Novel CSF β-synuclein-specific assays signal early synaptic degeneration in Alzheimer's disease

Background Beta-synuclein (beta-syn), measured at N-terminal epitopes, is an emerging cerebrospinal fluid (CSF) biomarker for synaptic degeneration in Alzheimer's disease (AD). Targeting the mid-region or C-terminus of beta-syn may enhance analytical specificity due to the distinct structures of these regions across the synuclein protein family, unlike targeting the N-terminus, which is conserved across the family. This study aimed to confirm that beta-syn is a promising CSF biomarker in AD, using novel assays designed to target different regions of beta-syn, to investigate whether these regions are differentially affected in AD. Methods We developed two novel CSF beta-syn-specific ELISAs targeting mid-region and C-terminus epitopes and assessed their analytical performance. Using these novel assays in combination with the established N-terminus ELISA, we analyzed a proof-of-concept cohort comprising biomarker-confirmed AD (n = 25) and non-AD subjects (n = 25) and a larger clinical cohort (n = 160) from the Amsterdam Dementia Cohort, wich included 41 individuals with subjective cognitive decline (SCD, controls; AD biomarker negative; 64.3 +/- 3.3 years, 23 females), 39 with SCD (AD biomarker positive; 65.7 +/- 3.1 years, 17 females), 40 with mild cognitive impairment due to AD (MCI-AD; 66.2 +/- 2.9 years, 20 females), and 40 with AD dementia (AD-dem; 65.3 +/- 3.4 years, 20 females). Results Both the mid-region and C-terminus assays demonstrated reliable analytical performance. All assays consistently detected beta-syn in all clinical samples above their limits of detection, with a good average intra-assay coefficient of variation (range of the three assays: 2.7-6.5%CV) in the proof-of-concept cohort and clinical cohort (range of the three assays: 3.9-7.5%CV). CSF beta-syn levels, with all the assays, were significantly elevated in all the AD groups compared with the controls in both cohorts. The diagnostic performance of the assays for distinguishing AD patients from controls was comparable (Delong's p > 0.05, AUC 0.71-0.80). Notably, mid-region beta-syn significantly differentiated SCD-AD patients from AD-dem patients (p = 0.035) and MCI-AD patients at a trend level. Only mid-region and C-terminal levels correlated with MMSE scores (mid-region rho = -0.22, p = 0.006; C-terminal rho = -0.19, p = 0.016; N-terminus rho = -0.14, p = 0.069). Conclusion Our novel assays demonstrated good analytical and clinical performance. CSF beta-syn reliably indicates early synaptic degeneration in AD. The mid-region assay uniquely differentiated SCD-AD from AD-dem, showing promise for early disease detection.