Proteomic profiling of neuronal mitochondria reveals modulators of synaptic architecture

Abstract

BackgroundNeurons are highly polarized cells consisting of three distinct functional domains: the cell body (and associated dendrites), the axon and the synapse. Previously, it was believed that the clinical phenotypes of neurodegenerative diseases were caused by the loss of entire neurons, however it has recently become apparent that these neuronal sub-compartments can degenerate independently, with synapses being particularly vulnerable to a broad range of stimuli. Whilst the properties governing the differential degenerative mechanisms remain unknown, mitochondria consistently appear in the literature, suggesting these somewhat promiscuous organelles may play a role in affecting synaptic stability. Synaptic and non-synaptic mitochondrial subpools are known to have different enzymatic properties (first demonstrated by Lai et al., 1977). However, the molecular basis underpinning these alterations, and their effects on morphology, has not been well documented.MethodsThe current study has employed electron microscopy, label-free proteomics and in silico analyses to characterize the morphological and biochemical properties of discrete sub-populations of mitochondria. The physiological relevance of these findings was confirmed in-vivo using a molecular genetic approach at the Drosophila neuromuscular junction.ResultsHere, we demonstrate that mitochondria at the synaptic terminal are indeed morphologically different to non-synaptic mitochondria, in both rodents and human patients. Furthermore, generation of proteomic profiles reveals distinct molecular fingerprints – highlighting that the properties of complex I may represent an important specialisation of synaptic mitochondria. Evidence also suggests that at least 30% of the mitochondrial enzymatic activity differences previously reported can be accounted for by protein abundance. Finally, we demonstrate that the molecular differences between discrete mitochondrial sub-populations are capable of selectively influencing synaptic morphology in-vivo. We offer several novel mitochondrial candidates that have the propensity to significantly alter the synaptic architecture in-vivo.ConclusionsOur study demonstrates discrete proteomic profiles exist dependent upon mitochondrial subcellular localization and selective alteration of intrinsic mitochondrial proteins alters synaptic morphology in-vivo.Electronic supplementary materialThe online version of this article (10.1186/s13024-017-0221-9) contains supplementary material, which is available to authorized users.

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.…”

A mouse model for spinal muscular atrophy provides insights into non-alcoholic fatty liver disease pathogenesis

“…The data set is then visualized as nodes (proteins) that are connected to each other in a network based on their expression levels (edges). This data set can further be subdivided into discreet "clusters" based on a Markov Clustering Algorithm (MCL), thus segregating data in an unbiased manner as previously described [66][67][68].…”