Transcallosal Projections Require Glycoprotein M6-Dependent Neurite Growth and Guidance.

The function of mature neurons critically relies on the developmental outgrowth and projection of their cellular processes. It has long been postulated that the neuronal glycoproteins M6a and M6b are involved in axon growth because these four-transmembrane domain-proteins of the proteolipid protein family are highly enriched on growth cones, but in vivo evidence has been lacking. Here, we report that the function of M6 proteins is required for normal axonal extension and guidance in vivo. In mice lacking both M6a and M6b, a severe hypoplasia of axon tracts was manifested. Most strikingly, the corpus callosum was reduced in thickness despite normal densities of cortical projection neurons. In single neuron tracing, many axons appeared shorter and disorganized in the double-mutant cortex, and some of them were even misdirected laterally toward the subcortex. Probst bundles were not observed. Upon culturing, double-mutant cortical and cerebellar neurons displayed impaired neurite outgrowth, indicating a cell-intrinsic function of M6 proteins. A rescue experiment showed that the intracellular loop of M6a is essential for the support of neurite extension. We propose that M6 proteins are required for proper extension and guidance of callosal axons that follow one of the most complex trajectories in the mammalian nervous system.

Actin-independent behavior and membrane deformation exhibited by the four-transmembrane protein M6a.

M6a is a four-transmembrane protein that is abundantly expressed in the nervous system. Previous studies have shown that over-expression of this protein induces various cellular protrusions, such as neurites, filopodia, and dendritic spines. In this detailed characterization of M6a-induced structures, we found their varied and peculiar characteristics. Notably, the M6a-induced protrusions were mostly devoid of actin filaments or microtubules and exhibited free random vibrating motion. Moreover, when an antibody bound to M6a, the membrane-wrapped protrusions were suddenly disrupted, leading to perturbation of the surrounding membrane dynamics involving phosphoinositide signaling. During single-molecule analysis, M6a exhibited cytoskeleton-independent movement and became selectively entrapped along the cell perimeter in an actin-independent manner. These observations highlight the unusual characteristics of M6a, which may have a significant yet unappreciated role in biological systems.

Induction of axon growth arrest without growth cone collapse through the N-terminal region of four-transmembrane glycoprotein M6a.

During development, axons elongate vigorously, carefully controlling their speed, to connect with their targets. In general, rapid axon growth is correlated with active growth cones driven by dynamic actin filaments. For example, when the actin-driven tip is collapsed by repulsive guidance molecules, axon growth is severely impaired. In this study, we report that axon growth can be suppressed, without destroying the actin-based structure or motility of the growth cones, when antibodies bind to the four-transmembrane glycoprotein M6a concentrated on the growth cone edge. Surprisingly, M6a-deficient axons grow actively but are not growth suppressed by the antibodies, arguing for an inductive action of the antibody. The binding of antibodies clusters and displaces M6a protein from the growth cone edge membrane, suggesting that the spatial rearrangement of this protein might underlie the unique growth cone behavior triggered by the antibodies. Molecular dissection of M6a suggested involvement for the N-terminal intracellular domain in this antibody-induced growth cone arrest.

Proteomics analysis of the temporal changes in axonal proteins during maturation.

After the initial primary projection, axons undergo various structural and functional changes to establish mature neural circuits. The changes in protein expression associated with this maturation were investigated in lateral olfactory tract axons using two-dimensional gel electrophoresis. The most prominent group upregulated during the period consisted of calcium-dependent membrane-binding proteins including VILIP1, neurocalcin delta, copine 6, and annexin A6 from three structurally different families. During maturation of primary cultured neurons, annexin A6 gradually became concentrated on the axon initial segment, and its overexpression significantly enhanced axon branching. On the other hand, overexpression of VILIP1 and neurocalcin delta reduced axon outgrowth and branching. The second group upregulated during axon maturation comprised tubulin- and microtubule-binding proteins including CRMP2, guanine deaminase, MAP1B, and fibronectin type3 SPRY domain-containing protein. Because the maturation of lateral olfactory axons involves massive extension of secondary collateral branches, the augmentation of these proteins during these stages may underlie the drastic restructuring of the axon cytoskeleton.

The effect of tert-butylhydroquinone-induced oxidative stress in MDBK cells using XTT assay: implication of tert-butylhydroquinone-induced NADPH generating enzymes.

Tetrazolium salts such as XTT and MTT are widely used to produce formazan for cell proliferation and cytotoxicity assays through bioreductase activity. However, the XTT assay showed significant increase in MDBK cell viability when cells were treated with both 50 and 100 muM of the pro-oxidant, tert-butylhydroquinone (t-BHQ), although the crystal violet assay showed no cytotoxic effect with these concentrations, and the induction of lipid peroxidation was not observed. We investigated the mechanism of enhancement of XTT substrate reduction after treatment of MDBK cells with t-BHQ, leading to apparent increase in cell viability. t-BHQ caused an increase in absorbance at 340 nm in culture medium, suggesting that t-BHQ increases cellular production and release of NADH and/or NADPH. Although t-BHQ did not change the NADH concentration in cell culture medium, the addition of NADP(+)-dependent glutathione reductase decreased the XTT reduction to the control level, indicating cellular release of NADPH. t-BHQ also increased intracellular glucose-6-phosphate dehydrogenase activity, producing NADPH. Taken together, our findings indicate that t-BHQ treatment activates NADPH generating enzymes such as glucose-6-phosphate dehydrogenase followed by release of NADPH in the cell culture medium, resulting in direct XTT reduction by NADPH.