Multiple lines of evidence suggest mitochondria play a role in Parkinson’s disease (PD) neurodegeneration (Parker and Swerdlow 1998). Alteration of the electron transport chain (ETC) enzyme complex I appears particularly relevant to this disease. Toxin-mediated complex I inhibition causes Parkinsonism and induces PD-relevant neuropathology in humans and animal models (Langston et al. 1983; Betarbet et al. 2000). Multiple tissues from PD patients show reduced complex I Vmax activity (Swerdlow 2007a).
Genetically altered mice are commonly used to model PD (Moore and Dawson 2008). These murine lines are created through transgenic expression of mutant genes that cause autosomal dominant PD variants or knock-out of genes that cause autosomal recessive PD variants. Mitochondrial physiology can be altered in these mice (Gispert et al. 2009), which is consistent with the view that mitochondria are relevant to PD. Most PD patients, though, do not show obvious Mendelian inheritance, do not carry currently identifiable PD-related nuclear gene mutations, and are felt to have a sporadic disease. It is unclear how rigorously mitochondrial alterations in the Mendelian models recapitulate sporadic PD patient mitochondrial alterations.
Human sporadic disease research has arguably been slowed by limitations in sporadic disease modeling. When it comes to the specific modeling of mitochondrial function in human sporadic diseases, although, approaches such as the cytoplasmic hybrid (cybrid) approach may prove useful (Swerdlow et al. 1997). Cybrids are cell lines created when the cytoplasmic contents of one cell are transferred to another cell. To date, multiple investigators have used different cybrid platforms to study sporadic PD mitochondrial function. Most studies of sporadic PD subject mitochondria show reduced complex I activity, and in most PD cybrid studies transfer of sporadic PD subject platelet mitochondria to mtDNA-depleted cells creates cell lines with persistently reduced complex I Vmax activities. The PD cybrid complex I defect is further associated with and may alter other physiologic parameters including oxidative stress, calcium homeostasis, toxin susceptibility, stress pathways and synuclein aggregation (Swerdlow 2007a; Esteves et al. 2008, 2009). In this study, we evaluated mitochondrial respiration in PD cybrid cell lines, as well as the status of certain proteins [SIRT1, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), and NF-kB] whose functions are influenced by cell aerobic-anaerobic balances.
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- Materials and methods
In this study, a cybrid approach was used to model sporadic PD mitochondrial function and evaluate its impact on certain proteins that are influenced by mitochondrial respiration. In these cell lines, it was found that the complex I defect seen in sporadic PD patients associates with a reduced MRC, increased mitochondrial proton leak, reduced SIRT1 phosphorylation, reduced PGC1α protein, and increased NF-kB activation. We conclude mitochondrial respiration and pathways influenced by aerobic metabolism are altered in NT2 cybrid cell lines generated through transfer of PD subject platelet mitochondria.
The cybrid approach was first applied to the study of PD mitochondria in 1996 (Swerdlow et al. 1996). A major intent of that study was to address the origin of the systemic complex I Vmax defect seen in sporadic PD subjects. Prior to that study, the cybrid approach had mostly been used to study the functional consequences of known mtDNA mutations (Swerdlow 2007a). A conclusion reached in the 1996 PD cybrid study was that mtDNA was likely to be at least partly responsible for the sporadic PD complex I defect. To date, however, no single unequivocal mtDNA signature has emerged as the definitive source of the complex I defect observed in that and subsequent other PD cybrid studies. One possible interpretation of this is the PD cybrid complex I defect arises independent of mtDNA. Another interpretation is simply that although mtDNA is responsible no single mtDNA mutation or polymorphism solely accounts for the defect. Reports that mtDNA deletions or microheteroplasmic mutations in the ND5 gene are found to a much greater degree in PD subject brains than in control subject brains are more consistent with the latter possibility, although studies looking for such mtDNA mutations in PD subject platelet mtDNA have not been published (Smigrodzki et al. 2004; Parker and Parks 2005; Bender et al. 2006; Kraytsberg et al. 2006). Our current study does not help to address this longstanding debate.
A second PD cybrid question relates to whether PD cybrids let investigators model sporadic PD mitochondrial dysfunction. Since 1996, over a dozen different PD cybrid studies have reported PD cybrid mitochondria or physiology directly affected by mitochondrial function differ between cybrids containing mitochondria transferred from PD subjects as compared to cybrids containing mitochondria transferred from control subjects (Swerdlow 2007a; Esteves et al. 2008, 2009). These ‘positive’ PD cybrid studies have been performed using SH-SY5Y neuroblastoma, A549 lung carcinoma, and NT2 teratocarcinoma nuclear backgrounds. To date, only one ‘negative’ cybrid study has been reported (Aomi et al. 2001). This study was performed using a HeLa cervical carcinoma nuclear background, and interestingly the negative conclusions of this study were partly based on a whole cell basal oxygen consumption analysis. In this study, we also found whole cell basal oxygen consumption between NT2 PD and control cybrids was equivalent. However, we also found the PD cybrid complex I Vmax was decreased, the PD cybrid maximum respiratory capacity was decreased, the mitochondrial proton leak was increased, and differences in SIRT1, PGC1α, and NF-kB levels or function were present. Therefore, although we confirm the respiratory analysis of Aomi et al., we conclude PD cybrids do facilitate modeling of sporadic PD subject mitochondrial function.
Because cybrids lines are generated using tumor cell lines it is perhaps not surprising that basal whole cell oxygen consumption is equivalent between PD and control cybrids. Tumor cells in general are subject to the Warburg Effect in which mitochondrial oxygen consumption is actively maintained at limited, relatively low levels (Feron 2009). A more intensive analysis of mitochondrial respiration did, however, reveal differences. Most importantly, we found the MRC of cybrid cell lines containing mitochondria transferred from PD subject platelets was reduced.
Whole cell respiratory analyses are becoming increasingly popular as the technology for assessing whole cell oxygen consumption improves. Along with improvements in respiratory analysis instruments comes refinement in whole cell respiratory analysis protocols. Some investigators recommend using a gradual FCCP titration to induce MRC states (Hutter et al. 2006) although when serial FCCP injections are not practical single FCCP injections are used (Amo et al. 2008). In deference to this technical issue, we compared the effects of FCCP titration to a single FCCP bolus. Both approaches produced similar results (data not shown). Using oligomycin to measure proton leak across the mitochondrial membrane is also becoming more common. With this approach, we found the PD cybrid proton leak rate was relatively high. An increased proton leak would predictably depolarize the mitochondrial membrane potential. Other PD cybrid studies report PD cybrid mitochondria are relatively depolarized (Esteves et al. 2008). We wonder whether the increased proton leak we observed is at least partly responsible for this.
We further extended our analyses to proteins such as SIRT1 that influence or are influenced by cell respiratory function. SIRT1 helps determine cell respiratory set points and because it is dependent on cytosolic NAD+/NADH ratios, SIRT1 activity is itself determined by the cell redox state (Yang and Sauve 2006; Arduino et al. 2009; Finkel et al. 2009). Although cytosolic and mitochondrial NAD+/NADH pools are not in direct contact, the two pools can influence each other through shuttles that permit the exchange of reducing equivalents. For example, if the mitochondrial NAD+/NADH ratio is high, cytosolic NADH can be converted to NAD+ via the transporter-mediated transfer of reducing equivalents from cytosolic NADH to mitochondrial NAD+. In general, high cytosolic NAD+/NADH ratios are associated with SIRT1 activation. We found no evidence that SIRT1 activity was increased and evidence consistent with a possible activity decrease. This suggests the cytosolic NAD+/NADH ratio in PD cybrids is either comparable to or slightly less than that of control cybrids. Although a complex I lesion could contribute to a low NAD+/NADH ratio, it is difficult to know whether this is an artifact of studying SIRT1 in a tumor cell model and we are unsure of the significance of this finding. Regardless, because SIRT1 activates PGC1α and PGC1α facilitates its own expression decreased SIRT1 activity is functionally consistent with the observed reduction in PD cybrid PGC1α levels (Nemoto et al. 2005; Handschin and Spiegelman 2006). In related studies, we have found increasing mitochondrial reactive oxygen species (ROS) production in neuroblastoma cells associates with reduced SIRT1 activity (in preparation). Multiple studies report PD cybrid mitochondria have increased ROS production. We speculate that in these tumor cell lines increased mitochondrial ROS production may actually stimulate the Warburg Effect, which could potentially manifest as a reduction in mitochondrial biogenesis signals.
ROS are known to activate NF-kB (Kaltschmidt et al. 1999; Kabe et al. 2005). Cells maintain a cytosolic pool of NF-kB and activation of this transcription factor associates with its nuclear translocation. Nuclear NF-kB translocation is observed in PD subject dopaminergic neurons (Hunot et al. 1997). Relevant to this, Onyango et al. (2005) previously reported immunochemistry data indicating NF-kB activity was increased in SH-SY5Y PD cybrids. Our current data obtained from studies of NT2 PD cybrids and using a transcription reporter system confirm and extend this report. Because SIRT1-mediated NF-kB deacetylation is known to inhibit NF-kB (Yeung et al. 2004), and our PD cybrids have a potential reduction in SIRT1 activity, it is possible that in addition to increased ROS decreased SIRT1 activity may also mediate NF-kB activation.
Whether or not the cybrid approach facilitates modeling of sporadic PD mitochondrial function is not simply an academic question. Although animal models based on rare Mendelian PD variants are increasingly relied upon by the PD research community, the ability to extrapolate findings from Mendelian PD model studies to sporadic PD may be limited. In Alzheimer’s disease and amyotrophic lateral sclerosis numerous therapies that showed efficacy in Mendelian disease models subsequently failed in human clinical trials, underscoring the importance of developing valid sporadic disease models (Swerdlow 2007b; Yamamoto et al. 2008). Our data argue that although the cybrid approach has limitations, it can facilitate the study of sporadic PD subject mitochondrial function and provide valuable insight into sporadic PD molecular physiology.