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The nervous system monitors the environment to maintain homeostasis, which can be affected by stressful conditions. Using mammalian models of chronic stress, we previously observed altered brain levels of GPM6A, a protein involved in neuronal morphology. However, GPM6A's role in systemic stress responses remains unresolved. The nematode Caenorhabditis elegans expresses a GPM6A ortholog, the neuronal membrane glycoprotein 1 (NMGP-1). Because of the shared features between nematode and mammalian nervous systems and the vast genetic tools available in C. elegans, we used the worm to elucidate the role of GPM6A in the stress response. We first identified nmgp-1 expression in different amphid and phasmid neurons. To understand the nmgp-1 role, we characterized the behavior of nmgp-1(RNAi) animals and two nmgp-1 mutant alleles. Compared to control animals, mutant and RNAi-treated worms exhibited increased recovery time from the stress-resistant dauer stage, altered SDS chemosensation and reduced egg-laying rate resulting in egg retention (bag-of-worms phenotype). Silencing of nmgp-1 expression induced morphological abnormalities in the ASJ sensory neurons, partly responsible for dauer exit. These results indicate that nmgp-1 is required for neuronal morphology and for behaviors associated with chemosensation. Finally, we propose nmgp-1 mutants as a tool to screen drugs for human nervous system pathologies., (© 2021 International Society for Neurochemistry.)

Stress-responsive neuronal membrane glycoprotein M6a (Gpm6a) functions in neurite extension, filopodium and spine formation and synaptogenesis. The mechanisms of Gpm6a action in these processes are incompletely understood. Previously, we identified the actin regulator coronin-1a (Coro1a) as a putative Gpm6a interacting partner. Here, we used co-immunoprecipitation assays with the anti-Coro1a antibody to show that Coro1a associates with Gpm6a in rat hippocampal neurons. By immunofluorescence microscopy, we demonstrated that in hippocampal neurons Coro1a localizes in F-actin-enriched regions and some of Coro1a spots co-localize with Gpm6a labeling. Notably, the over-expression of a dominant-negative form of Coro1a as well as its down-regulation by siRNA interfered with Gpm6a-induced filopodium formation. Coro1a is known to regulate the plasma membrane translocation and activation of small GTPase Rac1. We show that Coro1a co-immunoprecipitates with Rac1 together with Gpm6a. Pharmacological inhibition of Rac1 resulted in a significant decrease in filopodium formation by Gpm6a. The same was observed upon the co-expression of Gpm6a with the inactive GDP-bound form of Rac1. In this case, the elevated membrane recruitment of GDP-bound Rac1 was detected as well. Moreover, the kinase activity of the p21-activated kinase 1 (Pak1), a main downstream effector of Rac1 that acts downstream of Coro1a, was required for Gpm6a-induced filopodium formation. Taken together, our results provide evidence that a signaling pathway including Coro1a, Rac1, and Pak1 facilitates Gpm6a-induced filopodium formation. Formation of filopodia by membrane glycoprotein M6a (Gpm6a) requires actin regulator coronin-1a (Coro1a), known to regulate plasma membrane localization and activation of Rac1 and its downstream effector Pak1. Coro1a associates with Gpm6a. Blockage of Coro1a, Rac1, or Pak1 interferes with Gpm6a-induced filopodium formation. Moreover, Gpm6a facilitates Rac1 membrane recruitment. Altogether, a mechanistic insight into the process of Gpm6a-induced neuronal filopodium formation is provided., (© 2016 International Society for Neurochemistry.)

Membrane glycoprotein M6a, which belongs to the tetraspan proteolipid protein family, promotes structural plasticity in neurons and cell lines by unknown mechanisms. This glycoprotein is encoded by Gpm6a, a stress-regulated gene. The hippocampus of animals chronically stressed by either psychosocial or physical stressors shows decreased M6a expression. Stressed Gpm6a-null mice develop a claustrophobia-like phenotype. In humans, de novo duplication of GPM6A results in learning/behavioral abnormalities, and two single-nucleotide polymorphisms (SNPs) in the non-coding region are linked to mood disorders. Here, we studied M6a dimerization in neuronal membranes and its functional relevance. We showed that the self-interaction of M6a transmembrane domains (TMDs) might be driving M6a dimerization, which is required to induce filopodia formation. Glycine mutants located in TMD2 and TMD4 of M6a affected its dimerization, thus preventing M6a-induced filopodia formation in neurons. In silico analysis of three non-synonymous SNPs located in the coding region of TMDs suggested that these mutations induce protein instability. Indeed, these SNPs prevented M6a from being functional in neurons, owing to decreased stability, dimerization or improper folding. Interestingly, SNP3 (W141R), which caused endoplasmic reticulum retention, is equivalent to that mutated in PLP1, W161L, which causes demyelinating Pelizaeus-Merzbacher disease. In this work we analyzed the functional contribution of transmembrane domains (TMDs) of the neuronal membrane glycoprotein M6a. We determined that certain glycines present in TMD2 and TMD4 are critical for filopodia induction in neurons. In addition, three nsSNPs located in the coding region of TMD2 and TMD3 of GPM6A impair M6a function by affecting its stability, folding and dimer formation., (© 2015 International Society for Neurochemistry.)

A neuronal integral membrane glycoprotein M6a has been suggested to be involved in a number of biological processes, including neuronal remodeling and differentiation, trafficking of mu-opioid receptors, and Ca(2+) transportation. Moreover, pathological situations such as chronic stress in animals and depression in humans have been associated with alterations in M6a sequence and expression. The mechanism of action of M6a is essentially unknown. In this work, we analyze the relevance of M6a distribution in plasma membrane, namely its lipid microdomain targeting, for its biological function in filopodia formation. We demonstrate that M6a is localized in membrane microdomains compatible with lipid rafts in cultured rat hippocampal neurons. Removal of cholesterol from neuronal membranes with methyl-β-cyclodextrin decreases M6a-induced filopodia formation, an effect that is reversed by the addition of cholesterol. Inhibition of Src kinases and MAPK prevents filopodia formation in M6a-over-expressing neurons. Src-deficient SYF cells over-expressing M6a fail to promote filopodia formation. Taken together, our findings reveal that the association of M6a with lipid rafts is important for its role in filopodia formation and Src and MAPK kinases participate in M6a signal propagation., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)