Combined additive effects of neuronal membrane glycoprotein GPM6a and the intercellular cell adhesion molecule ICAM5 on neuronal morphogenesis.

The mechanisms underlying neuronal development and synaptic formation in the brain depend on intricate cellular and molecular processes. The neuronal membrane glycoprotein GPM6a promotes neurite elongation, filopodia/spine formation, and synapse development, yet its molecular mechanisms remain unknown. Since the extracellular domains of GPM6a (ECs) command its function, we investigated the interaction between ICAM5, the neuronal member of the intercellular adhesion molecule (ICAM) family, and GPM6a's ECs. Our study aimed to explore the functional relationship between GPM6a and ICAM5 in hippocampal culture neurons and cell lines. Immunostaining of 15 days in vitro (DIV) neurons revealed significant co-localization between endogenous GPM6a clusters and ICAM5 clusters in the dendritic shaft. These results were further corroborated by overexpressing GPM6a and ICAM5 in N2a cells and hippocampal neurons at 5 DIV. Moreover, results from the co-immunoprecipitations and cell aggregation assays prove the cis and trans interaction between both proteins in GPM6a/ICAM5 overexpressing HEK293 cells. Additionally, GPM6a and ICAM5 overexpression additively enhanced neurite length, the number of neurites in N2a cells, and filopodia formation in 5 DIV neurons, indicating their cooperative role. These findings highlight the dynamic association between GPM6a and ICAM5 during neuronal development, offering insights into their contributions to neurite outgrowth, filopodia formation, and cell-cell interactions.

The role of S-adenosylhomocysteine hydrolase-like 1 in cancer.

This integrative review aims to highlight the importance of investigating the functional role of AHCYL1, also known as IRBIT, in cancer cells. It has recently been suggested that AHCYL1 regulates cell survival/death, stemness capacity, and the host adaptive response to the tumor microenvironment. Despite this knowledge, the role of AHCYL1 in cancer is still controversial, probably due to its ability to interact with multiple factors in a tissue-specific manner. Understanding the mechanisms regulating the functional interplay between the tumor and the tumor microenvironment that controls the expression of AHCYL1 could provide a deeper comprehension of the regulation of tumor development. Addressing how AHCYL1 modulates cellular plasticity processes in a tumoral context is potentially relevant to developing translational approaches in cancer biology.

Independent Recruitment of Different Types of Phospholipases A2 to the Venoms of Caenophidian Snakes: The Rise of PLA2-IIE within Pseudoboini (Dipsadidae).

Snake venoms harbor a wide and diverse array of enzymatic and nonenzymatic toxic components, allowing them to exert myriad effects on their prey. However, they appear to trend toward a few optimal compositional scaffolds, dominated by four major toxin classes: SVMPs, SVSPs, 3FTxs, and PLA2s. Nevertheless, the latter appears to be restricted to vipers and elapids, as it has never been reported as a major venom component in rear-fanged species. Here, by investigating the original transcriptomes from 19 species distributed in eight genera from the Pseudoboini tribe (Dipsadidae: Xenodontinae) and screening among seven additional tribes of Dipsadidae and three additional families of advanced snakes, we discovered that a novel type of venom PLA2, resembling a PLA2-IIE, has been recruited to the venom of some species of the Pseudoboini tribe, where it is a major component. Proteomic and functional analyses of these venoms further indicate that these PLA2s play a relevant role in the venoms from this tribe. Moreover, we reconstructed the phylogeny of PLA2s across different snake groups and show that different types of these toxins have been recruited in at least five independent events in caenophidian snakes. Additionally, we present the first compositional profiling of Pseudoboini venoms. Our results demonstrate how relevant phenotypic traits are convergently recruited by different means and from homologous and nonhomologous genes in phylogenetically and ecologically divergent snake groups, possibly optimizing venom composition to overcome diverse adaptative landscapes.

Combining Protein Conformational Diversity and Phylogenetic Information Using CoDNaS and CoDNaS-Q.

CoDNaS (http://ufq.unq.edu.ar/codnas/) and CoDNaS-Q (http://ufq.unq.edu.ar/codnasq) are repositories of proteins with different degrees of conformational diversity. Following the ensemble nature of the native state, conformational diversity represents the structural differences between the conformers in the ensemble. Each entry in CoDNaS and CoDNaS-Q contains a redundant collection of experimentally determined conformers obtained under different conditions. These conformers represent snapshots of the protein dynamism. While CoDNaS contains examples of conformational diversity at the tertiary level, a recent development, CoDNaS-Q, contains examples at the quaternary level. In the emerging age of accurate protein structure prediction by machine learning approaches, many questions remain open regarding the characterization of protein dynamism. In this context, most bioinformatics resources take advantage of distinct features derived from protein alignments, however, the complexity and heterogeneity of information makes it difficult to recover reliable biological signatures. Here we present five protocols to explore tertiary and quaternary conformational diversity at the individual protein level as well as for the characterization of the distribution of conformational diversity at the protein family level in a phylogenetic context. These protocols can provide curated protein families with experimentally known conformational diversity, facilitating the exploration of sequence determinants of protein dynamism. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Assessing conformational diversity with CoDNaS Alternate Protocol 1: Assessing conformational diversity at the quaternary level with CoDNaS-Q Basic Protocol 2: Exploring conformational diversity in a protein family Alternate Protocol 2: Exploring quaternary conformational diversity in a protein family Basic Protocol 3: Representing conformational diversity in a phylogenetic context.

The Elusive Trypanosoma cruzi Disperse Gene Protein Family (DGF-1).

Chagas disease, caused by Trypanosoma cruzi infections, is included in the group of neglected diseases, and efforts to develop new therapeutic or immunoprevention approaches have not been successful. After the publication of the T. cruzi genome, the number of molecular and biochemical studies on this parasite has increased considerably, many of which are focused on families of variant surface proteins, especially trans-sialidases, mucins, and mucin-associated proteins. The disperse gene protein 1 family (DGF-1) is one of the most abundant families in the T. cruzi genome; however, the large gene size, high copy numbers, and low antibody titers detected in infected humans make it an unattractive study target. However, here we argue that given the ubiquitous presence in all T. cruzi species, and physicochemical characteristics, the DGF-1 gene family may play and important role in host-parasite interactions.

Independent Recruitment of Different Types of Phospholipases A2 to the Venoms of Caenophidian Snakes: The Rise of PLA2-IIE within Pseudoboini (Dipsadidae)

Snake venoms harbor a wide and diverse array of enzymatic and nonenzymatic toxic components, allowing them to exert myriad effects on their prey. However, they appear to trend toward a few optimal compositional scaffolds, dominated by four major toxin classes: SVMPs, SVSPs, 3FTxs, and PLA2s. Nevertheless, the latter appears to be restricted to vipers and elapids, as it has never been reported as a major venom component in rear-fanged species. Here, by investigating the original transcriptomes from 19 species distributed in eight genera from the Pseudoboini tribe (Dipsadidae: Xenodontinae) and screening among seven additional tribes of Dipsadidae and three additional families of advanced snakes, we discovered that a novel type of venom PLA2, resembling a PLA2-IIE, has been recruited to the venom of some species of the Pseudoboini tribe, where it is a major component. Proteomic and functional analyses of these venoms further indicate that these PLA2s play a relevant role in the venoms from this tribe. Moreover, we reconstructed the phylogeny of PLA2s across different snake groups and show that different types of these toxins have been recruited in at least five independent events in caenophidian snakes. Additionally, we present the first compositional profiling of Pseudoboini venoms. Our results demonstrate how relevant phenotypic traits are convergently recruited by different means and from homologous and nonhomologous genes in phylogenetically and ecologically divergent snake groups, possibly optimizing venom composition to overcome diverse adaptative landscapes.

Independent recruitment of different types of Phospholipases A2 to the venoms of Caenophidian snakes: the rise of PLA2-IIE within Pseudoboini (Dipsadidae)

Snake venoms harbor a wide and diverse array of enzymatic and nonenzymatic toxic components, allowing them to exert myriad effects on their prey. However, they appear to trend toward a few optimal compositional scaffolds, dominated by four major toxin classes: SVMPs, SVSPs, 3FTxs, and PLA2s. Nevertheless, the latter appears to be restricted to vipers and elapids, as it has never been reported as a major venom component in rear-fanged species. Here, by investigating the original transcriptomes from 19 species distributed in eight genera from the Pseudoboini tribe (Dipsadidae: Xenodontinae) and screening among seven additional tribes of Dipsadidae and three additional families of advanced snakes, we discovered that a novel type of venom PLA2, resembling a PLA2-IIE, has been recruited to the venom of some species of the Pseudoboini tribe, where it is a major component. Proteomic and functional analyses of these venoms further indicate that these PLA2s play a relevant role in the venoms from this tribe. Moreover, we reconstructed the phylogeny of PLA2s across different snake groups and show that different types of these toxins have been recruited in at least five independent events in caenophidian snakes. Additionally, we present the first compositional profiling of Pseudoboini venoms. Our results demonstrate how relevant phenotypic traits are convergently recruited by different means and from homologous and nonhomologous genes in phylogenetically and ecologically divergent snake groups, possibly optimizing venom composition to overcome diverse adaptative landscapes.

SlBBX28 positively regulates plant growth and flower number in an auxin-mediated manner in tomato.

KEY MESSAGE: SlBBX28 is a positive regulator of auxin metabolism and signaling, affecting plant growth and flower number in tomato B-box domain-containing proteins (BBXs) comprise a family of transcription factors that regulate several processes, such as photomorphogenesis, flowering, and stress responses. For this reason, attention is being directed toward the functional characterization of these proteins, although knowledge in species other than Arabidopsis thaliana remains scarce. Particularly in the tomato, Solanum lycopersicum, only three out of 31 SlBBX proteins have been functionally characterized to date. To deepen the understanding of the role of these proteins in tomato plant development and yield, SlBBX28, a light-responsive gene, was constitutively silenced, resulting in plants with smaller leaves and fewer flowers per inflorescence. Moreover, SlBBX28 knockdown reduced hypocotyl elongation in darkness-grown tomato. Analyses of auxin content and responsiveness revealed that SlBBX28 promotes auxin-mediated responses. Altogether, the data revealed that SlBBX28 promotes auxin production and signaling, ultimately leading to proper hypocotyl elongation, leaf expansion, and inflorescence development, which are crucial traits determining tomato yield.

Functional Classification and Characterization of the Fungal Glycoside Hydrolase 28 Protein Family.

Pectin is a major constituent of the plant cell wall, comprising compounds with important industrial applications such as homogalacturonan, rhamnogalacturonan and xylogalacturonan. A large array of enzymes is involved in the degradation of this amorphous substrate. The Glycoside Hydrolase 28 (GH28) family includes polygalacturonases (PG), rhamnogalacturonases (RG) and xylogalacturonases (XG) that share a structure of three to four pleated ß-sheets that form a rod with the catalytic site amidst a long, narrow groove. Although these enzymes have been studied for many years, there has been no systematic analysis. We have collected a comprehensive set of GH28 encoding sequences to study their evolution in fungi, directed at obtaining a functional classification, as well as at the identification of substrate specificity as functional constraint. Computational tools such as Alphafold, Consurf and MEME were used to identify the subfamilies' characteristics. A hierarchic classification defines the major classes of endoPG, endoRG and endoXG as well as three exoPG classes. Ascomycete endoPGs are further classified in two subclasses whereas we identify four exoRG subclasses. Diversification towards exomode is explained by loops that appear inserted in a number of turns. Substrate-driven diversification can be identified by various specificity determining positions that appear to surround the binding groove.

Low nitrogen availability inhibits the phosphorus starvation response in maize (Zea mays ssp. mays L.).

BACKGROUND: Nitrogen (N) and phosphorus (P) are macronutrients essential for crop growth and productivity. In cultivated fields, N and P levels are rarely sufficient, contributing to the gap between realized and potential production. Fertilizer application increases nutrient availability, but is not available to all farmers, nor are current rates of application sustainable or environmentally desirable. Transcriptomic studies of cereal crops have revealed dramatic responses to either low N or low P single stress treatments. In the field, however, levels of both N and P may be suboptimal. The interaction between N and P starvation responses remains to be fully characterized. RESULTS: We characterized growth and root and leaf transcriptomes of young maize plants under nutrient replete, low N, low P or combined low NP conditions. We identified 1555 genes to respond to our nutrient treatments, in one or both tissues. A large group of genes, including many classical P starvation response genes, were regulated antagonistically between low N and P conditions. An additional experiment over a range of N availability indicated that a mild reduction in N levels was sufficient to repress the low P induction of P starvation genes. Although expression of P transporter genes was repressed under low N or low NP, we confirmed earlier reports of P hyper accumulation under N limitation. CONCLUSIONS: Transcriptional responses to low N or P were distinct, with few genes responding in a similar way to the two single stress treatments. In combined NP stress, the low N response dominated, and the P starvation response was largely suppressed. A mild reduction in N availability was sufficient to repress the induction of P starvation associated genes. We conclude that activation of the transcriptional response to P starvation in maize is contingent on N availability.

Sequence similarity in 3D for comparison of protein families.

Homologous proteins are often compared by pairwise sequence alignment, and structure superposition if the atomic coordinates are available. Unification of sequence and structure data is an important task in structural biology. Here, we present the Sequence Similarity 3D (SS3D) method of integrating sequence and structure information. SS3D is a distance and substitution matrix-based method for straightforward visualization of regions of similarity and difference between homologous proteins. This work details the SS3D approach, and demonstrates its utility through case studies comparing members of several protein families. The examples show that SS3D can effectively highlight biologically important regions of similarity and dissimilarity. We anticipate that the method will be useful for numerous structural biology applications, including, but not limited to, studies of binding specificity, structure-function relationships, and evolutionary pathways. SS3D is available with a manual and tutorial at https://github.com/0x462e41/SS3D/.

Functional redundancy and compensation: Deletion of multiple murine Crisp genes reveals their essential role for male fertility.

Mammalian Cysteine-RIch Secretory Protein (CRISP) family includes four members present in sperm and reported to regulate Ca2+ channels and fertilization. Based on our previous observations using single knockouts models and suggesting the existence of functional compensation among CRISP proteins, we investigated their relevance for male fertility by generating multiple Crisp gene mutants by CRISPR/Cas9 technology. Whereas targeting of Crisp1 and Crisp3 yielded subfertile males with early embryo developmental defects, the same deletion in zygotes from fertile Crisp2-/- .Crisp4-/- mice led to the generation of both triple and quadruple knockout mice exhibiting a complete or severe disruption of male fertility due to a combination of sperm transport, fertilization, and embryo developmental defects linked to intracellular Ca2+ dysregulation. These observations reveal that CRISP proteins are essential for male fertility and organize in functional modules that contribute distinctly to fertility success, bringing insights into the mechanisms underlying functional redundancy/compensation in protein families and emphasizing the importance of generating multiple and not just single knockout which might be masking the true functional relevance of family genes.

Identification of Potential Interacting Proteins With the Extracellular Loops of the Neuronal Glycoprotein M6a by TMT/MS.

Nowadays, great efforts are made to gain insight into the molecular mechanisms that underlie structural neuronal plasticity. Moreover, the identification of signaling pathways involved in the development of psychiatric disorders aids the screening of possible therapeutic targets. Genetic variations or alterations in GPM6A expression are linked to neurological disorders such as schizophrenia, depression, and Alzheimer's disease. GPM6A encodes the neuronal surface glycoprotein M6a that promotes filopodia/spine, dendrite, and synapse formation by unknown mechanisms. A substantial body of evidence suggests that the extracellular loops of M6a command its function. However, the proteins that associate with them and that modulate neuronal plasticity have not been determined yet. To address this question, we generated a chimera protein that only contains the extracellular loops of M6a and performed a co-immunoprecipitation with rat hippocampus samples followed by TMT/MS. Here, we report 72 proteins, which are good candidates to interact with M6a's extracellular loops and modify its function. Gene ontology (GO) analysis showed that 63% of the potential M6a's interactor proteins belong to the category "synapse," at both sides of the synaptic cleft, "neuron projections" (51%) and "presynapse" (49%). In this sense, we showed that endogenous M6a interacts with piccolo, synaptic vesicle protein 2B, and synapsin 1 in mature cultured hippocampal neurons. Interestingly, about 28% of the proteins left were related to the "myelin sheath" annotation, suggesting that M6a could interact with proteins at the surface of oligodendrocytes. Indeed, we demonstrated the (cis and trans) interaction between M6a and proteolipid protein (PLP) in neuroblastoma N2a cells. Finally, the 72 proteins were subjected to disease-associated genes and variants screening by DisGeNET. Apart from the diseases that have already been associated with M6a, most of the proteins are also involved in "autistic disorder," "epilepsy," and "seizures" increasing the spectrum of disorders in which M6a could play a role. Data are available via ProteomeXchange with identifier PXD017347.

Replacement and Parallel Simplification of Nonhomologous Proteinases Maintain Venom Phenotypes in Rear-Fanged Snakes.

Novel phenotypes are commonly associated with gene duplications and neofunctionalization, less documented are the cases of phenotypic maintenance through the recruitment of novel genes. Proteolysis is the primary toxic character of many snake venoms, and ADAM metalloproteinases, named snake venom metalloproteinases (SVMPs), are largely recognized as the major effectors of this phenotype. However, by investigating original transcriptomes from 58 species of advanced snakes (Caenophidia) across their phylogeny, we discovered that a different enzyme, matrix metalloproteinase (MMP), is actually the dominant venom component in three tribes (Tachymenini, Xenodontini, and Conophiini) of rear-fanged snakes (Dipsadidae). Proteomic and functional analyses of these venoms further indicate that MMPs are likely playing an "SVMP-like" function in the proteolytic phenotype. A detailed look into the venom-specific sequences revealed a new highly expressed MMP subtype, named snake venom MMP (svMMP), which originated independently on at least three occasions from an endogenous MMP-9. We further show that by losing ancillary noncatalytic domains present in its ancestors, svMMPs followed an evolutionary path toward a simplified structure during their expansion in the genomes, thus paralleling what has been proposed for the evolution of their Viperidae counterparts, the SVMPs. Moreover, we inferred an inverse relationship between the expression of svMMPs and SVMPs along the evolutionary history of Xenodontinae, pointing out that one type of enzyme may be substituting for the other, whereas the general (metallo)proteolytic phenotype is maintained. These results provide rare evidence on how relevant phenotypic traits can be optimized via natural selection on nonhomologous genes, yielding alternate biochemical components.

Malaria: Paving the way to developing peptide-based vaccines against invasion in infectious diseases.

Malaria remains a large-scale public health problem, killing more than 400,000 people and infecting up to 230 million worldwide, every year. Unfortunately, despite numerous efforts and research concerning vaccine development, results to date have been low and/or strain-specific. This work describes a strategy involving Plasmodium falciparum Duffy binding-like (DBL) and reticulocyte-binding protein homologue (RH) family-derived minimum functional peptides, netMHCIIpan3.2 parental and modified peptides' in silico binding prediction and modeling some Aotus major histocompatibility class II (MHCII) molecules based on known human molecules' structure to understand their differences. These are used to explain peptides' immunological behaviour when used as vaccine components in the Aotus model. Despite the great similarity between human and Aotus immune system molecules, around 50% of Aotus allele molecules lack a counterpart in the human immune system which could lead to an Aotus-specific vaccine. It was also confirmed that functional Plasmodium falciparum' conserved proteins are immunologically silent (in both the animal model and in-silico prediction); they must therefore be modified to elicit an appropriate immune response. Some peptides studied here had the desired behaviour and can thus be considered components of a fully-protective antimalarial vaccine.

Replacement and parallel simplification of non-homologous proteinases maintain venom phenotypes in rear-fanged snakes

Novel phenotypes are commonly associated with gene duplications and neofunctionalization, less documented are the cases of phenotypic maintenance through the recruitment of novel genes. Proteolysis is the primary toxic character of many snake venoms, and ADAM metalloproteinases, named Snake Venom Metalloproteinases (SVMPs), are largely recognized as the major effectors of this phenotype. However, by investigating original transcriptomes from 58 species of advanced snakes (Caenophidia) across their phylogeny, we discovered that a different enzyme, matrix metalloproteinase (MMP), is actually the dominant venom component in three tribes (Tachymenini, Xenodontini, and Conophiini) of rear-fanged snakes (Dipsadidae). Proteomic and functional analyses of these venoms further indicate that MMPs are likely playing an ‘SVMP-like’ function in the proteolytic phenotype. A detailed look into the venom-specific sequences revealed a new highly expressed MMP subtype, named snake venom MMP (svMMP), which originated independently on at least three occasions from an endogenous MMP-9. We further show that by losing ancillary non-catalytic domains present in its ancestors, svMMPs followed an evolutionary path toward a simplified structure during their expansion in the genomes, thus paralleling what has been proposed for the evolution of their Viperidae counterparts, the SVMPs. Moreover, we inferred an inverse relationship between the expression of svMMPs and SVMPs along the evolutionary history of Xenodontinae, pointing out that one type of enzyme may be substituting for the other, while the general (metallo)proteolytic phenotype is maintained. These results provide rare evidence on how relevant phenotypic traits can be optimized via natural selection on non-homologous genes, yielding alternate biochemical components.

Gene Phylogenies and Orthologous Groups.

This chapter covers the theory and practice of ortholog gene set computation. In the theoretical part we give detailed and formal descriptions of the relevant concepts. We also cover the topic of graph-based clustering as a tool to compute ortholog gene sets. In the second part we provide an overview of practical considerations intended for researchers who need to determine orthologous genes from a collection of annotated genomes, briefly describing some of the most popular programs and resources currently available for this task.

The Membrane Glycoprotein M6a Endocytic/Recycling Pathway Involves Clathrin-Mediated Endocytosis and Affects Neuronal Synapses.

Single point mutations or variations in the expression of the gene encoding the neuronal glycoprotein M6a have been associated with psychiatric disorders such as Alzheimer's disease, depression and schizophrenia. In cultured neurons, M6a positively contributes to neurite extension, axon guidance, filopodia/spine outgrowth, and synapse formation. The endocytic processes of neuronal membrane proteins are linked to the differentiation, growth, signaling and plasticity of neurons. However, the roles of M6a and the precise mechanisms through which M6a internalizes and recycles back to the neuronal membrane are unknown. Here, by using a controlled in vitro assay, we showed that if 30-40% of M6a is endocytosed, the number of synapses in hippocampal neurons decreases. When re-establishing the levels of M6a at the cell surface, the number of synapses returned to normal values. M6a internalization involves clathrin-coated pits, probably by association between the adaptor protein 2 and the 251YEDI254 "tyrosine-based" motif located within the C-tail of M6a. Upon endocytosis, M6a is sorted to early endosome antigen 1- and Rab5-positive endosomes and then sorted back to the cell surface via Rab11-positive endosomes or to degradation via Rab7 and, finally LAMP-1-positive endosomes. Our results demonstrated that the levels of M6a at the cell surface modified the formation/maintenance of synapses, without altering the protein levels of synaptophysin or N-methyl-D-aspartate receptor type-1. This novel mechanism might be relevant during neuronal development, pruning and/or many of the neurological disorders in which the number of synapses is affected.

Performance of Hidden Markov Models in Recovering the Standard Classification of Glycoside Hydrolases.

Glycoside hydrolases (GHs) are carbohydrate-active enzymes that assist the hydrolysis of glycoside bonds of complex sugars into carbohydrates. The current standard GH family classification is available in the CAZy database, which is based on the similarities of amino acid sequences and curated semi-automatically. However, with the exponential increase in data availability from genome sequences, automated classification methods are required for the fast annotation of coding sequences. Currently, the dbCAN database offers automatic annotations of signature domains from CAZy-defined classifications using a statistical approach, the hidden Markov models (HMMs). However, dbCAN does not contain the entire set of CAZy GH families. Moreover, no evaluation has been conducted so far of the viability of using HMM profiles as a means of automatically assigning GH amino acid sequences to the standard CAZy GH family classification itself. In this work, we performed a meta-analysis in which amino acid sequences from CAZy-defined GH families were used to build HMM family-specific profiles. We then queried a set with ~300 000 GH sequences against our database of HMM profiles estimated from CAZy families. We conducted the same evaluation against the available dbCAN HMM profiles. Our analyses recovered 65% of matches with the standard CAZy classification, whereas dbCAN HMMs resulted in 61% of matches. We also provided an analysis of the types of errors commonly found when HMMs are used to recover CAZy-based classifications. Although the performance of HMM was good, further developments are necessary for a fully automated classification of GH, allowing the standardization of GH classification among protein databases.

Filopodia formation driven by membrane glycoprotein M6a depends on the interaction of its transmembrane domains.

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.