Proteomic Analysis of Post-synaptic Density Fractions from Shank3 Mutant Mice Reveals Brain Region Specific Changes Relevant to Autism Spectrum Disorder

Abstract

Disruption of the human SHANK3 gene can cause several neuropsychiatric disease entities including Phelan-McDermid syndrome, autism spectrum disorder (ASD), and intellectual disability. Although, a wide array of neurobiological studies strongly supports a major role for SHANK3 in organizing the post-synaptic protein scaffold, the molecular processes at synapses of individuals harboring SHANK3 mutations are still far from being understood. In this study, we biochemically isolated the post-synaptic density (PSD) fraction from striatum and hippocampus of adult Shank3Δ11^-/- mutant mice and performed ion-mobility enhanced data-independent label-free LC-MS/MS to obtain the corresponding PSD proteomes (Data are available via ProteomeXchange with identifier PXD005192). This unbiased approach to identify molecular disturbances at Shank3 mutant PSDs revealed hitherto unknown brain region specific alterations including a striatal decrease of several molecules encoded by ASD susceptibility genes such as the serine/threonine kinase Cdkl5 and the potassium channel K_Ca1.1. Being the first comprehensive analysis of brain region specific PSD proteomes from a Shank3 mutant line, our study provides crucial information on molecular alterations that could foster translational treatment studies for SHANK3 mutation-associated synaptopathies and possibly also ASD in general.

Keywords: Homer1; Shank3; autism spectrum disorder; proteome; striatum; synapse.

FIGURE 1

Large-scale proteomic analysis of wild type (WT) and Shank3Δ11^-/- mutant (KO) post-synaptic density (PSD) fractions from striatum and hippocampus. (A) Western Blot analysis of the subcellular fractions derived from PSD isolation: Homogenate (Ho), Purified homogenate (S1), Crude membrane fraction (P2), Cytosol (S2), Synaptosomes (Syn), PSD and Synaptic cytosol (S3) from WT and Shank3Δ11^-/- mutant (KO) tissue. Note enrichment of Shank3 and PSD95 and depletion of Synaptophysin (Syp) in the PSD fraction of both, striatum (STR) and hippocampus (HIP). Representative bands for indicated proteins are shown. (B) Total number of proteins and the significantly changed proteins (blue: down-regulated; orange: up-regulated) identified in the PSD fraction of WT and Shank3Δ11^-/- mutant (KO) striatum (STR) or hippocampus (HIP) as indicated. The remaining Shank3 protein is excluded from this analysis. (C) Volcano plots of all molecular alterations in the WT and Shank3Δ11^-/- mutant (KO) PSD fraction from striatum and hippocampus [log₂(KO/WT), x-axis] and the respective statistical significance [-log10(p-value), y-axis]. Significantly regulated proteins (above horizontal dashed line marked as “sign.”) are colored (blue and left of vertical dashed line: down-regulated; orange and right of vertical dashed line: up-regulated). Changes that did not reach statistical significance remained black. Statistical analysis was performed using a Bonferroni-corrected unpaired two-tailed t-test and a sample size of n = 5 independent biological replicates. (D) Venn Diagram showing the number of proteins with altered expression levels in the Shank3Δ11^-/- mutant PSD from striatum (STR) and hippocampus (HIP) overlapping with a Shank3 in vivo interactome from synaptosomes (Han et al., 2013) (blue: down-regulated).

FIGURE 2

Gene ontology analysis of significantly altered proteins in the Shank3Δ11^-/- mutant PSD from striatum and hippocampus. Functional annotation of proteins with significantly altered expression levels in the striatal (A,B) and hippocampal (C,D) Shank3Δ11^-/- mutant PSD was performed using DAVID (v6.7, https://david.ncifcrf.gov) with the data in Supplementary Table S2 used as input. Bar diagrams visualize the top five significantly enriched GO terms for ‘cellular compartment’ for molecular alterations in the striatal (A) or hippocampal (C) PSD. The top five significantly enriched GO terms for ‘biological process’ and ‘molecular function’ for molecular alterations in the striatal (B) or hippocampal (D) PSD are listed in table-like diagrams.

FIGURE 3

A relevant number of the significantly altered proteins in the Shank3Δ11^-/- mutant PSD from striatum is encoded by ASD susceptibility genes. Significantly altered proteins in the striatal (A) and hippocampal (B) Shank3Δ11^-/- mutant (KO) PSD match with entries of the SFARI autism gene database. Proteins further encoded by high-risk TADA genes are highlighted in bold. The KO/WT ratios are marked in blue for down- and in orange for up-regulated proteins. (C,D) Protein–protein interactions (lines) among the significantly altered proteins in the striatal (C) and hippocampal (D) Shank3Δ11^-/- mutant PSD that match with entries in the SFARI autism gene database. Proteins, which are further encoded by TADA genes, are marked in bold and are in boxes. Proteins significantly down-regulated in Shank3Δ11^-/- mutant mice are marked in blue, significantly up-regulated proteins in orange.