Live imaging of retinotectal mapping reveals topographic map dynamics and a previously undescribed role for Contactin 2 in map sharpening.

Organization of neuronal connections into topographic maps is essential for processing information. Yet, our understanding of topographic mapping has remained limited by our inability to observe maps forming and refining directly in vivo. Here, we used Cre-mediated recombination of a new colorswitch reporter in zebrafish to generate the first transgenic model allowing the dynamic analysis of retinotectal mapping in vivo. We found that the antero-posterior retinotopic map forms early but remains dynamic, with nasal and temporal retinal axons expanding their projection domains over time. Nasal projections initially arborize in the anterior tectum but progressively refine their projection domain to the posterior tectum, leading to the sharpening of the retinotopic map along the antero-posterior axis. Finally, using a CRISPR-mediated mutagenesis approach, we demonstrate that the refinement of nasal retinal projections requires the adhesion molecule Contactin 2. Altogether, our study provides the first analysis of a topographic map maturing in real time in a live animal and opens new strategies for dissecting the molecular mechanisms underlying precise topographic mapping in vertebrates.

Electrical stimulation applied during differentiation drives the hiPSC-CMs towards a mature cardiac conduction-like cells.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) resemble fetal cardiomyocytes and electrical stimulation (ES) has been explored to mature the differentiated cells. Here, we hypothesize that ES applied at the beginning of the differentiation process, triggers both differentiation of the hiPSC-CMs into a specialized conduction system (CS) phenotype and cell maturation. We applied ES for 15 days starting on day 0 of the differentiation process and found an increased expression of transcription factors and proteins associated with the development and function of CS including Irx3, Nkx2.5 and contactin 2, Hcn4 and Scn5a, respectively. We also found activation of intercalated disc proteins (Nrap and ß-catenin). We detected ES-induced CM maturation as indicated by increased Tnni1 and Tnni3 expression. Confocal micrographs showed a shift towards expression of the gap junction protein connexin 40 in ES hiPSC-CM compared to the more dominant expression of connexin 43 in controls. Finally, analysis of functional parameters revealed that ES hiPSC-CMs exhibited faster action potential (AP) depolarization, longer intracellular Ca2+ transients, and slower AP duration at 90% of repolarization, resembling fast conducting fibers. Altogether, we provided evidence that ES during the differentiation of hiPSC to cardiomyocytes lead to development of cardiac conduction-like cells with more mature cytoarchitecture. Thus, hiPSC-CMs exposed to ES during differentiation can be instrumental to develop CS cells for cardiac disease modelling, screening individual drugs on a precison medicine type platform and support the development of novel therapeutics for arrhythmias.

Using the Allen gene expression atlas of the adult mouse brain to gain further insight into the physiological significance of TAG-1/Contactin-2.

The anatomic gene expression atlas (AGEA) of the adult mouse brain of the Allen Institute for Brain Science is a transcriptome-based atlas of the adult C57Bl/6 J mouse brain, based on the extensive in situ hybridization dataset of the Institute. This spatial mapping of the gene expression levels of mice under baseline conditions could assist in the formation of new, reasonable transcriptome-derived hypotheses on brain structure and underlying biochemistry, which could also have functional implications. The aim of this work is to use the data of the AGEA (in combination with Tabula Muris, a compendium of single cell transcriptome data collected from mice, enabling direct and controlled comparison of gene expression among cell types) to provide further insights into the physiology of TAG-1/Contactin-2 and its interactions, by presenting the expression of the corresponding gene across the adult mouse brain under baseline conditions and to investigate any spatial genomic correlations between TAG-1/Contactin-2 and its interacting proteins and markers of mature and immature oligodendrocytes, based on the pre-existing experimental or bibliographical evidence. The across-brain correlation analysis on the gene expression intensities showed a positive spatial correlation of TAG-1/Contactin-2 with the gene expression of Plp1, Myrf, Mbp, Mog, Cldn11, Bace1, Kcna1, Kcna2, App and Nfasc and a negative spatial correlation with the gene expression of Cspg4, Pdgfra, L1cam, Ncam1, Ncam2 and Ptprz1. Spatially correlated genes are mainly expressed by mature oligodendrocytes (like Cntn2), while spatially anticorrelated genes are mainly expressed by oligodendrocyte precursor cells. According to the data presented in this work, we propose that even though Contactin-2 expression during development correlates with high plasticity events, such as neuritogenesis, in adulthood it correlates with pathways characterized by low plasticity.

TAG-1 Multifunctionality Coordinates Neuronal Migration, Axon Guidance, and Fasciculation.

Neuronal migration, axon fasciculation, and axon guidance need to be closely coordinated for neural circuit assembly. Spinal motor neurons (MNs) face unique challenges during development because their cell bodies reside within the central nervous system (CNS) and their axons project to various targets in the body periphery. The molecular mechanisms that contain MN somata within the spinal cord while allowing their axons to exit the CNS and navigate to their final destinations remain incompletely understood. We find that the MN cell surface protein TAG-1 anchors MN cell bodies in the spinal cord to prevent their emigration, mediates motor axon fasciculation during CNS exit, and guides motor axons past dorsal root ganglia. TAG-1 executes these varied functions in MN development independently of one another. Our results identify TAG-1 as a key multifunctional regulator of MN wiring that coordinates neuronal migration, axon fasciculation, and axon guidance.

Impact of anti-CASPR2 autoantibodies from patients with autoimmune encephalitis on CASPR2/TAG-1 interaction and Kv1 expression.

Autoantibodies against CASPR2 (contactin-associated protein-like 2) have been linked to autoimmune limbic encephalitis that manifests with memory disorders and temporal lobe seizures. According to the growing number of data supporting a role for CASPR2 in neuronal excitability, CASPR2 forms a molecular complex with transient axonal glycoprotein-1 (TAG-1) and shaker-type voltage-gated potassium channels (Kv1.1 and Kv1.2) in compartments critical for neuronal activity and is required for Kv1 proper positioning. Whereas the perturbation of these functions could explain the symptoms observed in patients, the pathogenic role of anti-CASPR2 antibodies has been poorly studied. In the present study, we find that patient autoantibodies alter Caspr2 distribution at the cell membrane promoting cluster formation. We confirm in a HEK cellular model that the anti-CASPR2 antibodies impede CASPR2/TAG-1 interaction and we identify the domains of CASPR2 and TAG-1 taking part in this interaction. Moreover, introduction of CASPR2 into HEK cells induces a marked increase of the level of Kv1.2 surface expression and in cultures of hippocampal neurons Caspr2-positive inhibitory neurons appear to specifically express high levels of Kv1.2. Importantly, in both cellular models, anti-CASPR2 patient autoAb increase Kv1.2 expression. These results provide new insights into the pathogenic role of autoAb in the disease.

miR-3075 Inhibited the Migration of Schwann Cells by Targeting Cntn2.

Peripheral nerve injury is a complex biological process that involves the expression changes of various coding and non-coding RNAs. Previously, a number of novel miRNAs that were dysregulated in rat sciatic nerve stumps after peripheral nerve injury were identified and functionally annotated by Solexa sequencing. In the current study, we studied one of these identified novel miRNAs, miR-3075, in depth. Results of transwell-based cell migration assay showed that increased expression of miR-3075 suppressed the migration rate of Schwann cells while decreased expression of miR-3075 elevated the migration rate of Schwann cells, demonstrating that miR-3075 inhibited Schwann cell migration. Results of BrdU cell proliferation assay showed that neither miR-3075 mimic nor miR-3075 inhibitor would affect Schwann cell proliferation. We further studied candidate target genes of miR-3075 by using bioinformatic tools and analyzing gene expression patterns and found that miR-3075 might target contactin 2 (Cntn2). Previous study showed that Cntn2 regulated cell migration and myelination. Our current observation suggested that the biological effects of miR-3075 on Schwann cell phenotype might by through the negative regulation of Cntn2. Overall, our study revealed the function of a novel miRNA, miR-3075, and expanded our current understanding of the molecular mechanisms underlying peripheral nerve injury and regeneration.

Genetic variants in autism-related CNTNAP2 impair axonal growth of cortical neurons.

The CNTNAP2 gene, coding for the cell adhesion glycoprotein Caspr2, is thought to be one of the major susceptibility genes for autism spectrum disorder (ASD). A large number of rare heterozygous missense CNTNAP2 variants have been identified in ASD patients. However, most of them are inherited from an unaffected parent, questioning their clinical significance. In the present study, we evaluate their impact on neurodevelopmental functions of Caspr2 in a heterozygous genetic background. Performing cortical neuron cultures from mouse embryos, we demonstrate that Caspr2 plays a dose-dependent role in axon growth in vitro. Loss of one Cntnap2 allele is sufficient to elicit axonal growth alteration, revealing a situation that may be relevant for CNTNAP2 heterozygosity in ASD patients. Then, we show that the two ASD variants I869T and G731S, which present impaired binding to Contactin2/TAG-1, do not rescue axonal growth deficits. We find that the variant R1119H leading to protein trafficking defects and retention in the endoplasmic reticulum has a dominant-negative effect on heterozygous Cntnap2 cortical neuron axon growth, through oligomerization with wild-type Caspr2. Finally, we identify an additional variant (N407S) with a dominant-negative effect on axon growth although it is well-localized at the membrane and properly binds to Contactin2. Thus, our data identify a new neurodevelopmental function for Caspr2, the dysregulation of which may contribute to clinical manifestations of ASD, and provide evidence that CNTNAP2 heterozygous missense variants may contribute to pathogenicity in ASD, through selective mechanisms.

Distinct roles for the cell adhesion molecule Contactin2 in the development and function of neural circuits in zebrafish.

Contactin2 (Cntn2)/Transient Axonal Glycoprotein 1 (Tag1), a neural cell adhesion molecule, has established roles in neuronal migration and axon fasciculation in chick and mouse. In zebrafish, antisense morpholino-based studies have indicated roles for cntn2 in the migration of facial branchiomotor (FBM) neurons, the guidance of the axons of the nucleus of the medial longitudinal fascicle (nucMLF), and the outgrowth of Rohon-Beard (RB) central axons. To study functions of Cntn2 in later stages of neuronal development, we generated cntn2 mutant zebrafish using CRISPR-Cas9. Using a null mutant allele, we detected genetic interactions between cntn2 and the planar cell polarity gene vangl2, as shown previously with cntn2 morphants, demonstrating a function for cntn2 during FBM neuron migration in a sensitized background of reduced planar cell polarity signaling. In addition, maternal-zygotic (MZ) cntn2 mutant larvae exhibited aberrant touch responses and swimming, suggestive of defects in sensorimotor circuits, consistent with studies in mice. However, the nucMLF axon convergence, FBM neuron migration, and RB outgrowth defects seen in morphants were not seen in the mutants, and we show here that they are likely off-target effects of morpholinos. However, MLF axons exhibited local defasciculation in MZcntn2 mutants, consistent with a role for Cntn2 in axon fasciculation. These data demonstrate distinct roles for zebrafish cntn2 in neuronal migration and axon fasciculation, and in the function of sensorimotor circuits.

The function of contactin-2/TAG-1 in oligodendrocytes in health and demyelinating pathology.

The oligodendrocyte maturation process and the transition from the pre-myelinating to the myelinating state are extremely important during development and in pathology. In the present study, we have investigated the role of the cell adhesion molecule CNTN2/TAG-1 on oligodendrocyte proliferation, differentiation, myelination, and function during development and under pathological conditions. With the combination of in vivo, in vitro, ultrastructural, and electrophysiological methods, we have mapped the expression of CNTN2 protein in the oligodendrocyte lineage during the different stages of myelination and its involvement on oligodendrocyte maturation, branching, myelin-gene expression, myelination, and axonal function. The cuprizone model of central nervous system demyelination was further used to assess CNTN2 in pathology. During development, CNTN2 can transiently affect the expression levels of myelin and myelin-regulating genes, while its absence results in reduced oligodendrocyte branching, hypomyelination of fiber tracts and impaired axonal conduction. In pathology, CNTN2 absence does not affect the extent of de- and remyelination. However during remyelination, a novel, CNTN2-independent mechanism is revealed that is able to recluster voltage gated potassium channels (VGKCs) resulting in the improvement of fiber conduction.

The Kv1-associated molecules TAG-1 and Caspr2 are selectively targeted to the axon initial segment in hippocampal neurons.

Caspr2 and TAG-1 (also known as CNTNAP2 and CNTN2, respectively) are cell adhesion molecules (CAMs) associated with the voltage-gated potassium channels Kv1.1 and Kv1.2 (also known as KCNA1 and KCNA2, respectively) at regions controlling axonal excitability, namely, the axon initial segment (AIS) and juxtaparanodes of myelinated axons. The distribution of Kv1 at juxtaparanodes requires axo-glial contacts mediated by Caspr2 and TAG-1. In the present study, we found that TAG-1 strongly colocalizes with Kv1.2 at the AIS of cultured hippocampal neurons, whereas Caspr2 is uniformly expressed along the axolemma. Live-cell imaging revealed that Caspr2 and TAG-1 are sorted together in axonal transport vesicles. Therefore, their differential distribution may result from diffusion and trapping mechanisms induced by selective partnerships. By using deletion constructs, we identified two molecular determinants of Caspr2 that regulate its axonal positioning. First, the LNG2-EGF1 modules in the ectodomain of Caspr2, which are involved in its axonal distribution. Deletion of these modules promotes AIS localization and association with TAG-1. Second, the cytoplasmic PDZ-binding site of Caspr2, which could elicit AIS enrichment and recruitment of the membrane-associated guanylate kinase (MAGuK) protein MPP2. Hence, the selective distribution of Caspr2 and TAG-1 may be regulated, allowing them to modulate the strategic function of the Kv1 complex along axons.

Phenotypically silent Cre recombination within the postnatal ventricular conduction system.

The cardiac conduction system (CCS) is composed of specialized cardiomyocytes that initiate and maintain cardiac rhythm. Any perturbation to the normal sequence of electrical events within the heart can result in cardiac arrhythmias. To understand how cardiac rhythm is established at the molecular level, several genetically modified mouse lines expressing Cre recombinase within specific CCS compartments have been created. In general, Cre driver lines have been generated either by homologous recombination of Cre into an endogenous locus or Cre expression driven by a randomly inserted transgene. However, haploinsufficiency of the endogenous gene compromises the former approach, while position effects negatively impact the latter. To address these limitations, we generated a Cre driver line for the ventricular conduction system (VCS) that preserves endogenous gene expression by targeting the Contactin2 (Cntn2) 3' untranslated region (3'UTR). Here we show that Cntn23'UTR-IRES-Cre-EGFP/+ mice recombine floxed alleles within the VCS and that Cre expression faithfully recapitulates the spatial distribution of Cntn2 within the heart. We further demonstrate that Cre expression initiates after birth with preservation of native Cntn2 protein. Finally, we show that Cntn23'UTR-IRES-Cre-EGFP/+ mice maintain normal cardiac mechanical and electrical function. Taken together, our results establish a novel VCS-specific Cre driver line without the adverse consequences of haploinsufficiency or position effects. We expect that our new mouse line will add to the accumulating toolkit of CCS-specific mouse reagents and aid characterization of the cell-autonomous molecular circuitry that drives VCS maintenance and function.

Molecular Architecture of Contactin-associated Protein-like 2 (CNTNAP2) and Its Interaction with Contactin 2 (CNTN2).

Contactin-associated protein-like 2 (CNTNAP2) is a large multidomain neuronal adhesion molecule implicated in a number of neurological disorders, including epilepsy, schizophrenia, autism spectrum disorder, intellectual disability, and language delay. We reveal here by electron microscopy that the architecture of CNTNAP2 is composed of a large, medium, and small lobe that flex with respect to each other. Using epitope labeling and fragments, we assign the F58C, L1, and L2 domains to the large lobe, the FBG and L3 domains to the middle lobe, and the L4 domain to the small lobe of the CNTNAP2 molecular envelope. Our data reveal that CNTNAP2 has a very different architecture compared with neurexin 1α, a fellow member of the neurexin superfamily and a prototype, suggesting that CNTNAP2 uses a different strategy to integrate into the synaptic protein network. We show that the ectodomains of CNTNAP2 and contactin 2 (CNTN2) bind directly and specifically, with low nanomolar affinity. We show further that mutations in CNTNAP2 implicated in autism spectrum disorder are not segregated but are distributed over the whole ectodomain. The molecular shape and dimensions of CNTNAP2 place constraints on how CNTNAP2 integrates in the cleft of axo-glial and neuronal contact sites and how it functions as an organizing and adhesive molecule.

Vitronectin promotes the progress of the initial differentiation stage in cerebellar granule cells.

Vitronectin (VN), which is an extracellular matrix protein, is known to be involved in the proliferation and differentiation of primary cultured cerebellar granule cell precursors (CGCPs); however, the effect of VN is not fully understood. In this study, we analyzed the effects of VN loss on the proliferation and differentiation of CGCPs in VN knockout (VNKO) mice in vivo. First, immunohistochemistry showed that VN was distributed in the region from the inner external granule layer (iEGL) through the internal granule layer (IGL) in wild-type (WT) mice. Next, we observed the formation of the cerebellar cortex using sagittal sections of VNKO mice at postnatal days (P) 5, 8 and 11. Loss of VN suppressed the ratio of NeuN, a neuronal differentiation marker, to positive cerebellar granule cells (CGCs) in the external granule layer (EGL) and the ratio of CGCs in the IGL at P8, indicating that the loss of VN suppresses the differentiation into CGCs. However, the loss of VN did not significantly affect the proliferation of CGCPs. Next, the effect of VN loss on the initial differentiation stage of CGCPs was examined. The loss of VN increased the expression levels of Transient axonal glycoprotein 1 (TAG1), a marker of neurons in the initial differentiation stage, in the cerebella of VNKO mice at P5 and 8 and increased the ratio of TAG1-positive cells in the primary culture of VNKO-derived CGCPs, indicating that the loss of VN accumulates the CGCPs in the initial differentiation stage. Taken together, these results demonstrate that VN promotes the progress of the initial differentiation stage of CGCPs.

Differential modulation of the juxtaparanodal complex in Multiple Sclerosis.

Myelinated fibers are divided into discrete subdomains around the Nav-enriched nodes of Ranvier: the paranodes, where axoglial interactions occur, the juxtaparanodes, where voltage-gated potassium channels (VGKCs) are aggregated, and the internode. Perinodal changes have been reported in Multiple Sclerosis (MS) with functional consequences for the axon. Here we report on alterations of the juxtaparanodal proteins TAG-1, Caspr2 and VGKCs in normal appearing white matter (NAWM), perilesion and chronic lesion areas in post-mortem white matter tissue from MS patients compared to control white matter. We show that the molecular organization and maintenance of juxtaparanodes is affected in lesions, perilesions and NAWM in chronic MS through protein and mRNA expression as well as immunohistochemistry. The three molecules analyzed were differentially altered. TAG-1 clustering at juxtaparanodes was reduced in NAWM; TAG-1 and Caspr2 are diffused in perilesions and absent in lesion areas. VGKCs were no longer enriched at juxtaparanodes either at the NAWM or the perilesion and demyelinated plaques. While the protein levels of the three molecules showed only a tendency of reduction in the plaques, there was a significant upregulation of Caspr2 mRNA in the lesions accompanied by a transcriptional increase of paranodal Caspr, indicating an axonal homeostatic mechanism.

Identification of differential splicing genes in gliomas using exon expression profiling.

Diffuse gliomas are the most common type of malignant primary brain tumor, and their initiation and/or progression are often associated with alternative splicing. They produce an enormous economic burden on society and greatly impair the quality of life of those affected. The aim of the current study was to explore the differentially expressed genes (DEGs) observed in glioblastoma (GBM) and oligodendroglioma (OD) at the splicing level, and to analyze their functions in order to identify the underlying molecular mechanisms of gliomas. The exon­level expression profile data GSE9385 was downloaded from the Gene Expression Omnibus database, and included 26 GBM samples, 22 OD samples and 6 control brain samples. The differentially expressed exon­level probes were analyzed using the microarray detection of alternative splicing algorithm combined with the splicing index method, and the corresponding DEGs were identified. Next, a Gene Ontology enrichment analysis of the DEGs was performed. Additionally, the protein­protein interaction (PPI) networks were constructed based on the depth­first search algorithm. A total of 300 DEGs were identified to be shared by GBM and OD, including 97 upregulated and 203 downregulated DEGs. Furthermore, screening with a defined threshold identified 6 genes that were highly expressed in GBM, including AFF2, CACNA2D3 and ARPP21, while the 6 highly expressed genes in OD notably included CNTN2. The TP53 and HIST1H3A genes were the hub nodes in the PPI network of DEGs from GBM, while CNTN2 was linked to the highest degree in the OD PPI network. The present study provides a comprehensive bioinformatics analysis of DEGs in GBM and OD, which may provide a basis for understanding the initiation and/or progression of glioma development.

Genome-wide linkage and exome analyses identify variants of HMCN1 for splenic epidermoid cyst.

BACKGROUND: Splenic epidermoid cyst is a benign tumor-like lesion affecting the spleen and sometimes occurs in familial form. The causality of such rare diseases remain challenging, however recently, with the emergence of exome re-sequencing, the genetics of many diseases have been unveiled. In the present study, we performed a combinatorial approach of genome-wide parametric linkage and exome analyses for a moderate-sized Japanese family with frequent occurrence of splenic epidermoid cyst to identify the genetic causality of the disease. METHODS: Twelve individuals from the family were subject to SNP typing and exome re-sequencing was done for 8 family members and 4 unrelated patients from Kosovo. Linkage was estimated using multi-point parametric linkage analysis assuming a dominant mode of inheritance. All of the candidate variants from exome analysis were confirmed by direct sequencing. RESULTS: The parametric linkage analysis suggested two loci on 1q and 14q with a maximal LOD score of 2.5 . Exome generated variants were prioritized based on; impact on the protein coding sequence, novelty or rareness in public databases, and position within the linkage loci. This approach identified three variants; variants of HMCN1 and CNTN2 on 1q and a variant of DDHD1 on 14q. The variant of HMCN1 (p.R5205H) showed the best co-segregation in the family after validation with Sanger sequencing. Additionally, rare missense variants (p.A4704V, p.T5004I, and p.H5244Q) were detected in three unrelated Kosovo patients. The identified variants of HMCN1 are on conserved domains, particularly the two variants on calcium-binding epidermal growth factor domain. CONCLUSIONS: The present study, by combining linkage and exome analyses, identified HMCN1 as a genetic causality of splenic epidermoid cyst. Understanding the biology of the disease is a key step toward developing innovative approaches of intervention.

TAG-1-assisted progenitor elongation streamlines nuclear migration to optimize subapical crowding.

Neural progenitors exhibit cell cycle-dependent interkinetic nuclear migration (INM) along the apicobasal axis. Despite recent advances in understanding its underlying molecular mechanisms, the processes to which INM contributes mechanically and the regulation of INM by the apicobasally elongated morphology of progenitors remain unclear. We found that knockdown of the cell-surface molecule TAG-1 resulted in retraction of neocortical progenitors' basal processes. Highly shortened stem-like progenitors failed to undergo basalward INM and became overcrowded in the periventricular (subapical) space. Surprisingly, the overcrowded progenitors left the apical surface and migrated into basal neuronal territories. These observations, together with the results of in toto imaging and physical tests, suggest that progenitors may sense and respond to excessive mechanical stress. Although, unexpectedly, the heterotopic progenitors remained stem-like and continued to sequentially produce neurons until the late embryonic period, histogenesis was severely disrupted. Thus, INM is essential for preventing overcrowding of nuclei and their somata, thereby ensuring normal brain histogenesis.

Exome capture sequencing reveals new insights into hepatitis B virus-induced hepatocellular carcinoma at the early stage of tumorigenesis.

Hepatocellular carcinoma (HCC), the most common type of liver cancer, is the third primary cause of cancer-related mortality worldwide. The molecular mechanisms underlying the initiation and formation of HCC remain obscure. In the present study, we performed exome sequencing using tumor and normal tissues from 3 hepatitis B virus (HBV)-positive BCLC stage A HCC patients. Bioinformatic analysis was performed to find candidate protein-altering somatic mutations. Eighty damaging mutations were validated and 59 genes were reported to be mutated in HBV-related HCCs for the first time here. Further analysis using whole genome sequencing (WGS) data of 88 HBV-related HCC patients from the European Genome-phenome Archive database showed that mutations in 33 of the 59 genes were also detected in other samples. Variants of two newly found genes, ZNF717 and PARP4, were detected in more than 10% of the WGS samples. Several other genes, such as FLNA and CNTN2, are also noteworthy. Thus, the exome sequencing analysis of three BCLC stage A patients provides new insights into the molecular events governing the early steps of HBV-induced HCC tumorigenesis.