Cysteine Residues in the Large Extracellular Loop (EC2) Are Essential for the Function of the Stress-regulated Glycoprotein M6a

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. Glycoprotein M6a is a neuronally expressed member of the proteolipid protein (PLP/DM20) family (1) whose gene has been identified as a stress-responsive gene in the hippocampal formation. In several animal models of chronic stress, expression levels for M6a in hippocampal tissue were found to be diminished by chronic stress exposure and this effect was counteracted by treatment with antidepressants (2-3). Recently, an association of the M6A gene with the subgroup of schizophrenia patients with high levels of depression has been reported (4). These findings suggest that M6a plays a role in the stress-induced hippocampal alterations that are found in psychiatric disorders in general. M6a is prominently expressed in the central nervous system as early as embryonic day 10 and remains detectable in adulthood (5). Originally, it was identified as an antigen reacting with the monoclonal M6 antibody and its role as a modulator of neurite outgrowth was postulated (6). Lagenaur et al. (6) demonstrated that IgG or Fab fragments of M6 antibody interfere with the extension of neurites by cultured cerebellar neurons. A recent study by Zhao et al. (7) shows that M6a expressed in the murine neural retina also regulates neurite extension. The neurite outgrowth of M6a-overexpressing retinal cells was strikingly enhanced, although M6a did not affect differentiation and proliferation.Even though the precise biological function of M6a still remains unclear, there is a growing body of evidence indicating the importance of M6a in the processes of neural development such as neurite extension and differentiation. For example, a study by Mukobata et al. (8) reported that M6a expression enhances nerve growth factor-primed neurite extension in rat pheochromocytoma PC12 cells. They show that it also induces an increase in the intracellular Ca 2ϩ concentration of PC12 cells and that the anti-M6a antibody efficiently interferes with both nerve growth factor-triggered Ca 2ϩ influx and neurite extension (8). Next, inhibition of mouse M6a expression was found to lead to decreased differentiation of neurons derived from mouse embryonic stem cells (9). Furthermore, it has been demonstrated that M6a plays an important role in neurite/ filopodium outgrowth and synapse formation (10). This study shows that M6a overexpression induces neurite formation and increases filopodia density in hippocampal neurons. M6a knockdown with small interference RNA methodology showed that M6a low expressing neurons display decreased filopodia number and a lower density of synaptophysin clusters. Th...

Altered expression of neuroplasticity-related genes in the brain of depressed suicides

“…GPM6A functions in processes of neuronal remodeling and plasticity, such as neurite outgrowth, filopodium formation, and synaptogenesis (Alfonso et al, 2005; Fuchsova et al, 2009; Brocco et al, 2010; Scorticati et al, 2011). Its overexpression induces neurite formation and increases filopodia density in hippocampal neurons.…”

“…Twenty-four hours later, cells were lysed and the protein samples for SDS–PAGE were prepared as described (Fuchsova et al, 2009). The protein samples were separated on 10% SDS–PAGE gels and transferred to a nitro-cellulose membrane by electroblotting.…”

“…Remarkably, the myelin proteolipid protein (PLP/DM20) family members, such as GPM6A, GPM6B, and PLP1 transcript variant DM20, but not PLP1 (Fernandez et al, 2010), have been shown to be involved in the processes of neurite outgrowth and filopodium formation (Lagenaur et al, 1992; Mukobata et al, 2002; Alfonso et al, 2005; Michibata et al, 2008; Zhao et al, 2008; Fuchsova et al, 2009; Brocco et al, 2010; Scorticati et al, 2011). GPM6A, in particular, is also required for filopodium motility and synaptogenesis (Fuchsova et al, 2009; Brocco et al, 2010), and it has been implicated in neuronal differentiation of human stem cells (Michibata et al, 2009) and PC12 cells (Mukobata et al, 2002). When siRNA methodology is used, GPM6A low-expressing neurons display decreased filopodia numbers and a lower density of synaptophysin clusters (Alfonso et al, 2005).…”

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

“…5(E)]. Because the three deletions constructs maintained the intact extracellular domains, all of them were still recognizable by mAbs 1B4 and M6, which extracellularly bind M6a (Fuchsova et al, 2009).…”

M6 proteins regulate axon outgrowth in mouse callosal neurons

“…For the formation of filopodia/spine, the second extracellular domain (C174 and C192) is essential (Fuchsova et al, 2009). When the anti-M6a antibody stalls axon alongation, a short N-terminus sequence (amino acid 1-25) is important (Sato et al, 2011a).…”