Anti-SOD1 Nanobodies That Stabilize Misfolded SOD1 Proteins Also Promote Neurite Outgrowth in Mutant SOD1 Human Neurons
Abstract
:1. Introduction
2. Results
2.1. Nanobodies with Selectivity for ALS-Linked SOD1
2.2. Anti-SOD1 Nanobodies Lead to Enhanced, Rather Than Reduced, Levels of Ectopic SOD1 in Cellulo
2.3. The Subcellular Localization of Mutant SOD1 Is Restored by Co-Expression of Anti-SOD1 Nanobodies
2.4. Anti-SOD1 Nanobodies Stabilize Mutant SOD1 in Cellulo
2.5. Anti-SOD1 Nanobody Expression Is Non-Toxic and Induces Enhanced Neurite Outgrowth in Human SOD1 A4V Motor Neurons
2.6. Anti-SOD1 Nanobodies Detect Human SOD1 G93A in Lysates from an ALS Mouse Model
3. Discussion
4. Materials and Methods
4.1. Generation of Anti-SOD1 Nanobodies
4.2. Plasmid Construction
4.3. Recombinant Nanobody Expression and Purification
4.4. ELISA
4.5. Transient Transfection of HEK293T Cells
4.6. Image Acquisition and Analysis of Transfected HEK293T Cells
4.6.1. HEK293T N/C Ratio
4.6.2. HEK293T Signal Intensity Analysis
4.6.3. Western Blot Analysis
4.7. iPSC Culture, Motor Neuron Differentiation, and Lentiviral Transduction
4.8. Immunofluorescence Microscopy Analysis of iPSC-Derived Motor Neurons
4.8.1. Neurite-Tracing Analysis
4.8.2. SOD1-Intensity Analysis
4.9. Differential Scanning Fluorimetry (DSF)
4.10. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Kumar, M.S.; Fowler-Magaw, M.E.; Kulick, D.; Boopathy, S.; Gadd, D.H.; Rotunno, M.; Douthwright, C.; Golebiowski, D.; Yusuf, I.; Xu, Z.; et al. Anti-SOD1 Nanobodies That Stabilize Misfolded SOD1 Proteins Also Promote Neurite Outgrowth in Mutant SOD1 Human Neurons. Int. J. Mol. Sci. 2022, 23, 16013. https://doi.org/10.3390/ijms232416013
Kumar MS, Fowler-Magaw ME, Kulick D, Boopathy S, Gadd DH, Rotunno M, Douthwright C, Golebiowski D, Yusuf I, Xu Z, et al. Anti-SOD1 Nanobodies That Stabilize Misfolded SOD1 Proteins Also Promote Neurite Outgrowth in Mutant SOD1 Human Neurons. International Journal of Molecular Sciences. 2022; 23(24):16013. https://doi.org/10.3390/ijms232416013
Chicago/Turabian StyleKumar, Meenakshi Sundaram, Megan E. Fowler-Magaw, Daniel Kulick, Sivakumar Boopathy, Del Hayden Gadd, Melissa Rotunno, Catherine Douthwright, Diane Golebiowski, Issa Yusuf, Zuoshang Xu, and et al. 2022. "Anti-SOD1 Nanobodies That Stabilize Misfolded SOD1 Proteins Also Promote Neurite Outgrowth in Mutant SOD1 Human Neurons" International Journal of Molecular Sciences 23, no. 24: 16013. https://doi.org/10.3390/ijms232416013