Frontotemporal dysregulation of the SNARE protein interactome is associated with faster cognitive decline in old age

Abstract

The molecular underpinnings associated with cognitive reserve remain poorly understood. Because animal models fail to fully recapitulate the complexity of human brain aging, postmortem studies from well-designed cohorts are crucial to unmask mechanisms conferring cognitive resistance against cumulative neuropathologies. We tested the hypothesis that functionality of the SNARE protein interactome might be an important resilience factor preserving cognitive abilities in old age. Cognition was assessed annually in participants from the Rush "Memory and Aging Project" (MAP), a community-dwelling cohort representative of the overall aging population. Associations between cognition and postmortem neurochemical data were evaluated in functional assays quantifying various species of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) machinery in samples from the inferior temporal (IT, n = 154) and middle-frontal (MF, n = 174) gyri. Using blue-native gel electrophoresis, we isolated and quantified several types of complexes containing the three SNARE proteins (syntaxin-1, SNAP25, VAMP), as well as the GABAergic/glutamatergic selectively expressed complexins-I/II (CPLX1/2), in brain tissue homogenates and reconstitution assays with recombinant proteins. Multivariate analyses revealed significant associations between IT and MF neurochemical data (SNARE proteins and/or complexes), and multiple age-related neuropathologies, as well as with multiple cognitive domains of MAP participants. Controlling for demographic variables, neuropathologic indices and total synapse density, we found that temporal 150-kDa SNARE species (representative of pan-synaptic functionality) and frontal CPLX1/CPLX2 ratio of 500-kDa heteromeric species (representative of inhibitory/excitatory input functionality) were, among all the immunocharacterized complexes, the strongest predictors of cognitive function nearest death. Interestingly, these two neurochemical variables were associated with different cognitive domains. In addition, linear mixed effect models of global cognitive decline estimated that both 150-kDa SNARE levels and CPLX1/CPLX2 ratio were associated with better cognition and less decline over time. The results are consistent with previous studies reporting that synapse dysfunction (i.e. dysplasticity) may be initiated early, and relatively independent of neuropathology-driven synapse loss. Frontotemporal dysregulation of the GABAergic/glutamatergic stimuli might be a target for future drug development.

Keywords: Aging; Alzheimer's disease; Cognitive decline; Double dissociation; Excitatory/inhibitory balance; Native PAGE; Postmortem brain; Protein-protein interactions; SNARE complex; Synaptic pathology.

Fig. 1.

Characterization of the presynaptic complexes targeted in the present study. (a) Solubilized brain protein complexes from human inferior temporal cortex (IT) were resolved by blue-native (BN)-PAGE and immunoblotted (IB) with specific antibodies against syntaxin-1 (STX1), SNAP25 (S25) and complexins I (CPLX1) and II (CPLX2) (see Supplementary Table S1). The same representative case is shown in all individual immunoblots, with two exposure (exp.) times (ranging 20 s to 3 min; indicated at the bottom) per probing antibody. Arrowheads point to the identified and/or quantified complexes for each antibody. (b) Reconstitution assays were achieved by sequentially adding, from top to bottom, 1 μm of the recombinant proteins indicated (+) beneath the immunoblots. Abbreviations of the recombinant protein names and tags, and other key information of these constructs, are indicated in Supplementary Table S2. After incubation at 37°C for 30 min, the resulting protein complexes were resolved by BN-PAGE followed by Coomassie de-staining (following manufacturer’s instructions), or immunolabeled as above. Antibodies against munc18–1 (long variant; M18L) and synaptotagmin (STG) were unable to react against their corresponding recombinant constructs under the present experimental conditions (not shown). Arrowheads point to the identified SNARE (purple) or SNARE+complexin (tangerine) heteromers, or the monomeric species (green) for each immunoblot. Of note, recombinant SNAP25 alone, but not syntaxin-1 or VAMP, mimicked all of the above bands, suggesting that SNAP25 has the ability to self-assemble into aggregates with similar stoichiometries as those in SNARE heteromers in vitro and ex vivo. While this observation represents a novel finding with potential implications for the biochemistry of SNARE dynamics, it is also beyond the aim and scope of the present work, and future studies should determine the physiological relevance (if any) of SNAP25 aggregates. (c) Human IT solubilized complexes were immunoprecipitated (IP) with anti-mouse IgG (negative control), anti-STX1, anti-SNAP25, anti-CPLX1, or anti-CPLX2 specific antibodies, and the resulting IP products resolved, along the IP input sample, by BN-PAGE followed by immunoblotting standard procedures. (d) Anti-SNAP25 (left panel) and anti-CPLX2 (right panel) IP products were resolved by one- (1-D) followed by two-dimensional (2-D) BN+SDS-PAGE. Proteins were transferred to PVDF membranes and sequentially immunoblotted with anti-STX1 (red), anti-SNAP25 (blue), and anti-CPLX2 (green) specific antibodies. Top and left arrows indicate the directions of BN- and SDS-PAGE, respectively. Note the presence of SDS-resistant STX1/SNAP25 complexes/aggregates at 75–250 kDa, as well as the immunostaining for the primary antibodies used in IP at 50 kDa. For unknown reasons, the antibody-antigen reaction between the anti-SNAP25 antibody (i.e. SP12) used in the co-IP reactions and the anti-mouse IgG1 secondary antibody and was faint in 1-D BN-PAGE (c), while strong upon 2-D SDS-PAGE separation (d). Note, however, that the same secondary antibody reacted as expected against anti-complexin-I/II antibodies (i.e. SP33 and LP27, both IgG1), but not against anti-syntaxin-1 (i.e. SP7 IgG2a), in both 1-D and 2-D BN/SDS-PAGE. (a–d) Molecular masses (in kDa) of native and SDS-PAGE prestained standards are shown on the left and above.

Fig. 2.

Heatmap of Pearson’s R (left panel) and P (right panel) values following multiple pairwise correlations between the pathologic, stereological and cognitive variables of MAP participants on top, and the neurochemical data obtained in the inferior temporal (IT) or middle frontal (MF) gyri by either direct (d) or capture (c) ELISA, or BN-PAGE, as indicated on the left. The burden of both local (i.e. IT or MF) and global (Glob) brain AD (AD) pathology is evaluated. The ratios of complexin-I to complexin-II were also calculated for each of the equivalent BN-PAGE complexes as an index of functional inhibitory/excitatory input balance. For practical reasons, clinical diagnoses were coded as 0 (for NCI), 1 (MCI), or 2 (dementia). Abbreviations: Aβ, amyloid-β; Arterioloscl, arteriolosclerosis; CPLX1, complexin-I; CPLX2, complexin-II; DP, diffuse plaques; Hippscl, hippocampal sclerosis; k, kDa (kilodaltons); LBD, Lewy body disease; μglia, microglia; MMSE, mini-mental state examination; NFT, neurofibrillary tangles; NP, neuritic plaques; P-tau, phosphotau; PPIs, protein-protein interactions; S25, SNAP25; STX1, syntaxin-1; SYP, synaptophysin; VAMP, vesicle-associated membrane protein.

Fig. 3.

Representative images of human hippocampal sections in (a) epifluorescence and (b and c) confocal microscopy following triple immunostaining with antibodies against (a and b) HLA-DR (red; clone CR3/43; targeting activated microglia), SNAP25 (S25; blue; clone SP12) and complexin-I (CPLX1; green; clone SP33), or (c) HLA-DR (red), VAMP (green; clone SP10) and misfolded tau (blue; clone Alz-50). (a) Overview of the hippocampal subfields in a paraffin-embedded, 6-μm-thick section, showing the expected localizations for HLA-DR (activated microglia-like cells), SNAP25 (labeling the neuropil with strongest staining throughout the perforant pathway), and CPLX1 (intense staining of the neuropil attributable to GABAergic terminals surrounding granule cells of the dentate gyrus [DG] and pyramidal cells within the CA regions). (b) Example of a confocal micrograph of an HLA-DR-positive cell with clear inclusions of presynaptic material, in a free-floating, 40-μm-thick hippocampal section. (c) Positive colocalization analysis of both VAMP and Alz-50 with HLA-DR, using the method by Costes et al. [41,43]. Upper panels are single or merged channel confocal images, while bottom panels are ImageJ-built bitmaps resulting from pairwise colocalization analyses between the indicated antibodies, and represent those pixels where the overlapping between the channels are above an unbiased threshold of intensities

Fig. 4.

Temporal (IT) and frontal (MF) immnudensities of the major complexes quantified in the study in brains of MAP participants stratified by (a) CERAD scale, (b) Braak stage, or (c) clinical diagnosis. Bars are mean ± standard error. Datasets were analyzed by ANCOVA, using each protein complex measure as the dependent variable in separate models, CERAD/Braak/clinical diagnoses as the dependent variables, and sex, age, and PMI as covariates. *P < 0.05, **P < 0.01, and ***P < 0.001, FDR-corrected P-values in ANCOVA followed by Dunnet’s post hoc test.

Fig. 5.

(a) Interaction plots resulting from the double dissociation model predicting MAP participants’ cognitive function nearest death (and controlling for typical demographic and pathologic variables), in which the amounts of temporal (IT) and frontal (MF) pathologic (amyloid-β and phosphotau) and presynaptic function (150-kDa syntaxin-1 and 500-kDa complexin I/II ratio) indices were each crossed by a term identifying the brain area where they were measured. Lines represent the best fit for the association between cognition and the amounts of each of these brain indices in IT (red) and MF (blue). After false discovery rate (FDR) correction, the interaction terms of presynaptic indices by brain area were both highly significant, whereas those crossing the pathologic indices by brain area were not (beta-estimates and p-values for each interaction term are shown in each scatterplot; full model not shown). (b) Trajectory of cognitive decline (residual values after adjusting for demographics, neuropathologies, synaptic density and cognitive function nearest death) associated with MAP participants within the high (blue), middle (green), and low (red) tertiles of 150-kDa STX1 immunodensities in IT (left) or the 500-kDa CPLX1/CPLX2 ratio values in MF (right). Models included fixed change-point at 3 years prior to death indicating period of terminal cognitive decline.

Fig. 6.

Cartoon summarizing the cycle of vesicle trafficking and neurotransmitter release, and the steps where the characterized and quantified protein complexes (tagged with their corresponding molecular weights in red boxes) are hypothesized to participate in the process. Abbreviations: CPLX1/2, complexins-I/II; k, kDa; S25, SNAP25; STG, synaptotagmin; STX1, syntaxin-1; t-SNARE, target SNARE; VAMP, vesicle-associated membrane protein; VGCC, voltage-gated calcium channel; v-SNARE, vesicle SNARE.