Synaptic markers of cognitive decline in neurodegenerative diseases: a proteomic approach

Abstract

See Attems and Jellinger (doi:10.1093/brain/awx360) for a scientific commentary on this article.Cognitive changes occurring throughout the pathogenesis of neurodegenerative diseases are directly linked to synaptic loss. We used in-depth proteomics to compare 32 post-mortem human brains in the prefrontal cortex of prospectively followed patients with Alzheimer's disease, Parkinson's disease with dementia, dementia with Lewy bodies and older adults without dementia. In total, we identified 10 325 proteins, 851 of which were synaptic proteins. Levels of 25 synaptic proteins were significantly altered in the various dementia groups. Significant loss of SNAP47, GAP43, SYBU (syntabulin), LRFN2, SV2C, SYT2 (synaptotagmin 2), GRIA3 and GRIA4 were further validated on a larger cohort comprised of 92 brain samples using ELISA or western blot. Cognitive impairment before death and rate of cognitive decline significantly correlated with loss of SNAP47, SYBU, LRFN2, SV2C and GRIA3 proteins. Besides differentiating Parkinson's disease dementia, dementia with Lewy bodies, and Alzheimer's disease from controls with high sensitivity and specificity, synaptic proteins also reliably discriminated Parkinson's disease dementia from Alzheimer's disease patients. Our results suggest that these particular synaptic proteins have an important predictive and discriminative molecular fingerprint in neurodegenerative diseases and could be a potential target for early disease intervention.

Keywords: Alzheimer’s disease; Lewy body dementias; cognitive impairment; mass spectrometry; synaptic proteins.

Figure 1

Proteomic data analyses. Thirty-two post-mortem brain samples underwent proteome profile comparison. (A) Samples were labelled at peptide level with four sets of isobaric tags (TMT10plex), each containing eight channels with randomized samples and two channels with the internal reference sample (Ref), followed by fractionation into 72 fractions by HiRIEF with the broad range IPG 3-10 strip prior to LC-MS analysis. (B) Schematic representation of the number of differentially regulated proteins across disease groups. Differentially regulated proteins in dementia with Lewy bodies were further analysed for KEGG pathways (C) and gene ontology (GO) terms (D). From the significantly altered synaptic proteins GRIA3, SNAP47, LRFN2, SYBU, SYT2, GAP43, GRIA4 and SV2C were chosen for further validation with ELISA or western blot analyses in a larger cohort (E) (two synaptic proteins neurogranin, and CAMK2 were previously found to be altered by us within the same cohort). AD = Alzheimer’s disease; C = non-demented controls; DLB = dementia with Lewy bodies; LC-MS = liquid chromatography-mass spectrometry; PDD = Parkinson’s disease with dementia.

Figure 2

Changes in synaptic protein levels and their contribution to discriminating patient groups. (A) Synaptic protein levels differed between the dementia groups. (B–E) Univariate statistical analyses were performed using Kruskal-Wallis test followed by post hoc Dunn’s multiple comparison test. Multivariate analyses show the contribution of synaptic proteins to discriminate controls from the different patient groups. Plots showing the variables of importance and their corresponding jack-knifed confidence intervals for the separation between controls (C) and Parkinson’s disease dementia patients (PDD, B), controls and dementia with Lewy bodies patients (DLB, C), controls and Alzheimer’s disease patients (AD, D) and Parkinson’s disease dementia patients and Alzheimer’s disease patients (E). A measure with high covariance is more likely to have an impact on group separation than a variable with low covariance. Measures with confidence intervals that include zero have low reliability.

Figure 3

Correlations between synaptic proteins and cognitive impairment in Parkinson’s disease dementia (A), dementia with Lewy bodies (B) and Alzheimer’s disease (C). Decreased SNAP47, SV2C and GRIA3 concentrations (A) correlated with cognitive impairment in Parkinson’s disease dementia (PDD). SV2C and GRIA3 concentrations are negatively correlated with the rate of MMSE decline in dementia with Lewy bodies (DLB, B) showing, along with SYBU levels, positive correlations with the last MMSE scores (B). Negative correlations between LRFN2 concentrations and the rate of MMSE decline as well as positive correlations with the last MMSE scores were observed in Alzheimer’s disease (AD, C). Associations were analysed using Spearman correlations.

Figure 4

Schematic overview of synaptic proteins with altered levels in dementia. The diagram depicts the proteins involved in the synaptic vesicle cycle focusing on the docking and priming proteins (VAMP2, Syntaxin-1, SNAP, Munc18a), along with proteins involved in the recycling of synaptic vesicles (Rab3A, SV2C) as well as postsynaptic proteins (NRGN, PSD95, LRFN2) and receptor proteins (GRIA3, 4) found to be differentially regulated in the various dementias. AMPAR = AMPA receptor/GRIA; GAP43 = neuromodulin; NMDAR = N-methyl-d-aspartic acid receptor; NRGN = neurogranin; VDCC = voltage-dependent calcium channel.