Primary Neurons and Differentiated NSC-34 Cells Are More Susceptible to Arginine-Rich ALS Dipeptide Repeat Protein-Associated Toxicity than Non-Differentiated NSC-34 and CHO Cells
Abstract
:1. Introduction
2. Results
2.1. Arginine-Rich DRPs Exhibit Greater Cytotoxicity in a Neuron-Like Cell Line
2.2. Arginine-Rich DRPs Induce Cytotoxicity in NSC-34 in a Significant, Dose-Dependent Manner
2.3. All DRPs Induce Apoptosis in a Significant, Dose-Dependent Manner
2.4. Arginine-Rich DRPs Interfere with NSC-34 Proliferation Activity
2.5. Arginine-Rich FITC-Labeled DRP Incubation Results in Increased Labeling of NSC-34 Cells Compared to Non-Arginine-Rich DRPs
2.6. NSC-34 Sensitivity to Arginine-Rich DRPs Increases with Neuronal Differentiation
2.7. Neonatal Mouse Derived Spinal Neurons are Most Sensitive to Arginine-Rich DRPs
3. Discussion
4. Materials and Methods
4.1. CHO and NSC-34 Cell Culture and NSC-34 Differentiation
4.2. Dipeptide Repeat Proteins for Testing
4.3. WST-1 Assay
4.4. LDH Assay
4.5. Caspase-3 Assay
4.6. BrdU ELISA
4.7. Flow Cytometry
4.8. Immunohistochemistry and Slide Preparation for Microscopy
4.9. Confocal Laser Scanning Microscopy
4.10. Isolation and Culture of Primary Neurons from Spinal Cords of Neonatal Mice
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
DRP | Dipeptide Repeat Protein |
HRE | Hexanucleotide Repeat Expansion |
C9HRE | C9orf72 Hexanucleotide Repeat Expansion Mutation |
RAN | Repeat-Associated non-ATG |
ALS | Amyotrophic Lateral Sclerosis |
FTD | Frontotemporal Dementia |
C9ALS/FTD | C9orf72 Amyotrophic Lateral Sclerosis/Frontotemporal Dementia |
CHO | Chinese Hamster Ovary cell line |
NSC-34 | Mouse Spinal Cord x Neuroblastoma Hybrid cell line |
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DRP | Endpoint | 3 µM | 1 µM | 300 nM | 100 nM | 30 nM | 10 nM |
---|---|---|---|---|---|---|---|
Day 14 | **** | ns | **** | **** | **** | **** | |
GR15 | Day 21 | **** | **** | **** | **** | **** | **** |
Day 28 | **** | **** | **** | **** | **** | **** | |
Day 14 | ** | ns | **** | **** | *** | **** | |
PR15 | Day 21 | **** | **** | **** | **** | **** | **** |
Day 28 | **** | **** | **** | **** | **** | **** | |
Day 14 | ns | ns | ns | ns | *** | ns | |
PA15 | Day 21 | ns | ns | ns | ns | ** | ns |
Day 28 | ns | ns | ns | ns | **** | ns | |
Day 14 | ns | ns | ns | * | ns | ns | |
GP15 | Day 21 | ns | ns | ns | ns | ns | ns |
Day 28 | ns | * | ns | * | ns | ns |
DRP | Comparison | 3 µM | 1 µM | 300 nM | 100 nM | 30 nM | 10 nM |
---|---|---|---|---|---|---|---|
D7 vs. D14 | ** | ns | **** | **** | **** | **** | |
GR15 | D14 vs. D21 | **** | **** | **** | **** | **** | ns |
D21 vs. D28 | **** | **** | **** | **** | **** | *** | |
D7 vs. D14 | ** | ns | **** | **** | *** | **** | |
PR15 | D14 vs. D21 | **** | **** | **** | **** | **** | ns |
D21 vs. D28 | **** | ** | ns | ns | *** | **** | |
D7 vs. D14 | ns | ns | ns | ns | *** | ns | |
PA15 | D14 vs. D21 | ns | ns | ns | ns | ns | ns |
D21 vs. D28 | ns | ns | ns | ns | ns | ns | |
D7 vs. D14 | ns | ns | ns | * | ns | ns | |
GP15 | D14 vs. D21 | ns | ns | ns | ns | ns | ns |
D21 vs. D28 | ns | ns | ns | ns | ns | ns |
Supplier | Sequence | Purity (%) | Molecular Weight (g) | Solubilized in | [Stock] | Stored at (°C) |
---|---|---|---|---|---|---|
GenicBio | (GR)15 | 94.53 | 3216.57 | Sterile DMSO | 10 mM | 4 |
GenicBio | (PR)15 | 93.17 | 3817.53 | Sterile DMSO | 10 mM | 4 |
GenicBio | (GP)15 | 94.49 | 2330.56 | Sterile DMSO | 10 mM | 4 |
GenicBio | (PA)15 | 90.75 | 2540.91 | Sterile DMSO | 10 mM | 4 |
GenicBio | FITC-(GR)15 | 92.15 | 3719.11 | Sterile DMSO | 10 mM | 4 |
GenicBio | FITC-(PR)15 | 94.52 | 4320.07 | Sterile DMSO | 10 mM | 4 |
GenicBio | FITC-(GP)15 | 95.50 | 2833.05 | Sterile DMSO | 10 mM | 4 |
GenicBio | FITC-(PA)15 | 94.69 | 3043.44 | Sterile DMSO | 10 mM | 4 |
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Gill, A.L.; Wang, M.Z.; Levine, B.; Premasiri, A.; Vieira, F.G. Primary Neurons and Differentiated NSC-34 Cells Are More Susceptible to Arginine-Rich ALS Dipeptide Repeat Protein-Associated Toxicity than Non-Differentiated NSC-34 and CHO Cells. Int. J. Mol. Sci. 2019, 20, 6238. https://doi.org/10.3390/ijms20246238
Gill AL, Wang MZ, Levine B, Premasiri A, Vieira FG. Primary Neurons and Differentiated NSC-34 Cells Are More Susceptible to Arginine-Rich ALS Dipeptide Repeat Protein-Associated Toxicity than Non-Differentiated NSC-34 and CHO Cells. International Journal of Molecular Sciences. 2019; 20(24):6238. https://doi.org/10.3390/ijms20246238
Chicago/Turabian StyleGill, Anna L., Monica Z. Wang, Beth Levine, Alan Premasiri, and Fernando G. Vieira. 2019. "Primary Neurons and Differentiated NSC-34 Cells Are More Susceptible to Arginine-Rich ALS Dipeptide Repeat Protein-Associated Toxicity than Non-Differentiated NSC-34 and CHO Cells" International Journal of Molecular Sciences 20, no. 24: 6238. https://doi.org/10.3390/ijms20246238