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.     

Schwann cell autophagy,myelinophagy, initiates myelin clearance from injured nerves

Although Schwann cell myelin breakdown is the universal outcome of a remarkably wide range of conditions that cause disease or injury to peripheral nerves, the cellular and molecular mechanisms that make Schwann cell–mediated myelin digestion possible have not been established. We report that Schwann cells degrade myelin after injury by a novel form of selective autophagy, myelinophagy. Autophagy was up-regulated by myelinating Schwann cells after nerve injury, myelin debris was present in autophagosomes, and pharmacological and genetic inhibition of autophagy impaired myelin clearance. Myelinophagy was positively regulated by the Schwann cell JNK/c-Jun pathway, a central regulator of the Schwann cell reprogramming induced by nerve injury. We also present evidence that myelinophagy is defective in the injured central nervous system. These results reveal an important role for inductive autophagy during Wallerian degeneration, and point to potential mechanistic targets for accelerating myelin clearance and improving demyelinating disease.     

The homeotic protein dlk is expressed during peripheral nerve development.

To investigate the molecular events controlling myelination of the peripheral nervous system, we compared gene expression of normal mouse sciatic nerves to that of the trembler mouse, whose Schwann cells are blocked in a pre-myelinating phenotype. Using cDNA array, we assessed expression levels of 1176 genes, and we found that delta-like protein (dlk), an epidermal growth factor-like homeotic protein, was expressed in the normal developing nerves, but at a low level in the dysmyelinating mutant trembler. Moreover, dlk expression was down-regulated when myelin protein expression was up-regulated, and no expression was observed in the developing brain. These results suggest that dlk expression is required for Schwann cell acquisition of the myelinating phenotype.     

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.     

The origin and development of glial cells in peripheral nerves

During the development of peripheral nerves, neural crest cells generate myelinating and non-myelinating glial cells in a process that parallels gliogenesis from the germinal layers of the CNS. Unlike central gliogenesis, neural crest development involves a protracted embryonic phase devoted to the generation of, first, the Schwann cell precursor and then the immature Schwann cell, a cell whose fate as a myelinating or non-myelinating cell has yet to be determined. Embryonic nerves therefore offer a particular opportunity to analyse the early steps of gliogenesis from transient multipotent stem cells, and to understand how this process is integrated with organogenesis of peripheral nerves.     

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.