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Circular RNAs (circRNAs) have been reported to play vital regulatory roles in human diseases. However, the functions of circRNAs in ischemic stroke (IS) are limited. In this study, we aimed to explore the functions and mechanisms of circRNA DLG associated protein 4 (circDLGAP4) in IS development. Oxygen-glucose deprivation (OGD)-treated HCN-2 cells were used to mimic IS environment in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to detect the levels of circDLGAP4, microRNA-503-3p (miR-503-3p) and neuronal growth regulator 1 (NEGR1) mRNA. RNase R assay was conducted to analyze the stability of circDLGAP4. Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis were adopted for cell viability and death, respectively. Western blot assay was performed for protein levels. Enzyme-linked immunosorbent assay (ELISA) kits were used to examine the concentrations of inflammatory cytokines. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay were employed to analyze the relationships among circDLGAP4, miR-503-3p and NEGR1. CircDLGAP4 level was declined in HCN-2 cells after OGD treatment. CircDLGAP4 overexpression promoted cell viability and suppressed cell death and inflammatory cytokine concentrations in OGD-treated HCN-2 cells. CircDLGAP4 acted as the sponge for miR-503-3p and the impacts of circDLGAP4 overexpression on cell viability, death and inflammation in OGD-treated HCN-2 cells were reversed by miR-503-3p elevation. Furthermore, NEGR1 was the target gene of miR-503-3p. MiR-503-3p inhibition ameliorated OGD-induced HCN-2 cell impairments, but NEGR1 knockdown abolished the effects. CircDLGAP4 alleviated OGD-induced HCN-2 cell damage by regulating miR-503-3p/NEGR1 axis.

Neuronal growth regulator 1 (NEGR1) and neurotrimin (NTM) are abundant cell-surface proteins found in the brain and form part of the IgLON (Immunoglobulin LSAMP, OBCAM, Neurotrimin) family. In humans, NEGR1 is implicated in obesity and mental disorders, while NTM is linked to intelligence and cognitive function. IgLONs dimerize homophilically and heterophilically, and they are thought to shape synaptic connections and neural circuits by acting in trans (spanning cellular junctions) and/or in cis (at the same side of a junction). Here, we reveal homodimeric structures of NEGR1 and NTM. They assemble into V-shaped complexes via their Ig1 domains, and disruption of the Ig1–Ig1 interface abolishes dimerization in solution. A hydrophobic ridge from one Ig1 domain inserts into a hydrophobic pocket from the opposing Ig1 domain producing an interaction interface that is highly conserved among IgLONs but remarkably plastic structurally. Given the high degree of sequence conservation at the interaction interface, we tested whether different IgLONs could elicit the same biological effect in vivo. In a small-scale study administering different soluble IgLONs directly into the brain and monitoring feeding, only NEGR1 altered food intake significantly. Taking NEGR1 as a prototype, our studies thus indicate that while IgLONs share a conserved mode of interaction and are able to bind each other as homomers and heteromers, they are structurally plastic and can exert unique biological action.

Cell adhesion molecules (CAMs) play crucial roles in the genesis and progress of tumor. We investigated the effects of single nucleotide polymorphisms (SNPs) of CAMs, neuronal growth regulator 1 (NEGR1), and Otoancorin (OTOA) on lung cancer susceptibility in Chinese nonsmoking females. Logistic regression and Cox regression analyses were conducted to investigate the effects of SNPs and environmental factors. For rs3102911, genotype TT carriers decreased the risk of lung cancer with an odds ratio (OR) of 0.635. AA genotypes of rs741718 increased the risk of lung cancer with an OR of 3.527. In stratified analysis, genotype AA carriers of rs741718 had a high susceptibility to lung adenocarcinoma compared with GG and AG genotypes. Analyses of association between SNPs and clinical characteristics revealed that rs3102911 as a protective factor and rs741718 as a risk factor influenced the lung cancer occurrence and progression in nonsmoking females.

The use of mass spectrometry for protein identification and quantification in cerebrospinal fluid (CSF) is at the forefront of research efforts to identify and explore biomarkers for the early diagnosis and prognosis of neurologic disorders. Here we implemented a 4-plex N,N-dimethyl leucine (DiLeu) isobaric labeling strategy in a longitudinal study aiming to investigate protein dynamics in children with B-cell acute lymphoblastic leukemia (B-cell ALL) undergoing chemotherapy. The temporal profile of CSF proteome during chemotherapy treatment at weeks 5, 10-14, and 24-28 highlighted many differentially expressed proteins, such as neural cell adhesion molecule, neuronal growth regulator 1, and secretogranin-3, all of which play important roles in neurodegenerative diseases. A total of 63 proteins were significantly altered across all of the time points investigated. The most over-represented biological processes from gene ontology analysis included platelet degranulation, complement activation, cell adhesion, fibrinolysis, neuron projection, regeneration, and regulation of neuron death. We expect that results from this and future studies will provide a means to monitor neurotoxicity and develop strategies to prevent central nervous system injury in response to chemotherapy in children.

Background Genome-wide association studies (GWAS) have identified several genetic variants associated with major depression disorder (MDD). However, pinpointing the causal variants which are responsible for the association signal at a risk locus remains a major challenge. Methods We used Summary data-based Mendelian Randomization (SMR) with Psychiatric Genomics Consortium (PGC) GWAS summary and brain expression quantitative trait loci (eQTL) data to identify genes whose expression levels are causally associated with MDD. Then we performed differential expression analysis, methylation quantitative trait loci analysis, and cognitive genetics analysis to investigate the potential roles of risk genes in the pathogenesis of MDD. Results Through SMR integrative analysis, we identified the SNP rs10789336 located in Neuronal growth regulator 1 (NEGR1) gene significantly affected the expression level of RPL31P12 in brain tissues and contributed to the risk of MDD (P = 1.96 × 10−6). Consistently, the SNP rs10789336 was associated with the methylation levels of three nearby DNA methylation sites, including cg09256413 (NEGR1, P=1.72 × 10−10), cg11418303 (prostaglandin E receptor 3 [PTGER3], P = 4.78 × 10−6), and cg23032215 (ZRANB2 antisense RNA 2 [ZRANB2-AS2], P = 1.23 × 10−4). Differential expression analysis suggested that the NEGR1 gene was upregulated in prefrontal cortex (P = 5.14 × 10−3). Cognitive genetics analysis showed that the SNP rs10789336 was associated with cognitive performance (P = 2.41 × 10−16), educational attainment (P = 1.75 × 10−14), general cognitive function (P = 2.65 × 10−12), and verbal numerical reasoning (P = 1.36 × 10−12). Conclusion Collectively, our results revealed that the SNP rs10789336 in NEGR1 might confer risk to MDD. Further investigation of the roles of NEGR1 in the pathogenesis of MDD is warranted.

Cell-to-cell adhesion is important for maintenance of brain structure and function. Abnormal neuronal cell adhesion and loss of its connectivity are considered a main cause of psychiatric disorders such as major depressive disorder (MDD). Various cell adhesion molecules (CAMs) are involved in neuronal cell adhesions and thereby affect brain functions such as learning and memory, cognitive functions, and psychiatric functions. Compared with other CAMs, neuronal growth regulator 1 (Negr1) has a distinct functioning mechanism in terms of its cross-talk with cytokine receptor signaling. Negr1 is a member of the immunoglobulin LON (IgLON) family of proteins and is involved in neuronal outgrowth, dendritic arborization, and synapse formation. In humans, Negr1 is a risk gene for obesity based on a genome-wide association study. More recently, accumulating evidence supports that it also plays a critical role in psychiatric disorders. In this review, we discuss the recent findings on the role of Negr1 in MDD, focusing on its regulatory mechanism. We also provide evidence of putative involvement of Negr1 in other psychiatric disorders based on the novel behavioral phenotypes of Negr1 knockout mice.

Neuronal growth regulator 1 (NEGR1) is a glycosylphosphatidylinositol-anchored membrane protein that mediates neural cell communication and synapse formation. Multiple genome-wide association studies have reported that variations in NEGR1 are associated with human body weight control. Recently, we found that NEGR1 is involved in intracellular cholesterol trafficking, suggesting that it performs a non-central nervous system (CNS) function associated with human obesity. We compared peripheral tissues such as the adipose, liver, and skeletal muscle tissues of Negr1−/− and Negr1+/+ (wild-type [WT]) C57BL/6 mice (n = 5–14). Intracellular lipid content was measured, and lipid accumulation was visualized by staining tissue cross-sections with lipid-specific stains. Muscle capacity of the WT and Negr1−/− mice was determined by performing a treadmill endurance test, and muscle fiber size was examined. Plasma glucose and insulin levels were measured, and glucose and insulin tolerance tests were performed. The Negr1−/− mice showed a significant increase in fat mass (~1.5-fold increase in the epididymal white adipose tissue, p = 0.000002), with abnormally enlarged adipose cells, compared with the WT mice. Primary adipocytes of the Negr1−/− mice contained enlarged cytosolic lipid droplets (p = 0.049). Moreover, these mice showed significant hepatic lipid accumulation (~2.3-fold increase, p = 0.043). Although the Negr1−/− mice did not show a significant change in plasma lipoprotein level, they showed a >1.3-fold increase in a serum glucose (p = 0.0002) and insulin (p = 0.016) levels. Moreover, the Negr1−/− mice showed decreased muscle capacity, as indicated by a decrease in muscle mass (p = 0.000003). These results indicate that NEGR1 deficiency induces abnormal fat deposition in various peripheral cells, especially fat and liver tissue cells, and suggest that NEGR1 is a potential molecular target for designing anti-obesity drugs to regulate body weight both centrally and peripherally.

Resumen Objetivo: Analizar la complejidad de la expresión génica en tejido adiposo de genes asociados con obesidad, mediante simulación computacional con diferentes herramientas bioinformáticas. Métodos: Después de una búsqueda bibliográfica en PubMed, se seleccionaron 37 genes asociados con obesidad con fold change mayor a 1,5. A partir del cálculo de valores de los z-score obtenidos de experimentos de micromatrices de ADN de muestras de tejido adiposo de personas obesas y de control, se construyó una red de interacción con el programa Cytoscape 3.2. La información detallada sobre las características genómicas de estos genes se extrajo de las bases de datos Genome Browser de la UCSC y del NCBI. Utilizando herramientas de análisis de multivariado, se hizo un análisis de componentes principales y uno de agrupación. Resultados: La red construida mostró que los genes con mayor número de interacciones fueron: 1) el factor nuclear respiratorio (NRF1), 2) el canal activado de potasio activado por calcio alfa 1 (KCNMA1) y 3) la sintasa de ácidos grasos (FASN). Los que tuvieron mayores valores de expresión fueron: 1) el factor de crecimiento endotelial vascular A (VEGFA), 2) la dioxigenasa dependiente de alfa-cetoglutarato (FTO) y 3) el regulador de crecimiento neuronal 1 (NEGR1). Las proteínas IL6, BDNF y HLC tuvieron los mayores valores de interacción con IL6R, NRF1 y ACACB, respetivamente. Las categorías ontológicas más importantes se relacionaron con procesos metabólicos de lipoproteínas, el ciclo de los ácidos tricarboxílicos, la activación de las MAP-quinasas y la cascada JNK. Conclusiones: En su conjunto los resultados obtenidos de sobreexpresión diferencial de genes asociados con el metabolismo de lípidos en el tejido adiposo de personas obesas podría ser un criterio para discriminar a nivel de diagnóstico esta patología. Summary Objective: To analyze the complexity of gene expression in the adipose tissue of genes associated with obesity, by computer simulation with different bioinformatics tools. Methods: After conducting a PubMed literature search, 37 genes associated with obesity with a fold change greater than 1.5 were selected. An interaction network was cons tructed using the Cytoscape 3.2 program from the calculation of z-score values obtained from DNA microarray experiments of adipose tissue samples collected from obese and control people. Detailed information on the genomic characteristics of these genes was extracted from the UCSC and NCBI Genome Browser databases. Using multivariate analysis tools, a principal component analysis and a cluster analysis were carried out. Results: The constructed network showed that the genes with the greatest number of interactions were: 1) the nuclear respiratory factor (NRF1), 2) the activated channel of potassium activate by calcium alpha 1 (KCNMA1), and 3) the fatty acid synthase (FASN). Those with the higher expression values were: 1) vascular endothelial growth factor A (VEGFA), 2) alpha-ketoglutarate-dependent dioxygenase (FTO), and 3) neuronal growth regulator 1 (NEGR1). The IL6, BDNF and HLC proteins had the highest interaction values with IL6R, NRF1 and ACACB, respectively. The most important ontological categories were related to lipoprotein metabolic processes, the tricarboxylic acid cycle, the activation of the MAP kinases, and the JNK cascade. Conclusions: As a whole, the results obtained from differential overexpression of genes associated with lipid metabolism in the adipose tissue of obese people could be a criterion to discriminate this pathology at a diagnostic level. Resumo Objetivo: Analisar a complexidade da expressão gênica no tecido adiposo de genes associados com obesidade, por meio de simulação por computador com diferentes ferramentas bioinformáticas. Métodos: Após uma busca bibliográfica em PubMed, foram selecionados 37 genes associados com obesidade com fold change superior a 1,5. A partir do cálculo de valores dos z-score obtidos de experimentos de micro matrizes de ADN de amostras de tecido adiposo de pessoas obesas e de controle, construiu-se uma rede de interação com o programa Cytoscape 3.2. A informação detalhada sobre as características genômicas destes genes foi obtida das bases de dados Genome Browser da UCSC e do NCBI. Utilizando ferramentas de análise de multivariado, fez-se uma análise de componentes principais e um de agrupação. Resultados: A rede construída mostrou que os genes com maior número de interações foram: 1) o fator nuclear respiratório (NRF1), 2) o canal ativado de potássio ativado por cálcio alfa 1 (KCNMA1) e 3) ácido graxo sintase (FASN). Os que tiveram maiores valores de expressão foram: 1) o fator de crescimento endo-telial vascular A (VEGFA), 2) a dioxigenase dependente de alfa-cetoglutarato (FTO) e 3) o regulador de crescimento neuronal 1 (NEGR1). As proteínas IL6, BDNF e HLC tiveram os maiores valores de interação com IL6R, NRF1 e ACACB, respectivamente. As categorias ontológicas mais importantes se relacionaram com processos metabólicos de lipoproteínas, o ciclo dos ácidos tri carboxílicos, a ativação das MAP-quinases e a cascata JNK. Conclusões: Em seu conjunto, os resultados obtidos de superexpressão diferencial de genes associados com o metabolismo de lipídios no tecido adiposo de pessoas obesas poderia ser um critério para discriminar a nível de diagnóstico esta patologia.

Lung cancer has been a leading cause of cancer-related death for decades and therapeutic strategies for non-driver mutation lung cancer are still lacking. A novel approach for this type of lung cancer is an emergent requirement. Here we find that loss of LSAMP (Limbic System Associated Membrane Protein), compared to other IgLON family of proteins NTM (Neurotrimin) and OPCML (OPioid-binding Cell adhesion MoLecule), exhibits the strongest prognostic and therapeutic significance in predicting lung adenocarcinoma (LUAD) progression. Lower expression of LSAMP and NTM, but not OPCML, were found in tumor parts compared with normal parts in six LUAD patients, and this was validated by public datasets, Oncomine® and TCGA. The lower expression of LSAMP, but not NTM, was correlated to shorter overall survival. Two epigenetic regulations, including hypermethylation and miR-143-3p upregulation but not copy number variation, were associated with downregulation of LSAMP in LUAD patients. Pathway network analysis showed that NEGR1 (Neuronal Growth Regulator 1) was involved in the regulatory loop of LSAMP. The biologic functions by LSMAP knockdown in lung cancer cells revealed LSMAP was linked to cancer cell migration via epithelial-mesenchymal transition (EMT) but not proliferation nor stemness of LUAD. Our result showed for the first time that LSAMP acts as a potential tumor suppressor in regulating lung cancer. A further deep investigation into the role of LSAMP in lung cancer tumorigenesis would provide therapeutic hope for such affected patients.

Obesity is associated with an increased risk of various diseases and mortality. Although nearly 50 % of adults have been reported trying to lose weight, the prevalence of obesity has increased. One factor that hinders weight loss-induced decrease in obesity prevalence is weight regain. Although behavioural, psychological and physiological factors associated with weight regain have been reviewed, the information regarding the relationship between weight regain and genetics has not been previously summarised. In this paper, we comprehensively review the association between genetic polymorphisms and weight regain in adults and children with obesity after weight loss. Based on this information, identification of genetic polymorphism in patients who undergo weight loss intervention might be used to estimate their risks of weight regain. Additionally, the genetic-based risk estimation may be used as a guide for physicians and dietitians to provide each of their patients with the most appropriate strategies for weight loss and weight maintenance.

Introduction: The variant NEGR1 rs2815752 has recently been linked with obesity in Caucasians. However, a very limited number of studies have examined the association of the NEGR1 rs2815752 with overweight/obesity in non-Caucasians with no such study ever performed in Pakistani population. Therefore, the present study was undertaken to seek the association of the rs2815752 with overweight, obesity, and related traits in Pakistanis. Subjects and methods: The study involved 112 overweight/control pairs (total 224) and 194 obese/control pairs (total 388). Anthropometric parameters were measured by employing standard procedures. Metabolic parameters were determined by biochemical assays. Behavioral information was collected through a questionnaire. The rs2815752 was genotyped via TaqMan allelic discrimination assay. Regression analyses were employed to analyze the data in SPSS software. Results: The study revealed significant gender-specific association of the rs2815752 with obesity (OR 3.03; CI 1.19–7.72, p = 0.020) and some obesity-related anomalous anthropometric traits (weight, BMI, waist circumference, hip circumference, and abdominal and supra-iliac skinfold thicknesses) in females according to dominant model (h = 0.023). However, no association of the rs2815752 with obesity-related behavioral and metabolic parameters was observed. Conclusion: The NEGR1 rs2815752 may be associated with obese phenotype and some of the related anthropometric traits in Pakistani females.

Emerging evidence has indicated that long non‑coding RNAs (lncRNAs) are closely associated with the pathogenesis of ischemic stroke. It has been reported that small nucleolar RNA host gene 12 (SNHG12) serves a critical role in ischemic stroke by acting as a competitive endogenous RNA (ceRNA). SNHG12 competes with various microRNAs (miRs) to regulate RNA transcription of specific targets. However, the effect of SNHG12 on oxygen‑glucose deprivation (OGD)‑induced neuronal apoptosis has rarely been reported. The present study demonstrated that SNHG12 expression was downregulated in OGD‑injured SH‑SY5Y cells. Furthermore, miR‑181a‑5p was reported as a target of SNHG12 and was negatively regulated by SNHG12. Moreover, NEGR1 was a target of miR‑181a‑5p, which functions as a negative regulator of NEGR1 in OGD‑induced neuronal apoptosis. In summary, the results strongly confirmed the hypothesis that SNHG12 functions as a ceRNA for miR‑181a‑5p and regulates the expression of NEGR1 thus inhibiting OGD‑induced apoptosis of SH‑SY5Y cells. Neuronal apoptosis aggravates brain damage during ischemic stroke, indicating that the activation of SNHG12 and NEGR1 expression and inhibition of miR‑181a‑5p may be a novel strategy for the clinical treatment of ischemic stroke.

Common fragile sites (CFSs) are particularly vulnerable regions of the genome that become visible as breaks, gaps, or constrictions on metaphase chromosomes when cells are under replicative stress. Impairment in DNA replication, late replication timing, enrichment of A/T nucleotides that tend to form secondary structures, the paucity of active or inducible replication origins, the generation of R-loops, and the collision between replication and transcription machineries on particularly long genes are some of the reported characteristics of CFSs that may contribute to their tissue-specific fragility. Here, we validated the induction of two CFSs previously found in the human fetal lung fibroblast line, Medical Research Council cell strain 5 (MRC-5), in another cell line derived from the same fetal tissue, Institute for Medical Research-90 cells (IMR-90). After induction of CFSs through aphidicolin, we confirmed the expression of the CFS 1p31.1 on chromosome 1 and CFS 3q13.3 on chromosome 3 in both fetal lines. Interestingly, these sites were found to not be fragile in lymphocytes, suggesting a role for epigenetic or transcriptional programs for this tissue specificity. Both these sites contained late-replicating genes NEGR1 (neuronal growth regulator 1) at 1p31.1 and LSAMP (limbic system-associated membrane protein) at 3q13.3, which are much longer, 0.880 and 1.4 Mb, respectively, than the average gene length. Given the established connection between long genes and CFS, we compiled information from the literature on all previously identified CFSs expressed in fibroblasts and lymphocytes in response to aphidicolin, including the size of the genes contained in each fragile region. Our comprehensive analysis confirmed that the genes found within CFSs are longer than the average human gene, interestingly, the two longest genes in the human genome are found within CFSs: Contactin Associated Protein 2 gene (CNTNAP2) in a lymphocytes&rsquo, CFS, and Duchenne muscular dystrophy gene (DMD) in a CFS expressed in both lymphocytes and fibroblasts. This indicates that the presence of very long genes is a unifying feature of all CFSs. We also obtained replication profiles of the 1p31.1 and 3q13.3 sites under both perturbed and unperturbed conditions using a combination of fluorescent in situ hybridization (FISH) and immunofluorescence against bromodeoxyuridine (BrdU) on interphase nuclei. Our analysis of the replication dynamics of these CFSs showed that, compared to lymphocytes where these regions are non-fragile, fibroblasts display incomplete replication of the fragile alleles, even in the absence of exogenous replication stress. Our data point to the existence of intrinsic features, in addition to the presence of long genes, which affect DNA replication of the CFSs in fibroblasts, thus promoting chromosomal instability in a tissue-specific manner.

Obesity is a complex disorder with nearly epidemic proportions in many parts of the world. Genome-wide association studies have demonstrated high heritability for obesity and body mass, with associations of certain candidate genes and their variations with respect to race, geographical location/country of origin. However, the functional mechanisms and different ethnic data of these loci are still poorly understood. In this case-control study, we investigated two single nucleotide polymorphisms, rs2815752 in the neuronal growth regulator 1 (NEGR1) gene and rs6548238 in the transmembrane protein 18 (TMEM18) gene, for association in a group of obese residents of Afyonkarahisar province (Turkey). Polymorphisms were genotyped in 172 obese subjects and 77 healthy controls. The results showed no significant differences between the obese subjects and the controls in terms of the allele and genotype frequencies of the NEGR1 gene rs2815752 and the TMEM18 gene rs6548238 polymorphisms. There were no significant associations of the rs2815752 polymorphism in obese subjects and controls with regard to anthropometric measurements and body composition parameters. However, several significant associations were found for the rs6548238 polymorphism with regard to anthropometric measurements and body composition. Consequently, there were no significant differences between the genotype and allele frequencies of NEGR1 gene rs2815752 and TMEM18 gene rs6548238 polymorphisms in the obese group and the controls. There were significant associations for the rs6548238 polymorphism, but not the rs2815752 polymorphism, with the anthropometric measurements and body composition parameters in the group of obese subjects.

Neuronal growth regulator 1 (NEGR1) belongs to the immunoglobulin (IgLON) superfamily of cell adhesion molecules involved in cortical layering. Recent functional and genomic studies implicate the role of NEGR1 in a wide spectrum of psychiatric disorders, such as major depression, schizophrenia and autism. Here, we investigated the impact of Negr1 deficiency on brain morphology, neuronal properties and social behavior of mice. In situ hybridization shows Negr1 expression in the brain nuclei which are central modulators of cortical-subcortical connectivity such as the island of Calleja and the reticular nucleus of thalamus. Brain morphological analysis revealed neuroanatomical abnormalities in Negr1−/− mice, including enlargement of ventricles and decrease in the volume of the whole brain, corpus callosum, globus pallidus and hippocampus. Furthermore, decreased number of parvalbumin-positive inhibitory interneurons was evident in Negr1−/− hippocampi. Behaviorally, Negr1−/− mice displayed hyperactivity in social interactions and impairments in social hierarchy. Finally, Negr1 deficiency resulted in disrupted neurite sprouting during neuritogenesis. Our results provide evidence that NEGR1 is required for balancing the ratio of excitatory/inhibitory neurons and proper formation of brain structures, which is prerequisite for adaptive behavioral profiles. Therefore, Negr1−/− mice have a high potential to provide new insights into the neural mechanisms of neuropsychiatric disorders.

Long non-coding RNAs and microRNAs control gene expression to determine central nervous system development and function. Neuronal growth regulator 1 (NEGR1) is a cell adhesion molecule that plays an important role in neurite outgrowth during neuronal development and its precise expression is crucial for correct brain development. The data described here is related to the research article titled “A long non-coding RNA, BC048612 and a microRNA, miR-203 coordinate the gene expression of Neuronal growth regulator 1 (NEGR1) adhesion protein” [1]. This data article contains detailed bioinformatics analysis of genetic signatures at the Negr1 gene locus retrieved from the UCSC genome browser. This approach could be adopted to identify putative regulatory non-coding RNAs in other tissues and diseases. Keywords: Bioinformatics, Long non-coding RNA, MicroRNA, Negr1, Regulation

The regulatory roles for non-coding RNAs, the long non-coding RNAs and microRNAs, are emerging as crucial determinants of central nervous system development and function. Neuronal growth regulator 1 (NEGR1) is a cell adhesion molecule that has been shown to play an important role in neurite outgrowth during neuronal development. Precise expression of the Negr1 gene is crucial for proper brain development and is dysregulated during brain injury. Hence, we attempted to elucidate the non-coding RNAs that control Negr1 gene expression. A long non-coding RNA, BC048612, transcribed from the bidirectional GC-rich Negr1 gene promoter was found to influence Negr1 mRNA expression. In vitro knockdown of the long non-coding RNA resulted in significant down-regulation of Negr1 mRNA expression, NEGR1 protein levels and neurite length whereas over-expression enhanced Negr1 mRNA expression, NEGR1 protein levels and increased neurite length. Meanwhile, another non-coding RNA, microRNA-203, was found to target the 3' untranslated region of the Negr1 mRNA. Inhibition of microRNA-203 led to increased expression of Negr1 mRNA, elevated NEGR1 protein levels and increased neurite length. Conversely, microRNA-203 over-expression decreased the level of Negr1 mRNA, NEGR1 protein and neurite length. Neither microRNA-203 nor the long non-coding RNA, BC048612 could influence each other's expression. Hence, the long non-coding RNA, BC048612, and microRNA-203 were determined to be positive and negative regulators of Negr1 gene expression respectively. These processes have a direct effect on NEGR1 protein levels and neurite length, thus highlighting the importance of the regulatory non-coding RNAs in modulating Negr1 gene expression for precise neuronal development.

Endothelial-mesenchymal transition (EndoMT) plays a critical role in the dysfunction of the blood-brain barrier (BBB). Circular RNAs (circRNAs) function as crucial regulatory factors in EndoMT. Nevertheless, the underlying mechanisms of circRNA HECW2 (circ_HECW2, hsa_circ_0057583) in lipopolysaccharide (LPS)-induced EndoMT remain largely unclear. The levels of circ_HECW2, miR-30e-5p and neuronal growth regulator 1 (NEGR1) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Ribonuclease (RNase) R and Actinomycin D assays were performed to validate the stability of circ_HECW2. Cell colony formation, proliferation and apoptosis were tested by a standard colony formation assay, the 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and flow cytometry, respectively. Targeted relationships among circ_HECW2, miR-30e-5p and NEGR1 were verified by a dual-luciferase reporter assay. Our data indicated that LPS increased circ_HECW2 expression and reduced miR-30e-5p expression in human brain microvascular endothelial cells (HBMECs). Circ_HECW2 silencing promoted cell proliferation and suppressed cell apoptosis and EndoMT in LPS-treated HBMECs. Mechanistically, circ_HECW2 directly interacted with miR-30e-5p by binding to miR-30e-5p. MiR-30e-5p was a functional mediator of circ_HECW2 in regulating LPS-induced cell EndoMT. Furthermore, Circ_HECW2 regulated NEGR1 expression through functioning as a miR-30e-5p sponge. Moreover, miR-30e-5p overexpression repressed the EndoMT of LPS-treated HBMECs by targeting NEGR1. Collectively, our current study demonstrated that circ_HECW2 silencing suppressed LPS-triggered HBMEC EndoMT at least in part through the regulation of the miR-30e-5p/NEGR1 axis, illuminating a promising strategy for EndoMT inhibition.

NEGR1 is a cell adhesion molecule present within synapses at the pre- and postsynaptic terminals, making it likely to play a role in synaptogenesis and synaptic neurotransmission. NEGR1 has been associated with obesity as well as various neurodevelopmental disorders. In this thesis, we study the effect of NEGR1-overexpression as well as NEGR1 silencing in cortical and hypothalamic neurons. Our results indicate that NEGR1-overexpression reduces synapse formation in cortical and hypothalamic neurons. Numbers of spines and filopodia along the dendrites of NEGR1-overexpressing cortical neurons are reduced. Repetitive stimulation results in reduced synaptic exocytosis with subsequent stimulation in axons of NEGR1-overexpressing cortical neurons. Concurrently, NEGR1-overexpressing neurons present with increasing rates of endocytosis across subsequent stimulations. Neurons overexpressing NEGR1 show lower levels of synaptophysin in synaptic boutons suggesting a reduction in the pool of synaptic vesicles available for exocytosis. Levels of CSP, a synaptic chaperone protein involved in chaperoning SNARE proteins, are reduced in synapses of NEGR1-overexpressing neurons, suggesting that NEGR1-overexpression potentiates the degradation of CSP. NEGR1 silencing with NEGR1 siRNA-transfection in cortical neurons also results in reduced synapse formation. Actin polymerization and dendritic spine and filopodium formation along dendrites of NEGR1-deficient neurons are reduced. The pool of synaptic vesicles available for exocytosis in axons of NEGR1 siRNA-transfected neurons is reduced and repetitive stimulation results in reduced exocytosis and endocytosis of synaptic vesicles during subsequent stimulations. Synaptic levels of CSP and its substrate SNAP25 are reduced in NEGR1 silenced neurons. Furthermore, NEGR1 knock down causes the synaptic activity-dependent reduction in the synaptic pool of CSP and synaptophysin. Hence, we propose that NEGR1 regulates targeting of CSP to synaptic vesicle, most likely, by regulating CSP palmitoylation, which plays a key role in targeting of CSP to synaptic vesicles. Our results show that NEGR1 has an important role in the regulation of synaptogenesis and synaptic vesicle recycling with both processes being important in neuronal development and continual synaptic maintenance. Future research will investigate the role that NEGR1 plays in regulating palmitoylation of CSP and other synaptic substrates and changes in this regulation in neurodevelopmental and neurodegenerative disorders.

Nowadays, diagnosis of neurodegenerative disorders is mainly based on neuroimaging and clinical symptoms, although postmortem neuropathological confirmation remains the gold standard diagnostic technique. Therefore, cerebrospinal fluid (CSF) proteome is considered a valuable molecular repository for diagnosing and targeting the neurodegenerative process. It is well known that olfactory dysfunction is among the earliest features of synucleinopathies such as Parkinson's disease (PD). Consequently, we consider that the application of tissue proteomics in primary olfactory structures is an ideal approach to explore early pathophysiological changes, detecting olfactory proteins that might be tested in CSF as potential biomarkers. Data mining of mass spectrometry-generated datasets has revealed that 30% of the olfactory bulb (OB) proteome is also localized in CSF. In this chapter, we describe a method that utilizes label-free quantitative proteomics and computational analysis to characterize human OB proteomes and potential cerebrospinal fluid (CSF) biomarkers associated with neurodegenerative syndromes. For that, we applied peptide fractionation methods, followed by tandem mass spectrometry (nanoLC-MS/MS), in silico analysis, and semi-quantitative orthogonal techniques in OB derived from PD subjects. After obtaining the differential OB proteome across Lewy-type alpha-synucleinopathy (LTS) stages and further validating the method, this workflow was applied to probe changes in NEGR1 (neuronal growth regulator 1) and GNPDA2 (glucosamine-6-phosphate deaminase 2) protein levels in CSF derived from parkinsonian subjects with respect to controls, observing an inverse correlation between both proteins and α-synuclein, the principal component analysis of Lewy pathology.

Neuronal growth regulator 1 (NEGR1) is a candidate gene for human obesity, which encodes the neural cell adhesion and growth molecule. The aim of the current study was to recognize the non-synonymous SNPs (nsSNPs) with the highest predicted deleterious effect on protein function of the NEGR1 gene. We have used five computational tools, namely, PolyPhen, SIFT, PROVEAN, MutPred and M-CAP, to predict the deleterious and pathogenic nsSNPs of the NEGR1 gene. Homology modeling approach was used to model the native and mutant NEGR1 protein models. Furthermore, structural validation was performed by the PROCHECK server to interpret the stability of the predicted models. We have predicted four potential deleterious nsSNPs, i.e., rs145524630 (Ala70Thr), rs267598710 (Pro168Leu), rs373419972 (Arg239Cys) and rs375352213 (Leu158Phe), which might be involved in causing obesity phenotypes. The predicted mutant models showed higher root mean square deviation and free energy values under the PyMoL and SWISS-PDB viewer, respectively. Additionally, the FTSite server predicted one nsSNP, i.e., rs145524630 (Ala70Thr) out of four identified nsSNPs found in the NEGR1 protein-binding site. There were four potential deleterious and pathogenic nsSNPs, i.e., rs145524630, rs267598710, rs373419972 and rs375352213, identified from the above-mentioned tools. In future, further functional in vitro and in vivo analysis could lead to better knowledge about these nsSNPs on the influence of the NEGR1 gene in causing human obesity. Hence, the present computational examination suggest that predicated nsSNPs may feasibly be a drug target and play an important role in contributing to human obesity.

In the developing brain, cell-surface proteins play crucial roles, but their protein-protein interaction network remains largely unknown. A proteomic screen identified 200 interactions, 89 of which were not previously published. Among these interactions, we find that the IgLONs, a family of five cell-surface neuronal proteins implicated in various human disorders, interact as homo- and heterodimers. We reveal their interaction patterns and report the dimeric crystal structures of Neurotrimin (NTRI), IgLON5, and the neuronal growth regulator 1 (NEGR1)/IgLON5 complex. We show that IgLONs maintain an extended conformation and that their dimerization occurs through the first Ig domain of each monomer and is Ca2+ independent. Cell aggregation shows that NTRI and NEGR1 homo- and heterodimerize in trans. Taken together, we report 89 unpublished cell-surface ligand-receptor pairs and describe structural models of trans interactions of IgLONs, showing that their structures are compatible with a model of interaction across the synaptic cleft. Many aspects of synapse formation, specification, and maturation rely on interactions among a rich repertoire of cell-surface glycoproteins with adhesive and repulsive properties. Although the identity of these proteins is known, their network of interactions remains largely untapped. Ranaivoson et al. have identified a number of protein-protein interactions and have determined the structures of three members of the IgLONs, a family of five proteins of the immunoglobulin superfamily that has recently been implicated in a wide range of human disease.

Neuronal growth regulator 1 (NEGR1), a member of the immunoglobulin superfamily cell adhesion molecule subgroup IgLON, has been involved in neuronal growth and connectivity. Genetic variants, in or near the NEGR1 locus, have been associated with obesity and, more recently, with learning difficulties, intellectual disability, and psychiatric disorders. Here, we described the only second report of NEGR1 gene disruption in 1p31.1 microdeletion in two patients. Patient 1 is a 14-year-old female with neurological and psychiatric features present also in her family. Patient 2 is a 5-month-old infant showing global hypotonia as unique neurological features till now. This patient also carries 7p22.1 duplication, of paternal origin, that could be responsible for some malformations present in the child. We hypothesize a role of NEGR1 in producing the phenotype of our patients and compare them with other cases previously reported in the literature and DECIPHER database to better identify a possible genotype-phenotype correlation.

Axon regeneration after spinal cord injury (SCI) is attenuated by growth inhibitory molecules associated with myelin. We report that rat myelin stimulated the growth of axons emerging from rat neural progenitor cells (NPCs) transplanted into sites of SCI in adult rat recipients. When plated on a myelin substrate, neurite outgrowth from rat NPCs and from human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) was enhanced threefold. In vivo, rat NPCs and human iPSC-derived NSCs extended greater numbers of axons through adult central nervous system white matter than through gray matter and preferentially associated with rat host myelin. Mechanistic investigations excluded Nogo receptor signaling as a mediator of stem cell-derived axon growth in response to myelin. Transcriptomic screens of rodent NPCs identified the cell adhesion molecule neuronal growth regulator 1 (Negr1) as one mediator of permissive axon-myelin interactions. The stimulatory effect of myelin-associated proteins on rodent NPCs was developmentally regulated and involved direct activation of the extracellular signal-regulated kinase (ERK). The stimulatory effects of myelin on NPC/NSC axon outgrowth should be investigated further and could potentially be exploited for neural repair after SCI.

Neuronal growth regulator 1 (NEGR1), a member of the immunoglobulin superfamily cell adhesion molecule subgroup IgLON, has been implicated in neuronal growth and connectivity. In addition, genetic variants in or near the NEGR1 locus have been associated with obesity and more recently with learning difficulties, intellectual disability and psychiatric disorders. However, experimental evidence is lacking to support a possible link between NEGR1, neuronal growth and behavioral abnormalities. Initial expression analysis of NEGR1 mRNA in C57Bl/6 wildtype (WT) mice by in situ hybridization demonstrated marked expression in the entorhinal cortex (EC) and dentate granule cells. In co-cultures of cortical neurons and NSC-34 cells overexpressing NEGR1, neurite growth of cortical neurons was enhanced and distal axons occupied an increased area of cells overexpressing NEGR1. Conversely, in organotypic slice co-cultures, Negr1-knockout (KO) hippocampus was less permissive for axons grown from EC of β-actin-enhanced green fluorescent protein (EGFP) mice compared to WT hippocampus. Neuroanatomical analysis revealed abnormalities of EC axons in the hippocampal dentate gyrus (DG) of Negr1-KO mice including increased numbers of axonal projections to the hilus. Neurotransmitter receptor ligand binding densities, a proxy of functional neurotransmitter receptor abundance, did not show differences in the DG of Negr1-KO mice but altered ligand binding densities to NMDA receptor and muscarinic acetylcholine receptors M1 and M2 were found in CA1 and CA3. Activity behavior, anxiety-like behavior and sensorimotor gating were not different between genotypes. However, Negr1-KO mice exhibited impaired social behavior compared to WT littermates. Moreover, Negr1-KO mice showed reversal learning deficits in the Morris water maze and increased susceptibility to pentylenetetrazol (PTZ)-induced seizures. Thus, our results from neuronal growth assays, neuroanatomical analyses and behavioral assessments provide first evidence that deficiency of the psychiatric disease-associated Negr1 gene may affect neuronal growth and behavior. These findings might be relevant to further evaluate the role of NEGR1 in cognitive and psychiatric disorders.

The arcuate nucleus of the hypothalamus contains neurons responsible for feeding. They control metabolic processes by secretion of neuropeptides in response to hunger and satiety signals from the gastrointestinal tract, pancreas and adipose tissue. Here we aim at addressing the challenge of non-availability of developing human brain/hypothalamic tissues for study. To bridge this gap, we generated human induced pluripotent stem cells (hiPSCs) from lymphoblasts of super obese (body mass index (BMI) ≥ 50) and normal (BMI ≤ 25) subjects and differentiated them into hypothalamic-like neuronal cultures (iHTNs), specifically those expressing pro-opiomelanocortin (POMC), agouti-related peptide (AgRP), cocaine and amphetamine regulating transcript (CART) and neurons capable of secreting neuropeptides such as neuropeptide Y (NPY) and alpha-melanocyte stimulating hormone (MSH). Of the total number of neurons (approx.75% of cells in culture), we saw 42% MSH , 44% NPY , 28% AgRP and 20% POMC/CART neurons. Bioinformatics analyses of transcriptomics from day 40 differentiated iHTNs reveal that these neuronal cultures maintain predominantly a fetal hypothalamic identity. We further show that these neurons are capable of relevantly responding to physiological (exogenous) metabolic stimuli e.g. ghrelin and leptin. Notably, transcriptomics of iHTNs obtained from super obese individuals retained signatures of obesity showing specific dysregulation of obesity-related genes such as neuronal growth regulator 1 (NEGR1) and secretogranin 3 (SCG3). Ingenuity Pathway Analysis also highlighted dysregulation of several obesity-related metabolic pathways like leptin signaling, GPCR-mediated enteroendocrine signaling, as well as the ER stress and inflammasome pathways in super obese iHTNs when compared to controls. The methods and characterization of iHTNs described here could serve as a cellular platform for modeling and testing gene-environment interactions involved in metabolic diseases and obesity.

Recent genome-wide association studies on major depressive disorder have implicated neuronal growth regulator 1 (Negr1), a GPI-anchored cell adhesion molecule in the immunoglobulin LON family. Although Negr1 has been shown to regulate neurite outgrowth and synapse formation, the mechanism through which this protein affects mood disorders is still largely unknown. In this research, we characterized Negr1-deficient (negr1-/-) mice to elucidate the function of Negr1 in anxiety and depression. We found that anxiety- and depression-like behaviors increased in negr1-/- mice compared with wild-type mice. In addition, negr1-/- mice had decreased adult hippocampal neurogenesis compared to wild-type mice. Concurrently, both LTP and mEPSC in the dentate gyrus (DG) region were severely compromised in negr1-/- mice. In our effort to elucidate the underlying molecular mechanisms, we found that lipocalin-2 (Lcn2) expression was decreased in the hippocampus of negr1-/- mice compared to wild-type mice. Heterologous Lcn2 expression in the hippocampal DG of negr1-/- mice rescued anxiety- and depression-like behaviors and restored neurogenesis and mEPSC frequency to their normal levels in these mice. Furthermore, we discovered that Negr1 interacts with leukemia inhibitory factor receptor (LIFR) and modulates LIF-induced Lcn2 expression. Taken together, our data uncovered a novel mechanism of mood regulation by Negr1 involving an interaction between Negr1 and LIFR along with Lcn2 expression.

Neuronal growth regulator 1 (NEGR1) has become a great interest based on the recent findings that its genetic alteration is implicated in human obesity and human dyslexia. By analyzing the gene expression profiles of tumor biopsies and normal tissues, we identified NEGR1 as a commonly down-regulated gene in many types of human cancer tissues. NEGR1 contains a C-terminal GPI anchor attachment site and is primarily localized to cell membrane rafts, especially in cell-to-cell contacting areas. The oncogenic phenotype was clearly attenuated when NEGR1 was overexpressed in the human ovarian cancer cell line SKOV-3. Furthermore, cell aggregation and neurite outgrowth was greatly increased by NEGR1 overexpression. On the contrary, cell migration and invasion was increased in NEGR1-depleted cells, suggesting that NEGR1 may contribute to tumor suppression. Taken together, we suggest that NEGR1 is a raft-associated extracellular protein that may participate in cell recognition and interaction, which is important in growth control and malignant transformation.

Common variants at only two loci, FTO and MC4R, have been reproducibly associated with body mass index (BMI) in humans. To identify additional loci, we conducted meta-analysis of 15 genome-wide association studies for BMI (n > 32,000) and followed up top signals in 14 additional cohorts (n > 59,000). We strongly confirm FTO and MC4R and identify six additional loci (P < 5 × 10⁻⁸): TMEM18, KCTD15, GNPDA2, SH2B1, MTCH2 and NEGR1 (where a 45-kb deletion polymorphism is a candidate causal variant). Several of the likely causal genes are highly expressed or known to act in the central nervous system (CNS), emphasizing, as in rare monogenic forms of obesity, the role of the CNS in predisposition to obesity. © 2009 Nature America, Inc. All rights reserved.

Background and objective Epilepsy is etiologically and genetically complex neurological disorder affecting millions of people worldwide. Juvenile myoclonic epilepsy (JME) is the most common epilepsy syndrome that starts in the teen age group commonly between ages 12, 18, and lasts till adulthood. One out of fourteen people with epilepsy suffers with JME. Myoclonic seizures and muscle twitching or uncontrolled jerking are the most common type of seizures in the people suffering with JME. Method To observe the novel CNVs involved in JME, we investigated a Saudi family with nine siblings including one male and one female affected members. In this study we used high density whole genome Agilent sure print G3 Hmn CGH 2x 400K array-CGH chips. Our results showed CNVs including the amplifications and deletions in different chromosomal regions in the patients as compared to the normal members of the family. Amplifications were observed in the chromosome 22 cytoband 22q11.23 with LDL receptor related protein 5 like (LRP5L), Immunoglobulin Lambda-Like Polypeptide 3 (IGLL3) and crystallin beta B2 pseudogene (CRYBB2P) genes respectively whereas the deletions were observed in the chromosomal regions 4q22.2 with Glutamate receptor, ionotropic, delta 2 (GRID2) as potential gene cytoband 1p31.1 with potential Neuronal Growth Regulator 1 gene (NEGR1) gene in this region and NME/NM23 family member (NME7) gene cytoband 1q24. Moreover, the array CGH resulting in deletions and duplication were also validated by using primer for simple PCR or also by using quantitative real time PCR analysis. We found deletions and duplication in JME patients in our study for the first time in Saudi population. Results & conclusion The findings in this study suggest that the array-CGH may be considered as a first line of genetic testing for diagnosis of epilepsy unless strong evidence is presented for a monogenic syndrome. The use of high throughput technique in this study will help to identify novel mechanisms underlying epileptic disorder in order to lower the burden of epilepsy in Saudi Arabia.

OBJECTIVE: To use a unique obesity-discordant sib-pair study design to combine differential expression analysis, expression quantitative trait loci (eQTLs) mapping and a coexpression regulatory network approach in subcutaneous human adipose tissue to identify genes relevant to the obese state.STUDY DESIGN: Genome-wide transcript expression in subcutaneous human adipose tissue was measured using Affymetrix U133 Plus 2.0 microarrays (Affymetrix, Santa Clara, CA, USA), and genome-wide genotyping data was obtained using an Applied Biosystems (Applied Biosystems; Life Technologies, Carlsbad, CA, USA) SNPlex linkage panel.SUBJECTS: A total of 154 Swedish families ascertained through an obese proband (body mass index (BMI) >30 kg m(-2)) with a discordant sibling (BMI>10 kg m(-2) less than proband).RESULTS: Approximately one-third of the transcripts were differentially expressed between lean and obese siblings. The cellular adhesion molecules (CAMs) KEGG grouping contained the largest number of differentially expressed genes under cis-acting genetic control. By using a novel approach to contrast CAMs coexpression networks between lean and obese siblings, a subset of differentially regulated genes was identified, with the previously GWAS obesity-associated neuronal growth regulator 1 (NEGR1) as a central hub. Independent analysis using mouse data demonstrated that this finding of NEGR1 is conserved across species.CONCLUSION: Our data suggest that in addition to its reported role in the brain, NEGR1 is also expressed in subcutaneous adipose tissue and acts as a central 'hub' in an obesity-related transcript network.

Introduction: Recent approvals of microsatellite instability (MSI) or mismatch repair (MMR) testing, in addition to PD-L1 expression, expand the tools available to identify tumor characteristics that may help predict the likelihood of patient response to immunotherapy treatment. However, even in MSI positive subgroups, not all subjects achieve a durable response and research continues to identify tumor characteristics that further predict the likelihood of patient response. To support and advance this area of research, new tools are being developed that provide deeper and unbiased views of the tumor proteome. Here, we characterize the protein expression profiles of 95 colorectal cancer tumors (CRC) using SWATH acquisition mass spectrometry (SWATH MS) to further probe tumor phenotypic characteristics. Experimental Methods: FFPE colon tissue samples (10 healthy, 95 cancer) were obtained from commercial biobanks. Proteins were extracted from the tissue, processed to peptides with trypsin, and prepared for LC-MS analysis. Peptides for each sample were injected on a Triart C18 column (YMC) coupled to a NanoLC 425 system (SCIEX) using a 43min gradient at a flow rate of 5µl/min. The eluted peptides were then analyzed with a TripleTOF® 6600 system (SCIEX) operated in SWATH mode. Total run time per sample was 1 hour. Data were analyzed in Spectronaut Pulsar X (Biognosys) with a project specific library. All data were filtered with a 1% FDR on peptide and protein level. Results: Across all samples, >4,500 protein groups were quantified (approximately 3,600 per sample). Data analysis revealed a large number of proteins (~1,000) were differentially expressed in the cancer cohort. Consistent with increased tumor cell proliferation, proteins involved in protein translation were upregulated in the tumor samples. Unsupervised clustering of the data separated the healthy and the cancer cohort. Clustering also revealed three main proteomic subtypes within in the cancer cohort (A, B and C), which were largely distinguished by expression of cell adhesion proteins, including neuronal growth regulator 1 (NEGR1), a potential tumor suppressor. Interestingly, hepatocyte nuclear factor 4-alpha (HNF4A), a transcription factor which is known to be elevated in CRC, was only overexpressed in subtype B. Further analysis of key protein networks related to CRC treatment and immunotherapy development will be presented. Conclusions: High-throughput proteomic profiling of FFPE tissues using SWATH-MS enables the deepest phenotypic characterization of tumor tissue. Ultimately, analyses of this type will enable a functional understanding of interplay between the tumor microenvironment, expression of protein networks and response to immune-directed therapies. Citation Format: Nicholas Dupuis, Jan Muntel, Roland Bruderer, Lukas Reiter. Expanding insights into the colorectal cancer tumor proteome; unbiased protein profiling reveals multiple proteomic-based tumor subtypes [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A066.

There is evidence that the obesity phenotype in the Caucasian populations is associated with variations in several genes, including neuronal growth regulator 1 (NEGR1), SEC16 homolog B (SCE16B), transmembrane protein 18 (TMEM18), ets variant 5 (ETV5), glucosamine-6-phosphate deaminase 2 (GNPDA2), prolactin (PRL), brain-derived neurotrophic factor (BDNF), mitochondrial carrier homolog 2 (MTCH2), Fas apoptotic inhibitory molecule 2 (FAIM2), SH2B adaptor protein 1 (SH2B1), v-maf musculoaponeurotic fibrosarcoma oncogene homolog (MAF), Niemann-Pick disease, type C1 (NPC1), melanocortin 4 receptor (MC4R) and potassium channel tetramerisation domain containing 15 (KCTD15). To investigate the relationship between obesity and these genes in the Japanese population, we genotyped 27 single-nucleotide polymorphisms (SNPs) in 14 genes from obese subjects (n=1129, body mass index (BMI) > or =30 kg m(-2)) and normal-weight control subjects (n=1736, BMI

Kilon is a member of the IgLON family belonging to the immunoglobulin superfamily of cell adhesion molecules. In the present study, we investigated temporal and spatial changes of Kilon expression and its modulatory functions for synapse number using hippocampal cultured neurons. Kilon was observed to localize chiefly at axons and presynaptic terminals at early culture stage, however, it was seen mainly at dendritic postsynaptic spine of mature neurons at late culture stages. Kilon was solubilized with detergent treatment at early culture stages, while it resisted to extraction of the detergent in mature neurons. The overexpression of Kilon gene using a plasmid vector decreased the number of dendritic synapses at early culture stages, whereas the overexpression increased the number of dendritic synapses at late culture. These results demonstrate the alteration of modulatory function of Kilon for the number of dendritic synapses concomitant with changes in its localization and detergent solubility during neuronal culture development.

IgLON proteins are GPI anchored adhesion molecules that control neurite outgrowth. In particular, Negr1 down-regulation negatively influences neuronal arborization in vitro and in vivo. In the present study, we found that the metalloprotease ADAM10 releases Negr1 from neuronal membrane. Ectodomain shedding influences several neuronal mechanisms, includingsurvival, synaptogenesis and the formation of neurite trees. By combining morphological analysis and virus-mediated selective protein silencing in primary murine cortical neurons, we found that pharmacologically inhibition of ADAM10 results in an impairment of neurite tree maturation that can be rescued upon treatment with soluble Negr1. Furthermore, we report that released Negr1 influences neurite outgrowth in a P-ERK1/2 and FGFR2 dependent manner. Together our findings suggest a role for Negr1 in regulating neurite outgrowth through the modulation of FGFR2 signalling pathway. Given the physiological and pathological role of ADAM10, Negr1 and FGFR2, the regulation of Negr1 shedding may play a crucial role in sustaining brain function and development.

The present study aimed to screen several differentially expressed genes (DEGs) and differentially expressed microRNAs (miRNAs) for two types of mesenchymal stem cell (MSC) differentiation. Bone morphogenetic protein 6 (BMP‑6) and dexamethasone were used to induce MSCs towards osteoblastic differentiation or adipocytic differentiation. The t‑test in the Bioconductor bioinformatics software tool was used to screen DEGs and differentially expressed miRNAs in the two samples. Subsequent gene ontology (GO) and pathway analyses on the DEGs were performed using the GO and Kyoto Encyclopedia of Genes and Genomes databases, respectively; potential target genes for the screened miRNAs were predicted using the TargetScan database. In addition, an interaction network between the DEGs and miRNAs was constructed. Numerous DEGs and miRNAs were screened during osteoblastic and adipocytic differentiation of MSCs. Important pathways, such as glutathione metabolism, pathogenic Escherichia coli infection and Parkinson's disease, and GO terms, including cytoskeletal protein binding and phospholipase inhibitor activity, were enriched in the screened DEGs from MSCs undergoing osteogenic differentiation and adipocytic differentiation. miRNAs, including miRNA (miR)‑382 and miR‑203, and DEGs, including neuronal growth regulator 1 (NEGR1), phosphatidic acid phosphatase 2B (PPAP2B), platelet‑derived growth factor receptor alpha (PDGFRA), interleukin 6 signal transducer (IL6ST) and sortilin 1 (SORT1), were demonstrated to be involved in osteoblastic differentiation. In addition, the downregulated miRNAs (including miR‑495, miR‑376a and miR‑543), the upregulated miR‑106a, the upregulated DEGs, including enabled homolog (ENAH), polypeptide N‑acetylgalactosaminyltransferase 1 and acyl‑CoA synthetase long‑chain family member 1, and the downregulated repulsive guidance molecule family member B and semaphorin SEMA7A were demonstrated to be involved in adipocytic differentiation. The results of the present study suggested that miRNAs (miR‑203 and miR‑382) and DEGs (NEGR1, PPAP2B, PDGFRA, IL6ST and SORT1) may serve pivotal functions in the osteoblastic differentiation of MSCs, whereas miR‑495, which is also involved in osteoblast differentiation and had four targets, including NEGR1, miR‑376a, miR‑543 and ENAH may have crucial roles in adipocytic differentiation of MSCs.

Background Meta-analysis of genome-wide association studies identified obesity-related genetic variants. Due to the pleiotropic effects of related phenotypes, we tested six of these obesity-related genetic variants for their association with physical activity: fat mass and obesity-associated (FTO)(rs9939609)T>A, potassium channel tetramerization domain containing (KCTD15) (rs11084753)G>A, melanocortin receptor4 (MC4R)(rs17782313)T>C, neuronal growth regulator 1 (NEGR1)(rs2815752)A>G, SH2B adapter protein 1 (SH2B1)(rs7498665)A>G, and transmembrane protein18 (TMEM18)(rs6548238)C>T. Method European-American women (n = 263) and men (n = 229) (23.5 ± 0.3 years, 24.6 ± 0.2 kg/m2) were genotyped and completed the Paffenbarger physical activity Questionnaire. Physical activity volume in metabolic energy equivalents [MET]-hour/week was derived from the summed time spent (hour/week) times the given MET value for vigorous, moderate, and light intensity physical activity, and sitting and sleeping, respectively. Multivariable adjusted [(age, sex, and body mass index (BMI)] linear regression tested associations among genotype (dominant/recessive model) and the log of physical activity volume. Result MC4R (rs17782313)T>C explained 1.1 % (p = 0.02), TMEM18(rs6548238)C>T 1.2 % (p = 0.01), and SH2B1 (rs7498665)A>G 0.6 % (p = 0.08) of the variability in physical activity volume. Subjects with the MC4R C allele spent 3.5 % less MET-hour/week than those with the TT genotype (p = 0.02). Subjects with the TMEM18 T allele spent 4.1 % less MET-hour/week than those with the CC genotype (p = 0.01). Finally, subjects with the SH2B1 GG genotype spent 3.6 % less MET-hour/week than A allele carriers (p = 0.08). Conclusion Our findings suggest a shared genetic influence among some obesity-related gene loci and physical activity phenotypes that should be explored further. Physical activity volume differences by genotype have public health importance equating to 11–13 lb weight difference annually.

Interstitial 1p deletions are rare events. Very few cases of 1p31.1p31.3 deletions characterized by variable phenotypes have been reported. No clear genotype-phenotype correlation has been determined yet. We present a child with a de novo interstitial 1p31.1p31.3 deletion, identified by array CGH, associated with intellectual disability and severe language impairment. The deleted region contains 20 OMIM genes, but we focused on GADD45A (MIM 126335; growth arrest- and DNA damage-inducible gene), LRRC7 (MIM 614453; leucine-rich repeat-containing protein 7), and NEGR1 (MIM 613173; neuronal growth regulator 1). We discuss whether these genes play a role in determining the phenotype of our patient in order to investigate the possibility of a genotype-phenotype correlation.

We present two siblings with a partial deletion of chromosome 1p31.1 involving only the neuronal growth regulator 1 (NEGR1) gene. The siblings had a history of neuropsychiatric and behavioral problems, learning difficulties, hypotonia, mild aortic root dilatation, hypermobility, and scoliosis. This is the first clinical report of a microdeletion of chromosome 1p31.1 involving only the NEGR1 gene.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders caused by the presence of an infectious prion protein. The primary site of pathology is the brain characterized by neuroinflammation, astrogliosis, prion fibrils, and vacuolation. The events preceding the observed pathology remain in question. We sought to identify biomarkers in the brain of TSE-infected and aged-matched control mice using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Since the brain proteome is too complex to resolve all proteins using 2D-DIGE, protein samples are initially filtered through either concanavalin A (ConA) or wheat-germ agglutinin (WGA) columns. Four differentially abundant proteins are identified through screening of the two different glycoproteomes: Neuronal growth regulator 1 (NEGR1), calponin-3 (CNN3), peroxiredoxin-6 (Prdx6), and glial fibrillary acidic protein (GFAP). Confirmatory Western blots are performed with samples from TSE-infected and comparative Alzheimer's disease (AD) affected brains and their respective controls from time points throughout the disease courses. The abundance of three of the four proteins increases significantly during later stages of prion disease whereas NEGR1 decreases in abundance. Comparatively, no significant changes are observed in later stages of AD. Our lab is the first to associate the glycosylated NEGR1 protein with prion disease pathology.