P01.53. Spheroid formation and axonal severing in adult neurons during oxidative stress: role of calcium

Purpose

Axonal severing is critical to the irreversible disability that occurs over the course of multiple sclerosis (MS). Reactive oxygen species (ROS) are implicated in neurodegenerative aspects of MS: axonal spheroid formation, severing, and axoplasmic Ca²⁺ elevation. However, the exact role of Ca²⁺ in spheroid formation remains unclear. The mechanism of action of natural anti-oxidants such as lipoic acid, which provide neuroprotection during oxidative stress in MS model, also remains unclear.

Methods

Primary cortical neurons from adult mice were subjected to physiologically-relevant levels of H₂O₂. Ca²⁺ dynamics and its sources were examined during spheroids formation using real time imaging, ratiometric Ca²⁺ indicators and immunocytochemistry.

Results

Exposure to ROS led to a 3.5 fold increase in axoplasmic Ca²⁺ by 30 min. Onset of axonal spheroid formation began at 15 min when Ca²⁺ increase was 2.2 fold. Axonal severing occurred at sites of spheroids around 90-120 min. Analysis of small axonal segments revealed an uneven distribution of Ca²⁺ during exposure to H₂O₂. Micrometers apart, focal Ca²⁺ increases in small axonal domains ranged from 2.8 to 4.4 fold. Domains with a 3.8 to 4.4-fold increase correlated with the sites of spheroids, suggesting high focal extracellular Ca²⁺ influx at these sites. Several treatments significantly attenuated Ca²⁺ increase and completely abolished spheroid formation under ROS: removal of extracellular Ca²⁺; N-type Ca²⁺ channel blocker omega-conotoxin GVIA; L-type Ca²⁺ channel blocker amlodipine; and reverse Na+/ Ca²⁺ exchanger (NCX1) blocker KB-R7943. Aggregation of reverse NCX1 and N-type voltage-gated Ca²⁺ channel was detected at spheroids.

Conclusion

Our results reveal a correlation between focal axoplasmic Ca²⁺ and spheroid formation and suggest that focal aggregation of the reverse NCX1 and N-type Ca²⁺ channel plays central role in high focal Ca²⁺ increase during oxidative stress. These findings provide a basis for investigating the neuroprotective mechanism of the natural anti-oxidant lipoic acid during oxidative stress.