Expression and localization of Ski determine cell type-specific TGFbeta signaling effects on the cell cycle

Transforming growth factor beta (TGFbeta) promotes epithelial cell differentiation but induces Schwann cell proliferation. We show that the protooncogene Ski (Sloan-Kettering viral oncogene homologue) is an important regulator of these effects. TGFbeta down-regulates Ski in epithelial cells but not in Schwann cells. In Schwann cells but not in epithelial cells, retinoblastoma protein (Rb) is up-regulated by TGFbeta. Additionally, both Ski and Rb move to the cytoplasm, where they partially colocalize. In vivo, Ski and phospho-Rb (pRb) appear to interact in the Schwann cell cytoplasm of developing sciatic nerves. Ski overexpression induces Rb hyperphosphorylation, proliferation, and colocalization of both proteins in Schwann cell and epithelial cell cytoplasms independently of TGFbeta treatment. Conversely, Ski knockdown in Schwann cells blocks TGFbeta-induced proliferation and pRb cytoplasmic relocalization. Our findings reveal a critical function of fine-tuned Ski levels in the control of TGFbeta effects on the cell cycle and suggest that at least a part of Ski regulatory effects on TGFbeta-induced proliferation of Schwann cells is caused by its concerted action with Rb.     

erbb3 and erbb2 are essential for schwann cell migration and myelination in zebrafish

BACKGROUND: Myelin is critical for efficient axonal conduction in the vertebrate nervous system. Neuregulin (Nrg) ligands and their ErbB receptors are required for the development of Schwann cells, the glial cells that form myelin in the peripheral nervous system. Previous studies have not determined whether Nrg-ErbB signaling is essential in vivo for Schwann cell fate specification, proliferation, survival, migration, or the onset of myelination. RESULTS: In genetic screens for mutants with disruptions in myelinated nerves, we identified mutations in erbb3 and erbb2, which together encode a heteromeric tyrosine kinase receptor for Neuregulin ligands. Phenotypic analysis shows that both genes are essential for development of Schwann cells. BrdU-incorporation studies and time-lapse analysis reveal that Schwann cell proliferation and migration, but not survival, are disrupted in erbb3 mutants. We show that Schwann cells can migrate in the absence of DNA replication. This uncoupling of proliferation and migration indicates that erbb gene function is required independently for these two processes. Pharmacological inhibition of ErbB signaling at different stages reveals a continuing requirement for ErbB function during migration and also provides evidence that ErbB signaling is required after migration for proliferation and the terminal differentiation of myelinating Schwann cells. CONCLUSIONS: These results provide in vivo evidence that Neuregulin-ErbB signaling is essential for directed Schwann cell migration and demonstrate that this pathway is also required for the onset of myelination in postmigratory Schwann cells.     

Autophagy Is Involved in the Reduction of Myelinating Schwann Cell Cytoplasm during Myelin Maturation of the Peripheral Nerve

Peripheral nerve myelination involves dynamic changes in Schwann cell morphology and membrane structure. Recent studies have demonstrated that autophagy regulates organelle biogenesis and plasma membrane dynamics. In the present study, we investigated the role of autophagy in the development and differentiation of myelinating Schwann cells during sciatic nerve myelination. Electron microscopy and biochemical assays have shown that Schwann cells remove excess cytoplasmic organelles during myelination through macroautophagy. Inhibition of autophagy via Schwann cell-specific removal of ATG7, an essential molecule for macroautophagy, using a conditional knockout strategy, resulted in abnormally enlarged abaxonal cytoplasm in myelinating Schwann cells that contained a large number of ribosomes and an atypically expanded endoplasmic reticulum. Small fiber hypermyelination and minor anomalous peripheral nerve functions are observed in this mutant. Rapamycin-induced suppression of mTOR activity during the early postnatal period enhanced not only autophagy but also developmental reduction of myelinating Schwann cells cytoplasm in vivo. Together, our findings suggest that autophagy is a regulatory mechanism of Schwann cells structural plasticity during myelination.     

Axonal regulation of Schwann cell integrin expression suggests a role for alpha 6 beta 4 in myelination

Ensheathment and myelination of axons by Schwann cells in the peripheral nervous system requires contact with a basal lamina. The molecular mechanism(s) by which the basal lamina promotes myelination is not known but is likely to reflect the activity of integrins expressed by Schwann cells. To initiate studies on the role of integrins during myelination, we characterized the expression of two integrin subunits, beta 1 and beta 4, in an in vitro myelination system and compared their expression to that of the glial adhesion molecule, the myelin-associated glycoprotein (MAG). In the absence of neurons, Schwann cells express significant levels of beta 1 but virtually no beta 4 or MAG. When Schwann cells are cocultured with dorsal root ganglia neurons under conditions promoting myelination, expression of beta 4 and MAG increased dramatically in myelinating cells, whereas beta 1 levels remained essentially unchanged. (In general agreement with these findings, during peripheral nerve development in vivo, beta 4 levels also increase during the period of myelination in sharp contrast to beta 1 levels which show a striking decrease.) In cocultures of neurons and Schwann cells, beta 4 and MAG appear to colocalize in nascent myelin sheaths but have distinct distributions in mature sheaths, with beta 4 concentrated in the outer plasma membrane of the Schwann cell and MAG localized to the inner (periaxonal) membrane. Surprisingly, beta 4 is also present at high levels with MAG in Schmidt-Lanterman incisures. Immunoprecipitation studies demonstrated that primary Schwann cells express beta 1 in association with the alpha 1 and alpha 6 subunits, while myelinating Schwann cells express alpha 6 beta 4 and possibly alpha 1 beta 1. beta 4 is also downregulated during Wallerian degeneration in vitro, indicating that its expression requires continuous Schwann cell contact with the axon. These results indicate that axonal contact induces the expression of beta 4 during Schwann cell myelination and suggest that alpha 6 beta 4 is an important mediator of the interactions of myelinating Schwann cells with the basal lamina.     

Identification of novel cell-adhesion molecules in peripheral nerves using a signal-sequence trap

The development and maintenance of myelinated nerves in the PNS requires constant and reciprocal communication between Schwann cells and their associated axons. However, little is known about the nature of the cell-surface molecules that mediate axon-glial interactions at the onset of myelination and during maintenance of the myelin sheath in the adult. Based on the rationale that such molecules contain a signal sequence in order to be presented on the cell surface, we have employed a eukaryotic-based, signal-sequence-trap approach to identify novel secreted and membrane-bound molecules that are expressed in myelinating and non-myelinating Schwann cells. Using cDNA libraries derived from dbcAMP-stimulated primary Schwann cells and 3-day-old rat sciatic nerve mRNAs, we generated an extensive list of novel molecules expressed in myelinating nerves in the PNS. Many of the identified proteins are cell-adhesion molecules (CAMs) and extracellular matrix (ECM) components, most of which have not been described previously in Schwann cells. In addition, we have identified several signaling receptors, growth and differentiation factors, ecto-enzymes and proteins that are associated with the endoplasmic reticulum and the Golgi network. We further examined the expression of several of the novel molecules in Schwann cells in culture and in rat sciatic nerve by primer-specific, real-time PCR and in situ hybridization. Our results indicate that myelinating Schwann cells express a battery of novel CAMs that might mediate their interactions with the underlying axons.     

The QKI-6 and QKI-7 RNA Binding Proteins Block Proliferation and Promote Schwann Cell Myelination

Background

The quaking viable (qk^v) mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS). The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qk^v mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination.

Methodology/Principal Findings

To define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27^KIP1 and myelin basic protein (MBP), markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27^KIP1 and Krox-20 mRNAs, as assessed by quantitative RT-PCR.

Conclusions/Significance

Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.     

P0 promoter directs expression of reporter and toxin genes to Schwann cells of transgenic mice.

We generated transgenic mice that specifically express foreign genes in myelinating Schwann cells. A 1.1 kb segment of 5' flanking sequence from the rat P0 gene was used to drive expression of the genes encoding human growth hormone (hGH) and bacterial diphtheria toxin A chain (DT-A). The P0-hGH mice expressed hGH in myelinating Schwann cells, but not in nonmyelinating Schwann cells, the central nervous system, or any other tissue assayed. This expression was activated on a developmental schedule comparable to that of endogenous myelin gene expression. One line of P0-DT-A mice developed a generalized hypomyelinating peripheral neuropathy, with Schwann cell deficiency apparent in newborn animals. Peripheral nerves from adult mice of this line displayed morphological alterations ranging from completely denuded axons to myelinated Schwann cells undergoing degeneration, although occasional Schwann cells were able to form apparently normal myelin sheaths. Pronounced secondary changes, including proliferation and retraction of processes, occurred in the nonmyelinating Schwann cells of these P0-DT-A mice.