns in AD patients with the position T181 being only slightly hyperphosphorylated in comparison to the position T217 which was the one with the most significant difference. Interestingly, the position S199 was rather hypophosphorylated in AD. Combined with the global increasing of CSF tau concentration, T217 detection achieved a total discrimination of the AD patients in our cohort suggesting that it represent a promising tool to discriminate AD. Conclusions: The MS quantification of CSF tau phosphopeptides using mass spectrometry is a promising analytical tool to study AD tau-opathy. It brings also new perspectives for AD diagnosis, therapy targeting and monitoring. Background: Due to the ever-increasing aging population, there is an urgent need to identify biomarkers that can accurately detect and diagnose Alzheimer's disease (AD). Our previous studies have not only demonstrated the ubiquity and abundance of autoan-tibodies in human sera, but have also determined that subsets of these blood-based autoantibodies can serve as disease-specific bio-markers capable of diagnosing mild-moderate stages of AD and Parkinson's disease with high sensitivity and specificity. Pathological changes linked to AD are known to precede overt clinical symptoms for up to a decade prior to clinical diagnosis, thereby providing a potential window of opportunity for early and even pre-symptomatic detection, which offer the most potential benefit to patients. In the present study, our objective was to identify a panel of autoantibody biomarkers that is useful for accurate diagnosis of patients with mild cognitive impairment (MCI) driven by early stages of AD pathology. Methods: Sera from a total of 236 subjects, including 50 MCI subjects with confirmed low CSF Ab42 levels enrolled in the Alzheimer's Disease Neuroimaging Initiative 2 (ADNI2) study, were screened with human protein mi-croarrays containing 9,486 potential antigen targets to identify autoantibody biomarkers for MCI. Autoantibodies with a higher prevalence in MCI were identified and then tested using Random Forest for their ability to distinguish MCI subjects from age-and sex-matched controls, as well as from individuals with other neuro-degenerative and non-neurodegenerative diseases. Autoantibody biomarker performance was further evaluated using Receiver Operating Characteristic (ROC) curves. Results: Results demonstrate that autoantibody biomarkers detected in serum can differentiate early-stage MCI patients from age-and sex-matched controls with an overall accuracy, sensitivity, and specificity of 100.0%. These biomarkers were also capable of differentiating MCI patients from those with mild-moderate AD with an overall accuracy of 98.7%. Additionally, they can also be used to distinguish MCI subjects from those with other neurological (e.g., Parkinson's disease and multiple sclerosis) and non-neurological (e.g., breast cancer) diseases. Conclusions: These results demonstrate, for the first time, that autoantibodies can be used as relatively non-invasive and effective blood-based biomarkers for early diagnosis and staging of AD.