Ashwin Woodhoo

c-Jun Reprograms Schwann Cells of Injured Nerves to Generate a Repair Cell Essential for Regeneration

Summary The radical response of peripheral nerves to injury (Wallerian degeneration) is the cornerstone of nerve repair. We show that activation of the transcription factor c-Jun in Schwann cells is a global regulator of Wallerian degeneration. c-Jun governs major aspects of the injury response, determines the expression of trophic factors, adhesion molecules, the formation of regeneration tracks and myelin clearance and controls the distinctive regenerative potential of peripheral nerves. A key function of c-Jun is the activation of a repair program in Schwann cells and the creation of a cell specialized to support regeneration. We show that absence of c-Jun results in the formation of a dysfunctional repair cell, striking failure of functional recovery, and neuronal death. We conclude that a single glial transcription factor is essential for restoration of damaged nerves, acting to control the transdifferentiation of myelin and Remak Schwann cells to dedicated repair cells in damaged tissue.

c-Jun is a negative regulator of myelination

Schwann cell myelination depends on Krox-20/Egr2 and other promyelin transcription factors that are activated by axonal signals and control the generation of myelin-forming cells. Myelin-forming cells remain remarkably plastic and can revert to the immature phenotype, a process which is seen in injured nerves and demyelinating neuropathies. We report that c-Jun is an important regulator of this plasticity. At physiological levels, c-Jun inhibits myelin gene activation by Krox-20 or cyclic adenosine monophosphate. c-Jun also drives myelinating cells back to the immature state in transected nerves in vivo. Enforced c-Jun expression inhibits myelination in cocultures. Furthermore, c-Jun and Krox-20 show a cross-antagonistic functional relationship. c-Jun therefore negatively regulates the myelinating Schwann cell phenotype, representing a signal that functionally stands in opposition to the promyelin transcription factors. Negative regulation of myelination is likely to have significant implications for three areas of Schwann cell biology: the molecular analysis of plasticity, demyelinating pathologies, and the response of peripheral nerves to injury.

Notch controls embryonic Schwann cell differentiation, postnatal myelination and adult plasticity

Notch signaling is central to vertebrate development, and analysis of Notch has provided important insights into pathogenetic mechanisms in the CNS and many other tissues. However, surprisingly little is known about the role of Notch in the development and pathology of Schwann cells and peripheral nerves. Using transgenic mice and cell cultures, we found that Notch has complex and extensive regulatory functions in Schwann cells. Notch promoted the generation of Schwann cells from Schwann cell precursors and regulated the size of the Schwann cell pool by controlling proliferation. Notch inhibited myelination, establishing that myelination is subject to negative transcriptional regulation that opposes forward drives such as Krox20. Notably, in the adult, Notch dysregulation resulted in demyelination; this finding identifies a signaling pathway that induces myelin breakdown in vivo. These findings are relevant for understanding the molecular mechanisms that control Schwann cell plasticity and underlie nerve pathology, including demyelinating neuropathies and tumorigenesis.

Development of the Schwann cell lineage: from the neural crest to the myelinated nerve

The myelinating and nonmyelinating Schwann cells in peripheral nerves are derived from the neural crest, which is a transient and multipotent embryonic structure that also generates the other main glial subtypes of the peripheral nervous system (PNS). Schwann cell development occurs through a series of transitional embryonic and postnatal phases, which are tightly regulated by a number of signals. During the early embryonic phases, neural crest cells are specified to give rise to Schwann cell precursors, which represent the first transitional stage in the Schwann cell lineage, and these then generate the immature Schwann cells. At birth, the immature Schwann cells differentiate into either the myelinating or nonmyelinating Schwann cells that populate the mature nerve trunks. In this review, we will discuss the biology of the transitional stages in embryonic and early postnatal Schwann cell development, including the phenotypic differences between them and the recently identified signaling pathways, which control their differentiation and maintenance. In addition, the role and importance of the microenvironment in which glial differentiation takes place will be discussed.

Novel signals controlling embryonic Schwann cell development, myelination and dedifferentiation

Abstract  Immature Schwann cells found in perinatal rodent nerves are generated from Schwann cell precursors (SCPs) that originate from the neural crest. Immature Schwann cells generate the myelinating and non‐myelinating Schwann cells of adult nerves. When axons degenerate following injury, Schwann cells demyelinate, proliferate and dedifferentiate to assume a molecular phenotype similar to that of immature cells, a process essential for successful nerve regeneration. Increasing evidence indicates that Schwann cell dedifferentiation involves activation of specific receptors, intracellular signalling pathways and transcription factors in a manner analogous to myelination. We have investigated the roles of Notch and the transcription factor c‐Jun in development and after nerve transection. In vivo, Notch signalling regulates the transition from SCP to Schwann cell, times Schwann cell generation, controls Schwann cell proliferation and acts as a brake on myelination. Notch is elevated in injured nerves where it accelerates the rate of dedifferentiation. Likewise, the transcription factor c‐Jun is required for Schwann cell proliferation and death and is down‐regulated by Krox‐20 on myelination. Forced expression of c‐Jun in Schwann cells prevents myelination, and in injured nerves, c‐Jun is required for appropriate dedifferentiation, the re‐emergence of the immature Schwann cell state and nerve regeneration. Thus, both Notch and c‐Jun are negative regulators of myelination. The growing realisation that myelination is subject to negative as well as positive controls and progress in molecular identification of negative regulators is likely to impact on our understanding of demyelinating disease and mechanisms that control nerve repair.

Gene profiling and bioinformatic analysis of Schwann cell embryonic development and myelination

To elucidate the molecular mechanisms involved in Schwann cell development, we profiled gene expression in the developing and injured rat sciatic nerve. The genes that showed significant changes in expression in developing and dedifferentiated nerve were validated with RT‐PCR, in situ hybridisation, Western blot and immunofluorescence. A comprehensive approach to annotating micro‐array probes and their associated transcripts was performed using Biopendium™, a database of sequence and structural annotation. This approach significantly increased the number of genes for which a functional insight could be found. The analysis implicates agrin and two members of the collapsin response‐mediated protein (CRMP) family in the switch from precursors to Schwann cells, and synuclein‐1 and αB‐crystallin in peripheral nerve myelination. We also identified a group of genes typically related to chondrogenesis and cartilage/bone development, including type II collagen, that were expressed in a manner similar to that of myelin‐associated genes. The comprehensive function annotation also identified, among the genes regulated during nerve development or after nerve injury, proteins belonging to high‐interest families, such as cytokines and kinases, and should therefore provide a uniquely valuable resource for future research.

The trunk neural crest and its early glial derivatives: a study of survival responses, developmental schedules and autocrine mechanisms

Regulation of survival during gliogenesis from the trunk neural crest is poorly understood. Using adapted survival assays, we directly compared crest cells and the crest-derived precursor populations that generate satellite cells and Schwann cells. A range of factors that supports Schwann cells and glial precursors does not rescue crest, with the major exception of neuregulin-1 that rescues crest cells provided they contact the extracellular matrix. Autocrine survival appears earlier in developing satellite cells than Schwann cells. Satellite cells also show early expression of S100beta, BFABP and fibronectin and early survival responses to IGF-1, NT-3 and PDGF-BB that in developing Schwann cells are not seen until the precursor/Schwann cell transition. These experiments define novel differences between crest cells and early glia and show that entry to the glial lineage is an important point for regulation of survival responses. They show that survival mechanisms among PNS glia differ early in development and that satellite cell development runs ahead of schedule compared to Schwann cells in several significant features.

Schwann cell precursors transplanted into the injured spinal cord multiply, integrate and are permissive for axon growth.

There is a strong current interest in the use of cell transplantation for the treatment of spinal cord injuries. We report here the novel and potentially useful properties of an early cell in the Schwann cell lineage, the Schwann cell precursor (SCP). The experiments reveal a striking difference between these cells and Schwann cells when transplanted into the CNS. Unlike Schwann cells, SCPs thrive in the CNS where they initially proliferate rapidly but then fall out of division, thus effectively filling up the large cystic cavities formed following crush injury, while avoiding tumor formation. By 8 weeks, SCPs had started to express S100β protein, a marker that differentiates Schwann cells from SCPs and had formed an apparently stable, vascularized cell mass, which created a continuous cellular bridge across the cystic cavities. The formation of the surrounding glial scar was reduced by local spread of the transplanted cells into the surrounding CNS tissue, where the cells integrated intimately with astrocytes and attenuated the physical barrier they normally form. SCP transplantation also altered and reduced the expression of chondroitin sulfate proteoglycans around the injury site. Caudal to the SCP transplants there was a large increase in the number of axons, compared with that seen in nontransplanted control tissue, showing that the implants effectively support axonal growth or sprouting. SCPs have advantageous attributes for CNS repair, despite the fact that sticky tape removal and ladder crossing tests at 8 weeks did not reveal significant functional improvements when compared with control animals.

Calcineurin–nuclear factor of activated t cells regulation of Krox-20 expression in Schwann cells requires elevation of intracellular cyclic AMP

The transcription factor Krox‐20 (Egr2) is a master regulator of Schwann cell myelination. In mice from which calcineurin B had been excised in cells of the neural crest lineage, calcineurin–nuclear factor of activated T cells (NFAT) signaling was required for neuregulin‐related Schwann cell myelination (Kao et al. [ 2009 ] Immunity 12:359–372). Whether NFAT signaling required simultaneous elevation of intracellular cAMP levels was not explored. In vivo, Krox‐20 expression requires continuous axon–Schwann cell signaling that in Schwann cell cultures can be mimicked by elevation of intracellular cAMP. We have investigated the role of the calcineurin–NFAT pathway in Krox‐20 induction in purified rat Schwann cell cultures. Activation of this pathway requires elevation of intracellular Ca2+ levels. The calcium ionophore A23187 or ionomycin was used to increase intracellular Ca2+ levels in Schwann cell cultures that had been treated with dibutyryl cAMP to induce Krox‐20. Increase in Ca2+ levels significantly potentiated Krox‐20 induction, determined by Krox‐20 immunolabeling of individual cells and Western blotting. Levels of the myelin proteins periaxin and P0 were also elevated. The potentiating effect was blocked by cyclosporin A, a specific blocker of the calcineurin–NFAT pathway. We found that, in the absence of cAMP elevation, treatment with A23187 alone failed to induce Krox‐20 expression, indicating that NFAT upregulation of Krox‐20 requires elevation of cAMP levels in Schwann cells. P‐VIVIT, another specific inhibitor of calcineurin–NFAT interaction, blocked Krox‐20 induction in response to dibutyryl cAMP and ionophore. HA‐NFAT1 (1–460)‐GFP translocated to the nucleus on treatment with dibutyryl cAMP with or without added ionophore. NFAT isoforms 1–4 were detected in purified Schwann cells by quantitative RT‐PCR.

The RNA-binding protein human antigen R controls global changes in gene expression during Schwann cell development.

An important prerequisite to myelination in peripheral nerves is the establishment of one-to-one relationships between axons and Schwann cells. This patterning event depends on immature Schwann cell proliferation, apoptosis, and morphogenesis, which are governed by coordinated changes in gene expression. Here, we found that the RNA-binding protein human antigen R (HuR) was highly expressed in immature Schwann cells, where genome-wide identification of its target mRNAs in vivo in mouse sciatic nerves using ribonomics showed an enrichment of functionally related genes regulating these processes. HuR coordinately regulated expression of several genes to promote proliferation, apoptosis, and morphogenesis in rat Schwann cells, in response to NRG1, TGFβ, and laminins, three major signals implicated in this patterning event. Strikingly, HuR also binds to several mRNAs encoding myelination-related proteins but, contrary to its typical function, negatively regulated their expression, likely to prevent ectopic myelination during development. These functions of HuR correlated with its abundance and subcellular localization, which were regulated by different signals in Schwann cells.