Daniela Salomon

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.

A Glial Signal Consisting of Gliomedin and NrCAM Clusters Axonal Na+ Channels during the Formation of Nodes of Ranvier

Saltatory conduction requires high-density accumulation of Na(+) channels at the nodes of Ranvier. Nodal Na(+) channel clustering in the peripheral nervous system is regulated by myelinating Schwann cells through unknown mechanisms. During development, Na(+) channels are first clustered at heminodes that border each myelin segment, and later in the mature nodes that are formed by the fusion of two heminodes. Here, we show that initial clustering of Na(+) channels at heminodes requires glial NrCAM and gliomedin, as well as their axonal receptor neurofascin 186 (NF186). We further demonstrate that heminodal clustering coincides with a second, paranodal junction (PNJ)-dependent mechanism that allows Na(+) channels to accumulate at mature nodes by restricting their distribution between two growing myelin internodes. We propose that Schwann cells assemble the nodes of Ranvier by capturing Na(+) channels at heminodes and by constraining their distribution to the nodal gap. Together, these two cooperating mechanisms ensure fast and efficient conduction in myelinated nerves.

Caspr regulates the processing of contactin and inhibits its binding to neurofascin

Three cell adhesion molecules are present at the axoglial junctions that form between the axon and myelinating glia on either side of nodes of Ranvier. These include an axonal complex of contacin-associated protein (Caspr) and contactin, which was proposed to bind NF155, an isoform of neurofascin located on the glial paranodal loops. Here, we show that NF155 binds directly to contactin and that surprisingly, coexpression of Caspr inhibits this interaction. This inhibition reflects the association of Caspr with contactin during biosynthesis and the resulting expression of a low molecular weight (LMw), endoglycosidase H–sensitive isoform of contactin at the cell membrane, which remains associated with Caspr but is unable to bind NF155. Accordingly, deletion of Caspr in mice by gene targeting results in a shift from the LMw- to a HMw-contactin glycoform. These results demonstrate that Caspr regulates the intracellular processing and transport of contactin to the cell surface, thereby affecting its ability to interact with other cell adhesion molecules.

Cntnap4 differentially contributes to GABAergic and dopaminergic synaptic transmission

Although considerable evidence suggests that the chemical synapse is a lynchpin underlying affective disorders, how molecular insults differentially affect specific synaptic connections remains poorly understood. For instance, Neurexin 1a and 2 (NRXN1 and NRXN2) and CNTNAP2 (also known as CASPR2), all members of the neurexin superfamily of transmembrane molecules, have been implicated in neuropsychiatric disorders. However, their loss leads to deficits that have been best characterized with regard to their effect on excitatory cells. Notably, other disease-associated genes such as BDNF and ERBB4 implicate specific interneuron synapses in psychiatric disorders. Consistent with this, cortical interneuron dysfunction has been linked to epilepsy, schizophrenia and autism. Using a microarray screen that focused upon synapse-associated molecules, we identified Cntnap4 (contactin associated protein-like 4, also known as Caspr4) as highly enriched in developing murine interneurons. In this study we show that Cntnap4 is localized presynaptically and its loss leads to a reduction in the output of cortical parvalbumin (PV)-positive GABAergic (γ-aminobutyric acid producing) basket cells. Paradoxically, the loss of Cntnap4 augments midbrain dopaminergic release in the nucleus accumbens. In Cntnap4 mutant mice, synaptic defects in these disease-relevant neuronal populations are mirrored by sensory-motor gating and grooming endophenotypes; these symptoms could be pharmacologically reversed, providing promise for therapeutic intervention in psychiatric disorders.

Caspr3 and Caspr4, Two Novel Members of the Caspr Family Are Expressed in the Nervous System and Interact with PDZ Domains

The NCP family of cell-recognition molecules represents a distinct subgroup of the neurexins that includes Caspr and Caspr2, as well as Drosophila Neurexin-IV and axotactin. Here, we report the identification of Caspr3 and Caspr4, two new NCPs expressed in nervous system. Caspr3 was detected along axons in the corpus callosum, spinal cord, basket cells in the cerebellum and in peripheral nerves, as well as in oligodendrocytes. In contrast, expression of Caspr4 was more restricted to specific neuronal subpopulations in the olfactory bulb, hippocampus, deep cerebellar nuclei, and the substantia nigra. Similar to the neurexins, the cytoplasmic tails of Caspr3 and Caspr4 interacted differentially with PDZ domain-containing proteins of the CASK/Lin2-Veli/Lin7-Mint1/Lin10 complex. The structural organization and distinct cellular distribution of Caspr3 and Caspr4 suggest a potential role of these proteins in cell recognition within the nervous system.

The molecular basis for the assembly and modulation of adherens-type junctions

Ultrastructural analyses of cells and tissues over the last several decades have indicated that cells commonly form contacts through specialized membrane domains. The cellular topology and fine structure of such adhesion sites varies considerably, reflecting the highly diverse physiological roles and cellular manifestations of adhesive interactions. In general, two major classes of adhesions may be distinguished, namely cell-matrix and cell-cell contacts. Among these distinct types of cell adhesions may be formed, attaching cells to the various extracellular matrix (ECM) networks on, or within which cells grow. Some of these attachment sites display a characteristic association with cytoskeletal filaments, providing an apparent mechanical linkage between intracellular force-generating systems and the pericellular connective tissue. It has been widely accepted that these contacts and their spatially- and temporallycontrolled reorganization, play cardinal roles in a large number of processes (which are often quite conflicting) such as cell anchorage and locomotion, growth stimulation and growth arrest, etc. Among these cytoskeleton-bound ECM contacts two major types were discerned on morpho

Genetic deletion of Cadm4 results in myelin abnormalities resembling Charcot-Marie-Tooth neuropathy.

The interaction between myelinating Schwann cells and the axons they ensheath is mediated by cell adhesion molecules of the Cadm/Necl/SynCAM family. This family consists of four members: Cadm4/Necl4 and Cadm1/Necl2 are found in both glia and axons, whereas Cadm2/Necl3 and Cadm3/Necl1 are expressed by sensory and motor neurons. By generating mice lacking each of the Cadm genes, we now demonstrate that Cadm4 plays a role in the establishment of the myelin unit in the peripheral nervous system. Mice lacking Cadm4 (PGK-Cre/Cadm4fl/fl), but not Cadm1, Cadm2, or Cadm3, develop focal hypermyelination characterized by tomacula and myelin outfoldings, which are the hallmark of several Charcot-Marie-Tooth neuropathies. The absence of Cadm4 also resulted in abnormal axon–glial contact and redistribution of ion channels along the axon. These neuropathological features were also found in transgenic mice expressing a dominant-negative mutant of Cadm4 lacking its cytoplasmic domain in myelinating glia Tg(mbp-Cadm4dCT), as well as in mice lacking Cadm4 specifically in Schwann cells (DHH-Cre/Cadm4fl/fl). Consistent with these abnormalities, both PGK-Cre/Cadm4fl/fl and Tg(mbp-Cadm4dCT) mice exhibit impaired motor function and slower nerve conduction velocity. These findings indicate that Cadm4 regulates the growth of the myelin unit and the organization of the underlying axonal membrane.

Zebrafish cyclin D1 is differentially expressed during early embryogenesis.

We have isolated and determined the nucleotide sequence of a cDNA containing the complete coding region of cyclin D1 from embryonic zebrafish cDNA library. The cyclin D1 gene is a single copy gene within the zebrafish genome, which undergoes an alternative polyadenylation process. The initial expression of cyclin D1 transcript occurs at the presumed onset of G1 phase in the developing zebrafish embryo.

Contact-dependent regulation of vinculin expression in cultured fibroblasts: a study with vinculin-specific cDNA probes.

Vinculin specific cDNA clones were isolated from chicken embryo fibroblast (CEF) cDNA library in lambda gt11. The clones, ranging in size from 2.8 to 5.0 kb, were initially selected by rabbit antibodies to vinculin. Their identity was further confirmed by their specific reactivities with a battery of different vinculin‐specific monoclonal antibodies. Southern blot analysis of restriction enzyme digested chicken spleen DNA suggested that all the isolated cDNA clones correspond to the same gene(s). Northern blot hybridization revealed that the vinculin‐specific cDNA clones react with a single 6.5 kb mRNA in total cellular RNA preparations of CEF, whole chicken embryos and chicken gizzard smooth muscle. Moreover, fractionation of CEF poly(A)+ RNA by sucrose gradient centrifugation followed by translation in cell free system indicated that the mRNA coding for vinculin has a size of about 6.0‐7.0 kb. The identity of these clones was finally confirmed by selection hybridization assay. The isolated vinculin‐specific cDNA probes were subsequently used in order to study the effect of substrate adhesiveness on the expression of vinculin. We show here that cells cultured on highly adhesive substrate, such as endothelial extracellular matrix (ECM), form large vinculin‐rich focal contacts, while cells grown on poorly adhesive substrate poly(2‐hydroxyethyl methacrylate) [poly(HEMA)] contain only small distorted vinculin spots. These morphological differences were accompanied by over 5‐fold reduction in vinculin synthesis in cells growing on poly(HEMA), compared to those cultured on the ECM and over 7.5‐fold decrease in the levels of vinculin‐specific mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Kv7.2 Regulates the Function of Peripheral Sensory Neurons

The Kv7 (KCNQ) family of voltage‐gated K+ channels regulates cellular excitability. The functional role of Kv7.2 has been hampered by the lack of a viable Kcnq2‐null animal model. In this study, we generated homozygous Kcnq2‐null sensory neurons using the Cre‐Lox system; in these mice, Kv7.2 expression is absent in the peripheral sensory neurons, whereas the expression of other molecular components of nodes (including Kv7.3), paranodes, and juxtaparanodes is not altered. The conditional Kcnq2‐null animals exhibit normal motor performance but have increased thermal hyperalgesia and mechanical allodynia. Whole‐cell patch recording technique demonstrates that Kcnq2‐null sensory neurons have increased excitability and reduced spike frequency adaptation. Taken together, our results suggest that the loss of Kv7.2 activity increases the excitability of primary sensory neurons. J. Comp. Neurol. 522:3262–3280, 2014.