Parkinson's disease (PD) brains exhibit decreased complex I activity (Schapira and Gegg 2011), which is important since this enzyme is both the rate-limiting step for mitochondrial respiratory chain activity, and a key site for generation of reactive oxygen species (Murphy 2009). Mitochondrial defects are widespread in both the substantia nigra and cortex in PD (Keeney et al. 2006; Parker et al. 2008), but the underlying mechanisms that lead to mitochondrial dysfunction are unclear. Meta-analyses of several studies indicated that pesticide exposure potentially increases PD risk on average by ~ 1.5–3 fold (Priyadarshi et al. 2000, 2001; van et al. 2012; Pezzoli and Cereda 2013). Also, a recent study reported that exposure to paraquat and rotenone in farming communities increased the incidence of PD with an odds ratio of ~ 2–3 fold (Tanner et al. 2011). Rotenone is an herbicide, insecticide, and piscicide, and is the most potent member of the rotenoid family of neurotoxins found in tropical plants. Rotenone can pass through the blood brain barrier and plasma membrane, bind to complex I in striatal sections from rodent brains with a Kd of ~ 55 nM, decreasing its activity, and inducing oxidative stress (Higgins and Greenamyre 1996; Panov et al. 2005). In animal models, dopaminergic neurons appear to be particularly susceptible to rotenone-induced degeneration (Betarbet et al. 2000). Oral administration of rotenone at 30 mg/kg for 56 days in mice has been shown to cause α-synuclein accumulation and dopaminergic neurodegeneration (Inden et al. 2011). In vitro studies have shown that short term (1 week), low concentrations of rotenone induce accumulation of soluble and insoluble α-synuclein, and after 4 weeks, increased apoptosis in SK-N-MC neuroblastoma cells (Sherer et al. 2002).
Autophagy plays an important role in mitochondrial quality control (Higdon et al. 2012; Hill et al. 2012; Lee et al. 2012; Benavides et al. 2013; Dodson et al. 2013a,b; Liang et al. 2013; Mitchell et al. 2013a,b; Zhang 2013; Boyer-Guittaut et al. 2014; Giordano et al. 2014; Levonen et al. 2014; Redmann et al. 2014), and changes in autophagy have been observed in postmortem PD brains (Anglade et al. 1997). In addition, some of the familial PD genes such as pink1, parkin, and DJ-1 play important roles in the process of autophagy of mitochondria, known as mitophagy (Youle and Narendra 2011). In undifferentiated SH-SY5Y cells, it has been shown that rotenone inhibits autophagic flux prior to inducing cell death, and that restoration of autophagic activity is partially protective (Pan et al. 2009; Xiong et al. 2011). Importantly, this transformed cell line is metabolically distinct from neurons, and in the undifferentiated state exhibits aerobic glycolysis that likely changes their susceptibility to mitochondrial neurotoxins (Schneider et al. 2011). Studies performed in primary cortical neurons have shown that concentrations of rotenone far in excess of that resulting in complete inhibition of complex I activity (e.g. 250 nM–1 μM) induce externalization of the mitochondrial-specific cardiolipin, which serves as a tag for the mitochondria to undergo mitophagy (Chu et al. 2013). How low nM concentrations of rotenone, which are more likely to occur during environmental exposure, affect mitochondrial function in primary neurons, and whether mitophagy is engaged as a protective response, have not been critically examined, and are the focus of the present study.
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- Acknowledgments and conflict of interest disclosure
- Supporting Information
In this study, we investigated changes to mitochondrial function in response to the environmental neurotoxin rotenone in primary neurons, and the impact of modulating autophagy on neuronal survival in response to this compound.
As expected, rotenone has a significant effect on mitochondrial function consistent with inhibition at complex I (Figs 1 and 6). Of particular note is the sensitivity of the key aspects of cellular mitochondrial function at only 10 nM rotenone. These data highlight a critical role of complex I in maintaining the bioenergetics of primary neurons, and an inability of other metabolic substrates to compensate. It is important to note that in a cellular setting, inhibition of complex I will also inhibit flux through the TCA cycle, increasing the impact of rotenone inhibition on mitochondrial electron transport. Autophagic flux was inhibited by rotenone (Fig. 2), suggesting that the cells could also exhibit deficits in removal of damaged cellular constituents. In contrast, despite the fact that autophagy is inhibited, rotenone only decreased MitoTracker fluorescence without affecting LysoTracker staining (Fig. 3). This might be as a result of partial inhibition of mitochondrial function rather than a decrease in mitochondrial mass, since there was no apparent change in mtDNA copy number (Figure S1).
Associated with the decrease in mitochondrial membrane potential, MitoTracker and LysoTracker colocalization studies (Fig. 3) demonstrated that 10 nM rotenone for 24 h increased the delivery of mitochondria to the lysosomes, indicating an increase in mitophagy. The observation that rotenone induces mitophagy while general autophagic flux is inhibited suggests that a mitochondrial-specific lysosomal degradation pathway may be activated, as has recently been suggested, via a formation of mitochondrial spheroids (Ding et al. 2012; Ni et al. 2013; Yin and Ding 2013). Potential signaling mechanisms include, but are not limited to, preferential inhibitory modification of general macroautophagy but not mitophagy proteins, or preferential fission/fusion machinery activities and PARKIN/PINK1 function in response to loss of mitochondrial membrane potential. Increasing mitophagy without changing mtDNA copy number may be because of the following possibilities: (i) at 24 h after rotenone, although the mitophagic events are increased, mtDNA has not been digested at this time point, (ii) there is concurrent mitochondrial biogenesis that maintains the mitochondrial population, or (iii) the population of mitochondria that colocalizes with lysosomes are mainly mitochondrial fragments that are devoid of mtDNA.
The clearance of damaged mitochondria may improve the quality of the mitochondrial population, and so protect the primary cortical neurons from rotenone toxicity. These data are supported by the finding that inhibition of autophagy exacerbated rotenone toxicity, whereas activation of autophagy was partially protective (Fig. 5). Since we observed that in the presence of rotenone, rapamycin no longer activates autophagic flux, the partially protective effect of rapamycin may be independent of general macroautophagy, but may be dependent on mitophagy. In addition, rapamycin inhibits protein translation by inhibition of mammalian target of rapamycin, thereby decreasing the levels of short-lived proteins without significantly changing long-lived proteins; this may lead to changes in cellular signaling pathways. In our study, rapamycin enhanced complex I activity and decreased complex II activity on its own. In the presence of 10 nM rotenone, rapamycin did not change complex I activity, but further decreased complex II activity. Whether the decreased complex II activity contributed to rapamycin's ability to partially protect rotenone toxicity is unclear. To conclusively determine this possibility would require specific means to selectively up-regulate complex II activity to restore them to control levels in the presence of rapamycin. Removal of damaged mitochondria may also contribute to the protective function of rapamycin. However, since the mtDNA damage assay did not reveal any significant differences between rotenone exposed cells and rotenone + rapamycin exposed cells, more extensive studies using mitochondrial proteomic and lipidomic analysis are needed.
The interpretation of the bioenergetic profiles in response to 3-MA and rapamycin are complicated, since both these compounds had an effect on cellular mitochondrial function and complex I and II activities in the absence of rotenone. Cellular mitochondrial parameters were most severely affected with the combination of both rotenone and 3-MA, which also incidentally exhibited the greatest toxicity (Fig. 5). This finding is consistent with the primary hypothesis that the prevention of autophagy is detrimental to cellular bioenergetics and viability in the presence of rotenone. Interestingly, the approximately 90% inhibition of basal respiration in the presence of both 3-MA and rotenone is greater than the additive effect of rotenone or 3-MA alone, thus suggesting that inhibition of autophagy can accelerate bioenergetic defects. The rapamycin data are more difficult to explain, since the increase in viability (Fig. 5) was not reflected by an obvious improvement in cellular bioenergetics (Fig. 6). It is likely that the bioenergetic function in the presence of rapamycin and rotenone is adequate to maintain cell viability. Taken together, these observations indicate that general autophagy and mitophagy are differentially regulated in neurons, and that enhancement of general autophagy by rapamycin may result in metabolic modulation of the surviving cells.