ALS-Associated SOD1(G93A) Decreases SERCA Pump Levels and Increases Store-Operated Ca2+ Entry in Primary Spinal Cord Astrocytes from a Transgenic Mouse Model
Abstract
:1. Introduction
2. Results
2.1. hSOD1(G93A)-Expressing Astrocytes Have Enhanced SOCE-Mediated Ca2+ Influx
2.2. SOCE-Mediating Proteins Are Equally Present in hSOD1(WT) and hSOD1(G93A) Primary Astrocytes
2.3. hSOD1(G93A) Astrocytes Have Reduced basal Ca2+ Levels in the Cytosol and the ER Lumen Compared to Control Astrocytes
2.4. SERCA2 Is Down-regulated in hSOD1(G93A) Astrocytes
2.5. Mitochondria of hSOD1(G93A) and hSOD1(WT) Astrocytes Equally Respond to SOCE Stimulation
2.6. Ca2+-related Mitochondrial Parameters Are Comparable in hSOD1(G93A) and hSOD1(WT) Astrocytes
3. Discussion
4. Materials and Methods
4.1. Mouse Models and Breeding
4.2. Preparation of Primary Cultures of Murine Spinal Cord Astrocytes
4.3. Ca2+ Imaging
4.4. Measurement of the Mitochondrial Membrane Potential
4.5. Analysis of Mitochondrial Morphology
4.6. Western Blot Analysis
4.7. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
Ab | antibody |
AEQ | aequorin |
ALS | amyotrophic lateral sclerosis |
BSA | bovine serum albumin |
CLNX | calnexin |
CRT | calreticulin |
cyt | cytosol |
CFP | cyan fluorescent protein |
ER | endoplasmic reticulum |
fALS | familial ALS |
FCCP | carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone |
FRET | fluorescence resonance energy transfer |
FUS/TLS | Fused in Sarcoma/Translocated in Sarcoma |
GECI | genetically-encoded Ca2+ indicators |
GFAP | glial fibrillary acidic protein |
h | human |
KRB | Krebs-Ringer buffer |
MCU | mitochondrial Ca2+ uniporter |
mit | mitochondrial matrix |
MN | motor neuron |
mtRFP | mitochondrially-targeted red-fluorescent protein |
n | number of replicates |
p | polyclonal |
PM | plasma membrane |
PMCA | PM Ca2+ ATPase |
NCX | Na+/Ca2+ exchanger |
PVDF | polyvinylidene difluoride |
ROI | region of interest |
SDS | sodium dodecyl sulfate |
SDS-PAGE | SDS-polyacrylamide gel electrophoresis |
SEM | standard error of the mean |
SERCA | sarco-endoplasmic reticulum Ca2+ ATPase |
SOCE | store-operated Ca2+ entry |
SOD1 | superoxide dismutase 1 |
STIM | stromal interaction molecule |
TBS-T | Tris-buffered saline added with Tween-20 |
TDP-43 | TAR DNA-binding protein 43 |
Tg | transgenic |
TMRM | tetramethylrhodamine methyl ester |
YFP | yellow fluorescent protein |
WB | Western blot |
WT | wild-type |
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Norante, R.P.; Peggion, C.; Rossi, D.; Martorana, F.; De Mario, A.; Lia, A.; Massimino, M.L.; Bertoli, A. ALS-Associated SOD1(G93A) Decreases SERCA Pump Levels and Increases Store-Operated Ca2+ Entry in Primary Spinal Cord Astrocytes from a Transgenic Mouse Model. Int. J. Mol. Sci. 2019, 20, 5151. https://doi.org/10.3390/ijms20205151
Norante RP, Peggion C, Rossi D, Martorana F, De Mario A, Lia A, Massimino ML, Bertoli A. ALS-Associated SOD1(G93A) Decreases SERCA Pump Levels and Increases Store-Operated Ca2+ Entry in Primary Spinal Cord Astrocytes from a Transgenic Mouse Model. International Journal of Molecular Sciences. 2019; 20(20):5151. https://doi.org/10.3390/ijms20205151
Chicago/Turabian StyleNorante, Rosa Pia, Caterina Peggion, Daniela Rossi, Francesca Martorana, Agnese De Mario, Annamaria Lia, Maria Lina Massimino, and Alessandro Bertoli. 2019. "ALS-Associated SOD1(G93A) Decreases SERCA Pump Levels and Increases Store-Operated Ca2+ Entry in Primary Spinal Cord Astrocytes from a Transgenic Mouse Model" International Journal of Molecular Sciences 20, no. 20: 5151. https://doi.org/10.3390/ijms20205151