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Updated: Aug 20, 2026
Quantification of Immunostained Caspase-9 in Retinal Tissue
Published on: July 25, 2022
Rotenone-induced caspase 9/3-independent and -dependent cell death in undifferentiated and differentiated human
Jiang Li1, Maria L Spletter, Delinda A Johnson
1School of Pharmacy, University of Wisconsin at Madison, 777 Highland Avenue, Madison, WI 53705-2222, USA.
Abstract:
We used human neural stem cells (hNSCs) and their differentiated cultures as a model system to evaluate the mechanism(s) involved in rotenone (RO)- and camptothecin (CA)-induced cytotoxicity. Results from ultrastructural damage and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining indicated that RO-induced cytotoxicity resembled CA-induced apoptosis more than H(2)O(2)-induced necrosis. However, unlike CA-induced, caspase 9/3-dependent apoptosis, there was no increased activity in caspase 9, caspase 3 or poly (ADP-ribose) polymerase (PARP) cleavage in RO-induced cytotoxicity, in spite of time-dependent release of cytochrome c and apoptosis-inducing factor (AIF) following mitochondrial membrane depolarization and a significant increase in reactive oxygen species generation. Equal doses of RO and CA used in hNSCs induced caspase 9/3-dependent apoptosis in differentiated cultures. Time-dependent ATP depletion occurred earlier and to a greater extent in RO-treated hNSCs than in CA-treated hNSCs, or differentiated cultures treated with RO or CA. In conclusion, these results represent a unique ultrastructural and molecular characterization of RO- and CA-induced cytotoxicity in hNSCs and their differentiated cultures. Intracellular ATP levels may play an important role in determining whether neural progenitors or their differentiated cells follow a caspase 9/3-dependent or -independent pathway in response to acute insults from neuronal toxicants.
Insights
Rotenone and camptothecin induce cytotoxicity in human neural stem cells. ATP depletion influences whether cell death is caspase-dependent or independent.
Area of Science:
- Neuroscience
- Toxicology
- Cell Biology
Background:
- Neuronal toxicants like rotenone (RO) and camptothecin (CA) can induce cell death.
- Understanding the mechanisms of cytotoxicity in neural stem cells (hNSCs) is crucial for neurodegenerative disease research.
Purpose of the Study:
- To investigate the mechanisms of RO- and CA-induced cytotoxicity in hNSCs and their differentiated cultures.
- To compare RO-induced cell death with camptothecin (CA)-induced apoptosis and H(2)O(2)-induced necrosis.
Main Methods:
- Utilized human neural stem cells (hNSCs) and differentiated cultures.
- Assessed ultrastructural damage and performed TUNEL staining.
- Measured caspase 9/3 activity, PARP cleavage, cytochrome c and AIF release, reactive oxygen species (ROS) generation, and ATP levels.
Main Results:
- RO-induced cytotoxicity showed features of apoptosis but lacked caspase 9/3 activation, unlike CA-induced apoptosis.
- RO and CA induced caspase 9/3-dependent apoptosis in differentiated hNSCs.
- RO caused earlier and more extensive ATP depletion in hNSCs compared to CA or differentiated cells.
Conclusions:
- RO and CA exhibit distinct cytotoxicity mechanisms in hNSCs.
- Intracellular ATP levels are critical in determining the apoptotic pathway (caspase-dependent or -independent) in response to neuronal toxicants.
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