Contactin 2 homophilic adhesion structure and conformational plasticity.

The cell-surface attached glycoprotein contactin 2 is ubiquitously expressed in the nervous system and mediates homotypic cell-cell interactions to organize cell guidance, differentiation, and adhesion. Contactin 2 consists of six Ig and four fibronectin type III domains (FnIII) of which the first four Ig domains form a horseshoe structure important for homodimerization and oligomerization. Here we report the crystal structure of the six-domain contactin 2Ig1-6 and show that the Ig5-Ig6 combination is oriented away from the horseshoe with flexion in interdomain connections. Two distinct dimer states, through Ig1-Ig2 and Ig3-Ig6 interactions, together allow formation of larger oligomers. Combined size exclusion chromatography with multiangle light scattering (SEC-MALS), small-angle X-ray scattering (SAXS) and native MS analysis indicates contactin 2Ig1-6 oligomerizes in a glycan dependent manner. SAXS and negative-stain electron microscopy reveals inherent plasticity of the contactin 2 full-ectodomain. The combination of intermolecular binding sites and ectodomain plasticity explains how contactin 2 can function as a homotypic adhesion molecule in diverse intercellular environments.

Syndecan-3 regulates the time of transition from cell cycle exit to initial differentiation stage in mouse cerebellar granule cell precursors.

To analyze the role of syndecan-3 (SDC3), a heparan sulfate proteoglycan, in cerebellum development, we examined the effect of SDC3 on the transition from cell cycle exit to the initial differentiation stage of cerebellar granule cell precursors (CGCPs). First, we examined SDC3 localization in the developing cerebellum. SDC3 was mainly localized to the inner external granule layer where the transition from the cell cycle exit to the initial differentiation of CGCPs occurs. To examine how SDC3 regulates the cell cycle exit of CGCPs, we performed SDC3-knockdown (SDC3-KD) and -overexpression (Myc-SDC3) assays using primary CGCPs. SDC3-KD significantly increased the ratio of p27Kip1+ cells to total cells at day 3 in vitro (DIV3) and 4, but Myc-SDC3 reduced that at DIV3. Regarding the cell cycle exit efficiency using 24 h-labelled bromodeoxyuridine (BrdU) and a marker of cell cycling, Ki67, SDC3-KD significantly increased cell cycle exit efficiency (Ki67-; BrdU+ cells/BrdU+ cells) in primary CGCP at DIV4 and 5, but Myc-SDC3 reduced that at DIV4 and 5. However, SDC3-KD and Myc-SDC3 did not affect the efficiency of the final differentiation from CGCPs to granule cells at DIV3-5. Furthermore, the ratio of CGCPs in the cell cycle exiting stage to total cells, identified by initial differentiation markers TAG1 and Ki67 (TAG1+; Ki67+ cells), was considerably decreased by SDC3-KD at DIV4, but increased by Myc-SDC3 at DIV4 and 5. Altogether, these results indicate that SDC3 regulates the timing of the transition from the cell cycle exit stage to the initial differentiation stage of CGCP.

Phosphacan acts as a repulsive cue in murine and rat cerebellar granule cells in a TAG-1/GD3 rafts-dependent manner.

Phosphacan, a chondroitin sulfate proteoglycan, is a repulsive cue of cerebellar granule cells. This study aims to explore the molecular mechanism. The glycosylphosphatidylinositol-anchored neural adhesion molecule TAG-1 is a binding partner of phosphacan, suggesting that the repulsive effect of phosphacan is possibly because of its interaction with TAG-1. The repulsive effect was greatly reduced on primary cerebellar granule cells of TAG-1-deficient mice. Surface plasmon resonance analysis confirmed the direct interaction of TAG-1 with chondroitin sulfate C. On postnatal days 1, 4, 7, 11, 15, and 20 and in adulthood, phosphacan was present in the molecular layer and internal granular layer, but not in the external granular layer. In contrast, transient TAG-1 expression was observed exclusively within the premigratory zone of the external granular layer on postnatal days 1, 4, 7, and 11. Boyden chamber cell migration assay demonstrated that phosphacan exerted its repulsive effect on the spontaneous and brain-derived neurotrophic factor (BDNF)-induced migration of cerebellar granule cells. The BDNF-induced migration was inhibited by MK-2206, an Akt inhibitor. The pre-treatment with a raft-disrupting agent, methyl-ß-cyclodextrin, also inhibited the BDNF-induced migration, suggesting that lipid rafts are involved in the migration of cerebellar granule cells. In primary cerebellar granule cells obtained on postnatal day 7 and cultured for 7 days, the ganglioside GD3 and TAG-1 preferentially localized in the cell body, whereas the ganglioside GD1b and NB-3 localized in not only the cell body but also neurites. Pre-treatment with the anti-GD3 antibody R24, but not the anti-GD1b antibody GGR12, inhibited the spontaneous and BDNF-induced migration, and attenuated BDNF-induced Akt activation. These findings suggest that phosphacan is responsible for the repulsion of TAG-1-expressing cerebellar granule cells via GD3 rafts to attenuate BDNF-induced migration signaling.

TMEM106B in humans and Vac7 and Tag1 in yeast are predicted to be lipid transfer proteins.

TMEM106B is an integral membrane protein of late endosomes and lysosomes involved in neuronal function, its overexpression being associated with familial frontotemporal lobar degeneration, and point mutation linked to hypomyelination. It has also been identified in multiple screens for host proteins required for productive SARS-CoV-2 infection. Because standard approaches to understand TMEM106B at the sequence level find no homology to other proteins, it has remained a protein of unknown function. Here, the standard tool PSI-BLAST was used in a nonstandard way to show that the lumenal portion of TMEM106B is a member of the late embryogenesis abundant-2 (LEA-2) domain superfamily. More sensitive tools (HMMER, HHpred, and trRosetta) extended this to predict LEA-2 domains in two yeast proteins. One is Vac7, a regulator of PI(3,5)P2 production in the degradative vacuole, equivalent to the lysosome, which has a LEA-2 domain in its lumenal domain. The other is Tag1, another vacuolar protein, which signals to terminate autophagy and has three LEA-2 domains in its lumenal domain. Further analysis of LEA-2 structures indicated that LEA-2 domains have a long, conserved lipid-binding groove. This implies that TMEM106B, Vac7, and Tag1 may all be lipid transfer proteins in the lumen of late endocytic organelles.

Vacuolar protein Tag1 and Atg1-Atg13 regulate autophagy termination during persistent starvation in S. cerevisiae.

Under starvation conditions, cells degrade their own components via autophagy in order to provide sufficient nutrients to ensure their survival. However, even if starvation persists, the cell is not completely degraded through autophagy, implying the existence of some kind of termination mechanism. In the yeast Saccharomyces cerevisiae, autophagy is terminated after 10-12 h of nitrogen starvation. In this study, we found that termination is mediated by re-phosphorylation of Atg13 by the Atg1 protein kinase, which is also affected by PP2C phosphatases, and the eventual dispersion of the pre-autophagosomal structure, also known as the phagophore assembly site (PAS). In a genetic screen, we identified an uncharacterized vacuolar membrane protein, Tag1, as a factor responsible for the termination of autophagy. Re-phosphorylation of Atg13 and eventual PAS dispersal were defective in the Δtag1 mutant. The vacuolar luminal domain of Tag1 and autophagic progression are important for the behaviors of Tag1. Together, our findings reveal the mechanism and factors responsible for termination of autophagy in yeast.

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.

NeuroD1 Dictates Tumor Cell Differentiation in Medulloblastoma.

Tumor cells are characterized by unlimited proliferation and perturbed differentiation. Using single-cell RNA sequencing, we demonstrate that tumor cells in medulloblastoma (MB) retain their capacity to differentiate in a similar way as their normal originating cells, cerebellar granule neuron precursors. Once they differentiate, MB cells permanently lose their proliferative capacity and tumorigenic potential. Differentiated MB cells highly express NeuroD1, a helix-loop-helix transcription factor, and forced expression of NeuroD1 promotes the differentiation of MB cells. The expression of NeuroD1 in bulk MB cells is repressed by trimethylation of histone 3 lysine-27 (H3K27me3). Inhibition of the histone lysine methyltransferase EZH2 prevents H3K27 trimethylation, resulting in increased NeuroD1 expression and enhanced differentiation in MB cells, which consequently reduces tumor growth. These studies reveal the mechanisms underlying MB cell differentiation and provide rationales to treat MB (potentially other malignancies) by stimulating tumor cell differentiation.

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.

Ablation of CNTN2+ Pyramidal Neurons During Development Results in Defects in Neocortical Size and Axonal Tract Formation.

Corticothalamic axons express Contactin-2 (CNTN2/TAG-1), a neuronal recognition molecule of the immunoglobulin superfamily involved in neurogenesis, neurite outgrowth, and fasciculation. TAG-1, which is expressed transiently by cortical pyramidal neurons during embryonic development, has been shown to be fundamental for axonal recognition, cellular migration, and neuronal proliferation in the developing cortex. Although Tag-1 -/- mice do not exhibit any obvious defects in the corticofugal system, the role of TAG-1+ neurons during the development of the cortex remains elusive. We have generated a mouse model expressing EGFP under the Tag-1 promoter and encompassing the coding sequence of Diptheria Toxin subunit A (DTA) under quiescence with no effect on the expression of endogenous Tag-1. We show that while the line recapitulates the expression pattern of the molecule, it highlights an extended expression in the forebrain, including multiple axonal tracts and neuronal populations, both spatially and temporally. Crossing these mice to the Emx1-Cre strain, we ablated the vast majority of TAG-1+ cortical neurons. Among the observed defects were a significantly smaller cortex, a reduction of corticothalamic axons as well as callosal and commissural defects. Such defects are common in neurodevelopmental disorders, thus this mouse could serve as a useful model to study physiological and pathophysiological cortical development.

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.

Selective Axonal Expression of the Kv1 Channel Complex in Pre-myelinated GABAergic Hippocampal Neurons.

In myelinated fibers, the voltage-gated sodium channels Nav1 are concentrated at the nodal gap to ensure the saltatory propagation of action potentials. The voltage-gated potassium channels Kv1 are segregated at the juxtaparanodes under the compact myelin sheath and may stabilize axonal conduction. It has been recently reported that hippocampal GABAergic neurons display high density of Nav1 channels remarkably in clusters along the axon before myelination (Freeman et al., 2015). In inhibitory neurons, the Nav1 channels are trapped by the ankyrinG scaffold at the axon initial segment (AIS) as observed in pyramidal and granule neurons, but are also forming "pre-nodes," which may accelerate conduction velocity in pre-myelinated axons. However, the distribution of the Kv1 channels along the pre-myelinated inhibitory axons is still unknown. In the present study, we show that two subtypes of hippocampal GABAergic neurons, namely the somatostatin and parvalbumin positive cells, display a selective high expression of Kv1 channels at the AIS and all along the unmyelinated axons. These inhibitory axons are also highly enriched in molecules belonging to the juxtaparanodal Kv1 complex, including the cell adhesion molecules (CAMs) TAG-1, Caspr2, and ADAM22 and the scaffolding protein 4.1B. Here, taking advantage of hippocampal cultures from 4.1B and TAG-1 knock-out mice, we observed that 4.1B is required for the proper positioning of Caspr2 and TAG-1 along the distal axon, and that TAG-1 deficiency induces alterations in the axonal distribution of Caspr2. However, the axonal expression of Kv1 channels and clustering of ankyrinG were not modified. In conclusion, this study allowed the analysis of the hierarchy between channels, CAMs and scaffolding proteins for their expression along hippocampal inhibitory axons before myelination. The early steps of channel compartmentalization preceding myelination may be crucial for stabilizing nerve impulses switching from a continuous to saltatory conduction during network development.

Contactin-associated protein-like 2, a protein of the neurexin family involved in several human diseases.

Contactin-associated protein-like 2 (CASPR2) is a cell adhesion protein of the neurexin family. Proteins of this family have been shown to play a role in the development of the nervous system, in synaptic functions, and in neurological diseases. Over recent years, CASPR2 function has gained an increasing interest as demonstrated by the growing number of publications. Here, we gather published data to comprehensively review CASPR2 functions within the nervous system in relation to CASPR2-related diseases in humans. On the one hand, studies on Cntnap2 (coding for CASPR2) knockout mice revealed its role during development, especially, in setting-up the inhibitory network. Consistent with this result, mutations in the CNTNAP2 gene coding for CASPR2 in human have been identified in neurodevelopmental disorders such as autism, intellectual disability, and epilepsy. On the other hand, CASPR2 was shown to play a role beyond development, in the localization of voltage-gated potassium channel (VGKC) complex that is composed of TAG-1, Kv1.1, and Kv1.2. This complex was found in several subcellular compartments essential for action potential propagation: the node of Ranvier, the axon initial segment, and the synapse. In line with a role of CASPR2 in the mature nervous system, neurological autoimmune diseases have been described in patients without neurodevelopmental disorders but with antibodies directed against CASPR2. These autoimmune diseases were of two types: central with memory disorders and temporal lobe seizures, or peripheral with muscular hyperactivity. Overall, we review the up-to-date knowledge on CASPR2 function and pinpoint confused or lacking information that will need further investigation.

Arabidopsis mutants may represent recombinant introgression lines.

OBJECTIVES: It is a common practice in Arabidopsis to transfer a mutation generated in one genetic background to other genetic background via crossing. However, the drawback of this methodology is unavoidable presence of genomic fragments from the donor parent being often replacing desirable genomic fragments of the recurrent parent. Here, we highlighted problem of Arabidopsis mutants being recombinant introgression lines that can lead to unreliable and misinterpreted results. RESULTS: We studied the regulation of low copy number transposable elements Tag1 and Evelknievel (EK), located at the end of the bottom arm of chromosome 1 and both are present in the Arabidopsis Landsberg erecta (Ler) but not in Columbia (Col) ecotype. Using various epigenetic mutants (cmt3, ddm1, kyp2, ago4, rdr2 hen1 etc.), we found that certain mutants in the Ler background are deficient of Tag1 or EK or both and represent recombinant introgression lines whereby chromosomal regions from Col have been recombined into the Ler genome. Our data support a recent proposal calling for formulating standards for authentication of plant lines that are used in plant research. Most important is to verify that a given trait or genomic locus under study is correctly identified, particularly when using mutants generated by crossing.

RNAi for contactin 2 inhibits proliferation of U87-glioma stem cells by downregulating AICD, EGFR, and HES1.

Glioblastoma is the most common form of malignant brain tumors and has a poor prognosis. Glioma stem cells (GSCs) are thought to be responsible for the aberrant proliferation and invasion. Targeting the signaling pathways that promote proliferation in GSCs is one of the strategies for glioma treatment. In this study, we found increased expression of contactin 2 (CNTN2) and amyloid ß precursor protein (APP) in U87-derived GSCs (U87-GSCs). RNA interference (RNAi) for CNTN2 downregulated the expression of APP intracellular domain (AICD), which is the proteolytic product of APP. Treatment with CNTN2 RNAi inhibited the proliferation of U87-GSCs. CNTN2 RNAi decreased the expression of epidermal growth factor receptor and HES1, which are potential targets of AICD. In summary, inhibition of the CNTN2/APP signaling pathway may repress the proliferation in U87-GSCs via downregulating the expression of HES1 and epidermal growth factor receptor. CNTN2/APP/AICD signaling pathway plays an important role in U87 glial tumorigenesis. Further studies are warranted to elucidate the role of these signaling pathways in other sources of GSCs. Depending on their role in proliferation in other sources of GSCs, members of the CNTN2/APP/AICD signaling pathway may provide novel targets for the development of therapy for glioblastomas.

Activation of Tag1 transposable elements in Arabidopsis dedifferentiating cells and their regulation by CHROMOMETHYLASE 3-mediated CHG methylation.

Dedifferentiation, that is, the acquisition of stem cell-like state, commonly induced by stress (e.g., protoplasting), is characterized by open chromatin conformation, a chromatin state that could lead to activation of transposable elements (TEs). Here, we studied the activation of the Arabidopsis class II TE Tag1, in which two copies, situated close to each other (near genes) on chromosome 1 are found in Landsberg erecta (Ler) but not in Columbia (Col). We first transformed protoplasts with a construct in which a truncated Tag1 (ΔTag1 non-autonomous) blocks the expression of a reporter gene AtMBD5-GFP and found a relatively high ectopic excision of ΔTag1 accompanied by expression of AtMBD5-GFP in protoplasts derived from Ler compared to Col; further increase was observed in ddm1 (decrease in DNA methylation1) protoplasts (Ler background). Ectopic excision was associated with transcription of the endogenous Tag1 and changes in histone H3 methylation at the promoter region. Focusing on the endogenous Tag1 elements we found low level of excision in Ler protoplasts, which was slightly and strongly enhanced in ddm1 and cmt3 (chromomethylase3) protoplasts, respectively, concomitantly with reduction in Tag1 gene body (GB) CHG methylation and increased Tag1 transcription; strong activation of Tag1 was also observed in cmt3 leaves. Notably, in cmt3, but not in ddm1, Tag1 elements were excised out from their original sites and transposed elsewhere in the genome. Our results suggest that dedifferentiation is associated with Tag1 activation and that CMT3 rather than DDM1 plays a central role in restraining Tag1 activation via inducing GB CHG methylation.

TOMATO AGAMOUS1 and ARLEQUIN/TOMATO AGAMOUS-LIKE1 MADS-box genes have redundant and divergent functions required for tomato reproductive development.

Within the tomato MADS-box gene family, TOMATO AGAMOUS1 (TAG1) and ARLEQUIN/TOMATO AGAMOUS LIKE1 (hereafter referred to as TAGL1) are, respectively, members of the euAG and PLE lineages of the AGAMOUS clade. They perform crucial functions specifying stamen and carpel development in the flower and controlling late fruit development. To gain insight into the roles of TAG1 and TAGL1 genes and to better understand their functional redundancy and diversification, we characterized single and double RNAi silencing lines of these genes and analyzed expression profiles of regulatory genes involved in reproductive development. Double RNAi lines did show cell abnormalities in stamens and carpels and produced extremely small fruit-like organs displaying some sepaloid features. Expression analyses indicated that TAG1 and TAGL1 act together to repress fourth whorl sepal development, most likely through the MACROCALYX gene. Results also proved that TAG1 and TAGL1 have diversified their functions in fruit development: while TAG1 controls placenta and seed formation, TAGL1 participates in cuticle development and lignin biosynthesis inhibition. It is noteworthy that both TAG1 and double RNAi plants lacked seed development due to abnormalities in pollen formation. This seedless phenotype was not associated with changes in the expression of B-class stamen identity genes Tomato MADS-box 6 and Tomato PISTILLATA observed in silencing lines, suggesting that other regulatory factors should participate in pollen formation. Taken together, results here reported support the idea that both redundant and divergent functions of TAG1 and TAGL1 genes are needed to control tomato reproductive development.

Inhibitory axons are targeted in hippocampal cell culture by anti-Caspr2 autoantibodies associated with limbic encephalitis.

Contactin-associated protein-like 2 (Caspr2), also known as CNTNAP2, is a cell adhesion molecule that clusters voltage-gated potassium channels (Kv1.1/1.2) at the juxtaparanodes of myelinated axons and may regulate axonal excitability. As a component of the Kv1 complex, Caspr2 has been identified as a target in neuromyotonia and Morvan syndrome, but also in some cases of autoimmune limbic encephalitis (LE). How anti-Caspr2 autoimmunity is linked with the central neurological symptoms is still elusive. In the present study, using anti-Caspr2 antibodies from seven patients affected by pure LE, we determined that IgGs in the cerebrospinal fluid of four out seven patients were selectively directed against the N-terminal Discoïdin and LamininG1 modules of Caspr2. Using live immunolabeling of cultured hippocampal neurons, we determined that serum IgGs in all patients strongly targeted inhibitory interneurons. Caspr2 was highly detected on GAD65-positive axons that are surrounding the cell bodies and at the VGAT-positive inhibitory presynaptic contacts. Functional assays indicated that LE autoantibodies may induce alteration of Gephyrin clusters at inhibitory synaptic contacts. Next, we generated a Caspr2-Fc chimera to reveal Caspr2 receptors on hippocampal neurons localized at the somato-dendritic compartment and post-synapse. Caspr2-Fc binding was strongly increased on TAG-1-transfected neurons and conversely, Caspr2-Fc did not bind hippocampal neurons from TAG-1-deficient mice. Our data indicate that Caspr2 may participate as a cell recognition molecule in the dynamics of inhibitory networks. This study provides new insight into the potential pathogenic effect of anti-Caspr2 autoantibodies in central hyperexcitability that may be related with perturbation of inhibitory interneuron activity.

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.

Long-term follow-up of TaG1 non-muscle-invasive bladder cancer.

OBJECTIVES: To retrospectively assess the long-term outcome of patients initially diagnosed with TaG1 non-muscle-invasive bladder cancer (NMIBC) with no immediate postoperative instillation of intravesical chemotherapy and evaluate the reproducibility of the European Organization for Research and Treatment of Cancer (EORTC) scoring system for predicting bladder cancer outcome. METHODS AND MATERIALS: A retrospective analysis of 481 consecutive cases of initially diagnosed TaG1 NMIBC according to the 1973 World Health Organization classification between 1995 and 2008 in a single institution was performed. Time to first recurrence, time to progression to T1 or G3 bladder cancer, and time to progression to muscle-invasive bladder cancer were studied. Time to event distributions was estimated by means of cumulative incidence functions to accurately take into account the patients who died (competing risk) before recurrence or progression. The Harrell c statistic calculation was used for our study's data results as well the original data from EORTC to compare the predictive power of a survival model. RESULTS: The median follow-up was 88 months (interquartile range: 51-135 mo). The 10-year recurrence-free, T1 or G3 NMIBC progression-free, and muscle-invasive bladder cancer progression-free survival rates were 64.2%, 96.6%, and 97%, respectively. In multivariate analysis, tumor size and number of lesions were prognostic variables of the risk of recurrence. In our study and EORTC data sets, the Harrell c values obtained were c = 0.85 (95% CI: [0.75, 0.93]) and c = 0.85 (95% CI: [0.75, 0.93]), respectively. CONCLUSION: Our study reports a detailed and extensive outcome of TaG1 NMIBC treated by TURB with no immediate postoperative intravesical instillation of chemotherapy. Our results suggest that the EORTC is a useful external validation scoring system for predicting bladder cancer outcome.

Protective effect of bone marrow mesenchymal stem cells on PC12 cells apoptosis mediated by TAG1.

OBJECTIVE: This study aims to explore the protection effect of bone marrow mesenchymal stem cells (BMSCs) on PC12 cells apoptosis mediated by transient axonal glycoprotein 1 (TAG1). METHODS: PC12 cells were divided into control group, Aß25-35 group and BMSCs + Aß25-35 group. The effects of BMSCs on PC12 cells treated by Aß25-35 were detected using MTT, Hoechst 33258 and Annexin V-FITC/PI staining methods. The expression levels of TAG1, ß-amyloid precursor protein (APP), AICD and p53 were determined by RT-PCR and Western blotting methods. The expression levels of Bax and Bcl-2 were determined by Western blotting method. The activity of Caspase 3 was detected by spectrophotometric method. RESULTS: MTT results showed that cell activity decreased after the treatment of 20 µM Aß25-35 for 48 h (P<0.01) while it increased in BMSCs + Aß25-35 group (P<0.01). Hoechst 33258 and Annexin V-FITC/PI staining results showed that Aß25-35 could induce the apoptosis of PC12 cells while the apoptosis of PC12 cells was inhibited in BMSCs + Aß25-35 group. RT-PCR and Western blotting methods showed that 20 µM Aß25-35 could increase the expression levels of TAG1, APP, AICD and p53 (P<0.01) while they decreased in BMSCs + Aß25-35 group (P<0.01). 20 µM Aß25-35 could increase the expression levels of Bax and decrease the expression levels of Bcl-2 (P<0.01), while the expression levels of Bax decreased and the expression levels of Bcl-2 increase in BMSCs + Aß25-35 group (P<0.01). 20 µM Aß25-35 could enhance Caspase 3 activity while it decreased in BMSCs + Aß25-35 group (P<0.01). Conclusions BMSCs with Aß25-35 could inhibit the apoptosis of PC12 cells, which maybe related with TAG1/APP/AICD signal pathway.