| Abstract No.: | C-C3109 |
| Country: | Canada |
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| Title: | MECHANISMS OF CALCIUM DYSREGULATION IN AMYOTROPHIC LATERAL SCLEROSIS |
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| Authors/Affiliations: | 1 Miranda Tradewell*; 1 Sandra Minotti; 1 Heather Durham;
1 Montreal Neurological Institute, QC, Canada
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| Content: | Dysfunction of the ubiquitin-proteasome system (UPS) and inclusion formation are hallmarks of ALS and experimental models. Motor neurons expressing ALS-associated mutant SOD1 (SOD1mut) are protected from toxicity and formation of inclusions by treatments that reduce basal cytoplasmic Ca2+ ([Ca2+]c).
Objectives:
1 - To establish a timeline of calcium dysregulation in a primary culture model of ALS.
2 – To understand the mechanisms by which Ca2+ homeostasis is dysregulated and its relationship to protein misfolding and aggregation.
Methods: Plasmid encoding wild type or mutant SOD1 (G93A) fused to eGFP was expressed in motor neurons of dissociated spinal cord cultures by intranuclear microinjection at 4 weeks in vitro. Formation of inclusions was monitored by epifluorescence microscopy. [Ca2+]c and mitochondrial Ca2+ [Ca2+]m were monitored by ratiometric imaging using fura-2 and a mitochondrial pericam, respectively.
Results: Motor neurons expressing SOD1mut-eGFP exhibited higher [Ca2+]c than those expressing SOD1wt-eGFP. [Ca2+]c was further elevated in motor neurons that possessed visible SOD1mut inclusions compared to those that did not, demonstrating a secondary rise in basal [Ca2+]c levels associated with inclusion formation. Elevations in [Ca2+]m occurred before the primary rise in [Ca2+]c, demonstrating the early involvement of mitochondria in [Ca2+]c buffering. Rounded mitochondria were observed shortly after the increase in [Ca2+]m. The secondary rise in [Ca2+]c was reduced by blocking calcium release from the endoplasmic reticulum (ER) using xestospongin C, indicating that the secondary rise is due to Ca2+ release from the ER through IP3 receptors (IP3R). 30μM ryanodine was ineffective in reducing [Ca2+]c, demonstrating that the release of calcium from the ER is IP3R-specific. Coexpression of the calcium-buffering protein, calbindin D-28K,not only reduced the percentage of motor neurons with SOD1mut-eGFP inclusions, but also the magnitude of [Ca2+]c increase in those that did develop inclusions, evidence that buffering [Ca2+]c can prevent formation of SOD1mut inclusions; however, calbindin D-28K failed to restore [Ca2+]c to normal even in neurons lacking inclusions. Increase in [Ca2+]c in motor neurons expressing SOD1mut-eGFP was not a direct result of impaired proteasome function; the effect was not reproduced by treating cultures with epoxomicin for 7 days, which resulted in 60-70% reduction in proteasomal activity.
Conclusion: Motor neuronal [Ca2+]c increased with expression of SOD1mut and a secondary rise was associated with formation of inclusions. Neither increase appeared to be a direct result of proteasomal inhibition. Rather, the ability of calbindin D-28K to reduce both inclusion formation and the magnitude of [Ca2+]c rise in motor neurons with inclusions suggests that a critical increase in [Ca2+]c promotes aggregation of SOD1mut. Early rises in basal [Ca2+]m followed by mitochondrial morphological abnormalities are in accordance with the observation that mitochondrial dysfunction occurs early in the disease process. Although mitochondria provide an early defense against SOD1mut toxicity by buffering Ca2+, once compromised, a secondary rise in [Ca2+]c results from release from the ER through IP3R. |
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