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Impaired spare respiratory capacity in cortical synaptosomes from Sod2 null mice.

ABSTRACT: 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.

SUBMITTER: Flynn JM

PROVIDER: S-EPMC3061438 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Impaired spare respiratory capacity in cortical synaptosomes from Sod2 null mice.

Flynn James M JM Choi Sung W SW Day Nicholas U NU Gerencser Akos A AA Hubbard Alan A Melov Simon S

Free radical biology & medicine 20110106 7

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 m ...[more]

PMID: 21215798

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Project description:Previous findings from our laboratory and others indicate that two-dimensional gel electrophoresis (2-DE) can be used to study protein expression in defined brain regions, but mainly the proteins which are present in high abundance in glia are readily detected. The current study was undertaken to determine the protein profile in a synaptosomal subcellular fraction isolated from the cerebral cortex of the rat. Both 2-DE and liquid chromatography - tandem mass spectrometry (LC-MS/MS) procedures were used to isolate and identify proteins in the synaptosomal fraction and accordingly >900 proteins were detected using 2-DE; the 167 most intense gel spots were isolated and identified with matrix-assisted laser desorption/ionization - time of flight peptide mass fingerprinting or LC-MS/MS. In addition, over 200 proteins were separated and identified with the LC-MS/MS "shotgun proteomics" technique, some in post-translationally modified form. The following classes of proteins associated with synaptic function were detected: (a) proteins involved in synaptic vesicle trafficking-docking (e.g., SNAP-25, synapsin I and II, synaptotagmin I, II, and V, VAMP-2, syntaxin 1A and 1B, etc.); (b) proteins that function as transporters or receptors (e.g., excitatory amino acid transporters 1 and 2, GABA transporter 1); (c) proteins that are associated with the synaptic plasma membrane (e.g., post-synaptic density-95/synapse-associated protein-90 complex, neuromodulin (GAP-43), voltage-dependent anion-selective channel protein (VDACs), sodium-potassium ATPase subunits, alpha 2 spectrin, septin 7, etc.); and (d) proteins that mediate intracellular signaling cascades that modulate synaptic function (e.g., calmodulin, calcium-calmodulin-dependent protein kinase subunits, etc.). Other identified proteins are associated with mitochondrial or general cytosolic function. Of the two proteins identified as endoplasmic reticular, both interact with the synaptic SNARE complex to regulate vesicle trafficking. Taken together, these results suggest that the integrity of the synaptosomes was maintained during the isolation procedure and that this subcellular fractionation technique enables the enrichment of proteins associated with synaptic function. The results also suggest that this experimental approach can be used to study the differential expression of multiple proteins involved in alterations of synaptic function.

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Impaired spare respiratory capacity in cortical synaptosomes from Sod2 null mice.

Publications

Impaired spare respiratory capacity in cortical synaptosomes from Sod2 null mice.

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