Single-Cell Transcriptomics of Parkinson's Disease Human In Vitro Models Reveals Dopamine Neuron-Specific Stress Responses
Authors: Fernandes, Hugo J. R.; Patikas, Nikolaos; Foskolou, Stefanie; Field, Sarah F.; Park, Jong-Eun; Byrne, Meg L.; Bassett, Andrew R.; Metzakopian, Emmanouil
The advent of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized Parkinson's disease (PD) research, but single-cell transcriptomic analysis suggests unresolved cellular heterogeneity within these models. Here, we perform the largest single-cell transcriptomic study of human iPSC-derived dopaminergic neurons to elucidate gene expression dynamics in response to cytotoxic and genetic stressors. We identify multiple neuronal subtypes with transcriptionally distinct profiles and differential sensitivity to stress, highlighting cellular heterogeneity in dopamine in vitro models. We validate this disease model by showing robust expression of PD GWAS genes and overlap with postmortem adult substantia nigra neurons. Importantly, stress signatures are ameliorated using felodipine, an FDA-approved drug. Using isogenic SNCA-A53T mutants, we find perturbations in glycolysis, cholesterol metabolism, synaptic signaling, and ubiquitin-proteasomal degradation. Overall, our study reveals cell type-specific perturbations in human dopamine neurons, which will further our understanding of PD and have implications for cell replacement therapies.
Covalent modification of Keap1 at Cys77 and Cys434 by pubescenoside a suppresses oxidative stress-induced NLRP3 inflammasome activation in myocardial ischemia-reperfusion injury
Authors: Cheng, Yuanyuan; Cheng, Liangkai; Gao, Xiang; Chen, Sixuan; Wu, Peng; Wang, Caiyan; Liu, Zhongqiu
Background and Purpose: Kelch ECH-associating protein 1 (Keap1) is a crucial chaperonin for E3 ubiquitin ligases. Modification of the key reactive cysteine residues in Keap1 affects the interaction between Keap1 and its substrate nuclear factor erythroid 2-related factor 2 (Nrf2), subsequently regulating oxidative stress and NLPR3 inflammasome activation, which are important factors for myocardial ischemia-reperfusion injury (MI/RI). Pubescenoside A (PBA), an active compound from Ilex pubescens, has antithrombotic and anti-inflammatory effects. However, the effect of PBA on MI/RI is still unknown. In the present study, we aimed to determine whether PBA can protect the heart against MI/RI and clarify the direct target and the underlying mechanism of PBA. Methods: The left anterior descending artery (LAD) ligation-induced MI/RI mice model or oxygen and glucose deprivation/reperfusion (OGD/R) were used to evaluate the cardioprotective effect of PBA. Pull-down assays, co-immunoprecipitation (Co-IP) assays, LC/MS/MS, isothermal calorimetry (ITC) experiments and covalent docking were used to identify the target of PBA. Results: PBA protected cardiomyocytes against OGD/R in vitro and LAD-induced MI/RI in vivo. PBA suppressed NLRP3 inflammation activation and induced the Nrf2 signaling pathway. Interestingly, PBA targeted Keap1 by selectively covalently binding to conserved cysteine residues, cysteine 77 (Cys77) in the BTB domain and cysteine 434 (Cys434) in the Kelch domain of Keap1, subsequently inhibiting ubiquitination of Nrf2 and activating antioxidant enzymes. Additionally, the cysteines of Keap1 has different degree of activation by PBA as follows: Cys77 > Cys434 > Cys23 > Cys38 > Cys226 > Cys273, which further elucidates the cysteine sensitivity of Keap1. Conclusions: Our results indicated that PBA might be a new Nrf2 activator that covalently binds to two critical domains of Keap1, and shows cardioprotective activities against ischemia-reperfusion injury.