Human iPSC-derived Dopaminergic Neurons

PB-II Activates the Nrf2/ARE Signaling Pathway and reduces Cellular ROS

Mechanism of PaBing-II (PB-II), a Chinese medicine for Parkinson's disease (PD) treatment, remains unclear. Wu's team employed hiPSC-derived dopaminergic neurons (DAn) and oxidative damage models to uncover the neuroprotective mechanism of PB-II.

To explore the mechanisms of PB-II protection on DAn from oxidative damage and inflammation, ROS level and inflammatory cytokines were measured in hiPSC-derived DAn after different treatments. While ROS levels remained similar between the Model and Blank Serum groups, PB-II Serum significantly reduced ROS levels under oxidative stress, indicating its antioxidant effect (Fig. 1A, B). Consistent with this, PB-II increased Nrf2 protein levels and the expression of downstream genes like NQO1, compared to no change in the Model group after H₂O₂ exposure (Fig. 1C–F). Additionally, PB-II increased Nrf2 nuclear translocation, similar to the Nrf2 activator artemisitene. In the ARE-luciferase reporter carrying MDA-MB-231 cells, PB-II activated ARE-mediated gene expression (Fig. 1G). ELISA results demonstrated that PB-II reduced the production of pro-inflammatory cytokines, including MCP-1, TNF-α, and IL-6, and increased anti-inflammatory IL-10 in DAn (Fig. 1H–K). In summary, the study demonstrated that PB-II protects DAn by activating the Nrf2/ARE pathway and reducing both oxidative damage and inflammation.

Fig. 1. Nrf2 pathway genes are activated in medicated cells and reduce ROS and inflammation (Wu S H, Rong C P, et al., 2024).

ET Protects Day 40 iDA Cultures against 6-OHDA-Induced Increase in mROS, Loss of MMP, Reduction in ATP Levels, and Loss of Dopamine Secretion

Parkinson's disease is a neurodegenerative disorder characterized by oxidative stress and mitochondrial defects in dopaminergic neurons. Ergothioneine (ET) is an endogenous antioxidant that can cross the blood-brain barrier, but it is unknown whether ET has neuroprotective effects in relevant human PD models. To establish this, Leow et al. modeled 6-OHDA-induced PD-like damage in human iPSC-derived dopaminergic neurons (iDA) and SH-SY5Y cells. Mitochondrial function, ROS levels, and protein damage were analyzed with or without ET treatment to see if ET could rescue 6-OHDA-induced neurotoxicity through OCTN1-mediated uptake.

MTT assay demonstrated that 6-OHDA induced 70–80% metabolic loss in iDA cultures (Fig. 2D). In a subset of cultures, 6-OHDA also induced neuronal degeneration, with the presence of fragmented neurons under the microscope (Fig. 3A), which is consistent with previous in vitro degeneration phenotypes. 6-OHDA increased the number of non-viable cells. iDA cultures were shown to express OCTN1, the transporter responsible for ET uptake (Fig. 2A). ET rescued iDA cultures in a concentration-dependent manner, and 1 mM ET was used in subsequent experiments (Fig. 2B). OCTN1 inhibitor VHCL was used to reduce intracellular ET levels (Fig. 2C). 6-OHDA increased the number of non-viable cells more in TH+ iDAs versus TH− non-iDAs (Fig. 3C). ET significantly reduced non-viable cells in both the TH+ and TH− populations except GM23720 non-iDAs (p = 0.233) (Fig. 3C). 6-OHDA decreased ATP levels, which was reversed by ET co-treatment (Fig. 4A). 6-OHDA also reduced dopamine secretion in iDA cultures, which was attenuated by ET co-treatment (Fig. 4B). 6-OHDA reduced mitochondrial membrane potential (MMP) and increased mitochondrial ROS levels in both cell populations, and both parameters were significantly more altered in iDAs versus non-iDAs (Fig. 4C–F). VHCL abolished ET-mediated rescue.

Fig. 2. Intracellular uptake of ET, mediated through OCTN1 transporters, protects iDA cultures against 6-OHDA-induced cell death in a concentration-dependent manner (Leow DM-K, Cheah IK-M, et al., 2024).

Fig. 3. ET-mediated protection of 6-OHDA-treated iDAs and non-iDAs in culture (Leow DM-K, Cheah IK-M, et al., 2024).

Fig. 4. Effect of ET on intracellular ATP levels and dopamine secretion in 6-OHDA-treated iDA cultures; TH+ Day 40 iDAs were also analysed for mitochondrial ROS and mitochondrial membrane potential levels (Leow DM-K, Cheah IK-M, et al., 2024).