A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells
Abstract
:1. Introduction
2. Results
2.1. Generation of Motor Neuron Models of ALS with Intracellular Accumulation of Misfolded and Aggregated SOD1
2.2. Total Protein Level of Extracellular Human and Murine SOD1 in Conditioned Medium
2.3. Involvement of Extracellular Oxidative Stress in the Thiol/Disulfide Redox Balance of Extracellular hSOD1
2.4. The Fraction of Non-Natively Folded Human SOD1, but not Murine SOD1, Is Increased in the Extracellular Environment
2.5. Metal-Free, Disulfide Oxidized SOD1 (apo-SOD1S-S) Is the Primary Misfolded Species in Conditioned Medium
2.6. Extracellular Misfolded apo-SOD1S-S Induces Intracellular Propagation of hSOD1WT Misfolding
2.7. Prion-Like Propagation of Extracellular Misfolded apo-SOD1S-S Induces Cell Death in Motor Neuron-Like NSC-34 Cells
3. Discussion
4. Materials and Methods
4.1. Expression Vectors
4.2. Cell Culture and Transfection
4.3. Fluorescent Imaging
4.4. Ultrafiltration of Conditioned Medium
4.5. Protein Extraction
4.6. Western Blotting
4.7. Enzyme-Linked Immunosorbent Assay
4.8. Measurement of SOD1 Enzymatic Activity
4.9. Immunoprecipitation
4.10. Intracellular Propagation of hSOD1WT Misfolding by Conditioned Medium and Purified hSOD1S-S Proteins
4.11. Assessment of Viability and Cytotoxicity of Cells Exposed to Conditioned Medium Containing Misfolded apo-SOD1S-S
4.12. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
4-HNE | 4-Hydroxynonenal |
ALS | Amyotrophic lateral sclerosis |
CNS | Central nervous system |
CSF | Cerebrospinal fluid |
DMEM | Dulbecco’s modified Eagle’s medium |
EIPA | 5-(N-ethyl-N-isopropyl) amiloride |
ELISA | Enzyme-linked immunosorbent assay |
GFP | Green fluorescent protein |
LDH | Lactate dehydrogenase |
HEPES | N-(2-hydroxyethyl) piperazine-N’-2-ethanesulfonic acid |
HRP | Horseradish peroxidase |
hSOD1 | Human superoxide dismutase 1 |
NP-40 | Nonidet P-40 |
PAGE | Polyacrylamide gel electrophoresis |
PBS | Phosphate-buffered saline |
SDS | Sodium dodecyl sulfate |
SOD1, | Superoxide dismutase 1 |
WT | Wild-type |
β-ME | Beta-mercaptoethanol |
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Takashima, C.; Kosuge, Y.; Inoue, M.; Ono, S.-I.; Tokuda, E. A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells. Int. J. Mol. Sci. 2021, 22, 4155. https://doi.org/10.3390/ijms22084155
Takashima C, Kosuge Y, Inoue M, Ono S-I, Tokuda E. A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells. International Journal of Molecular Sciences. 2021; 22(8):4155. https://doi.org/10.3390/ijms22084155
Chicago/Turabian StyleTakashima, Chika, Yasuhiro Kosuge, Masahisa Inoue, Shin-Ichi Ono, and Eiichi Tokuda. 2021. "A Metal-Free, Disulfide Oxidized Form of Superoxide Dismutase 1 as a Primary Misfolded Species with Prion-Like Properties in the Extracellular Environments Surrounding Motor Neuron-Like Cells" International Journal of Molecular Sciences 22, no. 8: 4155. https://doi.org/10.3390/ijms22084155