Bioenergetic adaptation in response to autophagy regulators during rotenone exposure

Abstract

Parkinson’s disease (PD) is the second most common neurodegenerative disorder with both mitochondrial dysfunction and insufficient autophagy playing a key role in its pathogenesis. Among the risk factors, exposure to the environmental neurotoxin rotenone increases the probability of developing PD. We previously reported that in differentiated SH-SY5Y cells, rotenone-induced cell death is directly related to inhibition of mitochondrial function. How rotenone at nM concentrations inhibits mitochondrial function, and whether it can engage the autophagy pathway necessary to remove damaged proteins and organelles, is unknown. We tested the hypothesis that autophagy plays a protective role against rotenone toxicity in primary neurons. We found that rotenone (10–100 nM) immediately inhibited cellular bioenergetics. Concentrations that decreased mitochondrial function at 2 hr, caused cell death at 24 hr with an LD50 of 10 nM. Overall autophagic flux was decreased by 10 nM rotenone at both 2 and 24 hr, but surprisingly mitophagy, or autophagy of the mitochondria, was increased at 24 hr, suggesting that a mitochondrial-specific lysosomal degradation pathway may be activated. Upregulation of autophagy by rapamycin protected against cell death while inhibition of autophagy by 3-methyladenine (3-MA) exacerbated cell death. Interestingly, while 3-MA exacerbated the rotenone-dependent effects on bioenergetics, rapamycin did not prevent rotenone-induced mitochondrial dysfunction, but caused reprogramming of mitochondrial substrate usage associated with both complex I and complex II activities. Taken together, these data demonstrate that autophagy can play a protective role in primary neuron survival in response to rotenone; moreover, surviving neurons exhibit bioenergetic adaptations to this metabolic stressor.

Inhibition of autophagy with bafilomycin and chloroquine decreases mitochondrial quality and bioenergetic function in primary neurons

“…These cells exhibited the typical profile for the mitochondrial stress test we have previously reported for primary neurons [38], [47]. As expected, after establishing a stable baseline measurement of oxygen consumption rate (OCR) the injection of oligomycin resulted in a decrease in OCR due to the inhibition of ATP synthase.…”

“…Quantitative real-time PCR was performed using SYBR green in an ABI 7500 machine. Mitochondrial DNA copy number was determined by normalizing results from primers targeted to mtDNA (Forward: 5'-CCAAGGAATTCCCCTACACA-3' and Reverse: 5′-GAAATTGCGAGAATGGTGGT-3′) against results from primers targeted to nuclear 18S DNA (Forward: 5'-CGAAAGCATTTGCCAAGAAT-3' and Reverse: [38], [39]. Mitochondrial DNA damage was determined by PCR as previously described [40].…”

Investigation into experimental toxicological properties of plant protection products having a potential link to Parkinson's disease and childhood leukaemia†

“…In PC12 cell, the concomitant treatment with trehalose (autophagic inducer) attenuated the rotenone-induced cell death while in-vivo trehalose treatment decreased the rotenoneinduced dopaminergic neurons loss . • Rotenone LD 50 of 10 nM in differentiated SH-SY5Y cells decreased autophagic flux at both 2 and 24 h. Upregulation of autophagy by rapamycin protected against cell death while inhibition of autophagy by 3-methyladenine exacerbated cell death (Giordano et al, 2014) • Treatment of embryonic midbrain neuronal cells with 0.1-10 lM rotenone for 30 min induces a decrease in polymerised tubulin and increased the number of apoptotic TH+ cells. Similar effects were observed with colchicine treatment, a well-known microtubule-depolarising agent and prevented by taxol, a well-known microtubule-stabilising agent.…”

Systematic literature review on Parkinson's disease and Childhood Leukaemia and mode of actions for pesticides

“…between 1-100 nM), one of the key downstream events is the increased cell death via activation of apoptosis induced by rotenone (Ahmadi et al, 2003;Arnold et al, 2011;Borland et al, 2008;Chang et al, 2011;Chen et al, 2008;Chiu et al, 2015;Choi et al, 2010b;Emmrich et al, 2013;Filomeni et al, 2012;Gao et al, 2012;Gao et al, 2002Gao et al, , 2003aGao et al, 2003b;Giordano et al, 2014;Giordano et al, 2012;Hsuan et al, 2006;Imamura et al, 2006;Jiang et al, 2006a;Kang et al, 2012a;Klintworth et al, 2007;Knaryan et al, 2014;Moon et al, 2005;Newhouse et al, 2004;Radad et al, 2006;Ramachandiran et al, 2006;Shamoto-Nagai et al, 2003;Sherer et al, 2003a;Takeuchi et al, 2009;Wang & Xu, 2005;Watabe & Nakaki, 2004;Zhou et al, 2015). This event has been observed as early as 6 h after 100 nM rotenone exposure in mouse dopaminergic MN9D cell line (Gao et al, 2012) up to 3 weeks after 1 nM rotenone exposure in rat differentiated dopaminergic PC6-3 cell line (Arnold et al, 2011).…”

“…al., 2015), proteasomal impairment (Betarbet et al, 2006;Shamoto-Nagai et al, 2003), increased intracellular calcium (Knaryan et al, 2014;Lupescu et al, 2012;Wang & Xu, 2005), ATP depletion (Kang et al, 2012a;Sherer et al, 2003a), activation of microglia Chang et al, 2011;Emmrich et al, 2013;Gao et al, 2013;Gao et al, 2002), impaired neurite formation Leung et al, 2007), impaired autophagy, namely disrupted autophagic flux (Filomeni et al, 2012;Gao et al, 2012;Garcia-Garcia et al, 2013;Giordano et al, 2014;Wu et al, 2015), decreased dopamine uptake (Gao et al, 2002;Mareysemper et al, 1993) as well as reduced cellular dopamine levels (Hirata & Nagatsu, 2005). Similar to oxidative stress, these events were usually analysed at the end of rotenone exposure, therefore making it difficult to extrapolate any temporal relationships among these events.…”