Human post-mortem synapse proteome integrity screening for proteomic studies of postsynaptic complexes

Abstract

Background: Synapses are fundamental components of brain circuits and are disrupted in over 100 neurological and psychiatric diseases. The synapse proteome is physically organized into multiprotein complexes and polygenic mutations converge on postsynaptic complexes in schizophrenia, autism and intellectual disability. Directly characterising human synapses and their multiprotein complexes from post-mortem tissue is essential to understanding disease mechanisms. However, multiprotein complexes have not been directly isolated from human synapses and the feasibility of their isolation from post-mortem tissue is unknown.

Results: Here we establish a screening assay and criteria to identify post-mortem brain samples containing well-preserved synapse proteomes, revealing that neocortex samples are best preserved. We also develop a rapid method for the isolation of synapse proteomes from human brain, allowing large numbers of post-mortem samples to be processed in a short time frame. We perform the first purification and proteomic mass spectrometry analysis of MAGUK Associated Signalling Complexes (MASC) from neurosurgical and post-mortem tissue and find genetic evidence for their involvement in over seventy human brain diseases.

Conclusions: We have demonstrated that synaptic proteome integrity can be rapidly assessed from human post-mortem brain samples prior to its analysis with sophisticated proteomic methods. We have also shown that proteomics of synapse multiprotein complexes from well preserved post-mortem tissue is possible, obtaining structures highly similar to those isolated from biopsy tissue. Finally we have shown that MASC from human synapses are involved with over seventy brain disorders. These findings should have wide application in understanding the synaptic basis of psychiatric and other mental disorders.

Figure 1

Method for rapid isolation of synaptic-enriched fractions from PM human cortex. GluN2B degradation and correlation with number of intact PSD proteins. A. Schematic representation of the subcellular fractionation method used to obtain postsynaptic protein enriched fractions (P2). Procedure time is indicated. H and S, homogenized cortex; P1, nucleus/cell debris; S1, cytosolic fraction; P2, triton insoluble fraction; S2, triton soluble fraction. B. Immunoblot showing protein enrichment or depletion between sample S and P2 from isolation protocol described in A. Postsynaptic markers: PSD95/DLG4, GluN2B and SAP102/DLG3. Pre-synaptic-markers: SYP and VGluT-1. A marker of mitochondria is also included: COXIV-1. C. Mean fold enrichment of proteins in final P2 fraction compared to starting S fraction analysed by immunoblotting (n = 5). Postsynaptic proteins were enriched in P2, while presynaptic and mitochondrial markers were depleted (S/P2 < 1). D. GluN2B immunoblot from control NSB and 28 PM samples (Additional file 1). PM samples show three main bands, band 1 corresponding with the full-length protein. The ratio of band 1 over band 2 intensities provides HUSPIR ratio. The antibody used was designed against the C-terminal region of GluN2B (BD Bioscience ref. 610416). E. For each PM sample the HUSPIR ratio is plotted against the number of intact PSD proteins. Significant positive Spearman’s coefficient of correlation (r) and p-value (p) are indicated. F. HUSPIR ratio for the set of 28 unselected samples and the set of 9 samples selected. Median and interquartile range shown. G. Comparison between percentage of PSD components observed in the set of 28 unselected samples and the prospective set of 9 selected samples. Median and interquartile range shown.

Figure 2

Comparative GluN2B degradation between brain areas. A. Representative immunoblot of GluN2B for 13 different cortical regions from brain SD025/13 (Additional file 1). These cortical regions included the Broadmann Areas (BA) 4, 11/12, 17, 19, 20/21, 37, 38, 39, 41/42 and 44/45 (Broca’s area), as well as three frontal cortex samples: frontal convexity (FC), anterior frontal convexity (Ant FC) and anterior frontal parasagital (Ant FPS). GluN2B degradation bands are indicated. B. Representative immunoblot of GluN2B for 5 limbic areas: Broadmann areas (BA) 23 and 24, anterior and posterior Hipocampus (HC) and amygdala (Amyg); and 8 sub-cortical regions: Pons, Midbrain (MB), Caudate nucleus (Caud nuc), Thalamus (Thal), Medulla (Med), Vermis, Basal ganglia mammillary body (BG mam) and Cerebellum (CB). GluN2B degradation bands are indicated. C. Plot summarizing HUSPIR values for 26 brain areas (13 cortical, 5 limbic and 8 sub-cortical) analysed by immunoblot for GluN2B degradation in 4 different brains (SD025/13, SD042/13, SD032/13 and SD023/13). HUSPIR values above one are indicated in green and below in red. Grey indicates no measure could be taken.

Figure 3

Affinity purification of the MAGUK Associated Signalling Complexes (MASC) from brain cortex and observed MASC composition in NSB or PM frontal cortex. A. Coomassie stained SDS-PAGE electrophoresis of MASC complexes isolated by affinity purification from mouse cortex, human NSB and PM frontal cortex. Human sample details in Additional file 1. MW: Molecular Weight markers; pep6, eluate from affinity purification using pep6 peptide; pep6δV, eluate from affinity purification using control peptide. B. Immunoblots of three members of the DLG4/PSD-95 family (DLG4/PSD-95, DLG3/SAP102 and DLG2/PSD-93) for 6 human MASC purifications (pep6), and corresponding controls (pep6δV); three from NSB and three from PM frontal cortex. C. Panther Protein Classes in the postsynaptic proteome (in green), the NSB MASC (in red) or the PM MASC (in blue) that are significantly enriched as compared with the human genome. Bar height corresponds with percentage of all proteins found in that Protein Class. (See Additional file 2 for methods and detailed bioinformatics functional analysis).

Figure 4

Involvement of MASC proteins in genetic diseases. A. Percentage of genes expressed in human NSB MASC, PM MASC and PSD causing central nervous system diseases included in OMIM. B. Proportion of CNS diseases caused by genes found in human NSB MASC (red), PM MASC (blue) and PSD (green). ICD-10 groups (Chapters) indicated on x-axis: Congenital Malformations refers to ICD-10 Chapter XVII, Nervous System Diseases refers to ICD-10 Chapter VI, Endocrine and metabolic refers to ICD-10 Chapter IV and Mental and Behavioural refer to ICD-10 Chapter V. Means compared by Student’s t-test. C. Genes expressed at the postsynapse causing any form of genetic Intellectual Disability (ID). For each postsynaptic protein complex pale columns indicate number of genes causing ID and dark columns the expected number to be found by chance. Binomial statistics are used to compute significance of the difference between observed and expected (Binomial Statistics, ***, p <1E-05; **, p <1E-03; *, p <0,05). D. Genes expressed at the postsynapse causing any form of genetic non-syndromic Intellectual Disability (NSID). For each postsynaptic protein complex pale columns indicate number of genes causing ID and dark columns the expected number to be found by chance. Binomial statistics are used to compute significance of the difference between observed and expected (Binomial Statistics, ***, p <1E-05; **, p <1E-03; *, p <0,05).