Transient Axonal Glycoprotein-1 Research Articles

Human CASPR2 Antibodies Reversibly Alter Memory and the CASPR2 Protein Complex.

The encephalitis associated with antibodies against contactin-associated proteinlike 2 (CASPR2) is presumably antibody-mediated, but the antibody effects and whether they cause behavioral alterations are not well known. Here, we used a mouse model of patients' immunoglobulin G (IgG) transfer and super-resolution microscopy to demonstrate the antibody pathogenicity. IgG from patients with anti-CASPR2 encephalitis or healthy controls was infused into the cerebroventricular system of mice. The levels and colocalization of CASPR2 with transient axonal glycoprotein 1 (TAG1) were determined with stimulated emission depletion microscopy (40-70μm lateral resolution). Hippocampal clusters of Kv1.1 voltage-gated potassium channels (VGKCs) and GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) were quantified with confocal microscopy. Behavioral alterations were assessed with standard behavioral paradigms. Cultured neurons were used to determine the levels of intracellular CASPR2 and TAG1 after exposure to patients' IgG. Infusion of patients' IgG, but not controls' IgG, caused memory impairment along with hippocampal reduction of surface CASPR2 clusters and decreased CASPR2/TAG1 colocalization. In cultured neurons, patients' IgG led to an increase of intracellular CASPR2 without affecting TAG1, suggesting selective CASPR2 internalization. Additionally, mice infused with patients' IgG showed decreased levels of Kv1.1 and GluA1 (two CASPR2-regulated proteins). All these alterations and the memory deficit reverted to normal after removing patients' IgG. IgG from patients with anti-CASPR2 encephalitis causes reversible memory impairment, inhibits the interaction of CASPR2/TAG1, and decreases the levels of CASPR2 and related proteins (VGKC, AMPAR). These findings fulfill the postulates of antibody-mediated disease and provide a biological basis for antibody-removing treatment approaches. ANN NEUROL 2022;91:801-813.

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.

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.

αvβ5 integrin mediates the effect of vitronectin on the initial stage of differentiation in mouse cerebellar granule cell precursors

Vitronectin (VN), one of the extracellular matrix proteins, controls the maturation of cerebellar granule cells (CGCs) through the promotion of the initial differentiation stage progress. However, the receptors of VN in the initial differentiation stage of CGC precursors (CGCPs) have not been clarified. In this study, we characterized the receptor candidates for VN in CGCPs. First, we confirmed that αvβ3 and αvβ5 integrins, which are receptor candidates for VN, were co-localized with VN in the developing cerebellum and primary cultured CGCPs. Next, the knockdown (KD) of αv, β3, and β5 integrins with small interference RNA (siRNA) for each integrin reduced the ratio of Tuj1, a final differentiation marker, -positive CGCPs. We further studied whether αvβ3 and αvβ5 integrins control the initial differentiation stage. The KD of αv and β5, but not β3, integrins significantly increased the ratio of transient axonal glycoprotein 1 (TAG1), an initial differentiation marker, -positive CGCPs, whereas the KD of αv and β3 integrins, not β5 integrin, stimulated the proliferation of CGCPs. Overexpression of β5 integrin stimulated the progress of the initial differentiation stage as well. To confirm the interaction between αvβ5 integrin and VN, VN was added to β5 integrin-KD CGCPs. The promotion of the progress of initial differentiation by VN was abrogated by β5 integrin KD using small hairpin RNA (shRNA). Taken together, our results indicated that αvβ5 integrin, as the very receptor of VN, is responsible for the progress of the initial differentiation stage in mouse CGCPs.

Expression of TAG1/APP signaling pathway in the proliferation and differentiation of glioma stem cells.

The aim of the present study was to examine the role of the expression of transient axonal glycoprotein-1 (TAG1)/precursor protein (APP) signaling pathway in the proliferation and differentiation of glioma stem cells. A glioma cell line (U373) was used as well as fluorescence quantitative PCR, western blot analysis, and enzyme-linked immunosorbent assay (ELISA), to examine the role of the expression of TAG1/APP signaling pathway in the proliferation and differentiation of glioma stem cells after five generations of in vitro culture. The results showed that compared to the normal glioma cells, the expression of TAG1 and APP was significantly increased in the proliferation of glioma stem cells. The results of ELISA and western blot analysis also confirmed a significant elevation in the protein expression of TAG1 in glioma stem cells compared to normal human glioma cells. When glioma stem cells were cultured in differentiation medium, as revealed by RT-PCR, the expression of TAG1 and APP in glioma stem cells initially increased and then decreased. In addition, the protein expression of TAG1 and APP was consistent with the RT-PCR results. Compared with undifferentiated glioma stem cells, the expression of TAG1 and APP decreased gradually with the extension of differentiation time. In conclusion, the expression of TAG1/APP signaling pathway in glioma cells was abnormal. Thus, this pathway is involved in the proliferation and differentiation of glioma cells and promotes the proliferation of glioma cells to inhibit the differentiation of glioma cells.

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.

Role of F3/contactin expression profile in synaptic plasticity and memory in aged mice

We have recently shown that overexpression of the F3/contactin adhesive glycoprotein (also known as Contactin-1) promotes neurogenesis in adult hippocampus, which correlates with improved synaptic plasticity and memory. Because F3/contactin levels physiologically decrease with age, here, we aim at investigating whether its overexpression might counteract the cognitive decline in aged animals. For this we use 20- to 24-month-old TAG/F3 transgenic mice in which F3/contactin overexpression is driven by regulatory sequences from the gene encoding the transient axonal glycoprotein TAG-1 throughout development. We show that aged TAG/F3 mice display improved hippocampal long-term potentiation and memory compared with wild-type littermates. The same mice undergo a decrease of neuronal apoptosis at the hippocampal level, which correlated to a decrease of active caspase-3; by contrast, procaspase-3 and Bax as well as the anti-apoptotic and plasticity-related pathway BDNF/CREB/Bcl-2 were rather increased. Interestingly, amyloid-precursor protein processing was shifted toward sAPPα generation, with a decrease of sAPPβ and amyloid-beta levels. Our data confirm that F3/contactin plays a role in hippocampal synaptic plasticity and memory also in aged mice, suggesting that it acts on molecular pathways related to apoptosis and amyloid-beta production.

Tag1 deficiency results in olfactory dysfunction through impaired migration of mitral cells.

The olfactory system provides mammals with the abilities to investigate, communicate and interact with their environment. These functions are achieved through a finely organized circuit starting from the nasal cavity, passing through the olfactory bulb and ending in various cortical areas. We show that the absence of transient axonal glycoprotein-1 (Tag1)/contactin-2 (Cntn2) in mice results in a significant and selective defect in the number of the main projection neurons in the olfactory bulb, namely the mitral cells. A subpopulation of these projection neurons is reduced in Tag1-deficient mice as a result of impaired migration. We demonstrate that the detected alterations in the number of mitral cells are well correlated with diminished odor discrimination ability and social long-term memory formation. Reduced neuronal activation in the olfactory bulb and the corresponding olfactory cortex suggest that Tag1 is crucial for the olfactory circuit formation in mice. Our results underpin the significance of a numerical defect in the mitral cell layer in the processing and integration of odorant information and subsequently in animal behavior.

Pioneering Axons Regulate Neuronal Polarization in the Developing Cerebral Cortex

The polarization of neurons, which mainly includes the differentiation of axons and dendrites, is regulated by cell-autonomous and non-cell-autonomous factors. In the developing central nervous system, neuronal development occurs in a heterogeneous environment that also comprises extracellular matrices, radial glial cells, and neurons. Although many cell-autonomous factors that affect neuronal polarization have been identified, the microenvironmental cues involved in neuronal polarization remain largely unknown. Here, we show that neuronal polarization occurs in a microenvironment in the lower intermediate zone, where the cell adhesion molecule transient axonal glycoprotein-1 (TAG-1) is expressed in cortical efferent axons. The immature neurites of multipolar cells closely contact TAG-1-positive axons and generate axons. Inhibition of TAG-1-mediated cell-to-cell interaction or its downstream kinase Lyn impairs neuronal polarization. These results show that the TAG-1-mediated cell-to-cell interaction between the unpolarized multipolar cells and the pioneering axons regulates the polarization of multipolar cells partly through Lyn kinase and Rac1.

Transient axonal glycoprotein-1 induces apoptosis-related gene expression without triggering apoptosis in U251 glioma cells.

Previous studies show that transient axonal glycoprotein-1, a ligand of amyloid precursor protein, increases the secretion of amyloid precursor protein intracellular domain and is involved in apoptosis in Alzheimer's disease. In this study, we examined the effects of transient axonal glycoprotein-1 on U251 glioma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that transient axonal glycoprotein-1 did not inhibit the proliferation of U251 cells, but promoted cell viability. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that transient axonal glycoprotein-1 did not induce U251 cell apoptosis. Real-time PCR revealed that transient axonal glycoprotein-1 substantially upregulated levels of amyloid precursor protein intracellular C-terminal domain, and p53 and epidermal growth factor receptor mRNA expression. Thus, transient axonal glycoprotein-1 increased apoptosis-related gene expression in U251 cells without inducing apoptosis. Instead, transient axonal glycoprotein-1 promoted the proliferation of these glioma cells.

Interaction of TAG1 and CASPR2, Proteins Associated to Autism Disorder?

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects brain development, social and communication skills. Although the causes of the disease are mostly unknown rare variations of the CANTNAP2, the gene that codes for contactin‐associated protein‐like 2 (CASPR2), have been implicated in ASD. We compared the cellular localization and glycosylation processing of wild type CASPR2 to twelve CASPR2 point mutations found in autistic individuals. In contrast to the wild‐type protein that localizes to the cell surface, mutants showed altered cellular disposition with endoplasmic reticulum retention and activation of one signaling pathway of the unfolded protein response. As CASPR2 is associated with a set of neuropsychiatric diseases, it is of interest to study the interaction of CASPR2 with proteins such as the transient axonal glycoprotein‐1 (TAG1). We are currently studying the interaction between different CASPR2 mutants and TAG1 by various biochemical techniques. Preliminary results show that we can measure the interaction between wild‐type TAG1 and wild type CASPR2, but not with one CASPR2 mutants. This data suggest that the interaction is disrupted by the mutation but also that another protein could be mediating this interaction. These studies will help understand better the biology of CASPR2 and its specific role in the development of autism and other neurodevelopmental disorders.

TAG1 Regulates the Endocytic Trafficking and Signaling of the Semaphorin3A Receptor Complex

Endocytic trafficking of membrane proteins is essential for neuronal structure and function. We show that Transient Axonal Glycoprotein 1 (TAG1 or CNTN2), a contactin-related adhesion molecule, plays a central role in the differential trafficking of components of the semaphorin3A (Sema3A) receptor complex into distinct endosomal compartments in murine spinal sensory neuron growth cones. The semaphorin3A receptor is composed of Neuropilin1 (NRP1), PlexinA4, and L1, with NRP1 being the ligand-binding component. TAG1 interacts with NRP1, causing a change in its association with L1 in the Sema3A response such that L1 is lost from the complex following Sema3A binding. Initially, however, L1 and NRP1 endocytose together and only become separated intracellularly, with NRP1 becoming associated with endosomes enriched in lipid rafts and colocalizing with TAG1 and PlexinA4. When TAG1 is missing, NRP1 and L1 fail to separate and NRP1 does not become raft associated; colocalization with PlexinA4 is reduced and Plexin signaling is not initiated. These observations identify a novel role for TAG1 in modulating the intracellular sorting of signaling receptor complexes.

Single‐nucleotide polymorphism of transient axonal glycoprotein‐1 and its correlation with clinical features and prognosis in chronic inflammatory demyelinating polyneuropathy

The single-nucleotide polymorphism (SNP) rs2275697 in the transient axonal glycoprotein-1 (TAG-1) gene was reported to be associated with responsiveness to intravenous immunoglobulin (IVIG) treatment in patients with chronic inflammatory demyelinating polyneuropathy (CIDP). However, it is not known if this SNP is associated with long-term prognosis. We examined the case records of 32 Chinese CIDP patients. The overall response rate to IVIG, prednisolone, or plasmapheresis was 83%. After 5.4 years, 57% of patients were on maintenance immunotherapy. Patients with higher modified Rankin score and more prolonged distal motor latencies in the upper limbs on presentation had a higher risk (odds ratio [OR] 3.86, 95% confidence interval [CI] 1.23-12.11 and OR 1.04, 95% CI 1.01-1.07, respectively) of being on maintenance immunotherapy. Blood samples from 24 patients and 147 controls were examined for their genotypes of four non-synonymous SNPs (rs41264871, rs36074532, rs5611135, and rs2275697) in the coding region of TAG-1. The G allelic frequency of rs2275697 was similar between CIDP patients and controls (56% and 50%, respectively) and was not associated with treatment responsiveness, treatment dependence, disability, or mortality.

Characterization of the axon initial segment (AIS) of motor neurons and identification of a para-AIS and a juxtapara-AIS, organized by protein 4.1B

BackgroundThe axon initial segment (AIS) plays a crucial role: it is the site where neurons initiate their electrical outputs. Its composition in terms of voltage-gated sodium (Nav) and voltage-gated potassium (Kv) channels, as well as its length and localization determine the neuron's spiking properties. Some neurons are able to modulate their AIS length or distance from the soma in order to adapt their excitability properties to their activity level. It is therefore crucial to characterize all these parameters and determine where the myelin sheath begins in order to assess a neuron's excitability properties and ability to display such plasticity mechanisms. If the myelin sheath starts immediately after the AIS, another question then arises as to how would the axon be organized at its first myelin attachment site; since AISs are different from nodes of Ranvier, would this particular axonal region resemble a hemi-node of Ranvier?ResultsWe have characterized the AIS of mouse somatic motor neurons. In addition to constant determinants of excitability properties, we found heterogeneities, in terms of AIS localization and Nav composition. We also identified in all α motor neurons a hemi-node-type organization, with a contactin-associated protein (Caspr)+ paranode-type, as well as a Caspr2+ and Kv1+ juxtaparanode-type compartment, referred to as a para-AIS and a juxtapara (JXP)-AIS, adjacent to the AIS, where the myelin sheath begins. We found that Kv1 channels appear in the AIS, para-AIS and JXP-AIS concomitantly with myelination and are progressively excluded from the para-AIS. Their expression in the AIS and JXP-AIS is independent from transient axonal glycoprotein-1 (TAG-1)/Caspr2, in contrast to juxtaparanodes, and independent from PSD-93. Data from mice lacking the cytoskeletal linker protein 4.1B show that this protein is necessary to form the Caspr+ para-AIS barrier, ensuring the compartmentalization of Kv1 channels and the segregation of the AIS, para-AIS and JXP-AIS.Conclusionsα Motor neurons have heterogeneous AISs, which underlie different spiking properties. However, they all have a para-AIS and a JXP-AIS contiguous to their AIS, where the myelin sheath begins, which might limit some AIS plasticity. Protein 4.1B plays a key role in ensuring the proper molecular compartmentalization of this hemi-node-type region.

Multifocal motor neuropathy is not associated with genetic variation in PTPN22, BANK1, Blk, FCGR2B, CD1A/E, and TAG‐1 genes

The contribution of genetic heterogeneity to the pathogenesis of multifocal motor neuropathy (MMN) has not been elucidated. We investigated frequencies of single nucleotide polymorphisms (SNPs) in the candidate genes protein tyrosine phosphatase, non-receptor type 22 (PTPN22), B-cell scaffold protein with ankyrin repeats (BANK1), B lymphocyte kinase (Blk), and Fc gamma receptor class IIB (FCGR2B), which have been found to be associated with other autoimmune diseases, CD1A and CD1E, important for antigen presentation of glycolipids, and transient axonal glycoprotein 1 (TAG-1), which is associated with responsiveness to intravenous immunoglobulin in patients with chronic inflammatory demyelinating polyneuropathy. SNP frequencies were determined by means of TaqMan SNP genotyping assay and direct sequencing of candidate genes in 92 Dutch patients with MMN and 1152 healthy controls. SNP frequencies did not differ between patients and controls (all p-values >0.15) and disease characteristics were not associated with SNP genotypes. Our results suggest that allelic variation in these genes does not play a major role in determining MMN susceptibility.

Clustering and Activity Tuning of Kv1 Channels in Myelinated Hippocampal Axons

Precise localization of axonal ion channels is crucial for proper electrical and chemical functions of axons. In myelinated axons, Kv1 (Shaker) voltage-gated potassium (Kv) channels are clustered in the juxtaparanodal regions flanking the node of Ranvier. The clustering can be disrupted by deletion of various proteins in mice, including contactin-associated protein-like 2 (Caspr2) and transient axonal glycoprotein-1 (TAG-1), a glycosylphosphatidylinositol-anchored cell adhesion molecule. However, the mechanism and function of Kv1 juxtaparanodal clustering remain unclear. Here, using a new myelin coculture of hippocampal neurons and oligodendrocytes, we report that tyrosine phosphorylation plays a critical role in TAG-1-mediated clustering of axonal Kv1.2 channels. In the coculture, myelin specifically ensheathed axons but not dendrites of hippocampal neurons and clustered endogenous axonal Kv1.2 into internodes. The trans-homophilic interaction of TAG-1 was sufficient to position Kv1.2 clusters on axonal membranes in a neuron/HEK293 coculture. Mutating a tyrosine residue (Tyr⁴⁵⁸) in the Kv1.2 C terminus or blocking tyrosine phosphorylation disrupted myelin- and TAG-1-mediated clustering of axonal Kv1.2. Furthermore, Kv1.2 voltage dependence and activation threshold were reduced by TAG-1 coexpression. This effect was eliminated by the Tyr⁴⁵⁸ mutation or by cholesterol depletion. Taken together, our studies suggest that myelin regulates both trafficking and activity of Kv1 channels along hippocampal axons through TAG-1.

Polymorphism of transient axonal glycoprotein‐1 in chronic inflammatory demyelinating polyneuropathy

Chronic inflammatory demyelinating polyneuropathy (CIDP) is characterized by immune-mediated peripheral demyelination. Although corticosteroid, intravenous immunoglobulin (IVIg), and plasma exchange have been established as the most effective therapeutics, sub-populations of patients show little or no response to either of these therapies. We identified the clinical, electrophysiological, and genetic features related to IVIg responsiveness in CIDP by conducting a multi-center study. Muscle atrophy and decreased compound muscle action potential (CMAP) were pronounced in IVIg non-responders, that is, features suggesting axonal dysfunction in peripheral nerves indicated IVIg unresponsiveness in CIDP. We then performed an association analysis using single nucleotide polymorphisms (SNPs) and haplotype studies between the IVIg responders and non-responders. We assessed SNPs of candidate genes that are particularly related to the function of Ranvier's node, paranode, or juxtaparanode. Two separate SNPs, corresponding to transient axonal glycoprotein-1 (TAG-1) and C-type lectin domain family 10, member A (CLEC10A), showed significant differences between responders and non-responders. Haplotype analysis of a series of expanded SNPs, from TAG-1 or CLEC10A, showed that only TAG-1 included a significant haplotype within one linkage disequilibrium block that accommodates IVIg responsiveness. Diplotype analysis of TAG-1 also supported this observation. In conclusion, SNPs in TAG-1, which is a key molecule for axon-Schwann cell interactions and is distributed at the juxtaparanode, are related to the IVIg responsiveness of CIDP patients.

A Combinatorial Semaphorin Code Instructs the Initial Steps of Sensory Circuit Assembly in the Drosophila CNS

Longitudinal axon fascicles within the Drosophila embryonic CNS provide connections between body segments and are required for coordinated neural signaling along the anterior-posterior axis. We show here that establishment of select CNS longitudinal tracts and formation of precise mechanosensory afferent innervation to the same CNS region are coordinately regulated by the secreted semaphorins Sema-2a and Sema-2b. Both Sema-2a and Sema-2b utilize the same neuronal receptor, plexin B (PlexB), but serve distinct guidance functions. Localized Sema-2b attraction promotes the initial assembly of a subset of CNS longitudinal projections and subsequent targeting of chordotonal sensory afferent axons to these same longitudinal connectives, whereas broader Sema-2a repulsion serves to prevent aberrant innervation. In the absence of Sema-2b or PlexB, chordotonal afferent connectivity within the CNS is severely disrupted, resulting in specific larval behavioral deficits. These results reveal that distinct semaphorin-mediated guidance functions converge at PlexB and are critical for functional neural circuit assembly.

The Expression of TAG-1 in Glial Cells Is Sufficient for the Formation of the Juxtaparanodal Complex and the Phenotypic Rescue of Tag-1 Homozygous Mutants in the CNS

Myelinated fibers are organized into specialized domains that ensure the rapid propagation of action potentials and are characterized by protein complexes underlying axoglial interactions. TAG-1 (Transient Axonal Glycoprotein-1), a cell adhesion molecule of the Ig superfamily, is expressed by neurons as well as by myelinating glia. It is essential for the molecular organization of myelinated fibers as it maintains the integrity of the juxtaparanodal region through its interactions with Caspr2 and the voltage-gated potassium channels (VGKCs) on the axolemma. Since TAG-1 is the only known component of the juxtaparanodal complex expressed by the glial cell, it is important to clarify its role in the molecular organization of juxtaparanodes. For this purpose, we generated transgenic mice that exclusively express TAG-1 in oligodendrocytes and lack endogenous gene expression (Tag-1(-/-);plp(Tg(rTag-1))). Phenotypic analysis clearly demonstrates that glial TAG-1 is sufficient for the proper organization and maintenance of the juxtaparanodal domain in the CNS. Biochemical analysis shows that glial TAG-1 physically interacts with Caspr2 and VGKCs. Ultrastructural and behavioral analysis of Tag-1(-/-);plp(Tg(rTag-1)) mice shows that the expression of glial TAG-1 is sufficient to restore the axonal and myelin deficits as well as the behavioral defects observed in Tag-1(-/-) animals. Together, these data highlight the pivotal role of myelinating glia on axonal domain differentiation and organization.

Myoblast-Derived Neuronal Cells Form Glutamatergic Neurons in the Mouse Cerebellum

Production of neurons from non-neural cells has far-reaching clinical significance. We previously found that myoblasts can be converted to a physiologically active neuronal phenotype by transferring a single recombinant transcription factor, REST-VP16, which directly activates target genes of the transcriptional repressor, REST. However, the neuronal subtype of M-RV cells and whether they can establish synaptic communication in the brain have remained unknown. M-RV cells engineered to express green fluorescent protein (M-RV-GFP) had functional ion channels but did not establish synaptic communication in vitro. However, when transplanted into newborn mice cerebella, a site of extensive postnatal neurogenesis, these cells expressed endogenous cerebellar granule precursors and neuron proteins, such as transient axonal glycoprotein-1, neurofilament, type-III β-tubulin, superior cervical ganglia-clone 10, glutamate receptor-2, and glutamate decarboxylase. Importantly, they exhibited action potentials and were capable of receiving glutamatergic synaptic input, similar to the native cerebellar granule neurons. These results suggest that M-RV-GFP cells differentiate into glutamatergic neurons, an important neuronal subtype, in the postnatal cerebellar milieu. Our findings suggest that although activation of REST-target genes can reprogram myoblasts to assume a general neuronal phenotype, the subtype specificity may then be directed by the brain microenvironment.