Proteomic mapping of differentially vulnerable pre-synaptic populations identifies regulators of neuronal stability in vivo

Abstract

Synapses are an early pathological target in many neurodegenerative diseases ranging from well-known adult onset conditions such as Alzheimer and Parkinson disease to neurodegenerative conditions of childhood such as spinal muscular atrophy (SMA) and neuronal ceroid lipofuscinosis (NCLs). However, the reasons why synapses are particularly vulnerable to such a broad range of neurodegeneration inducing stimuli remains unknown. To identify molecular modulators of synaptic stability and degeneration, we have used the Cln3 −/− mouse model of a juvenile form of NCL. We profiled and compared the molecular composition of anatomically-distinct, differentially-affected pre-synaptic populations from the Cln3 −/− mouse brain using proteomics followed by bioinformatic analyses. Identified protein candidates were then tested using a Drosophila CLN3 model to study their ability to modify the CLN3-neurodegenerative phenotype in vivo. We identified differential perturbations in a range of molecular cascades correlating with synaptic vulnerability, including valine catabolism and rho signalling pathways. Genetic and pharmacological targeting of key ‘hub’ proteins in such pathways was sufficient to modulate phenotypic presentation in a Drosophila CLN3 model. We propose that such a workflow provides a target rich method for the identification of novel disease regulators which could be applicable to the study of other conditions where appropriate models exist.

Comparative profiling of the synaptic proteome from Alzheimer’s disease patients with focus on the APOE genotype

“…Synaptic preparations were prepared and quality control for post-mortem protein degradation were confirmed as previously described 12, 13, 14 (Supplementary Figure 1 for example total protein stains and western blotting). Any synaptoneurosome preparations containing nuclear histone protein were discarded and fresh preparations made from the same case.…”

“…Having confirmed that the extracted protein is of appropriate quality, we then applied a comprehensive workflow to enable us to assess (at the protein level) the relative contribution of both regional vulnerability and APOE variants as a risk factors to AD pathogenesis (Figure 1C, Figure 2) 12, 13 . We performed a complex 8-plex TMT LC-MS/MS analysis.…”

“…Pools containing equal amounts of protein (25 μg per case) were prepared of each of the 8 groups (control APOE3/3 BA41/42, control APOE3/3 BA17, control APOEx/4 BA41/42, control APOEx/4 BA17, AD APOE3/3 BA41/42, AD APOE3/3 BA17, AD APOEx/4 BA41/42, AD APOEx/4 BA17). Preparation of the samples, quantification, and bioinformatics was carried out according to standardized protocols 12, 13, 14 .…”

“…The Database for Annotation Visualization and Integrated Discovery (DAVID) was used to test whether synaptic protein sets were enriched in the samples 51 . To obtain further insight into potential pathways changed in AD synapses, Ingenuity Pathway Analysis (IPA, Ingenuity Systems) was used as previously described 12, 13, 52 with the interaction data limited as follows: direct and indirect interactions; experimentally observed data only; 35 molecules per network; 10 networks per dataset. Prediction activation scores (z-scores) were calculated in IPA.…”

“…Prediction activation scores (z-scores) were calculated in IPA. Expression clustering was performed in Biolayout Express 3D software by applying Markov clustering algorithms to raw proteomic data (MCL 19 2.2) as previously described in 13 . All graphs were clustered using Pearson correlation r=0.96.…”