NTNG1 Research Articles

Molecular features distinguish ten neuronal types in the mouse superficial superior colliculus.

The superior colliculus (SC) is a midbrain center involved in controlling head and eye movements in response to inputs from multiple sensory modalities. Visual inputs arise from both the retina and visual cortex and converge onto the superficial layer of the SC (sSC). Neurons in the sSC send information to deeper layers of the SC and to thalamic nuclei that modulate visually guided behaviors. Presently, our understanding of sSC neurons is impeded by a lack of molecular markers that define specific cell types. To better understand the identity and organization of sSC neurons, we took a systematic approach to investigate gene expression within four molecular families: transcription factors, cell adhesion molecules, neuropeptides, and calcium binding proteins. Our analysis revealed 12 molecules with distinct expression patterns in mouse sSC: cadherin 7, contactin 3, netrin G2, cadherin 6, protocadherin 20, retinoid-related orphan receptor β, brain-specific homeobox/POU domain protein 3b, Ets variant gene 1, substance P, somatostatin, vasoactive intestinal polypeptide, and parvalbumin. Double labeling experiments, by either in situ hybridization or immunostaining, demonstrated that the 12 molecular markers collectively define 10 different sSC neuronal types. The characteristic positions of these cell types divide the sSC into four distinct layers. The 12 markers identified here will serve as valuable tools to examine molecular mechanisms that regulate development of sSC neuronal types. These markers could also be used to examine the connections between specific cell types that form retinocollicular, corticocollicular, or colliculothalamic pathways. J. Comp. Neurol. 524:2300-2321, 2016. © 2016 Wiley Periodicals, Inc.

Genetic Variations Associated with Brain Disorders: Focus on Synaptic Plasticity and Apoptosis Regulatory Genes in Schizophrenia, Posttraumatic Stress Disorder and Ischemic Stroke

Epidemiologic, clinical and experimental data indicates that a majority of brain disorders including schizophrenia (SCZ), posttraumatic stress disorder (PTSD), and ischemic stroke (IS) are multifactorial disorders with strong and complex genetic component. Identification of all genetic variations associated with these disorders may sufficiently contribute to understanding of their basic pathomechanisms and encourage development of new innovative approaches to their early diagnosis and treatment. The aim of this review article is to provide overview of our recent studies on evaluation of potential association of SCZ, PTSD and IS with functional single nucleotide polymorphisms (SNPs) of synaptic plasticity and apoptosis regulatory genes in Armenian population. Here, our attention was focused on genes encoding netrin G1 (NTNG1), brain-derived neurotrophic factor (BDNF), complexin-2 (CPLX2), nerve growth factor (NGF) and its receptor (NGFR), annexin family proteins - annexin A5 and annexin A11 (ANXAV, ANXA11), and B-cell lymphoma 2 (Bcl-2) family proteins - Bcl-2 proper and Bcl-2-associated X protein (BCL2, BAX). Genomic DNA samples of diseased and healthy individuals were genotyped for a number of SNPs of the mentioned genes using polymerase chain reaction with sequence-specific primers (PCR-SSP). The significance of differences in genotype and allele frequencies and minor allele carriage between patients and healthy control subjects was determined using Pearson’s Chi-square test. P-values less than 0.05 were considered statistically significant. Significant associations were found between: (1) SCZ and BDNF rs6265, CPLX2 rs1366116, rs3892909, NGF rs6330, rs4839435, NGFR rs734194, rs11466155, rs2072446, ANXAV rs11575945, BAX rs1057369 SNPs; (2) PTSD and CPLX2 rs1366116, BCL2 rs956572 SNPs; (3) IS and NTNG1 rs628117, CPLX2 rs1366116, ANXAV rs11575945 SNPs. The obtained results indicated the involvement of genetically determined alterations in synaptic plasticity and apoptosis in pathomechanisms of SCZ, PTSD and IS. The minor T allele of the CPLX2 gene rs1366116 polymorphism represents risk factor for all studied diseased conditions indicating important functional significance of this genetic variation in maintenance of synaptic plasticity. Another important conclusion of these studies is that minor alleles of some polymorphic variants of genes, encoding synaptic plasticity and apoptosis regulatory proteins, may play a protective role relative to SCZ decreasing the risk for development of this disorder. In summary, our studies emphasize the important contribution of changes in synaptic plasticity and apoptosis regulation to pathomechanisms of SCZ, PTSD, and IS as well as significant input of genetic factors to these changes.

FUS-mediated alternative splicing in the nervous system: consequences for ALS and FTLD

Mutations in fused in sarcoma (FUS) in a subset of patients with amyotrophic lateral sclerosis (ALS) linked this DNA/RNA-binding protein to neurodegeneration. Most of the mutations disrupt the nuclear localization signal which strongly suggests a loss-of-function pathomechanism, supported by cytoplasmic inclusions. FUS-positive neuronal cytoplasmic inclusions are also found in a subset of patients with frontotemporal lobar degeneration (FTLD). Here, we discuss recent data on the role of alternative splicing in FUS-mediated pathology in the central nervous system. Several groups have shown that FUS binds broadly to many transcripts in the brain and have also identified a plethora of putative splice targets; however, only ABLIM1, BRAF, Ewing sarcoma protein R1 (EWSR1), microtubule-associated protein tau (MAPT), NgCAM cell adhesion molecule (NRCAM), and netrin G1 (NTNG1) have been identified in at least three of four studies. Gene ontology analysis of all putative targets unanimously suggests a role in axon growth and cytoskeletal organization, consistent with the altered morphology of dendritic spines and axonal growth cones reported upon loss of FUS. Among the axonal targets, MAPT/tau and NTNG1 have been further validated in biochemical studies. The next challenge will be to confirm changes of FUS-mediated alternative splicing in patients and define their precise role in the pathophysiology of ALS and FTLD.

Molecular and cell biological effects of 3,5,3′-triiodothyronine on progenitor cells of the enteric nervous system in vitro

IntroductionThyroid hormones play important roles in the development of neural cells in the central nervous system. Even minor changes to normal thyroid hormone levels affect dendritic and axonal outgrowth, sprouting and myelination and might even lead to irreversible damages such as cretinism. Despite our knowledge of the influence on the mammalian CNS, the role of thyroid hormones in the development of the enteric nervous system (ENS) still needs to be elucidated.In this study we have analyzed for the first time the influence of 3,5,3′-triiodothyronine (T3) on ENS progenitor cells using cell biological assays and a microarray technique. ResultsIn our in vitro model, T3 inhibited cell proliferation and stimulated neurite outgrowth of differentiating ENS progenitor cells. Microarray analysis revealed a group of 338 genes that were regulated by T3 in differentiating enterospheres. 67 of these genes are involved in function and development of the nervous system. 14 of them belong to genes that are involved in axonal guidance or neurite outgrowth. Interestingly, T3 regulated the expression of netrin G1 and endothelin 3, two guidance molecules that are involved in human enteric dysganglionoses. ConclusionThe results of our study give first insights how T3 may affect the enteric nervous system. T3 is involved in proliferation and differentiation processes in enterospheres. Microarray analysis revealed several interesting gene candidates that might be involved in the observed effects on enterosphere differentiation. Future studies need to be conducted to better understand the gene to gene interactions.

The netrin G1 gene rs628117 polymorphism is associated with ischemic stroke

Recent studies demonstrated that naturally occurring genetic alterations in synaptic plasticity-related genes may influence both stroke progression and poor functional recovery after stroke. Netrin G1 is an axonal protein involved in synaptic plasticity. In the present study, we evaluated the potential association of the netrin G1 gene rs628117 single nucleotide polymorphism with ischemic stroke in an Armenian population. In total, 127 patients with ischemic stroke and 128 healthy subjects (controls) were involved in this study. Genomic DNA samples of ischemic stroke patients and controls were genotyped for netrin G1 gene (NTNG1) rs628117 single nucleotide polymorphism using polymerase chain reaction with sequence-specific primers. Data were analyzed by Pearson's χ2 test. The results obtained implicated rs628117 single nucleotide polymorphism of NTNG1 in pathogenesis of ischemic stroke. In particular, it was shown that the NTNG1 rs628117*G minor allele is positively associated with ischemic stroke and the carriers of this allele were overrepresented in ischemic stroke patients compared with controls. Our finding nominates the minor G allele of the NTNG1 rs628117 single nucleotide polymorphism as a risk factor for ischemic stroke at least in Armenian population.

ITRAQ and multiple reaction monitoring as proteomic tools for biomarker search in cerebrospinal fluid of patients with Parkinson's disease dementia

About 30% of patients with Parkinson's disease (PD) develop Parkinson's disease dementia (PDD) in the course of the disease. Until now, diagnosis is based on clinical and neuropsychological examinations, since so far there is no laboratory marker.In this study we aimed to find a neurochemical marker which would allow a risk assessment for the development of a dementia in PD patients. For this purpose, we adopted a gel-free proteomic approach (iTRAQ-method) to identify biomarker-candidates in the cerebrospinal fluid (CSF) of patients with PD, PDD and non-demented controls (NDC). Validation of these candidates was then carried out by multiple-reaction-monitoring (MRM) optimised for CSF.Using the iTRAQ-approach, we were able to identify 16 differentially regulated proteins. Fourteen out of these 16 proteins could then be followed-up simultaneously in our optimised MRM-measurement protocol.However only Tyrosine-kinase-non-receptor-type 13 and Netrin-G1 differed significantly between PDD and NDC cohorts. In addition, a significant difference was found for Golgin-160 and Apolipoprotein B-100 between PD and NDC.Apart from possible pathophysiological considerations, we propose that Tyrosine-kinase non-receptor-type 13 and Netrin G1 are biomarker candidates for the development of a Parkinson's disease dementia. Furthermore we suggest that iTRAQ and MRM are valuable tools for the discovery of biomarker in cerebrospinal fluid. However further validation studies need to be done with larger patient cohorts and other proteins need to be checked as well.

Crystal Structure of the Ligand Binding Domain of Netrin G2

Netrin G proteins represent a small family of synaptic cell adhesion molecules related to netrins and to the polymerization domains of laminins. Two netrin G proteins are encoded in vertebrate genomes, netrins G1 and G2, which are known to bind the leucine-rich repeat proteins netrin G ligand (NGL)-1 and NGL-2, respectively. Netrin G proteins share a common multi-domain architecture comprising a laminin N-terminal (LN) domain followed by three laminin epidermal growth factor-like (LE) domains and a C′ region containing a glycosylphosphatidylinositol anchor. Here, we use deletion analysis to show that the LN domain region of netrin Gs contains the binding site for NGLs to which they bind with 1:1 stoichiometry and sub-micromolar affinity. Netrin Gs are alternatively spliced in their LE domain regions, but the binding region, the LN domain, is identical in all splice forms. We determined the crystal structure for a fragment comprising the LN domain and domain LE1 of netrin G2 by sulfur single-wavelength anomalous diffraction phasing and refined it to 1.8 Å resolution. The structure reveals an overall architecture similar to that of laminin α chain LN domains but includes significant differences including a Ca2+ binding site in the LN domain. These results reveal the minimal binding unit for interaction of netrin Gs with NGLs, define structural features specific to netrin Gs, and suggest that netrin G alternative splicing is not involved in NGL recognition.

Curcumin is a potent modulator of microglial gene expression and migration

BackgroundMicroglial cells are important effectors of the neuronal innate immune system with a major role in chronic neurodegenerative diseases. Curcumin, a major component of tumeric, alleviates pro-inflammatory activities of these cells by inhibiting nuclear factor kappa B (NFkB) signaling. To study the immuno-modulatory effects of curcumin on a transcriptomic level, DNA-microarray analyses were performed with resting and LPS-challenged microglial cells after short-term treatment with curcumin.MethodsResting and LPS-activated BV-2 cells were stimulated with curcumin and genome-wide mRNA expression patterns were determined using DNA-microarrays. Selected qRT-PCR analyses were performed to confirm newly identified curcumin-regulated genes. The migration potential of microglial cells was determined with wound healing assays and transwell migration assays. Microglial neurotoxicity was estimated by morphological analyses and quantification of caspase 3/7 levels in 661W photoreceptors cultured in the presence of microglia-conditioned medium.ResultsCurcumin treatment markedly changed the microglial transcriptome with 49 differentially expressed transcripts in a combined analysis of resting and activated microglial cells. Curcumin effectively triggered anti-inflammatory signals as shown by induced expression of Interleukin 4 and Peroxisome proliferator activated receptor α. Several novel curcumin-induced genes including Netrin G1, Delta-like 1, Platelet endothelial cell adhesion molecule 1, and Plasma cell endoplasmic reticulum protein 1, have been previously associated with adhesion and cell migration. Consequently, curcumin treatment significantly inhibited basal and activation-induced migration of BV-2 microglia. Curcumin also potently blocked gene expression related to pro-inflammatory activation of resting cells including Toll-like receptor 2 and Prostaglandin-endoperoxide synthase 2. Moreover, transcription of NO synthase 2 and Signal transducer and activator of transcription 1 was reduced in LPS-triggered microglia. These transcriptional changes in curcumin-treated LPS-primed microglia also lead to decreased neurotoxicity with reduced apoptosis of 661W photoreceptor cultures.ConclusionsCollectively, our results suggest that curcumin is a potent modulator of the microglial transcriptome. Curcumin attenuates microglial migration and triggers a phenotype with anti-inflammatory and neuroprotective properties. Thus, curcumin could be a nutraceutical compound to develop immuno-modulatory and neuroprotective therapies for the treatment of various neurodegenerative disorders.

Functional variants of the genes involved in neurodevelopment and susceptibility to schizophrenia in an Armenian population

Aberrant neurodevelopment contributes to the etiology of schizophrenia. This study aimed to investigate the potential association of netrin G1 ( NTNG1) rs628117 and brain-derived neurotrophic factor (BDNF) Val66Met (rs6265) genetic polymorphisms with susceptibility to schizophrenia. One hundred three Armenian patients with schizophrenia and 105 healthy control subjects were genotyped by polymerase chain reaction with sequence-specific primers. Whereas the NTNG1 rs628117 genotypes were equally distributed in the groups, the carriers of the less common BDNF 66Met allele were overrepresented among patients with schizophrenia when compared with healthy controls (55% vs 35%, odds ratio = 2.28, 95% confidence interval 1.14–1.98, p corrected = 0.006). Furthermore, the 66Met/Met genotype correlated with earlier disease onset ( p = 0.024). In conclusion, our single-cohort study nominates the BDNF 66Met allele as a risk factor for schizophrenia in an Armenian population. This must be confirmed in other Armenian cohorts.

Nonobese Diabetic Congenic Strain Analysis of Autoimmune Diabetes Reveals Genetic Complexity of the Idd18 Locus and Identifies Vav3 as a Candidate Gene

We have used the public sequencing and annotation of the mouse genome to delimit the previously resolved type 1 diabetes (T1D) insulin-dependent diabetes (Idd)18 interval to a region on chromosome 3 that includes the immunologically relevant candidate gene, Vav3. To test the candidacy of Vav3, we developed a novel congenic strain that enabled the resolution of Idd18 to a 604-kb interval, designated Idd18.1, which contains only two annotated genes: the complete sequence of Vav3 and the last exon of the gene encoding NETRIN G1, Ntng1. Targeted sequencing of Idd18.1 in the NOD mouse strain revealed that allelic variation between NOD and C57BL/6J (B6) occurs in noncoding regions with 138 single nucleotide polymorphisms concentrated in the introns between exons 20 and 27 and immediately after the 3' untranslated region. We observed differential expression of VAV3 RNA transcripts in thymocytes when comparing congenic mouse strains with B6 or NOD alleles at Idd18.1. The T1D protection associated with B6 alleles of Idd18.1/Vav3 requires the presence of B6 protective alleles at Idd3, which are correlated with increased IL-2 production and regulatory T cell function. In the absence of B6 protective alleles at Idd3, we detected a second T1D protective B6 locus, Idd18.3, which is closely linked to, but distinct from, Idd18.1. Therefore, genetic mapping, sequencing, and gene expression evidence indicate that alteration of VAV3 expression is an etiological factor in the development of autoimmune beta-cell destruction in NOD mice. This study also demonstrates that a congenic strain mapping approach can isolate closely linked susceptibility genes.

Association of polymorphisms in the haplotype block spanning the alternatively spliced exons of the NTNG1 gene at 1p13.3 with schizophrenia in Japanese populations

Chromosome 1p13 is linked with schizophrenia in Japanese families, and one of the candidate genes in this region is the netrin G1 (NTNG1) gene at 1p13.3. Associations of 56 tag single-nucleotide polymorphisms (SNPs) with schizophrenia were explored by transmission disequilibrium analysis in 160 Japanese trios and by case-control analysis in 2,174 Japanese cases and 2,054 Japanese controls. An association between SNP rs628117 and schizophrenia was identified by case-control comparison (nominal allelic p=0.0009; corrected p=0.006). The associated polymorphism is located in intron 9 and in the haplotype block encompassing the alternatively spliced exons of the gene. Allelic association of a different SNP in the same haplotype block in Japanese families was previously reported. These findings support that the NTNG1 gene is associated with schizophrenia in the Japanese.

Netrin G1 Mutations Are an Uncommon Cause of Atypical Rett Syndrome With or Without Epilepsy

Mutations in the methyl-cytosine-phosphate-guanosine dinucleotide (CpG) binding protein 2 gene are identified in up to 90% of patients with classic Rett syndrome. However, the lack of methyl-CpG binding protein 2 mutations in a small group of classic Rett syndrome cases, and the low frequency of these mutations in atypical Rett syndrome patients, suggest that other gene defects may play a role in this disorder. One report described a patient with atypical Rett syndrome who presented with early epilepsy and a de novo translocation which disrupted the Netrin G1 gene. This study tested a sample of 91 female patients with a clinically heterogeneous phenotype ranging from encephalopathy with epilepsy to atypical Rett syndrome without epilepsy for mutations in the Netrin G1 gene, to evaluate its involvement in this condition. Nine sequence variations (including six novel variations) were identified, all of which were unlikely to be pathogenic. One was a novel C to G transversion, resulting in a p.Leu537Val amino-acid substitution in one patient. The same substitution was detected in the asymptomatic mother, suggesting an absence of biological significance. Our study suggests that Netrin G1 is not involved in atypical Rett syndrome or in unexplained encephalopathy with epilepsy, but in specific forms to be delineated better in the future.

Interstitial deletion of the short arm of chromosome 1 (1p13.1p21.1) in a girl with mental retardation, short stature and colobomata

Interstitial deletions on the short arm of chromosome 1 are rare. We describe a girl with severe mental retardation, short stature and dysmorphic features including colobomata where high-resolution comparative genomic hybridization revealed an interstitial deletion with breakpoints in band 1p13.1 and 1p21.1. The deletion was further characterized by real-time polymerase chain reaction. We hypothesize that haploinsufficiency of WNT2B (wingless-type MMTV integration site family, member 2B) and NTNG1 (Netrin G1) contributed to the patient's phenotype.

NTNG1 mutations are a rare cause of Rett syndrome

A translocation that disrupted the netrin G1 gene (NTNG1) was recently reported in a patient with the early seizure variant of Rett syndrome (RTT). The netrin G1 protein (NTNG1) has an important role in the developing central nervous system, particularly in axonal guidance, signalling and NMDA receptor function and was a good candidate gene for RTT. We recruited 115 patients with RTT (females: 25 classic and 84 atypical; 6 males) but no mutation in the MECP2 gene. For those 52 patients with epileptic seizure onset in the first 6 months of life, CDKL5 mutations were also excluded. We aimed to determine whether mutations in NTNG1 accounted for a significant subset of patients with RTT, particularly those with the early onset seizure variant and other atypical presentations. We sequenced the nine coding exons of NTNG1 and identified four sequence variants, none of which were likely to be pathogenic. Mutations in the NTNG1 gene appear to be a rare cause of RTT but NTNG1 function demands further investigation in relation to the central nervous system pathophysiology of the disorder.

Disruption of Netrin G1 by a balanced chromosome translocation in a girl with Rett syndrome

We have identified a girl with characteristic features of Rett syndrome (RTT) who carries a de novo balanced translocation involving chromosomes 1 and 7. Both breakpoints were mapped by fluorescence in situ hybridization with selected genomic clones from the regions of interest. Southern blot hybridisations, utilizing probes derived from breakpoint spanning BACs, detected several aberrant fragments specific for the patient. Sequence analysis of the cloned junction fragment indicated that on chromosome 1 the predominantly brain-expressed Netrin G1 (NTNG1) gene is disrupted, whereas on chromosome 7 there was no indication for a truncated gene. The chromosome 1 breakpoint lies within the 3' part of NTNG1 and affects alternatively spliced transcripts, suggesting that the phenotype in this patient is the result of disturbed NTNG1 expression. In silico translation of the NTNG1 splice variants predicted protein isoforms with different C-termini: one membrane bound through a glycosylphosphatidylinositol anchor and the other soluble. The membrane-bound protein isoform would be affected by the breakpoint, whereas the soluble form would remain intact. Our results suggest that the central nervous system is sensitive to NTNG1 expression levels and that NTNG1 is a novel candidate disease gene for RTT.