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Updated: May 15, 2026
Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing
Published on: June 9, 2017
Rotenone inhibits autophagic flux prior to inducing cell death
Burton J Mader1, Violetta N Pivtoraiko, Hilary M Flippo
1Department of Pathology, Neuropathology Division, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Abstract:
Rotenone, which selectively inhibits mitochondrial complex I, induces oxidative stress, α-synuclein accumulation, and dopaminergic neuron death, principal pathological features of Parkinson's disease. The autophagy-lysosome pathway degrades damaged proteins and organelles for the intracellular maintenance of nutrient and energy balance. While it is known that rotenone causes autophagic vacuole accumulation, the mechanism by which this effect occurs has not been thoroughly investigated. Treatment of differentiated SH-SY5Y cells with rotenone (10 μM) induced the accumulation of autophagic vacuoles at 6 h and 24 h as indicated by Western blot analysis for microtubule associated protein-light chain 3-II (MAP-LC3-II). Assessment of autophagic flux at these time points indicated that autophagic vacuole accumulation resulted from a decrease in their effective lysosomal degradation, which was substantiated by increased levels of autophagy substrates p62 and α-synuclein. Inhibition of lysosomal degradation may be explained by the observed decrease in cellular ATP levels, which in turn may have caused the observed concomitant increase in acidic vesicle pH. The early (6 h) effects of rotenone on cellular energetics and autophagy-lysosome pathway function preceded the induction of cell death and apoptosis. These findings indicate that the classical mitochondrial toxin rotenone has a pronounced effect on macroautophagy completion that may contribute to its neurotoxic potential.
Insights
Rotenone exposure impairs the completion of macroautophagy by inhibiting lysosomal degradation. This disruption in cellular waste removal precedes cell death, suggesting a key role in Parkinson's disease pathogenesis.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Rotenone is a mitochondrial complex I inhibitor linked to Parkinson's disease pathology, including oxidative stress and dopaminergic neuron loss.
- The autophagy-lysosome pathway is crucial for cellular homeostasis, degrading damaged components.
- While rotenone causes autophagic vacuole accumulation, its precise mechanism remains unclear.
Purpose of the Study:
- To investigate the mechanism underlying rotenone-induced autophagic vacuole accumulation.
- To determine the impact of rotenone on autophagic flux and lysosomal function.
- To explore the relationship between rotenone's effects on cellular energetics and autophagy.
Main Methods:
- Differentiated SH-SY5Y cells were treated with rotenone (10 μM).
- Western blot analysis was used to detect microtubule-associated protein-light chain 3-II (MAP-LC3-II) levels.
- Autophagic flux was assessed by measuring levels of autophagy substrates p62 and α-synuclein, alongside cellular ATP levels and acidic vesicle pH.
Main Results:
- Rotenone treatment led to autophagic vacuole accumulation at 6 and 24 hours.
- Autophagic flux was impaired due to decreased lysosomal degradation, evidenced by increased p62 and α-synuclein.
- Rotenone decreased cellular ATP levels and increased acidic vesicle pH, preceding cell death induction.
Conclusions:
- Rotenone significantly disrupts the completion of macroautophagy by inhibiting lysosomal degradation.
- Impaired autophagic flux and compromised cellular energetics are early events in rotenone neurotoxicity.
- These findings highlight a potential mechanism contributing to rotenone's neurotoxic effects in Parkinson's disease models.
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