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Dopaminergic neurons of the substantia nigra pars compacta are defective in Parkinson's disease, but the specificity of this dysfunction is not understood. One hypothesis is that mitochondrial bioenergetic capacity is intrinsically lower in striatal dopaminergic presynaptic nerve varicosities, making them unusually susceptible to inhibition of electron transport by oxidative damage. To test this hypothesis, we separated isolated synaptosomes bearing dopamine transporters using immunomagnetic beads and compared their respiration with that of the residual nondopaminergic synaptosomes. As predicted, dopaminergic synaptosomes from striatum had lower respiratory rates. However, so did dopaminergic synaptosomes from cortex, indicating a lack of the predicted striatal specificity. We used fluorescent probes to analyze the bioenergetic competence of individual synaptosomes in the two fractions. The respiratory differences became nonsignificant when respiration rates were normalized to the number of respiration-competent synaptosomes, suggesting that differences reflected the quality of the different fractions. To circumvent damage induced by synaptosomal separation, we monitored membrane potentials in whole unseparated single synaptosomes using fluorescent imaging, and then identified the dopaminergic subpopulation using a fluorescent dopamine transporter substrate (ASP+[4-(4-diethylaminostyryl)-N-methylpyridinium iodide]). The capacity of dopaminergic and nondopaminergic synaptosomes to maintain plasma membrane and mitochondrial membrane potential under several stresses did not differ. In addition, this capacity did not decline in either subpopulation with age, a risk factor for Parkinson's disease. We conclude that the intrinsic bioenergetic capacities of dopaminergic and nondopaminergic presynaptic synaptosomes from mice do not differ.

Parkinson's disease (PD) involves both motor and non-motor disturbances. Non-motor features include alterations in sensory olfactory function which may constitute a viable biomarker for the disorder. It is not clear what causes olfactory dysfunction but it appears to coincide with the development of synucleopathy within the olfactory bulb (OB). Elevation in alpha-synuclein (a-syn) is indeed a risk factor for development of the sporadic disorder. The multifactorial nature of the idiopathic disease combined with variability in its presentation suggests that it is likely to be influenced by several factors and that in vivo models that explore the synergistic effect of alpha-synuclein elevation with other potential contributing factors are likely to be of importance in understanding the disease etiology. Using a dual transgenic (DTg) mouse model of dopaminergic alpha-synuclein overexpression coupled with doxycycline (Dox)-inducible glutathione (GSH) depletion in these same cells, we demonstrate an age-related loss in behavioral olfactory function coupled with a significant neurodegeneration of glomerular dopaminergic neurons. This is accompanied by increase in alpha-synuclein levels in non-dopaminergic cells in the granule cell layer (GCL). In addition, isolated olfactory bulb synaptosomes from dual transgenic lines with Dox consistently showed a slight but significant reduction in maximum mitochondrial respiration compared to controls. These results suggest that in the presence of increased oxidative stress, increased alpha-synuclein expression within dopaminergic OB neurons results in neurodegeneration in the glomerular layer (GL) and increased alpha-synuclein levels in the granular cell layer which coincide with olfactory dysfunction.

BACKGROUND Low power ultrasound delivered through an angioplasty-like guidewire may be effective for intracoronary thrombolysis. We evaluated the preclinical feasibility and safety of such wire. METHODS AND RESULTS In 15 anesthetized Yucatan minipigs, the ultrasonic wire was advanced percutaneously into all three coronaries. Each coronary was randomized to long activation (6 minutes), short activation (3 minutes), or control (3 minutes indwelling, no activation). The energy delivered was 0.14 +/- 0.01 W/cm of active length (20 kHz). No changes in heart rate, rhythm, or arterial pressure occurred during wire positioning or activation. Mean lumen diameter (MLD) by quantitative angiography was not significantly different pre- and postintervention (2.36 +/- 0.12 mm vs 2.36 +/- 0.11 mm for long activation, P = 0.96; 2.33 +/- 0.15 mm vs 2.34 +/- 0.14 mm for short activation, P = 0.54; 2.30 +/- 0.12 mm vs 2.33 +/- 0.12 mm for control, P = 0.21). There were no angiographic stenoses at 60 or 90 days follow-up. Compared with baseline, MLD at follow-up increased in all the three groups (2.40 +/- 0.13 mm vs 2.53 +/- 0.11 mm, P = 0.004 for long activation; 2.37 +/- 0.17 mm vs 2.52 +/- 0.14 mm, P = 0.023 for short activation; 2.20 +/- 0.12 mm vs 2.33 +/- 0.11 mm, P = 0.001 for the control group). By histology, there were no clinically significant pathologic changes in coronary morphology. CONCLUSION Use of a transverse cavitation therapeutic wire is feasible and well tolerated acutely in the normal porcine coronary. At 60 and 90 days, no angiographically apparent damage, no clinically significant pathologic changes, and no adverse events were seen. This technology may be safely used during percutaneous coronary intervention. Further studies are justified to evaluate its efficacy for intracoronary thrombus ablation.

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.

Respirometry using modified cell culture microplates offers an increase in throughput and a decrease in biological material required for each assay. Plate based respirometers are susceptible to a range of diffusion phenomena; as O(2) is consumed by the specimen, atmospheric O(2) leaks into the measurement volume. Oxygen also dissolves in and diffuses passively through the polystyrene commonly used as a microplate material. Consequently the walls of such respirometer chambers are not just permeable to O(2) but also store substantial amounts of gas. O(2) flux between the walls and the measurement volume biases the measured oxygen consumption rate depending on the actual [O(2)] gradient. We describe a compartment model-based correction algorithm to deconvolute the biological oxygen consumption rate from the measured [O(2)]. We optimize the algorithm to work with the Seahorse XF24 extracellular flux analyzer. The correction algorithm is biologically validated using mouse cortical synaptosomes and liver mitochondria attached to XF24 V7 cell culture microplates, and by comparison to classical Clark electrode oxygraph measurements. The algorithm increases the useful range of oxygen consumption rates, the temporal resolution, and durations of measurements. The algorithm is presented in a general format and is therefore applicable to other respirometer systems.

Objective Our objective was to determine whether autologous endothelial progenitor cells (EPCs) delivered into the pericardial space will migrate to and incorporate into ischemic myocardium in a porcine model. Background Use of EPCs to enhance neovascularization and preserve myocardial function in ischemic tissue is undergoing intense scrutiny as a potential therapy. Delivery into the pericardial sac may overcome some of the limitations of currently employed cell delivery techniques. Methods EPCs were immunopurified from peripheral blood of Yorkshire pigs by selecting for the CD31 surface antigen, and adherent cells were cultured for 3–5 days. After myocardial ischemia was induced in the left anterior descending (LAD) artery, either autologous DiI (1,1′-dioctadecyl-1-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)-labeled EPCs ( n =10) or serum-free medium (SFM; n =8) was delivered into the pericardial space using a percutaneous transatrial approach. Animals were sacrificed on Day 7 or 21. Echocardiography was performed at baseline, during ischemia, and on Day 7 in six SFM group animals and six EPC group animals. Results On Day 7, EPCs were identified in the left ventricular (LV) anterior wall or anterior septum in all six EPC-treated animals (cell density of 626±122/mm 2 ). On Day 21, EPCs were identified in the LV anterior wall or anterior septum in three of four EPC-treated animals (cell density of 267±167/mm 2 ). These cells showed dual staining for DiI and Bandeiraea simplicifolia lectin I (a marker of both native and exogenous endothelial cells). At the Day 7 follow-up, echocardiography demonstrated that fractional shortening in the EPC-treated group was 30.6±3.4, compared with 22.6±2.8 in SFM controls ( P =.05). Conclusions EPCs can migrate from the pericardial space to incorporate exclusively into areas of ischemic myocardium and may have favorable effects on LV function.