Neuron Navigator Research Articles

Concerted transcriptional regulation of the morphogenesis of hypothalamic neurons by ONECUT3

Acquisition of specialized cellular features is controlled by the ordered expression of transcription factors (TFs) along differentiation trajectories. Here, we find a member of the Onecut TF family, ONECUT3, expressed in postmitotic neurons that leave their Ascl1+/Onecut1/2+ proliferative domain in the vertebrate hypothalamus to instruct neuronal differentiation. We combined single-cell RNA-seq and gain-of-function experiments for gene network reconstruction to show that ONECUT3 affects the polarization and morphogenesis of both hypothalamic GABA-derived dopamine and thyrotropin-releasing hormone (TRH)+ glutamate neurons through neuron navigator-2 (NAV2). In vivo, siRNA-mediated knockdown of ONECUT3 in neonatal mice reduced NAV2 mRNA, as well as neurite complexity in Onecut3-containing neurons, while genetic deletion of Onecut3/ceh-48 in C. elegans impaired neurocircuit wiring, and sensory discrimination-based behaviors. Thus, ONECUT3, conserved across neuronal subtypes and many species, underpins the polarization and morphological plasticity of phenotypically distinct neurons that descend from a common pool of Ascl1+ progenitors in the hypothalamus.

Microtubule-associated NAV3 regulates invasive phenotypes in glioblastoma cells.

Glioblastomas are aggressive brain tumors for which effective therapy is still lacking, resulting in dismal survival rates. These tumors display significant phenotypic plasticity, harboring diverse cell populations ranging from tumor core cells to dispersed, highly invasive cells. Neuron navigator 3 (NAV3), a microtubule-associated protein affecting microtubule growth and dynamics, is downregulated in various cancers, including glioblastoma, and has thus been considered a tumor suppressor. In this study, we challenge this designation and unveil distinct expression patterns of NAV3 across different invasion phenotypes. Using glioblastoma cell lines and patient-derived glioma stem-like cell cultures, we disclose an upregulation of NAV3 in invading glioblastoma cells, contrasting with its lower expression in cells residing in tumor spheroid cores. Furthermore, we establish an association between low and high NAV3 expression and the amoeboid and mesenchymal invasive phenotype, respectively, and demonstrate that overexpression of NAV3 directly stimulates glioblastoma invasive behavior in both 2D and 3D environments. Consistently, we observed increased NAV3 expression in cells migrating along blood vessels in mouse xenografts. Overall, our results shed light on the role of NAV3 in glioblastoma invasion, providing insights into this lethal aspect of glioblastoma behavior.

Variants of NAV3, a neuronal morphogenesis protein, cause intellectual disability, developmental delay, and microcephaly

Microtubule associated proteins (MAPs) are widely expressed in the central nervous system, and have established roles in cell proliferation, myelination, neurite formation, axon specification, outgrowth, dendrite, and synapse formation. We report eleven individuals from seven families harboring predicted pathogenic biallelic, de novo, and heterozygous variants in the NAV3 gene, which encodes the microtubule positive tip protein neuron navigator 3 (NAV3). All affected individuals have intellectual disability (ID), microcephaly, skeletal deformities, ocular anomalies, and behavioral issues. In mouse brain, Nav3 is expressed throughout the nervous system, with more prominent signatures in postmitotic, excitatory, inhibiting, and sensory neurons. When overexpressed in HEK293T and COS7 cells, pathogenic variants impaired NAV3 ability to stabilize microtubules. Further, knocking-down nav3 in zebrafish led to severe morphological defects, microcephaly, impaired neuronal growth, and behavioral impairment, which were rescued with co-injection of WT NAV3 mRNA and not by transcripts encoding the pathogenic variants. Our findings establish the role of NAV3 in neurodevelopmental disorders, and reveal its involvement in neuronal morphogenesis, and neuromuscular responses.

Mutated neuron navigator 3 as a candidate gene for a rare neurodevelopmental disorder.

Neuron navigator 3 (NAV3) is characterized as one of the neuron navigator family (NAV1, NAV2, NAV3) proteins predominantly expressed in the nervous system. The NAV3-encoded protein comprises a conserved AAA and coiled-coil domains characteristic of ATPases, which are associated with different cellular activities. We describe a Saudi proband presenting a complex recessive neurodevelopmental disorder (NDD). Whole exome sequencing (WES) followed by Sanger sequencing, 3D protein modeling and RT-qPCR was performed. WES revealed a bi-allelic frameshift variant (c.2604_2605delAG; p.Val870SerfsTer12) in exon 12 of the NAV3 gene. Furthermore, RT-qPCR revealed a significant decrease in the NAV3 mRNA expression in the patient sample, and 3D protein modeling revealed disruption of the overall secondary structure. For the time, we associate a bi-allelic variant in the NAV3 gene causing NDD in humans.

Landscape of extrachromosomal circular DNAs, transcriptome, and proteome analysis reveals insights into alcoholic liver cirrhosis

Alcoholic liver cirrhosis (ALC) is a result of excessive and chronic alcohol consumption. Because alchol can cause DNA damage, extrachromosomal circular DNA (eccDNA) was investigated in ALC liver due to it can be a result of DNA damage. Considering eccDNA has ability to lead to genomic instability as an enhancer of gene transcription, we utilized Circle-Seq to identify differences in eccDNA profiles and gene expression patterns in liver samples obtained from ALC patients (n = 3) and healthy controls (n = 3) to investigate the role of eccDNA in the development of ALC. The abundance of eccDNA in ALC (mean = 13,349) were higher than the healthy control (mean = 11,557) without significant difference (pvalue = 0.6530). We observed 1,032 eccDNA containing genes showed higher expression in ALC patients compared to healthy controls (p < 0.05, log2FC > 1). Notably, we discovered seven genes that exhibited a significant positive correlation between eccDNA abundance and gene expression levels. These genes include A disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS2), Voltage-dependent L-type calcium channel subunit alpha-1C (CACNA1C), Protein TANC1 (TANC1), Integrin alpha-2 (ITGA2), EH domain-containing protein 4 (EHD4), Phosphofurin acidic cluster sorting protein 1 (PACS1), and Neuron navigator 2 (NAV2). Through mass spectrometry proteomics, ITGA2 were found to have significantly higher abbudance in ALC. Integrins are a family of proteins plays key roles in the fibrosis development of liver. Thus, our study opens a new perspective for liver fibrosis development.

Identification of novel genes including NAV2 associated with isolated tall stature.

Very tall people attract much attention and represent a clinically and genetically heterogenous group of individuals. Identifying the genetic etiology can provide important insights into the molecular mechanisms regulating linear growth. We studied a three-generation pedigree with five isolated (non-syndromic) tall members and one individual with normal stature by whole exome sequencing; the tallest man had a height of 211cm. Six heterozygous gene variants predicted as damaging were shared among the four genetically related tall individuals and not present in a family member with normal height. To gain insight into the putative role of these candidate genes in bone growth, we assessed the transcriptome of murine growth plate by microarray and RNA Seq. Two (Ift140, Nav2) of the six genes were well-expressed in the growth plate. Nav2 (p-value 1.91E-62) as well as Ift140 (p-value of 2.98E-06) showed significant downregulation of gene expression between the proliferative and hypertrophic zone, suggesting that these genes may be involved in the regulation of chondrocyte proliferation and/or hypertrophic differentiation. IFT140, NAV2 and SCAF11 have also significantly associated with height in GWAS studies. Pathway and network analysis indicated functional connections between IFT140, NAV2 and SCAF11 and previously associated (tall) stature genes. Knockout of the all-trans retinoic acid responsive gene, neuron navigator 2 NAV2, in Xenopus supports its functional role as a growth promotor. Collectively, our data expand the spectrum of genes with a putative role in tall stature phenotypes and, among other genes, highlight NAV2 as an interesting gene to this phenotype.

Transcranial direct current stimulation of the right temporoparietal junction facilitates hippocampal spatial learning in Alzheimer’s disease and mild cognitive impairment

ObjectiveSpatial memory deficits are an early symptom in Alzheimer’s disease (AD), reflecting the neurodegenerative processes in the neuronal navigation network such as in hippocampal and parietal cortical areas. As no effective treatment options are available, neuromodulatory interventions are increasingly evaluated. Against this backdrop, we investigated the neuromodulatory effect of anodal transcranial direct current stimulation (tDCS) on hippocampal place learning in patients with AD or mild cognitive impairment (MCI). MethodsIn this randomized, double-blind, sham-controlled study with a cross-over design anodal tDCS of the right temporoparietal junction (2 mA for 20 minutes) was applied to 20 patients diagnosed with AD or MCI and in 22 healthy controls while they performed a virtual navigation paradigm testing hippocampal place learning. ResultsWe show an improved recall performance of hippocampal place learning after anodal tDCS in the patient group compared to sham stimulation but not in the control group. ConclusionsThese results suggest that tDCS can facilitate spatial memory consolidation via stimulating the parietal-hippocampal navigation network in AD and MCI patients. SignificanceOur findings suggest that tDCS of the temporoparietal junction may restore spatial navigation and memory deficits in patients with AD and MCI.

Neuron Navigator 1 (Nav1) regulates the response to cocaine in mice

Genetic variation accounts for much of the risk for developing a substance use disorder, but the underlying genetic factors and their genetic effector mechanisms are mostly unknown. Inbred mouse strains exhibit substantial and heritable differences in the extent of voluntary cocaine self-administration. Computational genetic analysis of cocaine self-administration data obtained from twenty-one inbred strains identified Nav1, a member of the neuron navigator family that regulates dendrite formation and axonal guidance, as a candidate gene. To test this genetic hypothesis, we generated and characterized Nav1 knockout mice. Consistent with the genetic prediction, Nav1 knockout mice exhibited increased voluntary cocaine intake and had increased motivation for cocaine consumption. Immunohistochemistry, electrophysiology, and transcriptomic studies were performed as a starting point for investigating the mechanism for the Nav1 knockout effect. Nav1 knockout mice had a reduced inhibitory synapse density in their cortex, increased excitatory synaptic transmission in their cortex and hippocampus, and increased excitatory neurons in a deep cortical layer. Collectively, our results indicate that Nav1 regulates the response to cocaine, and we identified Nav1 knockout induced changes in the excitatory and inhibitory synaptic balance in the cortex and hippocampus that could contribute to this effect.

Netrin-1 blockade inhibits tumor associated Myeloid-derived suppressor cells, cancer stemness and alleviates resistance to chemotherapy and immune checkpoint inhibitor.

Drug resistance and cancer relapse represent significant therapeutic challenges after chemotherapy or immunotherapy, and a major limiting factor for long-term cancer survival. Netrin-1 was initially identified as a neuronal navigation cue but has more recently emerged as an interesting target for cancer therapy, which is currently clinically investigated. We show here that netrin-1 is an independent prognostic marker for clinical progression of breast and ovary cancers. Cancer stem cells (CSCs)/Tumor initiating cells (TICs) are hypothesized to be involved in clinical progression, tumor relapse and resistance. We found a significant correlation between netrin-1 expression and cancer stem cell (CSC) markers levels. We also show in different mice models of resistance to chemotherapies that netrin-1 interference using a therapeutic netrin-1 blocking antibody alleviates resistance to chemotherapy and triggers an efficient delay in tumor relapse and this effect is associated with CSCs loss. We also demonstrate that netrin-1 interference limits tumor resistance to immune checkpoint inhibitor and provide evidence linking this enhanced anti-tumor efficacy to a decreased recruitment of a subtype of myeloid-derived suppressor cells (MDSCs) called polymorphonuclear (PMN)-MDSCs. We have functionally demonstrated that these immune cells promote CSCsfeatures and, consequently, resistance to anti-cancer treatments. Together, these data support the view of both a direct and indirect contribution of netrin-1 to cancer stemness and we propose that this may lead to therapeutic opportunities by combining conventional chemotherapies and immunotherapies with netrin-1 interfering drugs.

Revealing the correlations between brain cortical characteristics and susceptibility genes for Alzheimer disease: a cross-sectional study.

Alzheimer disease (AD) is a progressive neurodegenerative disease closely related to genes and characterized by the atrophy of the cerebral cortex. Correlations between imaging phenotypes and the susceptibility genes for AD, as demonstrated in the findings of genome-wide association studies (GWASs), still need to be addressed due to the complicated structure of the human cortex. In our study, an improved GWAS method, whole cortex characteristics GWAS (WCC-GWAS), was proposed. The WCC-GWAS uses multiple cortex characteristics of gray-matter volume (GMV), cortical thickness (CT), cortical surface area (CSA), and local gyrification index (LGI). A cohort of 496 participants was enrolled and divided into 4 groups: normal control (NC; n=122), early mild cognitive impairment (EMCI; n=196), late mild cognitive impairment (LMCI; n=62), and AD (n=116). Based on the Desikan-Killiany atlas, the brain was parcellated into 68 brain regions, and the WCC of each brain region was individually calculated. Four cortex characteristics of GMV, CT, CSA, and LGI across the 4 groups optimized with multiple comparisons and the ReliefF algorithm were taken as magnetic resonance imaging (MRI) brain phenotypes. Under the model of multiple linear additive genetic regression, the correlations between the MRI brain phenotypes and single-nucleotide polymorphisms (SNPs) were deduced. The findings identified 2 prominent correlations. First, rs7309929 of neuron navigator 3 (NAV3) located on chromosome 12 correlated with the decreased GMV for the left middle temporal gyrus (P=0.0074). Second, rs11250992 of long intergenic non-protein-coding RNA 700 (LINC00700) located on chromosome 10 correlated with the decreased CT for the left supramarginal gyrus (P=0.0019). The findings suggested that the correlations between phenotypes and genotypes could be effectively evaluated. The strategy of extracting MRI phenotypes as endophenotypes provided valuable indications in AD GWAS.

Tumour Suppressor Neuron Navigator 3 and Matrix Metalloproteinase 14 are Co-expressed in Most Melanomas but Downregulated in Thick Tumours.

Melanoma is a highly metastatic tumour originating from neural crest-derived melanocytes. The aim of this study was to analyse the expression of neuron navigator 3 (NAV3) in relation to membrane type-1 matrix metalloproteinase MMP14, a major regulator of invasion, in 40 primary melanomas, 15 benign naevi and 2 melanoma cell lines. NAV3 copy number changes were found in 18/27 (67%) primary melanomas, so that deletions dominated (16/27 of samples, 59%). NAV3 protein was found to be localized at the leading edge of migrating melanoma cells in vitro. Silencing of NAV3 reduced both melanoma cell migration in 2-dimensional conditions, as well as sprouting in 3-dimensional collagen I. NAV3 protein expression correlated with MMP14 in 26/37 (70%) primary melanomas. NAV3 and MMP14 were co-expressed in all tumours with Breslow thickness < 1 mm, in 11/23 of mid-thickness tumours (1-5 mm), but in only 1/6 samples of thick (> 5 mm) melanomas. Altogether, NAV3 number changes are frequent in melanomas, and NAV3 and MMP14, while expressed in all thin melanomas, are often downregulated in thicker tumours, suggesting that the lack of both NAV3 and MMP14 favours melanoma progression.

NAV3 is a genetic determinant of myocardial recovery in dilated cardiomyopathy and attenuates cardiac fibrosis

Abstract Background A genome-wide association study (GWAS) assessing change in left ventricular ejection fraction (LVEF) in dilated cardiomyopathy (DCM), a surrogate marker of morbidity and mortality in heart failure (HF), had not been performed previously and could provide insight into novel biological pathways that could lead to the development of new drugs that might target myocardial recovery. The presence and extent of cardiac fibrosis in DCM is independently associated with myocardial recovery and cardiovascular mortality. Purpose To identify the biological relevance of genetic targets that are associated with change in LVEF in patients with DCM. Methods A GWAS was performed using DNA from 686 patients with recent onset DCM who were on standard HF therapy using change in LVEF at a median of 6 months after initial diagnosis. Cultured human cardiac fibroblasts (HCFs) were used as an in vitro model to study the functional and biological relevance of the gene target identified in the GWAS. Specifically, HCFs were transfected with siRNA by using the Lipofectamine™ RNAiMAX Transfection Reagent for gene knockdown (KD). RNA-seq was performed using the Illumina TruSeq protocol with expression analysis conducted with the EdgeR package. Ingenuity Pathway Analysis was used. Results A single-nucleotide polymorphism, rs11105445(G&amp;gt;A), mapping to the neuron navigator 3 (NAV3) gene (rs11105445, p=2.37E-07; beta 2.74±0.53) was associated with improvement in LVEF. We performed a phenome-wide association study using data from the UK Biobank and demonstrated that genetic variation in NAV3 was significantly associated with HF mortality (p=3.2E-28), highlighting the potential importance of this gene in HF. Using GTEx data we demonstrated that in LV tissue the minor allele A was associated with ↓NAV3 expression (p=0.03) suggesting that ↓NAV3 expression might be associated with improvement in LVEF. We demonstrated that NAV3 KD significantly ↓TGF-β1 mediated HCF transdifferentiation into myofibroblasts, ↓α-smooth muscle actin (ACTA2) and ↓collagen I (COL1A1), therefore NAV3 KD was anti-fibrotic (see Figure 1), 1a. HCFs treated by vehicle/TGF-β1 after KD of NAV3/ctrl, and ACTA2 and COL1A1 were analyzed by qPCR; 1b. Representative immunofluorescence staining for α-SMA (in green), RNA-seq after NAV3 KD followed by pathway analysis suggested that NAV3 exerted its effect by regulating cell cycle related proteins (Figure 2), 2a. Volcano plot shows significant differentially expressed genes identified by RNA-seq analysis (down-regulated in blue, up-regulated in red); 2b. NAV3 KD significantly increased expression of cell cycle related proteins, which was validated by Western blot. Conclusions Decreased expression of NAV3 is associated with myocardial recovery in DCM, most likely due to its anti-fibrotic effect via direct regulation of cell cycle proteins. The role of NAV3 as a novel therapeutic target in DCM needs to be explored. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Mayo Clinic Center for Individualized MedicineMayo Clinic Department of Cardiovascular Medicine

342. GENOMIC ANALYSIS OF RESPONSE TO NEOADJUVANT CHEMOTHERAPY IN OESOPHAGEAL ADENOCARCINOMA

Abstract Oesophageal adenocarcinoma (OAC) is the ninth most common cancer worldwide, with a mortality of &amp;gt;500,000 deaths yearly. Neoadjuvant chemotherapy (NAC) followed by surgery is the standard of care (SOC) for locally advanced OAC. Although almost all patients receive chemotherapy as SOC, but &amp;lt;20% respond and benefit from chemotherapy before surgery. The OAC genome is complex and heterogeneous between patients, and it is not yet understood whether specific mutational patterns may result in chemotherapy sensitivity or resistance. To identify associations between genomic events and response to NAC in OAC, a comparative genomic analysis was performed in 65 patients using whole-genome sequencing. We defined response to NAC using Mandard Tumour Regression Grade TRG), with responders classified as TRG1–2 (n = 27) and non-responders classified as TRG4–5 (n = 38). We report a higher non-synonymous mutation burden in responders (median 2.08/Mb vs 1.70/Mb, P = 0.036) and elevated copy number variation (CNV) in non-responders (282 vs 136/patient, P &amp;lt; 0.001). We identified CNVs unique to each group, with cell cycle (CDKN2A, CCND1), c-Myc (MYC), RTK/PIK3 (KRAS, EGFR) and gastrointestinal differentiation (GATA6) pathway genes being specifically altered in non-responders. Of particular interest was the identification of the Neuron Navigator-3 (NAV3), a known tumour suppressor downstream of EGFR, which was mutated exclusively in 22% of non-responders. Our work characterises genetic features and mutations that are uniquely associated with response to NAC. We envision a treatment pipeline that incorporates driver mutation profiling in OAC, combining response prediction with targeted therapies to enhance response to NAC and improve survival outcomes.

Genetic expression and immunofluorescence mapping of NaV1 highlight potential therapeutic targets in anti-inflammatory treatment of aortic valves stenosis

Background and Aims : Aortic valve stenosis (AVS) is the most prevalent heart valve disease worldwide. Despite better interventional treatment strategies, AVS is associated with a high morbidity and mortality. The aortic valve is innervated, but the neural regulation of these AVS processes is unknown. Recently, a genome wide analysis study (GWAS) associated neuron navigator 1 (NAV1) with AVS. Since NAV1 is a microtubule-associated protein involved in developmental neural migration, the aim of the study was to establish the relation of NAV1 expression to AVS disease stages and AVS pathophysiological pathways, and to map the neural NAV1 to demonstrate its possible role in guiding the valvular anti-inflammatory cholinergic pathway.

O086 Genomic analysis of response to neoadjuvant chemotherapy in oesophageal adenocarcinoma

Abstract Introduction Oesophageal adenocarcinoma (OAC) is the ninth most common cancer worldwide with a mortality of over 500,000 deaths yearly. Neoadjuvant chemotherapy (NAC) followed by surgery is the standard of care (SOC) for locally advanced OAC. Although almost all patients receive chemotherapy as SOC, fewer than 20% obtain a clinically meaningful response and benefit before surgery. The OAC genome is complex and heterogeneous between patients, and it is not yet understood whether specific mutational patterns result in chemotherapy sensitivity or resistance. Methods To identify associations between genomic events and response to NAC in OAC, a comparative genomic analysis was performed in 65 patients using whole-genome sequencing. We defined response to NAC using Mandard Tumour Regression Grade TRG), with responders classified as TRG1-2 (n=27) and non-responders classified as TRG4- 5 (n=38). Results We report a higher non-synonymous mutation burden in responders (median 2.08/Mb vs 1.70/Mb, P=0.036) and elevated copy number variation (CNV) in non-responders (282 vs 136/patient, P&amp;lt;0.001). We identified CNVs unique to each group, with cell cycle (CDKN2A, CCND1), c-Myc (MYC), RTK/PIK3 (KRAS, EGFR) and gastrointestinal differentiation (GATA6) pathway genes being specifically altered in non-responders. Of particular interest was the identification of the Neuron Navigator-3 (NAV3), a known tumour suppressor downstream of EGFR, which was mutated exclusively in 22% of non-responders. Conclusion We characterise genetic features and mutations that are uniquely associated with response to NAC. We envision a treatment pipeline that incorporates driver mutation profiling in OAC, combining response prediction with targeted therapies enhancing response to NAC and improving survival outcomes. Take-home message Developing a method of determining an OAC patient's response to neoadjuvant chemotherapy before treatment is administered is desperately needed and will improve patient outcome and quality of life. We identified a number of aberrations in the genome that were unique to non-responders to chemotherapy compared to responders, particularly a known tumour suppressor gene namely Neuron Navigator-3, suggesting that these events may contribute to chemoresistance in these patients. Our work characterises pre-existing genomic alterations that have potential as biomarkers for resistance or sensitivity to NAC.

NAV3 Is a Novel Prognostic Biomarker Affecting the Immune Status of the Tumor Microenvironment in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common malignant tumors in the world. Tumor microenvironment (TME) plays a crucial role in the development of CRC. With the deep understanding of TME function, growing studies have demonstrated that alteration in tumor-infiltrating immune cells (TICs) and gene expressions are associated with clinical outcomes of various tumors. In this study, we aimed to recognize critical prognostic genes involved in immune states in TME of CRC. Hence, the proportion of TICs and the number of immune and stromal components in CRC samples from TCGA datasets were calculated by the use of CIBERSORT and ESTIMATE calculation methods. Different assays were applied to collect differential expression genes (DEGs) shared by the ImmuneScore and StromalScore. DEGs were further analyzed by the use of univariate Cox regression. Our attention focused on neuron navigator 3 (NAV3) which was highly expressed in CRC specimens and associated with an advanced clinical stage and poor prognosis of CRC patients. KEGG assays revealed that NAV3 may be involved in Alzheimer disease, amyotrophic lateral sclerosis, Huntington disease, FoxO signaling pathway, and human papillomavirus infection. Correlation assays showed that macrophage M0 and B cells memory, NK cells activated, dendritic cells resting, T cells CD4 memory activated, and T cells CD8 were correlated with NAV3 expression, indicating that NAV3 may represent the immune status of TME. Finally, RT-PCR confirmed that NAV3 expression was distinctly increased in CRC cells, and its knockdown suppressed the proliferation of CRC cells. Overall, NAV3 could be used as a novel predictor for TME of CRC and might be a novel prognostic biomarker. In the future, drugs targeting NAV3 might be developed as a potential immunotherapy for CRC patients.

Growth cone macropinocytosis of neurotrophin receptor and neuritogenesis are regulated by neuron navigator 1.

Neuron navigator 1 (Nav1) is a cytoskeleton-associated protein expressed during brain development that is necessary for proper neuritogenesis, but the underlying mechanisms are poorly understood. Here we show that Nav1 is present in elongating axon tracts during mouse brain embryogenesis. We found that depletion of Nav1 in cultured neurons disrupts growth cone morphology and neurotrophin-stimulated neuritogenesis. In addition to regulating both F-actin and microtubule properties, Nav1 promotes actin-rich membrane ruffles in the growth cone and promotes macropinocytosis at those membrane ruffles, including internalization of the TrkB receptor for the neurotrophin brain-derived neurotropic factor (BDNF). Growth cone macropinocytosis is important for downstream signaling, neurite targeting, and membrane recycling, implicating Nav1 in one or more of these processes. Depletion of Nav1 also induces transient membrane blebbing via disruption of signaling in the Rho GTPase signaling pathway, supporting the novel role of Nav1 in dynamic actin-based membrane regulation at the cell periphery. These data demonstrate that Nav1 works at the interface of microtubules, actin, and plasma membrane to organize the cell periphery and promote uptake of growth and guidance cues to facilitate neural morphogenesis during development.

Loss of Neuron Navigator 2 Impairs Brain and Cerebellar Development

Cerebellar hypoplasia and dysplasia encompass a group of clinically and genetically heterogeneous disorders frequently associated with neurodevelopmental impairment. The Neuron Navigator 2 (NAV2) gene (MIM: 607,026) encodes a member of the Neuron Navigator protein family, widely expressed within the central nervous system (CNS), and particularly abundant in the developing cerebellum. Evidence across different species supports a pivotal function of NAV2 in cytoskeletal dynamics and neurite outgrowth. Specifically, deficiency of Nav2 in mice leads to cerebellar hypoplasia with abnormal foliation due to impaired axonal outgrowth. However, little is known about the involvement of the NAV2 gene in human disease phenotypes. In this study, we identified a female affected with neurodevelopmental impairment and a complex brain and cardiac malformations in which clinical exome sequencing led to the identification of NAV2 biallelic truncating variants. Through protein expression analysis and cell migration assay in patient-derived fibroblasts, we provide evidence linking NAV2 deficiency to cellular migration deficits. In model organisms, the overall CNS histopathology of the Nav2 hypomorphic mouse revealed developmental anomalies including cerebellar hypoplasia and dysplasia, corpus callosum hypo-dysgenesis, and agenesis of the olfactory bulbs. Lastly, we show that the NAV2 ortholog in Drosophila, sickie (sick) is widely expressed in the fly brain, and sick mutants are mostly lethal with surviving escapers showing neurobehavioral phenotypes. In summary, our results unveil a novel human neurodevelopmental disorder due to genetic loss of NAV2, highlighting a critical conserved role of the NAV2 gene in brain and cerebellar development across species.

Transcriptional Control of Axon Guidance at Midline Structures

The development of the nervous system is a time-ordered and multi-stepped process that includes neurogenesis and neuronal specification, axonal navigation, and circuits assembly. During axonal navigation, the growth cone, a dynamic structure located at the tip of the axon, senses environmental signals that guide axons towards their final targets. The expression of a specific repertoire of receptors on the cell surface of the growth cone together with the activation of a set of intracellular transducing molecules, outlines the response of each axon to specific guidance cues. This collection of axon guidance molecules is defined by the transcriptome of the cell which, in turn, depends on transcriptional and epigenetic regulators that modify the structure and DNA accessibility to determine what genes will be expressed to elicit specific axonal behaviors. Studies focused on understanding how axons navigate intermediate targets, such as the floor plate of vertebrates or the mammalian optic chiasm, have largely contributed to our knowledge of how neurons wire together during development. In fact, investigations on axon navigation at these midline structures led to the identification of many of the currently known families of proteins that act as guidance cues and their corresponding receptors. Although the transcription factors and the regulatory mechanisms that control the expression of these molecules are not well understood, important advances have been made in recent years in this regard. Here we provide an updated overview on the current knowledge about the transcriptional control of axon guidance and the selection of trajectories at midline structures.

Neuron navigator 3 (NAV3) is required for heart development in zebrafish.

As a tightly controlled biological process, cardiogenesis requires the specification and migration of a suite of cell types to form a particular three-dimensional configuration of the heart. Many genetic factors are involved in the formation and maturation of the heart, and any genetic mutations may result in severe cardiac failures. The neuron navigator (NAV) family consists of three vertebrate homologs (NAV1, NAV2, and NAV3) of the neural guidance molecule uncoordinated-53 (UNC-53) in Caenorhabditis elegans. Although they are recognized as neural regulators, their expressions are also detected in many organs, including the heart, kidney, and liver. However, the functions of NAVs, regardless of neural guidance, remain largely unexplored. In our study, we found that nav3 gene was expressed in the cardiac region of zebrafish embryos from 24 to 48h post-fertilization (hpf) by means of in situ hybridization (ISH) assay. A CRISPR/Cas9-based genome editing method was utilized to delete the nav3 gene in zebrafish and loss of function of Nav3 resulted in a severe deficiency in its cardiac morphology and structure. The similar phenotypic defects of the knockout mutants could recur by nav3 morpholino injection and be rescued by nav3 mRNA injection. Dual-color fluorescence imaging of ventricle and atrium markers further confirmed the disruption of the heart development in nav3-deleted mutants. Although the heart rate was not affected by the deletion of nav3, the heartbeat intensity was decreased in the mutants. All these findings indicate that Nav3 was required for cardiogenesis in developing zebrafish embryos.