O5‐05‐03: Novel CSF Biomarkers Discriminating FTLD‐TDP from Non‐Demented Controls

Abstract

Background: A biomarker capable of detecting synapse loss, which occurs early in Alzheimer's disease (AD) pathophysiology, would greatly assist in preclinical diagnosis, when treatment would most likely be effective. The objective of this study was to identify and quantify synaptic protein levels in cerebrospinal fluid (CSF) in the search for preclinical biomarkers of synaptic loss. Methods: The CSF proteome database was constructed by non-targeted liquid chromatography mass spectrometry (LS-MS) on CSF samples from 50 cognitively healthy controls (CON) and 10 AD patients. The synaptic proteome database was constructed by literature curation of proteins detected in mouse, rat or human synaptic fractions (according to 16 published proteomic studies) or annotated with a synaptic function in online databases (Gene Ontology, KEGG, SynSysNet). Proteins without a neuropil-specific expression pattern in the cerebral cortex (Human Protein Atlas) were discarded. Array Tomography (AT) microscopy with fluorescent immunostaining for candidate biomarkers and core synaptic markers (synaptophysin and PSD-95) was performed on post-mortem AD and non-AD brains (n¼2). Quantification of up to 3 unlabeled peptides per protein was performed by targeted LC-MS on Phase 2 (15 CON and 15 AD) and Phase 3 (197 CON, 30 Preclinical AD, 56 Prodromal AD, 45 AD) CSF samples. Results: Phase 1) Cross-referencing the CSF (2,704 proteins) and synaptic (709 proteins) proteomes produced a list of 158 synaptic proteins detectable in the CSF (6% of the CSF proteome). Phase 2) A panel of 22 CSF proteins with a core synaptic function (neurotransmission, synaptogenesis or synaptic vesicle recycling) was selected for further evaluation. Co-localisation of the panel proteins with core synaptic markers was assessed by AT microscopy. ATconfirmed synaptic proteins were quantified in the pilot study of AD and CON CSF. Phase 3) Candidate biomarkers were quantified in CSF across the full CON and AD spectrum and evaluated as CSF stage biomarkers for AD. Conclusions:By performing a detailed characterization of the CSF and synaptic proteomes, we have identified a panel of synaptic proteins detectable in the CSF which, if confirmed as biomarkers of synaptic loss, could be invaluable stage biomarkers for AD. We analyzed the levels of YKL40, FABP4, MFGE8 and Catalase enzyme activity using specific assays in CSF cohorts from two different centers: the Emory university and Milan University. These cohorts included patients with FTD with confirmed Tau (n¼20) or TDP43 (n¼30) pathology, Alzheimer's disease (AD, n¼30), dementia with Lewy bodies (DLB, n¼29), vascular dementia (VaD, n¼7) and non-demented controls (n¼29). Results:YKL40 was increased in FTLD-TDP patients not only compared to controls (fold-change -f.c-:1.6, p<0.0001), confirming our previous study, but also compared to FTLD-Tau, DLB and VaD (fc:1.2-1.6; p<0.05). The highest levels of MFGE-8 were found in AD patients and were significantly different from those observed in both FTLD subtypes and DLB (f.c:0.8-1.4; p<...