Changes in Synaptic Proteins Precede Neurodegeneration Markers in Preclinical Alzheimer's Disease Cerebrospinal Fluid

Abstract

A biomarker of synapse loss, an early event in Alzheimer's disease (AD) pathophysiology that precedes neuronal death and symptom onset, would be a much-needed prognostic biomarker. With direct access to the brain interstitial fluid, the cerebrospinal fluid (CSF) is a potential source of synapse-derived proteins. In this study, we aimed to identify and validate novel CSF biomarkers of synapse loss in AD. Discovery: Combining shotgun proteomics of the CSF with an exhaustive search of the literature and public databases, we identified 251 synaptic proteins, from which we selected 22 for further study. Verification: Twelve proteins were discarded because of poor detection by Selected Reaction Monitoring (SRM). We confirmed the specific expression of 9 of the remaining proteins (Calsynytenin-1, GluR2, GluR4, Neurexin-2A, Neurexin-3A, Neuroligin-2, Syntaxin-1B, Thy-1, Vamp-2) at the human synapse using Array Tomography microscopy and biochemical fractionation methods. Exploration: Using SRM, we monitored these 9 synaptic proteins (20 peptides) in a cohort of CSF from cognitively normal controls and subjects in the pre-clinical and clinical AD stages (n = 80). Compared with controls, peptides from 8 proteins were elevated 1.3 to 1.6-fold (p < 0.04) in prodromal AD patients. Validation: Elevated levels of a GluR4 peptide at the prodromal stage were replicated (1.3-fold, p = 0.04) in an independent cohort (n = 60). Moreover, 7 proteins were reduced at preclinical stage 1 (0.6 to 0.8-fold, p < 0.04), a finding that was replicated (0.7 to 0.8-fold, p < 0.05) for 6 proteins in a third cohort (n = 38). In a cross-cohort meta-analysis, 6 synaptic proteins (Calsyntenin-1, GluR4, Neurexin-2A, Neurexin-3A, Syntaxin-1B and Thy-1) were reduced 0.8-fold (p < 0.05) in preclinical AD, changes that precede clinical symptoms and CSF markers of neurodegeneration. Therefore, these proteins could have clinical value for assessing disease progression, especially in preclinical stages of AD.

Keywords: Alzheimer's disease; Biomarker: Prognostic; Cerebrospinal fluid; Clinical proteomics; Selected reaction monitoring.

Graphical abstract

Fig. 1.

Expression of the panel proteins at the human cortical synapse. A, Using AT microscopy, ultrathin tissue slices (70 nm) from human post-mortem cortical tissue from one brain donor were immunostained for pre- (synaptophysin, red) and post- (PSD95, green) synaptic markers and the synaptic panel proteins (blue). A representative 3-D reconstruction of a representative synapse is shown for each panel protein. The segmented immunofluorescence of the 3 proteins in each individual stack (at 70 nm increments) is shown to the right of the reconstruction. Scale bars representing 1 μm are shown at the bottom of each z stack. B, Mean fold-enrichment plotted for each panel protein in homogenate (H), synaptosome-enriched (S) and PSD-enriched (PSD) fractions taken from post-mortem human cortex (n = 6). S/H and PSD/H; intensity in S or PSD fractions relative to H for the same sample. H/H; intensity in the H fraction for each sample relative to the mean intensity in the H fraction across all samples. Enrichment of the pre- (synaptophysin) and post (PSD-95) markers are also shown. Degrees of freedom (df), F statistic and p values for the ANOVA are shown at the top of each plot and Dunnett's p values are shown for significant pair-wise comparisons (α = 0.05).

Fig. 2.

Synaptic panel peptide levels in the CSF across the AD continuum. The log2 fold-change (± standard error; S.E.) in CSF levels of the synaptic panel peptides and summarized protein levels are plotted for each preclinical and clinical AD stage versus cognitively normal controls for (A) the exploratory cohort (Controls n = 20, Stage1 n = 10, Stage2 n = 10, Prodromal n = 20, AD dementia n = 20), (B) validation cohort-1 (Controls n = 18, Stage1 n = 9, Stage2 n = 8, Prodromal n = 10, AD dementia n = 15), (C) validation cohort-2 (Controls n = 20, Stage1 n = 18) and (D) the combined mean log2 fold-change across the 3 cohorts. For ease of interpretation, the natural values are labeled on the y axis on a log2 scale. The linestyle of the error bars were determined by p value cut-offs for pair-wise group comparisons using a mixed effect linear regression model (see legend). Stage1; preclinical AD stage 1, Stage2; preclinical stage 2,Prodromal; prodromal AD, Dementia; AD dementia.