Proteogenomics of synaptosomal mitochondrial oxidative stress

Oxidative stress is frequently implicated in the pathology of neurodegenerative disease. The chief source of this stress is from mitochondrial respiration, via the passage of reducing equivalents through the respiratory chain resulting in a small but potentially pathological production of superoxide. The superoxide that is produced during normal respiration is primarily detoxified within the mitochondria by superoxide dismutase 2 (Sod2), a key protein for maintaining mitochondrial function. Mitochondria are distributed throughout the soma of neurons, as well as along neuronal processes and at the synaptic terminus. This distribution of potentially independent mitochondria throughout the neuron, at distinct subcellular locations, allows for the possibility of regional subcellular deficits in mitochondrial function. There has been increasing interest in the quantification and characterization of messages and proteins at the synapse, due to its importance in neurodegenerative disease, most notably Alzheimer’s disease. Here, we report the transcriptomic and proteomic changes that occur in synaptosomes from frontal cortices of Sod2 null mice. Constitutively null Sod2 mice were differentially dosed with the synthetic catalytic antioxidant EUK-189, which can extend the lifespan of these mice, as well as uncover or prevent neurodegeneration due to endogenous oxidative stress. This approach facilitated insight into quantification of trafficked messages and proteins to the synaptosome. We used two complementary methods to investigate the nature of the synaptosome under oxidative stress; either whole genome gene expression microarrays or mass spectrometry-based proteomics using isobaric tagging for relative and absolute quantitation (iTRAQ) of proteins. We have characterized the relative enrichments of gene ontologies at both gene and protein expression that occur due to mitochondrial oxidative stress in the synaptosome, which may lead to new avenues of investigation in understanding the regulation of the synaptic function in normal and diseased states. As a result of using these approaches, we report for the first time an activation of the mTOR pathway in synaptosomes isolated from Sod2 null mice, confirmed by an upregulation of the phosphorylation of 4E-BP1.