Abstract
Gpm6a was identified as a stress-responsive gene in the hippocampal formation. This gene is down-regulated in the hippocampus of both socially and physically stressed animals, and this effect can be reversed by antidepressant treatment. Previously we showed that the stress-regulated protein M6a is a key modulator for neurite outgrowth and filopodium/spine formation. In the present work, mutational analysis was used to characterize the action of M6a at the molecular level. We show that four cysteines 162, 174, 192, and 202 within EC2 are functionally crucial sites. The presence of cysteines 162 and 202 is essential for the efficient cell surface expression of the M6a protein. In contrast, cysteines 174 and 192, which form a disulfide bridge as shown by biochemical analysis, are not required for the efficient surface expression of M6a. Their mutation to alanine does not interfere with the localization of M6a to filopodial protrusions in primary hippocampal neurons. The neurons expressing C174A and/or C192A mutants display decreased filopodia number. In non-permeabilized cells, these mutant proteins are not recognized by a function-blocking monoclonal antibody directed to M6a. Moreover, neurons in contact with axons expressing C174A/C192A mutant display significantly lower density of presynaptic clusters over their dendrites. Taken together, this study demonstrates that cysteines in the EC2 domain are critical for the role of M6a in filopodium outgrowth and synaptogenesis.
Highlights
Duced hippocampal alterations that are found in psychiatric disorders in general
We demonstrated that chronic stress induces a decrease in hippocampal mRNA levels for the neuronal membrane glycoprotein M6a shown to modulate neurite outgrowth and filopodium/spine formation [10]
We demonstrate that the second extracellular loop of M6a is critical for the role of the protein in filopodium outgrowth and synaptogenesis
Summary
M6a is prominently expressed in the central nervous system as early as embryonic day 10 and remains detectable in adulthood [5] It was identified as an antigen reacting with the monoclonal M6 antibody and its role as a modulator of neurite outgrowth was postulated [6]. A study by Mukobata et al [8] reported that M6a expression enhances nerve growth factor-primed neurite extension in rat pheochromocytoma PC12 cells. This study shows that M6a overexpression induces neurite formation and increases filopodia density in hippocampal neurons. The extracellular region of M6a contains cysteines residues that may be functionally crucial sites. We show that cysteines 174 and 192 within the EC2 are important for the role of the M6a protein in filopodia formation and synaptogenesis
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