Synergistic PXT3003 therapy uncouples neuromuscular function from dysmyelination in male Charcot-Marie-Tooth disease type 1A (CMT1A) rats.

Charcot-Marie-Tooth disease 1 A (CMT1A) is caused by an intrachromosomal duplication of the gene encoding for PMP22 leading to peripheral nerve dysmyelination, axonal loss, and progressive muscle weakness. No therapy is available. PXT3003 is a low-dose combination of baclofen, naltrexone, and sorbitol which has been shown to improve disease symptoms in Pmp22 transgenic rats, a bona fide model of CMT1A disease. However, the superiority of PXT3003 over its single components or dual combinations have not been tested. Here, we show that in a dorsal root ganglion (DRG) co-culture system derived from transgenic rats, PXT3003 induced myelination when compared to its single and dual components. Applying a clinically relevant ("translational") study design in adult male CMT1A rats for 3 months, PXT3003, but not its dual components, resulted in improved performance in behavioral motor and sensory endpoints when compared to placebo. Unexpectedly, we observed only a marginally increased number of myelinated axons in nerves from PXT3003-treated CMT1A rats. However, in electrophysiology, motor latencies correlated with increased grip strength indicating a possible effect of PXT3003 on neuromuscular junctions (NMJs) and muscle fiber pathology. Indeed, PXT3003-treated CMT1A rats displayed an increased perimeter of individual NMJs and a larger number of functional NMJs. Moreover, muscles of PXT3003 CMT1A rats displayed less neurogenic atrophy and a shift toward fast contracting muscle fibers. We suggest that ameliorated motor function in PXT3003-treated CMT1A rats result from restored NMJ function and muscle innervation, independent from myelination.

Transcriptional inhibition in Schwann cell development and nerve regeneration.

Schwann cells, the myelinating glial cells of the peripheral nervous system are remarkably plastic after nerve trauma. Their transdifferentiation into specialized repair cells after injury shares some features with their development from the neural crest. Both processes are governed by a tightly regulated balance between activators and inhibitors to ensure timely lineage progression and allow re-maturation after nerve injury. Functional recovery after injury is very successful in rodents, however, in humans, lack of regeneration after nerve trauma and loss of function as the result of peripheral neuropathies represents a significant problem. Our understanding of the basic molecular machinery underlying Schwann cell maturation and plasticity has made significant progress in recent years and novel players have been discovered. While the transcriptional activators of Schwann cell development and nerve repair have been well defined, the mechanisms counteracting negative regulation of (re-)myelination are less well understood. Recently, transcriptional inhibition has emerged as a new regulatory mechanism in Schwann cell development and nerve repair. This mini-review summarizes some of the regulatory mechanisms controlling both processes and the novel concept of "inhibiting the inhibitors" in the context of Schwann cell plasticity.

Peroxisomal dysfunctions cause lysosomal storage and axonal Kv1 channel redistribution in peripheral neuropathy.

Impairment of peripheral nerve function is frequent in neurometabolic diseases, but mechanistically not well understood. Here, we report a novel disease mechanism and the finding that glial lipid metabolism is critical for axon function, independent of myelin itself. Surprisingly, nerves of Schwann cell-specific Pex5 mutant mice were unaltered regarding axon numbers, axonal calibers, and myelin sheath thickness by electron microscopy. In search for a molecular mechanism, we revealed enhanced abundance and internodal expression of axonal membrane proteins normally restricted to juxtaparanodal lipid-rafts. Gangliosides were altered and enriched within an expanded lysosomal compartment of paranodal loops. We revealed the same pathological features in a mouse model of human Adrenomyeloneuropathy, preceding disease-onset by one year. Thus, peroxisomal dysfunction causes secondary failure of local lysosomes, thereby impairing the turnover of gangliosides in myelin. This reveals a new aspect of axon-glia interactions, with Schwann cell lipid metabolism regulating the anchorage of juxtaparanodal Kv1-channels.

Zeb2: Inhibiting the inhibitors in Schwann cells.

Development of Schwann cells is tightly regulated by concerted action of activating and inhibiting factors. Most of the regulatory feedback loops identified to date are transcriptional activators promoting induction of genes coding for integral myelin proteins and lipids. The mechanisms by which inhibitory factors are silenced during Schwann cell maturation are less well understood. We could recently show a pivotal function for the transcription factor zinc finger E-box binding homeobox 2 (Zeb2) during Schwann cell development and myelination as a transcriptional repressor of maturation inhibitors. Zeb2 belongs to a family of highly conserved 2-handed zinc-finger proteins and represses gene transcription by binding to E-box sequences in the regulatory region of target genes. The protein is known to repress E-cadherin during epithelial to mesenchymal transition (EMT) in tumor malignancy and mediates its functions by interacting with multiple co-factors. During nervous system development, Zeb2 is expressed in neural crest cells, the precursors of Schwann cells, the myelinating glial cells of peripheral nerves. Schwann cells lacking Zeb2 fail to fully differentiate and are unable to sort and myelinate peripheral nerve axons. The maturation inhibitors Sox2, Ednrb and Hey2 emerge as targets for Zeb2-mediated transcriptional repression and show persistent aberrant expression in Zeb2-deficient Schwann cells. While dispensible for adult Schwann cells, re-activation of Zeb2 is essential after nerve injury to allow remyelination and functional recovery. In summary, Zeb2 emerges as an "inhibitor of inhibitors," a novel concept in Schwann cell development and nerve repair.

Zeb2 is essential for Schwann cell differentiation, myelination and nerve repair.

Schwann cell development and peripheral nerve myelination require the serial expression of transcriptional activators, such as Sox10, Oct6 (also called Scip or Pou3f1) and Krox20 (also called Egr2). Here we show that transcriptional repression, mediated by the zinc-finger protein Zeb2 (also known as Sip1), is essential for differentiation and myelination. Mice lacking Zeb2 in Schwann cells develop a severe peripheral neuropathy, caused by failure of axonal sorting and virtual absence of myelin membranes. Zeb2-deficient Schwann cells continuously express repressors of lineage progression. Moreover, genes for negative regulators of maturation such as Sox2 and Ednrb emerge as Zeb2 target genes, supporting its function as an 'inhibitor of inhibitors' in myelination control. When Zeb2 is deleted in adult mice, Schwann cells readily dedifferentiate following peripheral nerve injury and become repair cells. However, nerve regeneration and remyelination are both perturbed, demonstrating that Zeb2, although undetectable in adult Schwann cells, has a latent function throughout life.

c-Jun activation in Schwann cells protects against loss of sensory axons in inherited neuropathy.

Charcot-Marie-Tooth disease type 1A is the most frequent inherited peripheral neuropathy. It is generally due to heterozygous inheritance of a partial chromosomal duplication resulting in over-expression of PMP22. A key feature of Charcot-Marie-Tooth disease type 1A is secondary death of axons. Prevention of axonal loss is therefore an important target of clinical intervention. We have previously identified a signalling mechanism that promotes axon survival and prevents neuron death in mechanically injured peripheral nerves. This work suggested that Schwann cells respond to injury by activating/enhancing trophic support for axons through a mechanism that depends on upregulation of the transcription factor c-Jun in Schwann cells, resulting in the sparing of axons that would otherwise die. As c-Jun orchestrates Schwann cell support for distressed neurons after mechanical injury, we have now asked: do Schwann cells also activate a c-Jun dependent neuron-supportive programme in inherited demyelinating disease? We tested this by using the C3 mouse model of Charcot-Marie-Tooth disease type 1A. In line with our previous findings in humans with Charcot-Marie-Tooth disease type 1A, we found that Schwann cell c-Jun was elevated in (uninjured) nerves of C3 mice. We determined the impact of this c-Jun activation by comparing C3 mice with double mutant mice, namely C3 mice in which c-Jun had been conditionally inactivated in Schwann cells (C3/Schwann cell-c-Jun(-/-) mice), using sensory-motor tests and electrophysiological measurements, and by counting axons in proximal and distal nerves. The results indicate that c-Jun elevation in the Schwann cells of C3 nerves serves to prevent loss of myelinated sensory axons, particularly in distal nerves, improve behavioural symptoms, and preserve F-wave persistence. This suggests that Schwann cells have two contrasting functions in Charcot-Marie-Tooth disease type 1A: on the one hand they are the genetic source of the disease, on the other, they respond to it by mounting a c-Jun-dependent response that significantly reduces its impact. Because axonal death is a central feature of much nerve pathology it will be important to establish whether an axon-supportive Schwann cell response also takes place in other conditions. Amplification of this axon-supportive mechanism constitutes a novel target for clinical intervention that might be useful in Charcot-Marie-Tooth disease type 1A and other neuropathies that involve axon loss.

c-Jun reprograms Schwann cells of injured nerves to generate a repair cell essential for regeneration.

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.

Critical time window of neuronal cholesterol synthesis during neurite outgrowth.

Cholesterol is an essential membrane component enriched in plasma membranes, growth cones, and synapses. The brain normally synthesizes all cholesterol locally, but the contribution of individual cell types to brain cholesterol metabolism is unknown. To investigate whether cortical projection neurons in vivo essentially require cholesterol biosynthesis and which cell types support neurons, we have conditionally ablated the cholesterol biosynthesis in these neurons in mice either embryonically or postnatally. We found that cortical projection neurons synthesize cholesterol during their entire lifetime. At all stages, they can also benefit from glial support. Adult neurons that lack cholesterol biosynthesis are mainly supported by astrocytes such that their functional integrity is preserved. In contrast, microglial cells support young neurons. However, compensatory efforts of microglia are only transient leading to layer-specific neuronal death and the reduction of cortical projections. Hence, during the phase of maximal membrane growth and maximal cholesterol demand, neuronal cholesterol biosynthesis is indispensable. Analysis of primary neurons revealed that neurons tolerate only slight alteration in the cholesterol content and plasma membrane tension. This quality control allows neurons to differentiate normally and adjusts the extent of neurite outgrowth, the number of functional growth cones and synapses to the available cholesterol. This study highlights both the flexibility and the limits of horizontal cholesterol transfer in vivo and may have implications for the understanding of neurodegenerative diseases.

A role for myelin-associated peroxisomes in maintaining paranodal loops and axonal integrity.

Demyelinating diseases of the nervous system cause axon loss but the underlying mechanisms are not well understood. Here we show by confocal and electron microscopy that in myelin-forming glia peroxisomes are associated with myelin membranes. When peroxisome biogenesis is experimentally perturbed in Pex5 conditional mouse mutants, myelination by Schwann cells appears initially normal. However, in nerves of older mice paranodal loops become physically unstable and develop swellings filled with vesicles and electron-dense material. This novel model of a demyelinating neuropathy demonstrates that peroxisomes serve an important function in the peripheral myelin compartment, required for long-term axonal integrity.

Cholesterol: a novel regulatory role in myelin formation.

Myelin consists of tightly compacted membranes that form an insulating sheath around axons. The function of myelin for rapid saltatory nerve conduction is dependent on its unique composition, highly enriched in glycosphingolipids and cholesterol. Cholesterol emerged as the only integral myelin component that is essential and rate limiting for the development of CNS and PNS myelin. Experiments with conditional mouse mutants that lack cholesterol biosynthesis in oligodendrocytes revealed that only minimal changes of the CNS myelin lipid composition are tolerated. In Schwann cells of the PNS, protein trafficking and myelin compaction depend on cholesterol. In this review, the authors summarize the role of cholesterol in myelin biogenesis and myelin disease.

Structure and composition of myelinated axons: a multimodal synchrotron spectro-microscopy study.

We report elemental mappings on the sub-cellular level of myelinated sciatic neurons isolated from wild type mice, with high spatial resolution. The distribution of P, S, Cl, Na, K, Fe, Mn, Cu was imaged in freeze-dried as well as cryo-preserved specimen, using the recently developed cryogenic sample environment at beamline ID21 at the European Synchrotron Radiation Facility (ESRF). In addition, synchrotron radiation based Fourier transform infrared (FTIR) spectromicroscopy was used as a chemically sensitive imaging method. Finally single fiber diffraction in highly focused hard X-ray beams, and soft X-ray microscopy and tomography in absorption contrast are demonstrated as novel techniques for the study of single nerve fibers.

Neuron-glia signaling and the protection of axon function by Schwann cells.

The interaction between neurons and glial cells is a feature of all higher nervous systems. In the vertebrate peripheral nervous system, Schwann cells ensheath and myelinate axons thereby allowing rapid saltatory conduction and ensuring axonal integrity. Recently, some of the key molecules in neuron-Schwann cell signaling have been identified. Neuregulin-1 (NRG1) type III presented on the axonal surface determines the myelination fate of axons and controls myelin sheath thickness. Recent observations suggest that NRG1 regulates myelination via the control of Schwann cell cholesterol biosynthesis. This concept is supported by the finding that high cholesterol levels in Schwann cells are a rate-limiting factor for myelin protein production and transport of the major myelin protein P0 from the endoplasmic reticulum into the growing myelin sheath. NRG1 type III activates ErbB receptors on the Schwann cell, which leads to an increase in intracellular PIP3 levels via the PI3-kinase pathway. Surprisingly, enforced elevation of PIP3 levels by inactivation of the phosphatase PTEN in developing and mature Schwann cells does not entirely mimic NRG1 type III stimulated myelin growth, but predominantly causes focal hypermyelination starting at Schmidt-Lanterman incisures and nodes of Ranvier. This indicates that the glial transduction of pro-myelinating signals has to be under tight and life-long control to preserve integrity of the myelinated axon. Understanding the cross talk between neurons and Schwann cells will help to further define the role of glia in preserving axonal integrity and to develop therapeutic strategies for peripheral neuropathies such as CMT1A.

Atorvastatin reduces the expression of aldo-keto reductases in HUVEC and PTEC. A new approach to influence the polyol pathway.

PURPOSE: Increased flux of glucose via the polyol pathway, oxidative stress and ischaemia lead to the upregulation of the aldose reductase (AR), the key enzyme of the polyol pathway. This adversely affects the organism and can in part be reduced by inhibition of the enzyme. METHODS: In this study, we examined the effect of the HMG-CoA-reductase inhibitor atorvastatin on the expression of aldose reductase (AR, AKR1B1), aldehyde reductase (AldR, AKR1A1) and small intestine reductase (SIR, AKR1B10) in human umbilical vein endothelial cells (HUVEC) and human proximal tubular epithelial cells (PTEC) by RT-PCR. RESULTS: In HUVEC, atorvastatin reduces the expression of aldehyde reductase and aldose reductase compared with control medium (-20% and -12% respectively, P < 0.05), while small intestine reductase is not expressed. In PTEC no regulation of aldehyde reductase and aldose reductase by atorvastatin could be measured, while the expression of small intestine reductase was reduced by 37% compared with control medium (P < 0.05). The reduction observed was not abolished by the addition of mevalonic acid. CONCLUSION: The reduction of members of the aldo-keto-reductase family by atorvastatin is a novel way to influence the polyol pathway and a new pleiotropic effect of atorvastatin.

Cholesterol regulates the endoplasmic reticulum exit of the major membrane protein P0 required for peripheral myelin compaction.

Rapid impulse conduction requires electrical insulation of axons by myelin, a cholesterol-rich extension of the glial cell membrane with a characteristic composition of proteins and lipids. Mutations in several myelin protein genes cause endoplasmic reticulum (ER) retention and disease, presumably attributable to failure of misfolded proteins to pass the ER quality control. Because many myelin proteins partition into cholesterol-rich membrane rafts, their interaction with cholesterol could potentially be part of the ER quality control system. Here, we provide in vitro and in vivo evidence that the major peripheral myelin protein P0 requires cholesterol for exiting the ER and reaching the myelin compartment. Cholesterol dependency of P0 trafficking in heterologous cells is mediated by a cholesterol recognition/interaction amino acid consensus (CRAC) motif. Mutant mice lacking cholesterol biosynthesis in Schwann cells suffer from severe hypomyelination with numerous uncompacted myelin stretches. This demonstrates that high-level cholesterol coordinates P0 export with myelin membrane synthesis, which is required for the correct stoichiometry of myelin components and for myelin compaction.