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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.

Presynaptic nerve terminals require high levels of ATP for the maintenance of synaptic function. Failure of synaptic mitochondria to generate adequate ATP has been implicated as a causative event preceding the loss of synaptic networks in neurodegenerative disease. Endogenous oxidative stress has often been postulated as an etiological basis for this pathology, but has been difficult to test in vivo. Inactivation of the superoxide dismutase gene (Sod2) encoding the chief defense enzyme against mitochondrial superoxide radicals results in neonatal lethality. However, intervention with an SOD mimetic extends the life span of this model and uncovers a neurodegenerative phenotype providing a unique model for the examination of in vivo oxidative stress. We present here studies on synaptic termini isolated from the frontal cortex of Sod2 null mice demonstrating impaired bioenergetic function as a result of mitochondrial oxidative stress. Cortical synaptosomes from Sod2 null mice demonstrate a severe decline in mitochondrial spare respiratory capacity in response to physiological demand induced by mitochondrial respiratory chain uncoupling with FCCP or by plasma membrane depolarization induced by 4-aminopyridine treatment. However, Sod2 null animals compensate for impaired oxidative metabolism in part by the Pasteur effect allowing for normal neurotransmitter release at the synapse, setting up a potentially detrimental energetic paradigm. The results of this study demonstrate that high-throughput respirometry is a facile method for analyzing specific regions of the brain in transgenic models and can uncover bioenergetic deficits in subcellular regions due to endogenous oxidative stress.