M6 proteins regulate axon outgrowth in mouse callosal neurons

Abstract

Corpus callosum (CC) is the largest fiber tract in the human brain that connects the left and right cerebral hemispheres. The absence of the CC results in impairment of higher-order cognitive functions because interhemispheric transfer of information is disrupted. The formation of the CC is intricately regulated by a large number of molecules, which have not been fully characterized, while actively searched. In this study, I showed that additional molecules, M6 proteins, are also involved in the CC development.M6 proteins consist of M6a and M6b, highly homologous four-transmembrane proteins. M6a was originally found by the antibody screening for candidate molecules that are involved in guidance of axons. Previously, we found that when anti-M6a antibody binds to M6a protein in cultured neurons, the axons stall the elongation. Therefore, it seemed that M6a is involved in axon elongation of developing neurons. However, no study has so far examined the function of M6 proteins in vivo. To address this issue, I analyzed brain morphology and axonal projections in knockout mice for the M6a and M6b genes.First, to assess whether the two M6 proteins have redundant functions, I investigated expression patterns of M6a and M6b proteins in the wildtype mouse brains during developmental stages, using a newly prepared anti-M6b specific antibody. M6a was expressed mainly in growing axons as previously reported. The new anti-M6b antibody revealed that M6b was well co-localized with M6a in these axons. The expression level of M6b on the axons appeared lower at E14.5 compared with M6a, and subsequently intensified at E16.5 and P0, displaying comparable overlapping patterns with M6a. I also examined the subcellular distribution of M6 proteins in neurons in dissociated culture. As described previously, M6a protein was enriched in the edge of growth cones and the shaft filopodia of axons in cultured neurons. M6b protein was also expressed in the growth cones and the shaft filopodia, and co- However, the size of the CC bundle was abnormally small in the M6a-/-M6b-/-knockout mice.This hypoplasia in the CC was not clear at early developmental stages (E14.5, E16.5 and P0), but became obvious by P7 and throughout life. I quantified the size of the CC in wild-type, single and double mutant mice at P7, and found that the defect was most pronounced in the double knockout mice, indicating the redundant function of the two M6 proteins.I considered the following three possibilities to explain the smaller CC bundles in the double knockout mice. 1) the number of the callosal neurons is reduced.2) The axons of the callosal neurons are misdirected from callosal pathway.3) The axon outgrowth is impaired in the callosal neurons.First, to evaluate the possibility 1), I measured the density of cortical neurons in the mutant cortex in the motor, somatosensory, and visual areas. The neural density of the double mutant cortex was comparable to that of the wild-type. This result suggests that the number of callosal neurons was not reduced in the M6a...