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.
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.
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.
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.