Our data indicate that intracellular ROS accumulation is an early event in T-peptide-induced neuronal cell death. We observed a significant increase in intracellular ROS levels at early-stage af ter treatment with T-peptide. The intracellular ROS levels dropped dramatically at late-stage in our cell culture model, which suggests that ROS accumulation may happen prior to other cytopathology in AD. The work presented above also shows that T-peptideinduced intracellular ROS levels increase in T-peptideinduced neuronal cells in a dose-dependent manner. Cell death was not observed in cells treated with T-peptide at low concentration. The concentration-dependent manner may provide the evidence that the treatment of exogenous T-peptide can increase the intracellular ROS to a toxic level, which can kill the cells or trigger other events that can initiate cell death, such as elevating intracellular Ca2+ levels. It has been suggested by others that mitochondria play a crucial role in age-related neurodegenerative tauopathies. Many lines of evidence show that mitochondrial DNA mutation and production of excess intracellular ROS contribute to neurodegenerative diseases. It has been suggested by others that ROS is generated at high mitochondrial membrane potential. However, we observed a decrease in mitochondrial membrane potential in T-peptide-treated HT-22 cells. We proposed that ROS accumulation happens before mitochondrial membrane potential decrease. To confirm this hypothesis, we compared the mitochondrial membrane potential in cells treated with T-peptide alone versus T-peptide together with glutathione. The mitochondrial membrane potential in cells treated with T-peptide together with glutathione was similar to the control cells, which indicates ROS accumulation may be the primary event that leads to mitochondrial membrane potential reduction. It has also been suggested by others that mitochondrial membrane potential decrease is a consequence of ROS accumulation.8
In our previous study, we have found that leupeptin, a lysosomal protease inhibitor, can rescue neuronal cells from T-peptide, which indicates that lysosomes are involved in T-peptide-induced neuronal cell death. It has been suggested that the ROS accumulation induced by intracellular amyloid aggregates can lead to lipid peroxidation, which can lead to lysosomal membrane leakage. The release of lysosomal hydrolases into the cytosol may be a key event that leads to neuronal cell death. We will further examine the role of ROS accumulation in lysosomal leakage by monitoring lysosomal membrane disruption in cells treated with T-peptide alone versus T-peptide together with glutathione using Acridine Orange.
References 1. Anne Eckert et al., “Mitochondrial Dysfunction, Apoptotic Cell Death, and Alzheimer ’s Disease,” Biochemical Pharmacology 66 (2003): 1627–1634. 2. Julie K. Andersen, “Oxidative Stress in Neurodegeneration: Cause or Consequence,” Nature Reviews Neuroscience 5 (2004): 18–25. 3. Kelly Zhao et al., “Neuron-selective Toxicity of Tau Peptide in a Cell Culture Model of Neurodegenerative Tauopathy: Essential Role for Aggregation in Neurotoxicity,” Journal of Neuroscience Research 88 (2010): 3399–3413. 4. Ibid. 5. Ibid. 6. Ibid. 7. Ibid. 8. Ibid.
The decline in intracellular glutathione concentration and increase of oxidative stress has been suggested to precede the signaling events associated in apoptotic cell death. Mitochondria are involved in apoptotic cell death by alternating the redox potential. It has been suggested by others that apoptotic cell death may be involved in neurodegenerative tauopathies. However, nuclear fragmentation, a hallmark feature of apoptosis, was not observed in T-peptide-treated neuronal cells, which indicates that the T-peptide-induced neuronal cell death is not apoptotic.
Spring 2014
The Exley
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