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Alterations in bioenergetic function induced by Parkinson’s disease mimetic compounds: Lack of correlation with superoxide generation

Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI, USA.
Journal of Neurochemistry (Impact Factor: 4.24). 06/2012; 122(5):941-51. DOI: 10.1111/j.1471-4159.2012.07836.x
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ABSTRACT J. Neurochem. (2012) 122, 941–951.
In vitro and in vivo models of Parkinson’s disease (PD) suggest that increased oxidant production leads to mitochondrial dysfunction in dopaminergic neurons and subsequent cell death. However, it remains unclear if cell death in these models is caused by inhibition of mitochondrial function or oxidant production. The objective of this study was to determine the relationship between mitochondrial dysfunction and oxidant production in response to multiple PD neurotoxicant mimetics. MPP+ caused a dose-dependent decrease in the basal oxygen consumption rate in dopaminergic N27 cells, indicating a loss of mitochondrial function. In parallel, we found that MPP+ only modestly increased oxidation of hydroethidine as a diagnostic marker of superoxide production in these cells. Similar results were found using rotenone as a mitochondrial inhibitor, or 6-hydroxydopamine (6-OHDA) as a mechanistically distinct PD neurotoxicant, but not with exposure to paraquat. In addition, the extracellular acidification rate, used as a marker of glycolysis, was stimulated to compensate for oxygen consumption rate inhibition after exposure to MPP+, rotenone, or 6-OHDA, but not paraquat. Together these data indicate that MPP+, rotenone, and 6-OHDA dramatically shift bioenergetic function away from the mitochondria and towards glycolysis in N27 cells.

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    • "We determined that 100 µM 6-OHDA is an optimal dose in N27 cells based on our preliminary dose-response studies. Also, we and many other laboratories [8], [33]–[37] showed that 6-OHDA selectively targets dopaminergic neurons at concentrations of 10–100 µM because it enters dopaminergic neurons through dopamine transporter. In this regard, dopamine transporters are expressed at much higher levels in primary mesencephalic dopaminergic neurons relative to N27 cells. "
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    ABSTRACT: Oxidative stress is a major pathophysiological mediator of degenerative processes in many neurodegenerative diseases including Parkinson's disease (PD). Aberrant cell signaling governed by protein phosphorylation has been linked to oxidative damage of dopaminergic neurons in PD. Although several studies have associated activation of certain protein kinases with apoptotic cell death in PD, very little is known about protein kinase regulation of cell survival and protection against oxidative damage and degeneration in dopaminergic neurons. Here, we characterized the PKD1-mediated protective pathway against oxidative damage in cell culture models of PD. Dopaminergic neurotoxicant 6-hydroxy dopamine (6-OHDA) was used to induce oxidative stress in the N27 dopaminergic cell model and in primary mesencephalic neurons. Our results indicated that 6-OHDA induced the PKD1 activation loop (PKD1S744/S748) phosphorylation during early stages of oxidative stress and that PKD1 activation preceded cell death. We also found that 6-OHDA rapidly increased phosphorylation of the C-terminal S916 in PKD1, which is required for PKD1 activation loop (PKD1S744/748) phosphorylation. Interestingly, negative modulation of PKD1 activation by RNAi knockdown or by the pharmacological inhibition of PKD1 by kbNB-14270 augmented 6-OHDA-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 (PKD1WT) or constitutively active PKD1 (PKD1S744E/S748E) attenuated 6-OHDA-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury. Collectively, our results demonstrate that PKD1 signaling plays a cell survival role during early stages of oxidative stress in dopaminergic neurons and therefore, positive modulation of the PKD1-mediated signal transduction pathway can provide a novel neuroprotective strategy against PD.
    PLoS ONE 05/2014; 9(5):e96947. DOI:10.1371/journal.pone.0096947 · 3.23 Impact Factor
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    • "6-Hydroxydopamine, an agent that has been thought to enhance O 2 ⋅ formation, did not induce 2-OH-E + formation, but only enhanced formation of non-specific oxidation products. Fluorescence microscopy experiments, however, showed increased red fluorescence under these and other conditions generating other one-electron oxidizing species [24] [25]. In the absence of HPLC results, one would have interpreted the fluorescence results incorrectly and equated the increase in red fluorescence to enhanced O 2 ⋅ formation from all the agents and ignoring the additional oxidation pathway(s) of the probe. "
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    ABSTRACT: Nearly ten years ago, we demonstrated that superoxide radical anion ( ) reacts with the hydroethidine dye (HE, also known as dihydroethidium, DHE) to form a diagnostic marker product, 2-hydroxyethidium (2-OH-E+). This particular product is not derived from reacting HE with other biologically relevant oxidants (hydrogen peroxide, hydroxyl radical, or peroxynitrite). This discovery negated the longstanding view that reacts with HE to form the other oxidation product, ethidium (E+). It became clear that due to the overlapping fluorescence spectra of E+ and 2-OH-E+, fluorescence-based techniques using the “red fluorescence” are not suitable for detecting and measuring in cells using HE or other structurally analogous fluorogenic probes (MitoSOXTM Red or hydropropidine). However, using HPLC-based assays, 2-OH-E+ and analogous hydroxylated products can be easily detected and quickly separated from other oxidation products.
    Biochimica et Biophysica Acta 05/2013; 1840(2). DOI:10.1016/j.bbagen.2013.05.008 · 4.66 Impact Factor
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    • "There was also no compensatory up-regulation in ECAR in PD63CLB. This was surprising because other studies have shown that loss of complex I activity as a result of neurotoxicity induces a loss of OCR with a corresponding increase in ECAR [38]. This loss of mtETC function and OCR is consistent with the abnormal morphology of mitochondria in PD63CLB cells (Figure 2D). "
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    ABSTRACT: Background Lewy bodies (LB) are a neuropathological hallmark of Parkinson’s disease (PD) and other synucleinopathies. The role their formation plays in disease pathogenesis is not well understood, in part because studies of LB have been limited to examination of post-mortem tissue. LB formation may be detrimental to neuronal survival or merely an adaptive response to other ongoing pathological processes. In a human cytoplasmic hybrid (cybrid) neural cell model that expresses mitochondrial DNA from PD patients, we observed spontaneous formation of intracellular protein aggregates (“cybrid LB” or CLB) that replicate morphological and biochemical properties of native, cortical LB. We studied mitochondrial morphology, bioenergetics and biogenesis signaling by creating stable sub-clones of three PD cybrid cell lines derived from cells expressing CLB. Results Cloning based on CLB expression had a differential effect on mitochondrial morphology, movement and oxygen utilization in each of three sub-cloned lines, but no long-term change in CLB expression. In one line (PD63CLB), mitochondrial function declined compared to the original PD cybrid line (PD63Orig) due to low levels of mtDNA in nucleoids. In another cell line (PD61Orig), the reverse was true, and cellular and mitochondrial function improved after sub-cloning for CLB expression (PD61CLB). In the third cell line (PD67Orig), there was no change in function after selection for CLB expression (PD67CLB). Conclusions Expression of mitochondrial DNA derived from PD patients in cybrid cell lines induced the spontaneous formation of CLB. The creation of three sub-cloned cybrid lines from cells expressing CLB resulted in differential phenotypic changes in mitochondrial and cellular function. These changes were driven by the expression of patient derived mitochondrial DNA in nucleoids, rather than by the presence of CLB. Our studies suggest that mitochondrial DNA plays an important role in cellular and mitochondrial dysfunction in PD. Additional studies will be needed to assess the direct effect of CLB expression on cellular and mitochondrial function.
    Molecular Neurodegeneration 01/2013; 8(1):6. DOI:10.1186/1750-1326-8-6 · 5.29 Impact Factor
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