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The aim of the study was to apply the TMTcalibrator+ MS approach to explore the possibility of detecting markers of microglial activation in the CSF, and comparing the levels of such proteins in the CSF of AD patients compared to non-AD controls. We hypothesise that an early event in neurodegenerative pathology involves activation of microglial cells that will secrete distinct proteins into the brain and subsequently into CSF and ultimately plasma. The challenge is how to find these molecules in a readily available biofluid. Traditional unbiased proteomic approaches have struggled to detect low abundance markers due to the high dynamic range of plasma and CSF proteins, and the predominance of plasma albumin and other hyper-abundant proteins. To improve biomarker discovery in peripheral fluids, we developed TMTcalibrator+; a novel MS workflow. Here the use of isobaric Tandem Mass Tags (TMT) allows reference tissue or cell line samples to be labelled and mixed into equivalently labelled samples of peripheral biofluids at a concentration sufficient to ensure the vast majority of MS/MS acquisitions are made on reference-derived peptides. In this study, we analysed the peptides present in the CSF from AD and non-AD patients that are common to activated microglia cells to determine which activated microglial proteins are also found in CSF and which, if any, are differentially expressed in AD compared to individuals with normal cognitive function.

The diagnosis and management of Alzheimer’s disease and related dementias remains challenging due to a lack of accurate biomarkers. Whilst changes in the cerebrospinal fluid (CSF) levels of amyloid beta and tau proteins perform well as diagnostic markers in established disease, they lack adequate sensitivity for use in early diagnosis and offer little prognostic value. To expand the utility of CSF in management of dementia we have developed a panel of 16 proteins based on our prior proteomics discovery projects, review of the literature and consultation with key opinion leaders.

Many neurodegenerative diseases are characterised by abnormal posttranslational modification (PTM) of one or more proteins leading to their aggregation and, by mechanisms not yet fully understood, ultimately to neuronal cell death. One of the most common forms of pathological PTM is phosphorylation, with phosphorylated forms of tau (AD), TDP-43 (ALS, FTLD) and alpha-synuclein (PD) all associated with disease, whereas decreased phosphorylation on huntingtin (HD) is thought to be protective. The role of cell signalling pathways, mostly regulated by kinase/phosphatase cascades is also increasingly being recognised as modulating key aspects of neurodegeneration such as re-activation of cell cycle in neuronal cells and activation of immune cells such as microglia and astrocytes. Accordingly, there is a strong rationale to develop and apply global phosphorylation profiling to neuronal cell cultures and brain tissues used in neurodegenerative disease research. We have recently reported SysQuant, a global phosphoproteomic workflow that employs isobaric Tandem Mass Tags (TMT) and differential chromatography to measure fractions of both enriched phosphopeptides and the un-enriched nonphosphorylated peptides derived from tryptic digestion of tumour cell lysates of up to 10 samples in a single mass spectrometry experiment(1). We have now applied SysQuant to frozen brain tissue from a mouse model of human tauopathy (TMHT Mouse, QPS Austria) treated with vehicle or vehicle + kinase inhibitors, and we are able to demonstrate significant, potentially protective effects on the phosphorylation of aggregating proteins, as well as regulation of key pathways such as glycolysis/gluconeogenesis, calcium signalling, oxidative phosphorylation, citrate cycle along with all major neurodegenerative KEGG pathways. By using a TMT 10plex labelling strategy we were able to generate a detailed, quantitative phosphoproteomic map for 3 x 3 x 3 animals treated with vehicle or one of two kinase inhibitors from hippocampus, cortex and rest of brain regions in approximately 8 weeks. Data from the hippocampus is presented here.

Background: Studies in humans have shown the potential role of alterations in DNA methylation in the pathogenesis of Late-Onset Alzheimer’s disease (LOAD). The epigenetic regulation of the genes involved in the Alzheimer’s pathogenesis may help to explain the complexity of the LOAD. Methods: DNAwas extracted from peripheral blood mononuclear cells of 100 Colombian AD patients (mean age 77.56 5 years) and 100 healthy subjects (mean age 75.36 5,5years). DNAwas bisulfite treated using EZ DNAMethylation Direct Kit (Zymo-research). Primers were manually designed for the 5’ region of the CpG Island in the APP promoter and APOE CpG Island on exon 4. The APP andAPOEMS-HRMswere performed in a CFX 96 device (BioRad), using 14 ng of bisulfite treated DNA. The quantitative results were obtained by interpolation of the first derived RFU normalized data, generated by the linear regression of the standard curve. Results: The correlation coefficient of the linear regression of the standard curves was r1⁄4 0.96 for APP gen and 0.898 for APOE gen The inter-assay and intra-assay reproducibilities were verified, having a coefficient of variance less than 0.5 between replicates. In a preliminary analysis we did not find differences in APP methylation levels between AD patients and control subjects (mean 2.14% +1.15 vs 2.05 +0.96). For APOE the difference was not significant inmethylation levels (patients vs. Controls 73.01 vs 70.9)Conclusions:The present study tested changes in DNA methylation of in blood samples from Colombian LOAD patients, which showed no significant differences in comparison with control samples for APP and APOE genes. This is the first report of the analysis of APP and APOE DNA methylation levels in LOAD using the quantitative and cost-effective in Latin American AD patients. It would be important to analyze, in further studies, the methylation levels for additional candidate regions in several tissues from AD patients.

Background:Non-invasive diagnostic markers are needed to ensure that current and future Alzheimer’s disease (AD) therapies are targeted to the right patient population. Previously, we identified plasma desmosterol as a candidate marker, with decreased levels observed (p value< 0.05, fold change1⁄4 0.36) in AD plasma samples versus controls plasma (Control/AD1⁄4 10/10). In this study, to validate this new plasma biomarker, we performed the assay using a larger commercially available Caucasian sample set and a large Asian cohort. In addition, we clarified if there are correlations between plasma desmosteroland well-established CSF biomarkers, like phosphorus Tau and Ab. Methods:We performed a validation study using a larger Caucasian sample set (Cohort 1: 109 patients, Control, MCI and AD) and a large Asian cohort (Cohort 2: 401 patients, AD and control) with LCMS. And the measurement of total Tau, phosphorus Tau (P181), Ab40 and42 were also performed using CSF from same patients. Results: At first, we performed the assay using a larger Caucasian sample set (Cohort 1: Control/MCI/AD1⁄4 42/26/41). This study validated our previous finding of significant decrease of both plasma desmosterol (p value < 0.001, fold change: 0.6) in AD and MCI patients (relative to Control), demonstrating their potential value as circulating biomarkers. The AUC of ROCcurve using plasma desmosterol was found to be 0.80, indicating high discriminating power of this marker in the two reference populations, confirming the validity of this potential biomarker in AD. Further analysis also showed significant correlations of plasma desmosterol with CSFAb 42/Ab 40 and Mini-Mental State Examination (MMSE) scores, suggesting that plasma desmosterolmight be reflective of disease progression. Next we performed a follow-up study using a large Asian cohort (Cohort 2: Control/AD1⁄4 201/200). Data analysis showed that desmosterol level in plasmawas found to be significantly different betweenADand control groups with p-values 2.3E-14 and comparableAUCofROCcurve.Moreover, like for cohort 1, high correlation between plasma desmosterol level andMMSE score was observed. Conclusions: Our study results validate the utility of desmosterol as a novel non-invasive AD biomarker candidate that should be further investigated in other dementia types.

In vivo effects of CK1d inhibitor effect on cognition and tau phosphorylation All compounds were well tolerated in this study with no overt signs of any adverse events. The TMHT model shows cognitive decline from 5 months but no motor effects and performance in the Morris Water Maze was therefore used to study compound effect on spatial learning and memory. All compounds showed some improvement relative to vehicletreated controls but only PS110 achieved statistical significance in both path length and escape latency (Figure 3).

which were combined with the PD and DS samples. Support Vector Machine analysiswas utilized to classify the 4 diagnostic groups using their overall biomarker profiles. The biomarker profiles yielded 100% accuracy in classifying all 4 diagnostic categories. When looking at individual proteins, pancreatic polypeptide was selectively dysregulated in PD, thrombopoeitin in DS, IL6 and TNFa in AD and IL7 in both AD and DS. Conclusions: To our knowledge, this study represents the first ever for a blood-based biomarker profile of AD to cross-validate on an independent assay platform. Additionally, our preliminary results suggest that the profile approach to analyzing multiple markers by disease states is a viable option for differential diagnosis between AD and non-AD neurological diseases. Additional analyses with larger samples of AD, PD and non-AD samples are underway.

Clusterin, complement C3, complement C3a, serum amyloid P component (SAP), alpha-2-macroglobulin (A2M), gelsolin, fibrinogen gamma-chain (FGG) and complement factor H (CFH) have emerged as candidate AD biomarkers from discovery exercises using twodimensional gel electrophoresis and TMT labeling technologies (Hye et al., 2006; Thambisetty et al., 2010; Guntert et al., 2010). Furthermore, possession of the apolipoprotein E (Apo E) E4 allele is the only unequivocal genetic risk factor known to-date for late-onset AD and the protein expression of this genetic association may translate as an AD biomarker. The expression levels and absolute quantitative amounts of these proteins across large patient populations have yet to be described. We have now developed two mass spectrometry assay methods based on using isotopically labelled synthetic peptides (TMT-SRM) or a whole plasma digest reference (TMTOrbi) to assess candidate biomarker levels in plasma and present the results from the analysis of a subset of samples from an extensive, well characterised cohort of AD, MCI and Control patients.

Background: Alzheimer’s disease (AD) is the most common form of dementia, at present there remains a need for accurate biomarkers that can predict disease progression. In recent years markers of prediction, early diagnosis and progression have been identified in plasma by our group. We have therefore selected a multiplexing approach using the immunobased MILLIPLEX MAP assays to validate our previously discovered biomarkers with the aim to identify a combination of markers that can predict disease progression. Methods: 430 Alzheimer’s disease subjects, 395 cognitively normal and 173 mild cognitively impairment subjects were recruited from 2 large multi-centre cohorts. EDTA plasma was collected and evaluated for 45 analytes, using immuno-based MILLIPLEX MAP assays. Samples were randomized, blinded to the clinical information and measured in duplicate. Results: The intra-assay precision was calculated as the coefficient of variation (%) for each analyte and was

Introduction Amyloid beta (Aβ) oligomers are one of the most toxic structural forms of the Aβ protein and are hypothesized to cause synaptotoxicity and memory failure as they build up in Alzheimer's disease (AD) patients' brain tissue. We previously demonstrated that antagonists of the sigma-2 receptor complex effectively block Aβ oligomer toxicity. CT1812 is an orally bioavailable, brain penetrant small molecule antagonist of the sigma-2 receptor complex that appears safe and well tolerated in healthy elderly volunteers. We tested CT1812's effect on Aβ oligomer pathobiology in preclinical AD models and evaluated CT1812's impact on cerebrospinal fluid (CSF) protein biomarkers in mild to moderate AD patients in a clinical trial (ClinicalTrials.gov NCT02907567). Methods Experiments were performed to measure the impact of CT1812 versus vehicle on Aβ oligomer binding to synapses in vitro, to human AD patient post mortem brain tissue ex vivo, and in living APPSwe /PS1dE9 transgenic mice in vivo. Additional experiments were performed to measure the impact of CT1812 versus vehicle on Aβ oligomer-induced deficits in membrane trafficking rate, synapse number, and protein expression in mature hippocampal/cortical neurons in vitro. The impact of CT1812 on cognitive function was measured in transgenic Thy1 huAPPSwe/Lnd+ and wild-type littermates. A multicenter, double-blind, placebo-controlled parallel group trial was performed to evaluate the safety, tolerability, and impact on protein biomarker expression of CT1812 or placebo given once daily for 28 days to AD patients (Mini-Mental State Examination 18-26). CSF protein expression was measured by liquid chromatography with tandem mass spectrometry or enzyme-linked immunosorbent assay in samples drawn prior to dosing (Day 0) and at end of dosing (Day 28) and compared within each patient and between pooled treated versus placebo-treated dosing groups. Results CT1812 significantly and dose-dependently displaced Aβ oligomers bound to synaptic receptors in three independent preclinical models of AD, facilitated oligomer clearance into the CSF, increased synaptic number and protein expression in neurons, and improved cognitive performance in transgenic mice. CT1812 significantly increased CSF concentrations of Aβ oligomers in AD patient CSF, reduced concentrations of synaptic proteins and phosphorylated tau fragments, and reversed expression of many AD-related proteins dysregulated in CSF. Discussion These preclinical studies demonstrate the novel disease-modifying mechanism of action of CT1812 against AD and Aβ oligomers. The clinical results are consistent with preclinical data and provide evidence of target engagement and impact on fundamental disease-related signaling pathways in AD patients, supporting further development of CT1812.