Gpr126 is essential for peripheral nerve development and myelination in mammals

In peripheral nerves, Schwann cells form the myelin sheath that insulates axons and allows rapid propagation of action potentials. Although a number of regulators of Schwann cell development are known, the signaling pathways that control myelination are incompletely understood. In this study, we show that Gpr126 is essential for myelination and other aspects of peripheral nerve development in mammals. A mutation in Gpr126 causes a severe congenital hypomyelinating peripheral neuropathy in mice, and expression of differentiated Schwann cell markers, including Pou3f1, Egr2, myelin protein zero and myelin basic protein, is reduced. Ultrastructural studies of Gpr126-/- mice showed that axonal sorting by Schwann cells is delayed, Remak bundles (non-myelinating Schwann cells associated with small caliber axons) are not observed, and Schwann cells are ultimately arrested at the promyelinating stage. Additionally, ectopic perineurial fibroblasts form aberrant fascicles throughout the endoneurium of the mutant sciatic nerve. This analysis shows that Gpr126 is required for Schwann cell myelination in mammals, and defines new roles for Gpr126 in axonal sorting, formation of mature non-myelinating Schwann cells and organization of the perineurium.     

Laminin gamma1 is critical for Schwann cell differentiation, axon myelination, and regeneration in the peripheral nerve

Laminins are heterotrimeric extracellular matrix proteins that regulate cell viability and function. Laminin-2, composed of alpha2, beta1, and gamma1 chains, is a major matrix component of the peripheral nervous system (PNS). To investigate the role of laminin in the PNS, we used the Cre-loxP system to disrupt the laminin gamma1 gene in Schwann cells. These mice have dramatically reduced expression of laminin gamma1 in Schwann cells, which results in a similar reduction in laminin alpha2 and beta1 chains. These mice exhibit motor defects which lead to hind leg paralysis and tremor. During development, Schwann cells that lack laminin gamma1 were present in peripheral nerves, and proliferated and underwent apoptosis similar to control mice. However, they were unable to differentiate and synthesize myelin proteins, and therefore unable to sort and myelinate axons. In mutant mice, after sciatic nerve crush, the axons showed impaired regeneration. These experiments demonstrate that laminin is an essential component for axon myelination and regeneration in the PNS.     

In vivo knockdown of ErbB3 in mice inhibits Schwann cell precursor migration

The myelin sheath insulates neuronal axons and markedly increases the nerve conduction velocity. In the peripheral nervous system (PNS), Schwann cell precursors migrate along embryonic neuronal axons to their final destinations, where they eventually wrap around individual axons to form the myelin sheath after birth. ErbB2 and ErbB3 tyrosine kinase receptors form a heterodimer and are extensively expressed in Schwann lineage cells. ErbB2/3 is thought to be one of the primary regulators controlling the entire Schwann cell development. ErbB3 is the bona fide Schwann cell receptor for the neuronal ligand neuregulin-1. Although ErbB2/3 is well known to regulate both Schwann cell precursor migration and myelination by Schwann cells in fishes, it still remains unclear whether in mammals, ErbB2/3 actually regulates Schwann cell precursor migration. Here, we show that knockdown of ErbB3 using a Schwann cell-specific promoter in mice causes delayed migration of Schwann cell precursors. In contrast, littermate control mice display normal migration. Similar results are seen in an in vitro migration assay using reaggregated Schwann cell precursors. Also, ErbB3 knockdown in mice reduces myelin thickness in sciatic nerves, consistent with the established role of ErbB3 in myelination. Thus, ErbB3 plays a key role in migration, as well as in myelination, in mouse Schwann lineage cells, presenting a genetically conservative role of ErbB3 in Schwann cell precursor migration.     

RETARDATION OF PERIPHERAL NERVE MYELINATION IN MICE TREATED WITH INHIBITORS OF CHOLESTEROL BIOSYNTHESIS : A Quantitative Electron Microscopic Study

The effect of two inhibitors of cholesterol biosynthesis, triparanol and AY 9944, on peripheral nerve myelination, was studied. Suckling mice were intraperitoneally injected with both drugs on 3 consecutive days and were sacrificed 6 hr after the last injection; others were suckled by an injected mother and sacrificed at 2½ days of age. A single mouse which had been injected with both drugs at 1, 2, and 3 days of age was sacrificed 2 wk after the last injection. Membranous and crystalline intracytoplasmic inclusions were observed in the Schwann cells of the sciatic nerves of all the experimental animals. Both the number of unmyelinated single axons and the number of myelin lamellae around each myelinating axon in the sciatic nerves were recorded for treated mice and of mice suckled by treated mothers. The sciatic nerve of the experimental mice contained a larger proportion of unmyelinated single axons and smaller numbers of myelin lamellae around the myelinating axons, when compared with age-matched controls. The results suggest that a decrease of endogenous cholesterol in suckling mice may affect peripheral nerve myelination in two ways: by retarding the "triggering" of myelination in unmyelinated axons and by decreasing the rate of myelination already in progress.     

N-WASP is required for membrane wrapping and myelination by Schwann cells

During peripheral nerve myelination, Schwann cells sort larger axons, ensheath them, and eventually wrap their membrane to form the myelin sheath. These processes involve extensive changes in cell shape, but the exact mechanisms involved are still unknown. Neural Wiskott-Aldrich syndrome protein (N-WASP) integrates various extracellular signals to control actin dynamics and cytoskeletal reorganization through activation of the Arp2/3 complex. By generating mice lacking N-WASP in myelinating Schwann cells, we show that N-WASP is crucial for myelination. In N-WASP-deficient nerves, Schwann cells sort and ensheath axons, but most of them fail to myelinate and arrest at the promyelinating stage. Yet, a limited number of Schwann cells form unusually short internodes, containing thin myelin sheaths, with the occasional appearance of myelin misfoldings. These data suggest that regulation of actin filament nucleation in Schwann cells by N-WASP is crucial for membrane wrapping, longitudinal extension, and myelination.     

P(0) glycoprotein overexpression causes congenital hypomyelination of peripheral nerves

We show that normal peripheral nerve myelination depends on strict dosage of the most abundantly expressed myelin gene, myelin protein zero (Mpz). Transgenic mice containing extra copies of Mpz manifested a dose-dependent, dysmyelinating neuropathy, ranging from transient perinatal hypomyelination to arrested myelination and impaired sorting of axons by Schwann cells. Myelination was restored by breeding the transgene into the Mpz-null background, demonstrating that dysmyelination does not result from a structural alteration or Schwann cell-extrinsic effect of the transgenic P(0) glycoprotein. Mpz mRNA overexpression ranged from 30-700%, whereas an increased level of P(0) protein was detected only in nerves of low copy-number animals. Breeding experiments placed the threshold for dysmyelination between 30 and 80% Mpz overexpression. These data reveal new points in nerve development at which Schwann cells are susceptible to increased gene dosage, and suggest a novel basis for hereditary neuropathy.     

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.     

FGF21 impedes peripheral myelin development by stimulating p38 MAPK/c‐Jun axis

Fibroblast growth factor 21 (FGF21) as a metabolic stress hormone, is mainly secreted by the liver. In addition to its well‐defined roles in energy homeostasis, FGF21 has been shown to promote remyelination after injury in the central nervous system. In the current study, we sought to examine the potential roles of FGF21 in the peripheral nervous system (PNS) myelination. In the PNS myelin development, Fgf21 expression was reversely correlated with myelin gene expression. In cultured primary Schwann cells (SCs), the application of recombinant FGF21 greatly attenuates myelination‐associated gene expression, including Oct6, Krox20, Mbp, Mpz, and Pmp22. Accordingly, the injection of FGF21 into neonatal rats markedly mitigates the myelination in sciatic nerves. On the contrary, the infusion of the anti‐FGF21 antibody accelerates the myelination. Mechanistically, both extracellular signal‐regulated kinase (ERK) and p38 mitogen‐activated protein kinase (MAPK) were stimulated by FGF21 in SCs and sciatic nerves. Following experiments including pharmaceutical intervention and gene manipulation revealed that the p38 MAPK/c‐Jun axis, rather than ERK, is targeted by FGF21 for mediating its repression on myelination in SCs. Taken together, our data provide a new aspect of FGF21 by acting as a negative regulator for the myelin development process in the PNS via activation of p38 MAPK/c‐Jun.     

Ndrg1-deficient mice exhibit a progressive demyelinating disorder of peripheral nerves

NDRG1 is an intracellular protein that is induced under a number of stress and pathological conditions, and it is thought to be associated with cell growth and differentiation. Recently, human NDRG1 was identified as a gene responsible for hereditary motor and sensory neuropathy-Lom (classified as Charcot-Marie-Tooth disease type 4D), which is characterized by early-onset peripheral neuropathy, leading to severe disability in adulthood. In this study, we generated mice lacking Ndrg1 to analyze its function and elucidate the pathogenesis of Charcot-Marie-Tooth disease type 4D. Histological analysis showed that the sciatic nerve of Ndrg1-deficient mice degenerated with demyelination at about 5 weeks of age. However, myelination of Schwann cells in the sciatic nerve was normal for 2 weeks after birth. Ndrg1-deficient mice showed muscle weakness, especially in the hind limbs, but complicated motor skills were retained. In wild-type mice, NDRG1 was abundantly expressed in the cytoplasm of Schwann cells rather than the myelin sheath. These results indicate that NDRG1 deficiency leads to Schwann cell dysfunction, suggesting that NDRG1 is essential for maintenance of the myelin sheaths in peripheral nerves. These mice will be used for future analyses of the mechanisms of myelin maintenance.     

A surface protein expressed by avian myelinating and nonmyelinating Schwann cells but not by satellite or enteric glial cells

Searching for specific markers of neural crest-derived cell lineages, we immunized mice with glycoproteins purified from adult quail peripheral myelin. We obtained a monoclonal antibody that reacts with myelin and peripheral glial cells. This antibody, to Schwann cell myelin protein (SMP), is specific for the membranes of all Schwann cells, irrespective of whether they are associated with myelinated nerves. SMP persists on Schwann cells in long-term cultures in vitro, but is absent from satellite cells of peripheral ganglia, both in vivo and in vitro. The antigen (a protein doublet of Mr 75,000-80,000) is present in, but not restricted to, the myelin lamellae, since it is distributed along the whole myelinating Schwann cell membrane. In the CNS, SMP appears as a single band of Mr 80,000. SMP is first detectable by immunofluorescence at E6 in the quail, which is at least 6 days earlier than the first appearance of already described markers related to myelination.     

A dual role of erbB2 in myelination and in expansion of the schwann cell precursor pool

Neuregulin-1 provides an important axonally derived signal for the survival and growth of developing Schwann cells, which is transmitted by the ErbB2/ErbB3 receptor tyrosine kinases. Null mutations of the neuregulin-1, erbB2, or erbB3 mouse genes cause severe deficits in early Schwann cell development. Here, we employ Cre-loxP technology to introduce erbB2 mutations late in Schwann cell development, using a Krox20-cre allele. Cre-mediated erbB2 ablation occurs perinatally in peripheral nerves, but already at E11 within spinal roots. The mutant mice exhibit a widespread peripheral neuropathy characterized by abnormally thin myelin sheaths, containing fewer myelin wraps. In addition, in spinal roots the Schwann cell precursor pool is not correctly established. Thus, the Neuregulin signaling system functions during multiple stages of Schwann cell development and is essential for correct myelination. The thickness of the myelin sheath is determined by the axon diameter, and we suggest that trophic signals provided by the nerve determine the number of times a Schwann cell wraps an axon.     

A small peptide mimetic of brain‐derived neurotrophic factor promotes peripheral myelination

The expression of the neurotrophins and their receptors is essential for peripheral nervous system development and myelination. We have previously demonstrated that brain‐derived neurotrophic factor (BDNF) exerts contrasting influences upon Schwann cell myelination in vitro – promoting myelination via neuronally expressed p75NTR, but inhibiting myelination via neuronally expressed TrkB. We have generated a small peptide called cyclo‐d PAKKR that structurally mimics the region of BDNF that binds p75NTR. Here, we have investigated whether utilizing cyclo‐d PAKKR to selectively target p75NTR is an approach that could exert a unified promyelinating response. Like BDNF, cyclo‐d PAKKR promoted myelination of nerve growth factor‐dependent neurons in vitro, an effect dependent on the neuronal expression of p75NTR. Importantly, cyclo‐d PAKKR also significantly promoted the myelination of tropomyosin‐related kinase receptor B‐expressing neurons in vitro, whereas BDNF exerted a significant inhibitory effect. This indicated that while BDNF exerted a contrasting influence upon the myelination of distinct subsets of dorsal root ganglion (DRG) neurons in vitro, cyclo‐d PAKKR uniformly promoted their myelination. Local injection of cyclo‐d PAKKR adjacent to the developing sciatic nerve in vivo significantly enhanced myelin protein expression and significantly increased the number of myelinated axons. These results demonstrate that cyclo‐d PAKKR promotes peripheral myelination in vitro and in vivo, suggesting it is a strategy worthy of further investigation for the treatment of peripheral demyelinating diseases.     

Effects of Irbesartan on the Growth and Differentiation of Adipocytes in Obese Zucker Rats

Objective: The aim of this study was to evaluate the effects of the selective angiotensin receptor 1 antagonist irbesartan on the growth and differentiation of the adipocytes in obese Zucker fa/fa rats. Research Methods and Procedures: Obese Zucker fa/fa rats were treated by oral route for 3 weeks with irbesartan at doses of 3–10‐30 mg/kg per day. The adipocyte differentiation was evaluated by analyzing tissue samples of white (retroperitoneal) or brown (interscapular) adipose tissue for the presence of peroxisome proliferator activated receptor γ, leptin, and the activity of glycerol‐3‐phosphate dehydrogenase. Results: This study showed that the treatment of obese Zucker fa/fa with irbesartan effectively reduced the differentiation of adipocytes within brown (interscapular) and white (retroperitoneal) adipose tissue. In fact, irbesartan significantly (p < 0.01) and dose‐dependently reduced the tissue levels of leptin, peroxisome proliferator activated receptor γ, and the activity of the enzyme glycerol‐3‐phoshate dehydrogenase accepted markers of adipocyte differentiation. None of the tested doses of irbesartan affected these markers in non‐obese rats. Discussion: The antagonism of the angiotensin receptor 1 receptors with irbesartan reduces the adipogenic activity of angiotensin II in obese Zucker rats, with the endpoint being reduction of the growth and differentiation of the adipocytes within the adipose tissue.     

Ablation of Dicer from Murine Schwann Cells Increases Their Proliferation while Blocking Myelination

The myelin sheaths that surround the thick axons of the peripheral nervous system are produced by the highly specialized Schwann cells. Differentiation of Schwann cells and myelination occur in discrete steps. Each of these requires coordinated expression of specific proteins in a precise sequence, yet the regulatory mechanisms controlling protein expression during these events are incompletely understood. Here we report that Schwann cell-specific ablation of the enzyme Dicer1, which is required for the production of small non-coding regulatory microRNAs, fully arrests Schwann cell differentiation, resulting in early postnatal lethality. Dicer^−/− Schwann cells had lost their ability to myelinate, yet were still capable of sorting axons. Both cell death and, paradoxically, proliferation of immature Schwann cells was markedly enhanced, suggesting that their terminal differentiation is triggered by growth-arresting regulatory microRNAs. Using microRNA microarrays, we identified 16 microRNAs that are upregulated upon myelination and whose expression is controlled by Dicer in Schwann cells. This set of microRNAs appears to drive Schwann cell differentiation and myelination of peripheral nerves, thereby fulfilling a crucial function for survival of the organism.     

IGF‐I deficient mice show reduced peripheral nerve conduction velocities and decreased axonal diameters and respond to exogenous IGF‐I treatment

Although insulin‐like growth factor‐I (IGF‐I) can act as a neurotrophic factor for peripheral neurons in vitro and in vivo following injury, the role IGF‐I plays during normal development and functioning of the peripheral nervous system is unclear. Here, we report that transgenic mice with reduced levels (two genotypes: heterozygous Igf1^+/− or homozygous insertional mutant Igf1^m/m) or totally lacking IGF‐I (homozygous Igf1^−/−) show a decrease in motor and sensory nerve conduction velocities in vivo. In addition, A‐fiber responses in isolated peroneal nerves from Igf1^+/− and Igf1^−/− mice are impaired. The nerve function impairment is most profound in Igf1^−/− mice. Histopathology of the peroneal nerves in Igf1^−/− mice demonstrates a shift to smaller axonal diameters but maintains the same total number of myelinated fibers as Igf1^+/+ mice. Comparisons of myelin thickness with axonal diameter indicate that there is no significant reduction in peripheral nerve myelination in IGF‐I–deficient mice. In addition, in Igf1^m/m mice with very low serum levels of IGF‐I, replacement therapy with exogenous recombinant hIGF‐I restores both motor and sensory nerve conduction velocities. These findings demonstrate not only that IGF‐I serves an important role in the growth and development of the peripheral nervous system, but also that systemic IGF‐I treatment can enhance nerve function in IGF‐I–deficient adult mice. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 142–152, 1999     

Immunocytological localization of the major peripheral nervous system glycoprotein P0 and the L2/HNK-1 and L3 carbohydrate structures in developing and adult mouse sciatic nerve

Immunocytological localization of the major glycoprotein of peripheral myelin P0 and its associated carbohydrate structures L2/HNK-1 and L3 was performed at the light- and electron-microscopic levels in mouse sciatic nerves at several developmental stages and in adulthood. P0 was first expressed on Schwann cells at the time that Schwann cells associated with axons on a 1:1 basis. P0 remains expressed at all times of myelin formation and in compact myelin. After cessation of myelination P0 is no longer detectable in the uncompacted parts of myelin, i.e., Schmidt-Lanterman incisures, paranodal loops, and outer and inner mesaxons. P0 is not detectable on basement membranes, interstitial collagens, and non-myelin-forming Schwann cells. The associated carbohydrate epitope L2 does not follow the expression of P0 at any developmental or adult stage. Until 21 days the L2 epitope is confined to nonmyelinated fibers. In sciatic nerves of mice older than 8 weeks, however, only a few nonmyelinated fibers remain L2-positive. L2 immunoreactivity is clearly seen in a subpopulation of compact myelin figures largely associated with motor fibers. The L3 epitope is never detectable on nonmyelinated fibers and becomes first visible when compact myelin is discerned. Unlike the L2 epitope L3 is present in most, if not all, compact myelin figures. These observations suggest that P0 may be involved in ensheathment of axons by Schwann cells at the decisive stages of initiation of myelination and later on, possibly in conjunction with the L3 carbohydrate structure, in maintenance of compact myelin. The appearance of the L2 carbohydrate epitopes in compact myelin of largely motor and fewer sensory nerve fibers at times when morphogenesis of myelin has ceased remains to be elucidated in functional terms.