John R. Bermingham

Gliomedin Mediates Schwann Cell-Axon Interaction and the Molecular Assembly of the Nodes of Ranvier

Accumulation of Na(+) channels at the nodes of Ranvier is a prerequisite for saltatory conduction. In peripheral nerves, clustering of these channels along the axolemma is regulated by myelinating Schwann cells through a yet unknown mechanism. We report the identification of gliomedin, a glial ligand for neurofascin and NrCAM, two axonal immunoglobulin cell adhesion molecules that are associated with Na+ channels at the nodes of Ranvier. Gliomedin is expressed by myelinating Schwann cells and accumulates at the edges of each myelin segment during development, where it aligns with the forming nodes. Eliminating the expression of gliomedin by RNAi, or the addition of a soluble extracellular domain of neurofascin to myelinating cultures, which caused the redistribution of gliomedin along the internodes, abolished node formation. Furthermore, a soluble gliomedin induced nodal-like clusters of Na+ channels in the absence of Schwann cells. We propose that gliomedin provides a glial cue for the formation of peripheral nodes of Ranvier.

ASF/SF2-regulated CaMKIIδ alternative splicing temporally reprograms excitation-contraction coupling in cardiac muscle

The transition from juvenile to adult life is accompanied by programmed remodeling in many tissues and organs, which is key for organisms to adapt to the demand of the environment. Here we report a novel regulated alternative splicing program that is crucial for postnatnal heart remodeling in the mouse. We identify the essential splicing factor ASF/SF2 as a key component of the program, regulating a restricted set of tissue-specific alternative splicing events during heart remodeling. Cardiomyocytes deficient in ASF/SF2 display an unexpected hypercontraction phenotype due to a defect in postnatal splicing switch of the Ca(2+)/calmodulin-dependent kinase IIdelta (CaMKIIdelta) transcript. This failure results in mistargeting of the kinase to sarcolemmal membranes, causing severe excitation-contraction coupling defects. Our results validate ASF/SF2 as a fundamental splicing regulator in the reprogramming pathway and reveal the central contribution of ASF/SF2-regulated CaMKIIdelta alternative splicing to functional remodeling in developing heart.

A POU domain transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system.

Axon pathfinding relies on the ability of the growth cone to detect and interpret guidance cues and to modulate cytoskeletal changes in response to these signals. We report that the murine POU domain transcription factor Brn-3.2 regulates pathfinding in retinal ganglion cell (RGC) axons at multiple points along their pathways and the establishment of topographic order in the superior colliculus. Using representational difference analysis, we identified Brn-3.2 gene targets likely to act on axon guidance at the levels of transcription, cell-cell interaction, and signal transduction, including the actin-binding LIM domain protein abLIM. We present evidence that abLIM plays a crucial role in RGC axon pathfinding, sharing functional similarity with its C. elegans homolog, UNC-115. Our findings provide insights into a Brn-3.2-directed hierarchical program linking signaling events to cytoskeletal changes required for axon pathfinding.

SC35 Plays a Role in T Cell Development and Alternative Splicing of CD45

Molecular diversity via alternative splicing is important for cellular function and development. SR proteins are strong candidate regulators of alternative splicing because they can modulate splice site selection. However, endogenous substrates for SR proteins are largely unknown, and their roles as splicing regulators in vertebrate development are unclear. Here we report that Cre-mediated conditional deletion of the prototypical SR protein SC35 in the thymus causes a defect in T cell maturation. Deletion of SC35 alters alternative splicing of CD45, a receptor tyrosine phosphatase known to be regulated by differential splicing during thymocyte development and activation. This study establishes a model to address the function of SR proteins in physiological settings and reveals a critical role of SC35 in a T cell-specific regulated splicing pathway.

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.

POU Domain Factors in Neural Development

Transcription factors serve critical roles in the progressive development of general body plan, organ commitment, and finally, specific cell types. Comparison of the biological roles of a series of individual members within a family permits some generalizations to be made regarding the developmental events that are likely to be regulated by a particular class of transcription factors. Here, we evidence that the developmental functions of the family of transcription factors characterized by the POU DNA binding motif exerts roles in mammalian development. The POU domain family of transcription factors was defined following the observation that the products of three mammalian genes, Pit-1, Oct-1, and Oct-2, and the protein encoded by the C. elegans gene unc-86, shared a region of homology, known as the POU domain. The POU domain is a bipartite DNA binding domain, consisting of two highly conserved regions, tethered by a variable linker. The approximately 75 amino acid N-terminal region was called the POU-specific domain and the C-terminal 60 amino acid region, the POU-homeodomain. High-affinity site-specific DNA binding by POU domain transcription factors requires both the POU-specific and the POU-homeodomain. Resolution of the crystal structures of Oct-1 and Pit-1 POU domains bound to DNA as a monomer and homodimer, respectively, confirmed several of the in vitro findings regarding interactions of this bipartite DNA binding domain with DNA and has provided important information regarding the flexibility and versatility of POU domain proteins. Overall the crystal structure of a monomer of the Oct-1 POU domain bound to the octamer element was similar to that predicted by the NMR solution structures of the POU-specific domain and the POU-homeodomain in isolation, with the POU-specific domain consists of four alpha helices, with the second and third helices forming a structure similar to the helix-turn-helix motif of the lambda and 434 repressors; several of the DNA base contacts are also conserved. A homodimer of the Pit-1 POU domain was crystallized bound to a Pit-1 dimer DNA element that is closely related to a site in the proximal promoter of the prolactin gene. The structure of the Pit-1 POU domain on DNA is very similar to that of Oct-1, and the Pit-1 POU-homeodomain/DNA structure is strikingly similar to that of other homeodomains, including the Oct-1 POU-homeodomain. The DNA contacts made by the Pit-1 POU-specific domain are also similar to those of Oct-1 and conserved with many made by the prokaryotic repressors. In the Oct-1 crystal, the POU-specific domain recognizes a GCAT half-site, while the corresponding sequence recognized by the Pit-1 POU-specific domain, GTAT, is on the opposing strand. As a result, the orientation of the Pit-1 POU-specific domain relative to the POU-homeodomain is flipped, as compared to the Oct-1 crystal structure, indicating the remarkable flexibility of the POU-specific domain in adapting to variations in sequence within the site. Also in contrast to the Oct-1 monomer structure is the observation that the POU-specific and POU-homeodomain of each Pit-1 molecule make major groove contacts on the same face of the DNA, consistent with the constraints imposed by its 15 amino acid linker. As a result, the Pit-1 POU domain homodimer essentially surrounds its DNA binding site. In the Pit-1 POU domain homodimer the dimerization interface is formed between the C-terminal end of helix 3 of the POU-homeodomain of one Pit-1 molecule and the N-terminus of helix 1 and the loop between helices 3 and 4 of the POU-specific domain of the other Pit-1 molecule. In contrast to other homeodomain crystal structures, the C-terminus of helix 3 in the Pit-1 POU-homeo-domain has an extended structure. (ABSTRACT TRUNCATED)

Modification of representational difference analysis applied to the isolation of forskolin-regulated genes from Schwann cells.

Many aspects of the response of Schwann cells to axonal cues can be induced in vitro by the adenylyl cyclase activator forskolin, yet the role of cAMP signaling in regulating Schwann cell differentiation remains unclear. To define better the relationship between cAMP signaling and Schwann cell differentiation, we used a modification of cDNA representational difference analysis (RDA) that permits the analysis of small amounts of mRNA and identified additional genes that are differentially expressed by forskolin‐treated and untreated Schwann cells. The genes that we have identified, including MKP3, a regulator of ERK signaling, and the sphingosine‐1‐phosphate receptor edg3/lpB3, may play important roles in mediating Schwann cell differentiation. J. Neurosci. Res. 63:516–524, 2001.

Concurrent Lpin1 and Nrcam mouse mutations result in severe peripheral neuropathy with transitory hindlimb paralysis.

Peripheral neuropathy is a broad category of disorders with a diverse etiology, grouped together by their common pathogenic effect on the peripheral nervous system (PNS). Because of the heterogeneity observed to be responsible for these disorders, a forward genetics method of gene discovery was used to identify additional affected pathways. In this report, we describe the mutant mouse line 20884, generated by N-ethyl-N-nitrosourea mutagenesis, which is characterized by adult-onset transitory hindlimb paralysis. Linkage mapping revealed that two point mutations are responsible for the phenotype: a partial loss-of-function mutation in the gene for phosphatidate phosphatase Lpin1 and a truncation mutation in the gene that encodes the neuronal cell adhesion molecule NrCAM. To investigate how the 20884 Lpin1 and Nrcam mutations interact to produce the paralysis phenotype, the double mutant and both single mutants were analyzed by quantitative behavioral, histological, and electrophysiological means. The Lpin120884 mutant and the double mutant are characterized by similar levels of demyelination and aberrant myelin structures. Nevertheless, the double mutant exhibits more severe electrophysiological abnormalities than the Lpin120884 mutant. The Nrcam20884 mutant is characterized by normal sciatic nerve morphology and a mild electrophysiological defect. Comparison of the double mutant phenotype with the two single mutants does not point to an additive relationship between the two defects; rather, the Lpin120884 and Nrcam20884 defects appear to act synergistically to produce the 20884 phenotype. It is proposed that the absence of NrCAM in a demyelinating environment has a deleterious effect, possibly by impairing the process of remyelination.

POU-er-ful developmental regulators revisited

The POU domain transcription factors have been proposed to be important regulators of key developmental processes. The characterization of naturally occurring mutations and targeted gene deletions has demonstrated that many of these proteins function in terminal differentiation events, including lineage determination, migration, proliferation, survival, and maintenance of mature cell types. POU domain factors can exert critical functions in cell types in which they exhibit either transient or sustained expression patterns, and it is likely that their target genes encode receptors, guidance molecules, and neuropeptides that define differentiated phenotypes.