Effect of inhibition of superoxide dismutase on motor neurons during growth: comparison of phosphorylated and non-phosphorylated neurofilament-containing spinal neurons by histogram distribution.

We reported recently that non-phosphorylated neurofilaments (NF)-positive neurons were more sensitive to the growth inhibitory effects of Cu/Zn superoxide dismutase (SOD1) than phosphorylated NF-positive neurons. The findings suggested that non-phosphorylated NF-positive neurons, presumed to represent spinal motor neurons, are more vulnerable to oxidative stress than other neurons, and thus explain in part the selective degeneration of motor neurons in amyotrophic lateral sclerosis. The present investigation is an extension to our previous study and examined the neurite growth process in the presence of diethyldithiocarbamate (DDC), an SOD1 inhibitor. Non-phosphorylated NF, representing spinal motor neurons, and phosphorylated NF, representing other spinal neurons, were stained with SMI-32 and SMI-31 antibodies, respectively. The distribution histogram of neurite length after treatment with 0 nM DDC (control) for 72 h appeared flatter compared with that of 24h. Although the addition of DDC (1 nM, 10 nM, 100 nM, or 1000 nM) to the culture medium for 72 h shifted the histogram of neurite length to a shorter range in a concentration-dependent manner, the neurite of SMI-31-immunoreactive neurons grew under DDC. On the other hand, DDC-treatment for 72 h altered the neurite growth of SMI-32-immunoreactive neurons compared with that for 24-h. The results suggest that SOD1 inhibition, representing accumulation of endogenous oxidative stress, suppresses neurite growth of spinal motor neurons, and that the growth of spinal motor neurons is more sensitive to oxidative stress than other types of neurons.