The role of Gpi-anchored axonal glycoproteins in neural development and neurological disorders.

This review article focuses on the Contactin (CNTN) subset of the Immunoglobulin supergene family (IgC2/FNIII molecules), whose components share structural properties (the association of Immunoglobulin type C2 with Fibronectin type III domains), as well as a general role in cell contact formation and axonal growth control. IgC2/FNIII molecules include 6 highly related components (CNTN 1-6), associated with the cell membrane via a Glycosyl Phosphatidyl Inositol (GPI)-containing lipid tail. Contactin 1 and Contactin 2 share ~50 (49.38)% identity at the aminoacid level. They are components of the cell surface, from which they may be released in soluble forms. They bind heterophilically to multiple partners in cis and in trans, including members of the related L1CAM family and of the Neurexin family Contactin-associated proteins (CNTNAPs or Casprs). Such interactions are important for organising the neuronal membrane, as well as for modulating the growth and pathfinding of axon tracts. In addition, they also mediate the functional maturation of axons by promoting their interactions with myelinating cells at the nodal, paranodal and juxtaparanodal regions. Such interactions also mediate differential ionic channels (both Na+ and K+) distribution, which is of critical relevance in the generation of the peak-shaped action potential. Indeed, thanks to their interactions with Ankyrin G, Na+ channels map within the nodal regions, where they drive axonal depolarization. However, no ionic channels are found in the flanking Contactin1-containing paranodal regions, where CNTN1 interactions with Caspr1 and with the Ig superfamily component Neurofascin 155 in cis and in trans, respectively, build a molecular barrier between the node and the juxtaparanode. In this region K+ channels are clustered, depending upon molecular interactions with Contactin 2 and with Caspr2. In addition to these functions, the Contactins appear to have also a role in degenerative and inflammatory disorders: indeed Contactin 2 is involved in neurodegenerative disorders with a special reference to the Alzheimer disease, given its ability to work as a ligand of the Alzheimer Precursor Protein (APP), which results in increased Alzheimer Intracellular Domain (AICD) release in a γ-secretase-dependent manner. On the other hand Contactin 1 drives Notch signalling activation via the Hes pathway, which could be consistent with its ability to modulate neuroinflammation events, and with the possibility that Contactin 1-dependent interactions may participate to the pathogenesis of the Multiple Sclerosis and of other inflammatory disorders.

F3/contactin and TAG1 play antagonistic roles in the regulation of sonic hedgehog-induced cerebellar granule neuron progenitor proliferation.

Modulation of the sonic hedgehog (SHH) pathway is a crucial factor in cerebellar morphogenesis. Stimulation of granule neuron progenitor (GNP) proliferation is a central function of SHH signalling, but how this is controlled locally is not understood. We show that two sequentially expressed members of the contactin (CNTN) family of adhesion molecules, TAG1 and F3, act antagonistically to control SHH-induced proliferation: F3 suppresses SHH-induced GNP proliferation and induces differentiation, whereas TAG1 antagonises F3. Production of GNPs in TAG1-null mice is delayed and reduced. F3 and TAG1 colocalise on GNPs with the related L1-like adhesion molecule NrCAM, and F3 fails to suppress the SHH-induced proliferation of NrCAM-deficient GNPs. We show that F3 and SHH both primarily affect a group of intermediate GNPs (IPs), which, though actively dividing, also express molecules associated with differentiation, including ß-tubulin III (TuJ1) and TAG1. In vivo, intermediate progenitors form a discrete layer in the middle of the external germinal layer (mEGL), while F3 becomes expressed on the axons of postmitotic granule neurons as they leave the inner EGL (iEGL). We propose, therefore, that F3 acts as a localised signal in the iEGL that induces SHH-stimulated cells in the overlying mEGL to exit cell cycle and differentiate. By contrast, expression of TAG1 on GNPs antagonises this signal in the mEGL, preventing premature differentiation and sustaining GNP expansion in a paracrine fashion. Together, these findings indicate that CNTN and L1-like proteins play a significant role in modulating SHH-induced neuronal precursor proliferation.

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.

F3/Contactin acts as a modulator of neurogenesis during cerebral cortex development.

The expression of the cell recognition molecule F3/Contactin (CNTN1) is generally associated with the functions of post-mitotic neurons. In the embryonic cortex, however, we find it expressed by proliferating ventricular zone (VZ) precursors. In contrast to previous findings in the developing cerebellum, F3/Contactin transgenic overexpression in the early cortical VZ promotes proliferation and expands the precursor pool at the expense of neurogenesis. At later stages, when F3/Contactin levels subside, however, neurogenesis resumes, suggesting that F3/Contactin expression in the VZ is inversely related to neurogenesis and plays a role in a feedback control mechanism, regulating the orderly progression of cortical development. The modified F3/Contactin profile therefore results in delayed corticogenesis, as judged by downregulation in upper and lower layer marker expression and by BrdU birth dating, indicating that, in this transgenic model, increased F3/Contactin levels counteract neuronal precursor commitment. These effects also occur in primary cultures and are reproduced by addition of an F3/Fc fusion protein to wild type cultures. Together, these data indicate a completely novel function for F3/Contactin. Parallel changes in the generation of the Notch Intracellular Domain and in the expression of the Hes-1 transcription factor indicate that activation of the Notch pathway plays a role in this phenotype, consistent with previous in vitro reports that F3/Contactin is a Notch1 ligand.

Juxtaparanodal clustering of Shaker-like K+ channels in myelinated axons depends on Caspr2 and TAG-1.

In myelinated axons, K+ channels are concealed under the myelin sheath in the juxtaparanodal region, where they are associated with Caspr2, a member of the neurexin superfamily. Deletion of Caspr2 in mice by gene targeting revealed that it is required to maintain K+ channels at this location. Furthermore, we show that the localization of Caspr2 and clustering of K+ channels at the juxtaparanodal region depends on the presence of TAG-1, an immunoglobulin-like cell adhesion molecule that binds Caspr2. These results demonstrate that Caspr2 and TAG-1 form a scaffold that is necessary to maintain K+ channels at the juxtaparanodal region, suggesting that axon-glia interactions mediated by these proteins allow myelinating glial cells to organize ion channels in the underlying axonal membrane.

Methodological standards, quality of reporting and regulatory compliance in animal research on amyotrophic lateral sclerosis: a systematic review.

OBJECTIVES: The amyotrophic lateral sclerosis (ALS) research community was one of the first to adopt methodology guidelines to improve preclinical research reproducibility. We here present the results of a systematic review to investigate how the standards in this field changed over the 10-year period during which the guidelines were first published (2007) and updated (2010). METHODS: We searched for papers reporting ALS research on SOD1 (superoxide dismutase 1) mice published between 2005 and 2015 on the ISI Web of Science database, resulting in a sample of 569 papers to review, after triage. Two scores-one for methodological quality, one for regulatory compliance-were built from weighted sums of separate sets of items, and subjected to multivariable regression analysis, to assess how these related to publication year, type of study, country of origin and journal. RESULTS: Reporting standards improved over time. Of papers published after the first ALS guidelines were made public, fewer than 9% referred specifically to these. Of key research parameters, only three (genetic background, number of transgenes and group size) were reported in >50% of the papers. Information on housing conditions, randomisation and blinding was absent in over two-thirds of the papers. Group size was among the best reported parameters, but the majority reported using fewer than the recommended sample size and only two studies clearly justified group size. CONCLUSIONS: General methodological standards improved gradually over a period of 8-10 years, but remained generally comparable with related fields with no specific guidelines, except with regard to severity. Only 11% of ALS studies were classified in the highest severity level (animals allowed to reach death or moribund stages), substantially below the proportion in studies of comparable neurodegenerative diseases such as Huntington's. The existence of field-specific guidelines, although a welcome indication of concern, seems insufficient to ensure adherence to high methodological standards. Other mechanisms may be required to improve methodological and welfare standards.

Tracking Differential Endocytosis and Trafficking of Semaphorin Receptor Complexes in Responding Nerve Growth Cones.

The study of receptor endocytosis is important to our understanding of the signal transduction events initiated by axon guidance cues in growth cones. Fab fragments of antibodies to guidance receptors and endocytic cargoes like transferrin and cholera toxin-B are the tools of choice for studying the dynamics of receptor internalization and intracellular trafficking by different pathways. We describe a method where in vitro cultures of growth cones are incubated with these ligands in the presence or absence of Sema3A, followed by stripping of remaining ligand on cell-surface and analysis by immunofluorescence techniques. These techniques can be employed for studying the endocytosis of any axon guidance receptor in response to attractive or repulsive guidance cues and, in particular, to allow the differential trafficking of specific receptor components to be revealed.

Distinct cis regulatory elements govern the expression of TAG1 in embryonic sensory ganglia and spinal cord.

Cell fate commitment of spinal progenitor neurons is initiated by long-range, midline-derived, morphogens that regulate an array of transcription factors that, in turn, act sequentially or in parallel to control neuronal differentiation. Included among these are transcription factors that regulate the expression of receptors for guidance cues, thereby determining axonal trajectories. The Ig/FNIII superfamily molecules TAG1/Axonin1/CNTN2 (TAG1) and Neurofascin (Nfasc) are co-expressed in numerous neuronal cell types in the CNS and PNS - for example motor, DRG and interneurons - both promote neurite outgrowth and both are required for the architecture and function of nodes of Ranvier. The genes encoding TAG1 and Nfasc are adjacent in the genome, an arrangement which is evolutionarily conserved. To study the transcriptional network that governs TAG1 and Nfasc expression in spinal motor and commissural neurons, we set out to identify cis elements that regulate their expression. Two evolutionarily conserved DNA modules, one located between the Nfasc and TAG1 genes and the second directly 5' to the first exon and encompassing the first intron of TAG1, were identified that direct complementary expression to the CNS and PNS, respectively, of the embryonic hindbrain and spinal cord. Sequential deletions and point mutations of the CNS enhancer element revealed a 130bp element containing three conserved E-boxes required for motor neuron expression. In combination, these two elements appear to recapitulate a major part of the pattern of TAG1 expression in the embryonic nervous system.

The neural adhesion molecule TAG-1 modulates responses of sensory axons to diffusible guidance signals.

When the axons of primary sensory neurons project into the embryonic mammalian spinal cord, they bifurcate and extend rostrocaudally before sending collaterals to specific laminae according to neuronal subclass. The specificity of this innervation has been suggested to be the result both of differential sensitivity to chemorepellants expressed in the ventral spinal cord and of the function of Ig-like neural cell adhesion molecules in the dorsal horn. The relationship between these mechanisms has not been addressed. Focussing on the pathfinding of TrkA+ NGF-dependent axons, we demonstrate for the first time that their axons project prematurely into the dorsal horn of both L1 and TAG-1 knockout mice. We show that axons lacking TAG-1, similar to those lacking L1, are insensitive to wild-type ventral spinal cord (VSC)-derived chemorepellants, indicating that adhesion molecule function is required in the axons, and that this loss of response is explained in part by loss of response to Sema3A. We present evidence that TAG-1 affects sensitivity to Sema3A by binding to L1 and modulating the endocytosis of the L1/neuropilin 1 Sema3A receptor complex. However, TAG-1 appears to affect sensitivity to other VSC-derived chemorepellants via an L1-independent mechanism. We suggest that this dependence of chemorepellant sensitivity on the functions of combinations of adhesion molecules is important to ensure that axons project via specific pathways before extending to their final targets.

Complete rescue of the nude mutant phenotype by a wild-type Foxn1 transgene.

In this paper we describe the production and analysis of mice carrying a 110-kb transgene that encompasses the wild-type Foxn1 genomic locus. Mutations in Foxn1 cause the nude phenotype. We show that in the hair follicles, transgenic mice with increased Foxn1 gene dosage exhibited increased Foxn1 expression that was restricted correctly to the nascent, post-mitotic cells of the differentiating hair cortex and hair cuticle lineages. We also demonstrate for the first time that a Foxn1 transgene rescues completely both the hair follicle and the thymus defects in animals that are also homozygous for the nude mutation at the endogenous Foxn1 locus, causing the development of a full coat of hair and a normal population of peripheral blood T lymphocytes. We conclude that sufficient cis-acting regulatory information resides within this 110-kb transgene to direct reliable and appropriate tissue-specific expression of the Foxn1 gene.