A Comprehensive Functional Investigation of the Human Translocator Protein 18 kDa (TSPO) in a Novel Human Neuronal Cell Knockout Model
Abstract
:1. Introduction
2. Results
2.1. Confirmation of TSPO Knockout in hiPSCs
2.2. Differentiation of hiPSC-Derived NPCs into Astrocytes
2.3. Differentiation of hiPSC-Derived NPCs into Neurons
2.4. Confirmation of TSPO Knockout in NPCs, Astrocytes, and Neurons
2.5. Effect of TSPO Expression on Steroid Synthesis in hiPSC-Derived Astrocytes
2.6. Impact of TSPO Expression on Mitochondrial Respiration
2.7. Role of TSPO in the Modulation of the Mitochondrial Membrane Potential
2.8. Involvement of TSPO in Ca2+ Homeostasis
2.9. Impact of TSPO on Cellular Bioenergetics and Glycolysis
2.10. TSPO-Deficient Neural Progenitors and Astrocytes Show Oxidative Stress
2.11. Effect of TSPO-Deficiency on mtDNA Copy Number, Mitochondrial Content, and Cell Size
2.12. Impact of TSPO on Mitochondrial Dynamics and Morphology
2.13. TSPO Expression in Major Depressive Disorder
3. Discussion
3.1. Role of TSPO in Neurosteroidogenesis
3.2. Impact of TSPO on Mitochondrial and Cellular Respiration
3.3. Mitochondrial Membrane Potential and Ca2+ and Redox Homeostasis
3.4. Involvement of TSPO in Mitochondrial Dynamics and Mitophagy
3.5. Possible Role for TSPO in Mitochondrial Dysfunction in Depression
4. Materials and Methods
4.1. Fibroblasts and Human-Induced Pluripotent Stem Cells
4.2. C20 Microglia and H295-R Cells
4.3. Generation of TSPO Knockout in iPSC Using CRISPR/Cas9 Genome Editing
4.4. Differentiation of iPSCs into Neural Progenitor Cells (NPCs), Neurons, and Astrocytes
4.5. Western Blotting
4.6. Immunofluorescence
4.7. Fluorescent Live-Cell Imaging
4.8. Quantitative Real-Time PCR and mtDNA Copy Number Analysis
4.9. Mitochondrial Respirometry
4.10. Total ATP Content Quantification
4.11. Scanning Electron Microscopy (SEM)
4.12. Pregnenolone Quantification
4.13. Flow Cytometry Analyses
4.14. Cytosolic and Mitochondrial Reactive Oxygen Species/Oxidative Stress
4.15. Mitochondrial Mass
4.16. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Bader, S.; Jahner, T.; Dörfelt, A.; Melchner, D.; Cardon, I.; Siegmund, H.I.; Brochhausen, C.; Rupprecht, R.; Milenkovic, V.M.; Wetzel, C.H. A Comprehensive Functional Investigation of the Human Translocator Protein 18 kDa (TSPO) in a Novel Human Neuronal Cell Knockout Model. Int. J. Mol. Sci. 2024, 25, 12882. https://doi.org/10.3390/ijms252312882
Bader S, Jahner T, Dörfelt A, Melchner D, Cardon I, Siegmund HI, Brochhausen C, Rupprecht R, Milenkovic VM, Wetzel CH. A Comprehensive Functional Investigation of the Human Translocator Protein 18 kDa (TSPO) in a Novel Human Neuronal Cell Knockout Model. International Journal of Molecular Sciences. 2024; 25(23):12882. https://doi.org/10.3390/ijms252312882
Chicago/Turabian StyleBader, Stefanie, Tatjana Jahner, Anett Dörfelt, Doris Melchner, Iseline Cardon, Heiko I. Siegmund, Christoph Brochhausen, Rainer Rupprecht, Vladimir M. Milenkovic, and Christian H. Wetzel. 2024. "A Comprehensive Functional Investigation of the Human Translocator Protein 18 kDa (TSPO) in a Novel Human Neuronal Cell Knockout Model" International Journal of Molecular Sciences 25, no. 23: 12882. https://doi.org/10.3390/ijms252312882
APA StyleBader, S., Jahner, T., Dörfelt, A., Melchner, D., Cardon, I., Siegmund, H. I., Brochhausen, C., Rupprecht, R., Milenkovic, V. M., & Wetzel, C. H. (2024). A Comprehensive Functional Investigation of the Human Translocator Protein 18 kDa (TSPO) in a Novel Human Neuronal Cell Knockout Model. International Journal of Molecular Sciences, 25(23), 12882. https://doi.org/10.3390/ijms252312882