LGI3/2-ADAM23 interactions cluster Kv1 channels in myelinated axons to regulate refractory period.

Along myelinated axons, Shaker-type potassium channels (Kv1) accumulate at high density in the juxtaparanodal region, directly adjacent to the paranodal axon-glia junctions that flank the nodes of Ranvier. However, the mechanisms that control the clustering of Kv1 channels, as well as their function at this site, are still poorly understood. Here we demonstrate that axonal ADAM23 is essential for both the accumulation and stability of juxtaparanodal Kv1 complexes. The function of ADAM23 is critically dependent on its interaction with its extracellular ligands LGI2 and LGI3. Furthermore, we demonstrate that juxtaparanodal Kv1 complexes affect the refractory period, thus enabling high-frequency burst firing of action potentials. Our findings not only reveal a previously unknown molecular pathway that regulates Kv1 channel clustering, but they also demonstrate that the juxtaparanodal Kv1 channels that are concealed below the myelin sheath, play a significant role in modifying axonal physiology.

Functional and molecular characterization of mouse Gata2-independent hematopoietic progenitors.

The Gata2 transcription factor is a pivotal regulator of hematopoietic cell development and maintenance, highlighted by the fact that Gata2 haploinsufficiency has been identified as the cause of some familial cases of acute myelogenous leukemia/myelodysplastic syndrome and in MonoMac syndrome. Genetic deletion in mice has shown that Gata2 is pivotal to the embryonic generation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). It functions in the embryo during endothelial cell to hematopoietic cell transition to affect hematopoietic cluster, HPC, and HSC formation. Gata2 conditional deletion and overexpression studies show the importance of Gata2 levels in hematopoiesis, during all developmental stages. Although previous studies of cell populations phenotypically enriched in HPCs and HSCs show expression of Gata2, there has been no direct study of Gata2 expressing cells during normal hematopoiesis. In this study, we generate a Gata2Venus reporter mouse model with unperturbed Gata2 expression to examine the hematopoietic function and transcriptome of Gata2 expressing and nonexpressing cells. We show that all the HSCs are Gata2 expressing. However, not all HPCs in the aorta, vitelline and umbilical arteries, and fetal liver require or express Gata2. These Gata2-independent HPCs exhibit a different functional output and genetic program, including Ras and cyclic AMP response element-binding protein pathways and other Gata factors, compared with Gata2-dependent HPCs. Our results, indicating that Gata2 is of major importance in programming toward HSC fate but not in all cells with HPC fate, have implications for current reprogramming strategies.

Chemical corrector treatment ameliorates increased seizure susceptibility in a mouse model of familial epilepsy.

Epilepsy is one of the most common and intractable brain disorders. Mutations in the human gene LGI1, encoding a neuronal secreted protein, cause autosomal dominant lateral temporal lobe epilepsy (ADLTE). However, the pathogenic mechanisms of LGI1 mutations remain unclear. We classified 22 reported LGI1 missense mutations as either secretion defective or secretion competent, and we generated and analyzed two mouse models of ADLTE encoding mutant proteins representative of the two groups. The secretion-defective LGI1(E383A) protein was recognized by the ER quality-control machinery and prematurely degraded, whereas the secretable LGI1(S473L) protein abnormally dimerized and was selectively defective in binding to one of its receptors, ADAM22. Both mutations caused a loss of function, compromising intracellular trafficking or ligand activity of LGI1 and converging on reduced synaptic LGI1-ADAM22 interaction. A chemical corrector, 4-phenylbutyrate (4PBA), restored LGI1(E383A) folding and binding to ADAM22 and ameliorated the increased seizure susceptibility of the LGI1(E383A) model mice. This study establishes LGI1-related epilepsy as a conformational disease and suggests new therapeutic options for human epilepsy.

The sodium channel isoform transition at developing nodes of Ranvier in the peripheral nervous system: dependence on a Genetic program and myelination-induced cluster formation.

Among sodium channel isoforms, Nav 1.6 is selectively expressed at nodes of Ranvier in both the CNS and the PNS. However, non-Nav 1.6 isoforms such as Nav 1.2 are also present at the CNS nodes in early development but gradually diminish later. It has been proposed that myelination is part of a glia-neuron signaling mechanism that produces this change in nodal isoform expression. The present study used isoform-specific antibodies to demonstrate that, in the PNS, four other neuronal sodium channel isoforms were also clustered at nodes in early development but eventually disappeared during maturation. To study possible roles of myelination in such transitions, we investigated the nodal expression of selected isoforms in the sciatic nerve of the transgenic mouse Oct6(ΔSCE/ßgeo) , whose PNS myelination is delayed in the first postnatal week but eventually resumes. We found that delayed myelination retarded the formation of nodal channel clusters and altered the expression-elimination patterns of sodium channel isoforms, resulting in significantly reduced expression levels of non-Nav 1.6 isoforms in such delayed nodes. However, delayed myelination did not significantly affect the gene expression, protein synthesis, or axonal trafficking of any isoform studied. Rather, we found evidence for a developmentally programmed increase in neuronal Nav 1.6 expression with constant or decreasing neuronal expression of other isoforms that were unaffected by delayed myelination. Thus our results suggest that, in the developmental isoform switch of the PNS, myelination does not play a signaling role as that proposed for the CNS but rather serves only to form nodal clusters from existing isoform pools.

Functional phylogenetic analysis of LGI proteins identifies an interaction motif crucial for myelination.

The cellular interactions that drive the formation and maintenance of the insulating myelin sheath around axons are only partially understood. Leucine-rich glioma-inactivated (LGI) proteins play important roles in nervous system development and mutations in their genes have been associated with epilepsy and amyelination. Their function involves interactions with ADAM22 and ADAM23 cell surface receptors, possibly in apposing membranes, thus attenuating cellular interactions. LGI4-ADAM22 interactions are required for axonal sorting and myelination in the developing peripheral nervous system (PNS). Functional analysis revealed that, despite their high homology and affinity for ADAM22, LGI proteins are functionally distinct. To dissect the key residues in LGI proteins required for coordinating axonal sorting and myelination in the developing PNS, we adopted a phylogenetic and computational approach and demonstrate that the mechanism of action of LGI4 depends on a cluster of three amino acids on the outer surface of the LGI4 protein, thus providing a structural basis for the mechanistic differences in LGI protein function in nervous system development and evolution.

Functional dissection of the Oct6 Schwann cell enhancer reveals an essential role for dimeric Sox10 binding.

The POU domain transcription factor Pou3f1 (Oct6/Scip/Tst1) initiates the transition from ensheathing, promyelinating Schwann cells to myelinating cells. Axonal and other extracellular signals regulate Oct6 expression through the Oct6 Schwann cell enhancer (SCE), which is both required and sufficient to drive all aspects of Oct6 expression in Schwann cells. Thus, the Oct6 SCE is pivotal in the gene regulatory network that governs the onset of myelin formation in Schwann cells and provides a link between myelin promoting signaling and activation of a myelin-related transcriptional network. In this study, we define the relevant cis-acting elements within the SCE and identify the transcription factors that mediate Oct6 regulation. On the basis of phylogenetic comparisons and functional in vivo assays, we identify a number of highly conserved core elements within the mouse SCE. We show that core element 1 is absolutely required for full enhancer function and that it contains closely spaced inverted binding sites for Sox proteins. For the first time in vivo, the dimeric Sox10 binding to this element is shown to be essential for enhancer activity, whereas monomeric Sox10 binding is nonfunctional. As Oct6 and Sox10 synergize to activate the expression of the major myelin-related transcription factor Krox20, we propose that Sox10-dependent activation of Oct6 defines a feedforward regulatory module that serves to time and amplify the onset of myelination in the peripheral nervous system.

N-WASP is required for membrane wrapping and myelination by Schwann cells.

During peripheral nerve myelination, Schwann cells sort larger axons, ensheath them, and eventually wrap their membrane to form the myelin sheath. These processes involve extensive changes in cell shape, but the exact mechanisms involved are still unknown. Neural Wiskott-Aldrich syndrome protein (N-WASP) integrates various extracellular signals to control actin dynamics and cytoskeletal reorganization through activation of the Arp2/3 complex. By generating mice lacking N-WASP in myelinating Schwann cells, we show that N-WASP is crucial for myelination. In N-WASP-deficient nerves, Schwann cells sort and ensheath axons, but most of them fail to myelinate and arrest at the promyelinating stage. Yet, a limited number of Schwann cells form unusually short internodes, containing thin myelin sheaths, with the occasional appearance of myelin misfoldings. These data suggest that regulation of actin filament nucleation in Schwann cells by N-WASP is crucial for membrane wrapping, longitudinal extension, and myelination.

Adam22 is a major neuronal receptor for Lgi4-mediated Schwann cell signaling.

The segregation and myelination of axons in the developing PNS, results from a complex series of cellular and molecular interactions between Schwann cells and axons. Previously we identified the Lgi4 gene (leucine-rich glioma-inactivated4) as an important regulator of myelination in the PNS, and its dysfunction results in arthrogryposis as observed in claw paw mice. Lgi4 is a secreted protein and a member of a small family of proteins that are predominantly expressed in the nervous system. Their mechanism of action is unknown but may involve binding to members of the Adam (A disintegrin and metalloprotease) family of transmembrane proteins, in particular Adam22. We found that Lgi4 and Adam22 are both expressed in Schwann cells as well as in sensory neurons and that Lgi4 binds directly to Adam22 without a requirement for additional membrane associated factors. To determine whether Lgi4-Adam22 function involves a paracrine and/or an autocrine mechanism of action we performed heterotypic Schwann cell sensory neuron cultures and cell type-specific ablation of Lgi4 and Adam22 in mice. We show that Schwann cells are the principal cellular source of Lgi4 in the developing nerve and that Adam22 is required on axons. Our results thus reveal a novel paracrine signaling axis in peripheral nerve myelination in which Schwann cell secreted Lgi4 functions through binding of axonal Adam22 to drive the differentiation of Schwann cells.

The claw paw mutation reveals a role for Lgi4 in peripheral nerve development.

Peripheral nerve development results from multiple cellular interactions between axons, Schwann cells and the surrounding mesenchymal tissue. The delayed axonal sorting and hypomyelination throughout the peripheral nervous system of claw paw (clp) mutant mice suggest that the clp gene product is critical for these interactions. Here we identify the clp mutation as a 225-bp insertion in the Lgi4 gene. Lgi4 encodes a secreted and glycosylated leucine-rich repeat protein and is expressed in Schwann cells. The clp mutation affects Lgi4 mRNA splicing, resulting in a mutant protein that is retained in the cell. Additionally, siRNA-mediated downregulation of Lgi4 in wild-type neuron-Schwann cell cocultures inhibits myelination, whereas exogenous Lgi4 restores myelination in clp/clp cultures. Thus, the abnormalities observed in clp mice are attributable to the loss of Lgi4 function, and they identify Lgi4 as a new component of Schwann cell signaling pathway(s) that controls axon segregation and myelin formation.

A generic tool for biotinylation of tagged proteins in transgenic mice.

The remarkable high affinity (Kd approximately 10(-15) M) of avidin/streptavidin for biotin has been extensively exploited in purification methodologies. Recently a small peptide sequence (Avi-tag) has been defined that can be specifically and efficiently biotinylated by the bacterial BirA biotin ligase. Fusion of this small peptide sequence to a protein of interest and co-expression with the BirA gene in mammalian cells allowed purification of the biotinylated protein together with its associated proteins and other molecules. Ideally, one would like to apply these technologies to purify tagged proteins directly from mouse tissues. To make this approach feasible for a large variety of proteins we developed a mouse strain that expresses the BirA gene ubiquitously by inserting it in the ROSA26 locus. We demonstrate that the BirA protein is indeed expressed in all tissues tested. In order to demonstrate functionality we show that it biotinylates the transgene-encoded Avi-tagged Gata1 and Oct6 transcription factors in erythroid cells of the foetal liver and Schwann cells of the peripheral nerve respectively. Therefore, this mouse can be crossed to any transgenic mouse to obtain efficient biotinylation of an Avi-tagged protein for the purpose of protein (complex) purification.

Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells.

Neural crest stem cells (NCSCs) persist in peripheral nerves throughout late gestation but their function is unknown. Current models of nerve development only consider the generation of Schwann cells from neural crest, but the presence of NCSCs raises the possibility of multilineage differentiation. We performed Cre-recombinase fate mapping to determine which nerve cells are neural crest derived. Endoneurial fibroblasts, in addition to myelinating and non-myelinating Schwann cells, were neural crest derived, whereas perineurial cells, pericytes and endothelial cells were not. This identified endoneurial fibroblasts as a novel neural crest derivative, and demonstrated that trunk neural crest does give rise to fibroblasts in vivo, consistent with previous studies of trunk NCSCs in culture. The multilineage differentiation of NCSCs into glial and non-glial derivatives in the developing nerve appears to be regulated by neuregulin, notch ligands, and bone morphogenic proteins, as these factors are expressed in the developing nerve, and cause nerve NCSCs to generate Schwann cells and fibroblasts, but not neurons, in culture. Nerve development is thus more complex than was previously thought, involving NCSC self-renewal, lineage commitment and multilineage differentiation.

Cell autonomy of the mouse claw paw mutation.

Mice homozygous for the autosomal recessive mutation claw paw (clp) are characterized by limb posture abnormalities and congenital hypomyelination, with delayed onset of myelination of the peripheral nervous system but not the central nervous system. Although this combination of limb and peripheral nerve abnormalities in clp/clp mice might suggest a common neurogenic origin of the syndrome, it is not clear whether the clp gene acts primarily in the neurone, the Schwann cell or both. In the work described here, we address this question of cell autonomy of the clp mutation through reciprocal nerve grafting experiments between wild-type and clp/clp animals. Our results demonstrate that the clp mutation affects the Schwann cell compartment and possibly also the neuronal compartment. These data suggest that the clp gene product is expressed in Schwann cells as well as neurones and is likely to be involved in direct axon--Schwann cell interactions. Within the Schwann cell, clp affects a myelin-related signaling pathway that regulates periaxin and Krox-20 expression, but not Oct-6.

A tissue-specific knockout reveals that Gata1 is not essential for Sertoli cell function in the mouse.

The transcription factor Gata1 is essential for the development of erythroid cells. Consequently, Gata1 null mutants die in utero due to severe anaemia. Outside the haematopoietic system, Gata1 is only expressed in the Sertoli cells of the testis. To elucidate the function of Gata1 in the testis, we made a Sertoli cell-specific knockout of the Gata1 gene in the mouse. We deleted a normally functioning 'floxed' Gata1 gene in pre-Sertoli cells in vivo through the expression of Cre from a transgene driven by the Desert Hedgehog promoter. Surprisingly, Gata1 null testes developed to be morphologically normal, spermatogenesis was not obviously affected and expression levels of putative Gata1 target genes, and other Gata factors, were not altered. We conclude that expression of Gata1 in Sertoli cells is not essential for testis development or spermatogenesis in the mouse.

The POU proteins Brn-2 and Oct-6 share important functions in Schwann cell development.

The genetic hierarchy that controls myelination of peripheral nerves by Schwann cells includes the POU domain Oct-6/Scip/Tst-1and the zinc-finger Krox-20/Egr2 transcription factors. These pivotal transcription factors act to control the onset of myelination during development and tissue regeneration in adults following damage. In this report we demonstrate the involvement of a third transcription factor, the POU domain factor Brn-2. We show that Schwann cells express Brn-2 in a developmental profile similar to that of Oct-6 and that Brn-2 gene activation does not depend on Oct-6. Overexpression of Brn-2 in Oct-6-deficient Schwann cells, under control of the Oct-6 Schwann cell enhancer (SCE), results in partial rescue of the developmental delay phenotype, whereas compound disruption of both Brn-2 and Oct-6 results in a much more severe phenotype. Together these data strongly indicate that Brn-2 function largely overlaps with that of Oct-6 in driving the transition from promyelinating to myelinating Schwann cells.

Targeted mutation of Cyln2 in the Williams syndrome critical region links CLIP-115 haploinsufficiency to neurodevelopmental abnormalities in mice.

Williams syndrome is a neurodevelopmental disorder caused by the hemizygous deletion of 1.6 Mb on human chromosome 7q11.23. This region comprises the gene CYLN2, encoding CLIP-115, a microtubule-binding protein of 115 kD. Using a gene-targeting approach, we provide evidence that mice with haploinsufficiency for Cyln2 have features reminiscent of Williams syndrome, including mild growth deficiency, brain abnormalities, hippocampal dysfunction and particular deficits in motor coordination. Absence of CLIP-115 also leads to increased levels of CLIP-170 (a closely related cytoplasmic linker protein) and dynactin at the tips of growing microtubules. This protein redistribution may affect dynein motor regulation and, together with the loss of CLIP-115-specific functions, underlie neurological alterations in Williams syndrome.

A cell type-specific allele of the POU gene Oct-6 reveals Schwann cell autonomous function in nerve development and regeneration.

While an important role for the POU domain transcription factor Oct-6 in the developing peripheral nerve has been well established, studies into its exact role in nerve development and regeneration have been hampered by the high mortality rate of newborn Oct-6 mutant animals. In this study we have generated a Schwann cell-specific Oct-6 allele through deletion of the Schwann cell-specific enhancer element (SCE) in the Oct-6 locus. Analysis of mice homozygous for this allele (deltaSCE allele) reveals that rate-limiting levels of Oct-6 in Schwann cells are dependent on the SCE and that this element does not contribute to Oct-6 regulation in other cell types. We demonstrate a Schwann cell autonomous function for Oct-6 during nerve development as well as in regenerating nerve. Additionally, we show that Krox-20, an important regulatory target of Oct-6 in Schwann cells, is activated, with delayed kinetics, through an Oct-6-independent mechanism in these mice.

Characterisation of cis-acting sequences reveals a biphasic, axon-dependent regulation of Krox20 during Schwann cell development.

In Schwann cells (SC), myelination is controlled by the transcription factor gene Krox20/Egr2. Analysis of cis-acting elements governing Krox20 expression in SC revealed the existence of two separate elements. The first, designated immature Schwann cell element (ISE), was active in immature but not myelinating SC, whereas the second, designated myelinating Schwann cell element (MSE), was active from the onset of myelination to adulthood in myelinating SC. In vivo sciatic nerve regeneration experiments demonstrated that both elements were activated during this process, in an axon-dependent manner. Together the activity of these elements reproduced the profile of Krox20 expression during development and regeneration. Genetic studies showed that both elements were active in a Krox20 mutant background, while the activity of the MSE, but likely not of the ISE, required the POU domain transcription factor Oct6 at the time of myelination. The MSE was localised to a 1.3 kb fragment, 35 kb downstream of Krox20. The identification of multiple Oct6 binding sites within this fragment suggested that Oct6 directly controls Krox20 transcription. Taken together, these data indicate that, although Krox20 is expressed continuously from 15.5 dpc in SC, the regulation of its expression is a biphasic, axon-dependent phenomenon involving two cis-acting elements that act in succession during development. In addition, they provide insight into the complexity of the transcription factor regulatory network controlling myelination.