RESUMEN
TFEB and TFE3 (TFEB/3), key regulators of lysosomal biogenesis and autophagy, play diverse roles depending on cell type. This study highlights a hitherto unrecognized role of TFEB/3 crucial for peripheral nerve repair. Specifically, they promote the generation of progenitor-like repair Schwann cells after axonal injury. In Schwann cell-specific TFEB/3 double knock-out mice of either sex, the TFEB/3 loss disrupts the transcriptomic reprogramming that is essential for the formation of repair Schwann cells. Consequently, mutant mice fail to populate the injured nerve with repair Schwann cells and exhibit defects in axon-regrowth, target reinnervation, and functional recovery. TFEB/3 deficiency inhibits the expression of injury-responsive repair Schwann cell genes, despite the continued expression of c-Jun, a previously identified regulator of repair Schwann cell function. TFEB/3 binding motifs are enriched in the enhancer regions of injury-responsive genes, suggesting their role in repair gene activation. Autophagy-dependent myelin breakdown is not impaired despite TFEB/3 deficiency. These findings underscore a unique role of TFEB/3 in adult Schwann cells that is required for proper peripheral nerve regeneration.Significance Statement Peripheral nerves have been recognized for their efficient regenerative capabilities compared to the central nervous system neurons. This is due to the remarkable ability of Schwann cells to undergo a reprogramming process, transforming into progenitor-like repair Schwann cells that actively contribute to axon regeneration and overall nerve repair. However, the specific transcriptional regulators responsible for initiating this transformation in the adult peripheral nerve have remained elusive. Our study elucidates a previously undescribed, injury-responsive function of TFEB/3 in adult Schwann cells, showcasing its ability to promote tissue repair. Our findings hold important implications for enhancing nerve regeneration by bolstering the regenerative capacity of glial cells, thereby contributing to advancements in the field of neural tissue repair.
RESUMEN
Myelin loss in the brain is a common occurrence in traumatic brain injury (TBI) that results from impact-induced acceleration forces to the head. Fast and abrupt head motions, either resulting from violent blows and/or jolts, cause rapid stretching of the brain tissue, and the long axons within the white matter tracts are especially vulnerable to such mechanical strain. Recent studies have shown that mechanotransduction plays an important role in regulating oligodendrocyte progenitors cell differentiation into oligodendrocytes. However, little is known about the impact of mechanical strain on mature oligodendrocytes and the stability of their associated myelin sheaths. We used an in vitro cellular stretch device to address these questions, as well as characterize a mechanotransduction mechanism that mediates oligodendrocyte responses. Mechanical stretch caused a transient and reversible myelin protein loss in oligodendrocytes. Cell death was not observed. Myelin protein loss was accompanied by an increase in intracellular Ca2+ and Erk1/2 activation. Chelating Ca2+ or inhibiting Erk1/2 activation was sufficient to block the stretch-induced loss of myelin protein. Further biochemical analyses revealed that the stretch-induced myelin protein loss was mediated by the release of Ca2+ from the endoplasmic reticulum (ER) and subsequent Ca2+ -dependent activation of Erk1/2. Altogether, our findings characterize an Erk1/2-dependent mechanotransduction mechanism in mature oligodendrocytes that de-stabilizes the myelination program.
Asunto(s)
Calcio/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Mecanotransducción Celular/fisiología , Proteínas de la Mielina/deficiencia , Oligodendroglía/metabolismo , Animales , Animales Recién Nacidos , Quelantes del Calcio/farmacología , Ionóforos de Calcio/farmacología , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Mecanotransducción Celular/efectos de los fármacos , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/metabolismo , Oligodendroglía/efectos de los fármacos , RatasRESUMEN
Interactions between axons and Schwann cells are essential for the acquisition of Schwann cell radial and longitudinal polarity and myelin sheath assembly. In the internode, the largest of these longitudinal domains, axon-Schwann cell interactions are mediated by the Nectin-like (Necl) cell adhesion proteins, also known as SynCAMs or Cadms. In particular, Necl-1/Cadm3 expressed on the axon surface binds to Necl-4/Cadm4 expressed along the adaxonal membrane of myelinating Schwann cells. Necl-4 promotes myelination in vitro and is required for the timely onset of myelination and the fidelity of the organization of the myelin sheath and the internode in vivo. A key question is the identity of the downstream effectors of Necl-4 that mediate its effects. The cytoplasmic terminal region (CTR) of Necl-4 contains a PDZ-domain binding motif. Accordingly, we used the CTR of Necl-4 in an unbiased proteomic screen of PDZ-domain proteins. We identify Par-3, a multi-PDZ domain containing protein of the Par-aPKC polarity complex previously implicated in myelination, as an interacting protein. Necl-4 and Par-3 are colocalized along the inner Schwann cell membrane and coprecipitate from Schwann cell lysates. The CTR of Necl-4 binds to the first PDZ domain of Par-3 thereby recruiting Par-3 to sites of Necl-4/Necl-1 interaction. Knockdown of Necl-4 perturbs Par-3 localization to the inner membrane of Schwann cells in myelinating co-cultures. These findings implicate interactions of Necl-1/Necl-4 in the recruitment of Par-3 to the Schwann cell adaxonal membrane and the establishment of Schwann cell radial polarity.
Asunto(s)
Axones/metabolismo , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Inmunoglobulinas/metabolismo , Dominios PDZ/fisiología , Células de Schwann/citología , Proteínas Adaptadoras Transductoras de Señales , Animales , Células CHO , Moléculas de Adhesión Celular/genética , Proteínas de Ciclo Celular , Membrana Celular/genética , Técnicas de Cocultivo , Cricetulus , Embrión de Mamíferos , Ganglios Espinales/citología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunoglobulinas/genética , Inmunoprecipitación , Técnicas In Vitro , Ratones , Neuronas , Dominios PDZ/genética , Ratas , Nervio Ciático/citología , TransfecciónRESUMEN
Nectin-like 4 (NECL4, CADM4) is a Schwann cell-specific cell adhesion molecule that promotes axo-glial interactions. In vitro and in vivo studies have shown that NECL4 is necessary for proper peripheral nerve myelination. However, the molecular mechanisms that are regulated by NECL4 and affect peripheral myelination currently remain unclear. We used an in vitro approach to begin identifying some of the mechanisms that could explain NECL4 function. Using mass spectrometry and Western blotting techniques, we have identified choline transporter-like 1 (CTL1) as a putative complexing partner with NECL4. We show that intracellular choline levels are significantly elevated in NECL4-deficient Schwann cells. The analysis of extracellular d9-choline uptake revealed a deficit in the amount of d9-choline found inside NECL4-deficient Schwann cells, suggestive of either reduced transport capabilities or increased metabolization of transported choline. An extensive lipidomic screen of choline derivatives showed that total phosphatidylcholine and phosphatidylinositol (but not diacylglycerol or sphingomyelin) are significantly elevated in NECL4-deficient Schwann cells, particularly specific subspecies of phosphatidylcholine carrying very long polyunsaturated fatty acid chains. Finally, CTL1-deficient Schwann cells are significantly impaired in their ability to myelinate neurites in vitro To our knowledge, this is the first demonstration of a bona fide cell adhesion molecule, NECL4, regulating choline homeostasis and lipid biogenesis. Phosphatidylcholines are major myelin phospholipids, and several phosphorylated phosphatidylinositol species are known to regulate key aspects of peripheral myelination. Furthermore, the biophysical properties imparted to plasma membranes are regulated by fatty acid chain profiles. Therefore, it will be important to translate these in vitro observations to in vivo studies of NECL4 and CTL1-deficient mice.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Colina/metabolismo , Lipogénesis , Proteínas de Transporte de Membrana/metabolismo , Células de Schwann/metabolismo , Animales , Adhesión Celular , Moléculas de Adhesión Celular Neuronal/genética , Células Cultivadas , Homeostasis , Proteínas de Transporte de Membrana/genética , Vaina de Mielina/metabolismo , Fosfatidilcolinas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Ratas Sprague-Dawley , Células de Schwann/citologíaRESUMEN
Axo-glial interactions are critical for myelination and the domain organization of myelinated fibers. Cell adhesion molecules belonging to the Cadm family, and in particular Cadm3 (axonal) and its heterophilic binding partner Cadm4 (Schwann cell), mediate these interactions along the internode. Using targeted shRNA-mediated knockdown, we show that the removal of axonal Cadm3 promotes Schwann cell myelination in the in vitro DRG neuron/Schwann cell myelinating system. Conversely, over-expressing Cadm3 on the surface of DRG neuron axons results in an almost complete inability by Schwann cells to form myelin segments. Axons of superior cervical ganglion (SCG) neurons, which do not normally support the formation of myelin segments by Schwann cells, express higher levels of Cadm3 compared to DRG neurons. Knocking down Cadm3 in SCG neurons promotes myelination. Finally, the extracellular domain of Cadm3 interferes in a dose-dependent manner with the activation of ErbB3 and of the pro-myelinating PI3K/Akt pathway, but does not interfere with the activation of the Mek/Erk1/2 pathway. While not in direct contradiction, these in vitro results shed lights on the apparent lack of phenotype that was reported from in vivo studies of Cadm3-/- mice. Our results suggest that Cadm3 may act as a negative regulator of PNS myelination, potentially through the selective regulation of the signaling cascades activated in Schwann cells by axonal contact, and in particular by type III Nrg-1. Further analyses of peripheral nerves in the Cadm-/- mice will be needed to determine the exact role of axonal Cadm3 in PNS myelination. GLIA 2016;64:2247-2262.
Asunto(s)
Moléculas de Adhesión Celular/deficiencia , Regulación de la Expresión Génica/genética , Inmunoglobulinas/deficiencia , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Células de Schwann/fisiología , Transducción de Señal/fisiología , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Moléculas de Adhesión Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo , Ganglios Espinales/citología , Regulación de la Expresión Génica/fisiología , Inmunoglobulinas/genética , Inmunoprecipitación , Proteína Básica de Mielina/metabolismo , Neuritas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Células de Schwann/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
In myelinating Schwann cells, E-cadherin is a component of the adherens junctions that stabilize the architecture of the noncompact myelin region. In other cell types, E-cadherin has been considered as a signaling receptor that modulates intracellular signal transduction and cellular responses. To determine whether E-cadherin plays a regulatory role during Schwann cell myelination, we investigated the effects of E-cadherin deletion and over-expression in Schwann cells. In vivo, Schwann cell-specific E-cadherin ablation results in an early myelination delay. In Schwann cell-dorsal root ganglia neuron co-cultures, E-cadherin deletion attenuates myelin formation and shortens the myelin segment length. When over-expressed in Schwann cells, E-cadherin improves myelination on Nrg1 type III(+/-) neurons and induces myelination on normally non-myelinated axons of sympathetic neurons. The pro-myelinating effect of E-cadherin is associated with an enhanced Nrg1-erbB receptor signaling, including activation of the downstream Akt and Rac. Accordingly, in the absence of E-cadherin, Nrg1-signaling is diminished in Schwann cells. Our data also show that E-cadherin expression in Schwann cell is induced by axonal Nrg1 type III, indicating a reciprocal interaction between E-cadherin and the Nrg1 signaling. Altogether, our data suggest a regulatory function of E-cadherin that modulates Nrg1 signaling and promotes Schwann cell myelin formation.
Asunto(s)
Axones/fisiología , Cadherinas/metabolismo , Vaina de Mielina/fisiología , Neurregulina-1/metabolismo , Células de Schwann/fisiología , Animales , Western Blotting , Cadherinas/genética , Técnicas de Cocultivo , Receptores ErbB/metabolismo , Técnica del Anticuerpo Fluorescente , Ganglios Espinales/crecimiento & desarrollo , Ganglios Espinales/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Ratones Transgénicos , Interferencia de ARN , Ratas , Nervio Ciático/crecimiento & desarrollo , Nervio Ciático/fisiología , Transducción de Señal/fisiologíaRESUMEN
Physical damage to the peripheral nerves triggers Schwann cell injury response in the distal nerves in an event termed Wallerian degeneration: the Schwann cells degrade their myelin sheaths and dedifferentiate, reverting to a phenotype that supports axon regeneration and nerve repair. The molecular mechanisms regulating Schwann cell plasticity in the PNS remain to be elucidated. Using both in vivo and in vitro models for peripheral nerve injury, here we show that inhibition of p38 mitogen-activated protein kinase (MAPK) activity in mice blocks Schwann cell demyelination and dedifferentiation following nerve injury, suggesting that the kinase mediates the injury signal that triggers distal Schwann cell injury response. In myelinating cocultures, p38 MAPK also mediates myelin breakdown induced by Schwann cell growth factors, such as neuregulin and FGF-2. Furthermore, ectopic activation of p38 MAPK is sufficient to induce myelin breakdown and drives differentiated Schwann cells to acquire phenotypic features of immature Schwann cells. We also show that p38 MAPK concomitantly functions as a negative regulator of Schwann cell differentiation: enforced p38 MAPK activation blocks cAMP-induced expression of Krox 20 and myelin proteins, but induces expression of c-Jun. As expected of its role as a negative signal for myelination, inhibition of p38 MAPK in cocultures promotes myelin formation by increasing the number as well as the length of individual myelin segments. Altogether, our data identify p38 MAPK as an important regulator of Schwann cell plasticity and differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Fibras Nerviosas Mielínicas/fisiología , Células de Schwann/metabolismo , Células de Schwann/fisiología , Degeneración Walleriana/fisiopatología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Animales , Técnicas de Cocultivo , Proteína 2 de la Respuesta de Crecimiento Precoz/biosíntesis , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/biosíntesis , Ratones , Ratones Endogámicos C57BL , Vaina de Mielina/metabolismo , Fibras Nerviosas Mielínicas/metabolismo , Neurregulina-1/farmacología , Traumatismos de los Nervios Periféricos/metabolismo , Traumatismos de los Nervios Periféricos/fisiopatología , Ratas , Nervio Ciático/metabolismo , Nervio Ciático/fisiopatología , Degeneración Walleriana/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Myelinated axons are organized into specialized domains critical to their function in saltatory conduction, i.e., nodes, paranodes, juxtaparanodes, and internodes. Here, we describe the distribution and role of the 4.1B protein in this organization. 4.1B is expressed by neurons, and at lower levels by Schwann cells, which also robustly express 4.1G. Immunofluorescence and immuno-EM demonstrates 4.1B is expressed subjacent to the axon membrane in all domains except the nodes. Mice deficient in 4.1B have preserved paranodes, based on marker staining and EM in contrast to the juxtaparanodes, which are substantially affected in both the PNS and CNS. The juxtaparanodal defect is evident in developing and adult nerves and is neuron-autonomous based on myelinating cocultures in which wt Schwann cells were grown with 4.1B-deficient neurons. Despite the juxtaparanodal defect, nerve conduction velocity is unaffected. Preservation of paranodal markers in 4.1B deficient mice is associated with, but not dependent on an increase of 4.1R at the axonal paranodes. Loss of 4.1B in the axon is also associated with reduced levels of the internodal proteins, Necl-1 and Necl-2, and of alpha-2 spectrin. Mutant nerves are modestly hypermyelinated and have increased numbers of Schmidt-Lanterman incisures, increased expression of 4.1G, and express a residual, truncated isoform of 4.1B. These results demonstrate that 4.1B is a key cytoskeletal scaffold for axonal adhesion molecules expressed in the juxtaparanodal and internodal domains that unexpectedly regulates myelin sheath thickness.
Asunto(s)
Proteínas de Microfilamentos/metabolismo , Fibras Nerviosas Mielínicas/metabolismo , Neuronas/citología , Células de Schwann/metabolismo , Animales , Ancirinas/metabolismo , Axones/metabolismo , Axones/ultraestructura , Molécula 1 de Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Estimulación Eléctrica , Embrión de Mamíferos , Conducta Exploratoria/fisiología , Ganglios Espinales/citología , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Proteína Básica de Mielina/metabolismo , Proteína P0 de la Mielina/metabolismo , Proteínas de la Mielina/metabolismo , Conducción Nerviosa/genética , Conducción Nerviosa/fisiología , Nódulos de Ranvier/metabolismo , Nódulos de Ranvier/ultraestructura , Células de Schwann/ultraestructura , Espectrina/metabolismoRESUMEN
Axon-glial interactions are critical for the induction of myelination and the domain organization of myelinated fibers. Although molecular complexes that mediate these interactions in the nodal region are known, their counterparts along the internode are poorly defined. We report that neurons and Schwann cells express distinct sets of nectin-like (Necl) proteins: axons highly express Necl-1 and -2, whereas Schwann cells express Necl-4 and lower amounts of Necl-2. These proteins are strikingly localized to the internode, where Necl-1 and -2 on the axon are directly apposed by Necl-4 on the Schwann cell; all three proteins are also enriched at Schmidt-Lanterman incisures. Binding experiments demonstrate that the Necl proteins preferentially mediate heterophilic rather than homophilic interactions. In particular, Necl-1 on axons binds specifically to Necl-4 on Schwann cells. Knockdown of Necl-4 by short hairpin RNA inhibits Schwann cell differentiation and subsequent myelination in cocultures. These results demonstrate a key role for Necl-4 in initiating peripheral nervous system myelination and implicate the Necl proteins as mediators of axo-glial interactions along the internode.
Asunto(s)
Axones/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Fibras Nerviosas Mielínicas/metabolismo , Isoformas de Proteínas/metabolismo , Nódulos de Ranvier , Células de Schwann/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Axones/ultraestructura , Células CHO , Adhesión Celular/fisiología , Moléculas de Adhesión Celular , Moléculas de Adhesión Celular Neuronal/genética , Células Cultivadas , Cricetinae , Cricetulus , Ganglios Espinales/metabolismo , Inmunoglobulinas , Ratones , Ratones Endogámicos C57BL , Vaina de Mielina/metabolismo , Vaina de Mielina/ultraestructura , Fibras Nerviosas Mielínicas/ultraestructura , Isoformas de Proteínas/genética , Interferencia de ARN , Nódulos de Ranvier/metabolismo , Nódulos de Ranvier/ultraestructura , Ratas , Células de Schwann/citología , Nervio Ciático/citología , Nervio Ciático/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Members of the neuregulin-1 (Nrg1) growth factor family play important roles during Schwann cell development. Recently, it has been shown that the membrane-bound type III isoform is required for Schwann cell myelination. Interestingly, however, Nrg1 type II, a soluble isoform, inhibits the process. The mechanisms underlying these isoform-specific effects are unknown. It is possible that myelination requires juxtacrine Nrg1 signaling provided by the membrane-bound isoform, whereas paracrine stimulation by soluble Nrg1 inhibits the process. To investigate this, we asked whether Nrg1 type III provided in a paracrine manner would promote or inhibit myelination. We found that soluble Nrg1 type III enhanced myelination in Schwann cell-neuron cocultures. It improved myelination of Nrg1 type III(+/-) neurons and induced myelination on normally nonmyelinated sympathetic neurons. However, soluble Nrg1 type III failed to induce myelination on Nrg1 type III(-/-) neurons. To our surprise, low concentrations of Nrg1 type II also elicited a similar promyelinating effect. At high doses, however, both type II and III isoforms inhibited myelination and increased c-Jun expression in a manner dependent on Mek/Erk (mitogen-activated protein kinase kinase/extracellular signal-regulated kinase) activation. These results indicate that paracrine Nrg1 signaling provides concentration-dependent bifunctional effects on Schwann cell myelination. Furthermore, our studies suggest that there may be two distinct steps in Schwann cell myelination: an initial phase dependent on juxtacrine Nrg1 signaling and a later phase that can be promoted by paracrine stimulation.
Asunto(s)
Vaina de Mielina/metabolismo , Neurregulina-1/metabolismo , Células de Schwann/metabolismo , Animales , Células Cultivadas , Técnicas de Cocultivo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ganglios Espinales/enzimología , Ganglios Espinales/metabolismo , Genes jun , Humanos , Ratones , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Vaina de Mielina/enzimología , Neurregulina-1/genética , Neuronas/enzimología , Neuronas/metabolismo , Isoformas de Proteínas/metabolismo , Ratas , Células de Schwann/enzimología , Nervio Ciático/enzimología , Nervio Ciático/metabolismoRESUMEN
The ability to understand in great details, at the molecular level, the process of myelination in the peripheral nervous system (PNS) is, in no minor part, due to the availability of an in vitro culture model of PNS myelination. This culture system is based on the ability to prepare large population of highly purified Schwann cells and dorsal root ganglia neurons that, once co-cultured, can be driven to form in vitro well-defined myelinated axon units. In this chapter, we present our detailed protocols to establish these cell cultures that are derived from modifications of procedures developed 35-40 years ago.
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Ganglios Espinales/citología , Vaina de Mielina/metabolismo , Neuronas/metabolismo , Células de Schwann/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Femenino , Ganglios Espinales/embriología , Embarazo , RatasRESUMEN
Lentiviral transduction is a gene delivery method that provides numerous advantages over direct transfection and traditional retroviral or adenoviral delivery methods. It facilitates for the transduction of primary cells inherently difficult to transfect, delivers constructs of interest to nondividing as well as dividing cells, and permits the long-term expression of sizable DNA inserts (e.g., <7 kb). The study of peripheral nerve myelination at the molecular level has long benefited from the Schwann cells/dorsal root ganglia (DRG) neurons myelinating co-culture system. As this culture system takes about a month to develop and perform experiments with, lentiviral-delivered constructs can be used to manipulate gene expression in Schwann cells and DRG neurons, primary cells that are otherwise resilient to direct transfection. Here we present our protocol for lentiviral production and purification and subsequent infection of large numbers of Schwann cells and/or DRG neurons for the molecular study of peripheral nerve myelination in vitro.
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Ganglios Espinales/citología , Neuronas/metabolismo , Células de Schwann/citología , Células de Schwann/metabolismo , Animales , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , Técnicas de Cocultivo/métodos , Humanos , Lentivirus/genética , Vaina de Mielina/metabolismo , RatasRESUMEN
OBJECTIVE: Polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE), which is a piezoelectric, biocompatible polymer, holds promise as a scaffold in combination with Schwann cells (SCs) for spinal cord repair. Piezoelectric materials can generate electrical activity in response to mechanical deformation, which could potentially stimulate spinal cord axon regeneration. Our goal in this study was to investigate PVDF-TrFE scaffolds consisting of aligned fibers in supporting SC growth and SC-supported neurite extension and myelination in vitro. APPROACH: Aligned fibers of PVDF-TrFE were fabricated using the electrospinning technique. SCs and dorsal root ganglion (DRG) explants were co-cultured to evaluate SC-supported neurite extension and myelination on PVDF-TrFE scaffolds. MAIN RESULTS: PVDF-TrFE scaffolds supported SC growth and neurite extension, which was further enhanced by coating the scaffolds with Matrigel. SCs were oriented and neurites extended along the length of the aligned fibers. SCs in co-culture with DRGs on PVDF-TrFE scaffolds promoted longer neurite extension as compared to scaffolds without SCs. In addition to promoting neurite extension, SCs also formed myelin around DRG neurites on PVDF-TrFE scaffolds. SIGNIFICANCE: This study demonstrated PVDF-TrFE scaffolds containing aligned fibers supported SC-neurite extension and myelination. The combination of SCs and PVDF-TrFE scaffolds may be a promising tissue engineering strategy for spinal cord repair.
Asunto(s)
Hidrocarburos Fluorados/química , Vaina de Mielina/fisiología , Neuritas/fisiología , Polivinilos/química , Células de Schwann/fisiología , Andamios del Tejido , Animales , Técnicas de Cocultivo , Colágeno , Combinación de Medicamentos , Ganglios Espinales/citología , Laminina , Proteoglicanos , Ratas , Ratas Sprague-DawleyRESUMEN
Tissue inhibitor of metalloproteinase-3 (TIMP-3) inhibits the activities of various metalloproteinases including matrix metalloproteinases and ADAM family proteins. In the peripheral nervous system, ADAM17, also known as TNF-α converting enzyme (TACE), cleaves the extracellular domain of Nrg1 type III, an axonal growth factor that is essential for Schwann cell myelination. The processing by ADAM17 attenuates Nrg1 signaling and inhibits Schwann cell myelination. TIMP-3 targets ADAM17, suggesting a possibility that TIMP-3 may elicit a promyelinating function in Schwann cells by relieving ADAM17-induced myelination block. To investigate this, we used a myelinating coculture system to determine the effect of TIMP-3 on Schwann cell myelination. Treatment with TIMP-3 enhanced myelin formation in cocultures, evident by an increase in the number of myelin segments and upregulated expression of Krox20 and myelin protein. The effect of TIMP-3 was accompanied by the inhibition of ADAM17 activity and an increase in Nrg1 type III signaling in cocultures. Accordingly, the N-terminus fragment of TIMP-3, which exhibits a selective inhibitory function toward ADAM17, elicited a similar myelination-promoting effect and increased Nrg1 type III activity. TIMP-3 also enhanced laminin production in cocultures, which is likely to aid Schwann cell myelination.
Asunto(s)
Vaina de Mielina/metabolismo , Células de Schwann/efectos de los fármacos , Nervio Ciático/citología , Inhibidor Tisular de Metaloproteinasa-3/farmacología , Proteína ADAM17/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Antioxidantes , Ácido Ascórbico/farmacología , Bromodesoxiuridina/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Transferencia Resonante de Energía de Fluorescencia , Ganglios Espinales/citología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteína Básica de Mielina/metabolismo , Neuronas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Ratas , Nervio Ciático/crecimiento & desarrollo , Nervio Ciático/metabolismoRESUMEN
The mechanisms that coordinate and balance a complex network of opposing regulators to control Schwann cell (SC) differentiation remain elusive. Here we demonstrate that zinc-finger E-box-binding homeobox 2 (Zeb2, also called Sip1) transcription factor is a critical intrinsic timer that controls the onset of SC differentiation by recruiting histone deacetylases HDAC 1 and 2 (HDAC1/2) and nucleosome remodeling and deacetylase complex (NuRD) co-repressor complexes in mice. Zeb2 deletion arrests SCs at an undifferentiated state during peripheral nerve development and inhibits remyelination after injury. Zeb2 antagonizes inhibitory effectors including Notch and Sox2. Importantly, genome-wide transcriptome analysis reveals a Zeb2 target gene encoding the Notch effector Hey2 as a potent inhibitor for Schwann cell differentiation. Strikingly, a genetic Zeb2 variant associated with Mowat-Wilson syndrome disrupts the interaction with HDAC1/2-NuRD and abolishes Zeb2 activity for SC differentiation. Therefore, Zeb2 controls SC maturation by recruiting HDAC1/2-NuRD complexes and inhibiting a Notch-Hey2 signaling axis, pointing to the critical role of HDAC1/2-NuRD activity in peripheral neuropathies caused by ZEB2 mutations.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Homeodominio/metabolismo , Fibras Nerviosas Mielínicas/ultraestructura , Nucleosomas/metabolismo , Proteínas Represoras/metabolismo , Células de Schwann/metabolismo , Animales , Diferenciación Celular/fisiología , Facies , Enfermedad de Hirschsprung/metabolismo , Histona Desacetilasa 1/genética , Discapacidad Intelectual/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Microcefalia/metabolismo , Neurogénesis/fisiología , Células de Schwann/citología , Caja Homeótica 2 de Unión a E-Box con Dedos de ZincRESUMEN
The Ig cell adhesion molecules (CAM) neurofascin (NF) and Nr-CAM are localized at developing nodes of Ranvier in peripheral myelinated axons prior to clustering of Na+ channels. Different isoforms of NF are expressed on neurons and glia, and NF binding on both cells has been suggested to play roles in node and paranode formation. To clarify the role of NF further, we analyzed effects of NF-Fc fusion proteins in Schwann cell-DRG neuron myelinating cocultures. NF-Fc significantly inhibited nodal clustering of Na+ channels, ankyrin G, and betaIV spectrin, and modestly reduced Caspr clustering at paranodal junctions; it did not significantly affect lengths or numbers of myelin-positive segments, axon initial segments, or accumulations of phosphorylated-ERM proteins in Schwann cell nodal microvilli. NF-Fc binds to Schwann cells but little or no binding to DRG neurons was detected. The results suggest a critical early role for axonal NF in clustering of Na+ channels at nodes of Ranvier via interactions with receptors on Schwann cells.
Asunto(s)
Ancirinas/metabolismo , Moléculas de Adhesión Celular/fisiología , Factores de Crecimiento Nervioso/fisiología , Proteínas del Tejido Nervioso/metabolismo , Nódulos de Ranvier/metabolismo , Canales de Sodio/metabolismo , Espectrina/metabolismo , Animales , Axones/metabolismo , Axones/fisiología , Moléculas de Adhesión Celular/biosíntesis , Comunicación Celular/fisiología , Técnicas de Cocultivo , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/fisiología , Factores de Crecimiento Nervioso/biosíntesis , Ratas , Receptores Fc/genética , Proteínas Recombinantes de Fusión/biosíntesis , Células de Schwann/metabolismoRESUMEN
The nodes of Ranvier are regularly spaced gaps between myelin sheaths that are markedly enriched in voltage-gated sodium channels and associated proteins. Myelinating glia play a key role in promoting node formation, although the requisite glial signals remain poorly understood. In this study, we have examined the expression of glial proteoglycans in the peripheral and central nodes. We report that the heparan sulfate proteoglycan, syndecan-3, becomes highly enriched with PNS node formation; its ligand, collagen V, is also concentrated at the PNS nodes and at lower levels along the abaxonal membrane. The V1 isoform of versican, a chondroitin sulfate proteoglycan, is also present in the nodal gap. By contrast, CNS nodes are enriched in versican isoform V2, but not syndecan-3. We have examined the molecular composition of the PNS nodes in syndecan-3 knockout mice. Nodal components are normally expressed in mice deficient in syndecan-3, suggesting that it has a nonessential role in the organization of nodes in the adult. These results indicate that the molecular composition and extracellular environment of the PNS and CNS nodes of Ranvier are significantly distinct.
Asunto(s)
Sistema Nervioso Central/metabolismo , Neuroglía/metabolismo , Sistema Nervioso Periférico/metabolismo , Proteoglicanos/metabolismo , Nódulos de Ranvier/metabolismo , Animales , Axones/metabolismo , Axones/ultraestructura , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Células Cultivadas , Sistema Nervioso Central/ultraestructura , Colágeno Tipo V/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/metabolismo , Sistema Nervioso Periférico/ultraestructura , Isoformas de Proteínas/metabolismo , Proteoglicanos/genética , Nódulos de Ranvier/ultraestructura , Células de Schwann/metabolismo , Sindecano-3 , VersicanosRESUMEN
We have used a monoclonal antibody to neurocan and specific polyclonal antibodies to the non-homologous glycosaminoglycan attachment regions of aggrecan and mRNA splice variants of versican to compare the localization and developmental changes of these structurally related hyaluronan-binding chondroitin sulfate proteoglycans in the rat retina and optic nerve. Staining for aggrecan and versican was first seen at embryonic day 16 in the optic nerve and retina, whereas neurocan was not detected in the embryonic eye. At postnatal day 0 (P0), beta-versican staining is largely confined to the inner plexiform layer whereas alpha-versican is also apparent in the neuroblastic layer. Both aggrecan and, much more weakly, neurocan immunoreactivity is present throughout the neonatal retina. At P9, aggrecan and versican immunoreactivity is most intense in the inner and outer plexiform and ganglion cell layers, accompanied by diffuse staining in the inner and outer nuclear layers. Aggrecan and alpha-versican are also present throughout the optic nerve and disk, whereas beta-versican and neurocan are confined to the laminar beams of the optic nerve. Between P0 and P9 there is a marked increase in beta-versican expression in the inner and outer nuclear layers and in the outer plexiform layer, whereas there is only weak staining of neurocan in the inner plexiform and ganglion cell layers of P9 retina. By 1 month postnatal the staining pattern of the fully differentiated retinal layers is essentially identical to that seen in the adult, where there is strong aggrecan and alpha-versican immunoreactivity in the retina and optic nerve, whereas beta-versican has essentially disappeared from the adult retina and, similarly to neurocan, is present only in the laminar beams of the optic nerve. The marked decrease of beta-versican in the retina is consistent with >90% decrease in its concentration in brain during postnatal development, suggesting that the developmental time-course for these proteoglycans in retina parallels that seen in other areas of the central nervous system.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/análisis , Proteínas de la Matriz Extracelular/análisis , Proteínas del Ojo/análisis , Proteínas del Tejido Nervioso/análisis , Nervio Óptico/crecimiento & desarrollo , Proteoglicanos/análisis , Retina/crecimiento & desarrollo , Agrecanos , Animales , Diferenciación Celular/inmunología , Matriz Extracelular/inmunología , Proteínas de la Matriz Extracelular/inmunología , Inmunohistoquímica/métodos , Lectinas Tipo C , Microscopía Fluorescente/métodos , Neurocano , Nervio Óptico/embriología , Nervio Óptico/inmunología , Ratas , Retina/embriología , Retina/inmunología , VersicanosRESUMEN
The localization of aggrecan and mRNA splice variants of versican in the developing rat central nervous system has been examined by using specific polyclonal antibodies to the nonhomologous glycosaminoglycan attachment regions of these hyaluronan-binding chondroitin sulfate proteoglycans. At embryonic day 16 (E16), aggrecan and versican splice variants containing either or both the alpha-and beta-domains are present in the marginal zone and subplate of the cerebral cortex and in the amygdala, internal capsule, and the optic and lateral olfactory tracts. There is strong staining of versican but not of aggrecan in the hippocampus and dentate gyrus by E19, whereas both aggrecan and alpha-versican are present in the fimbria. At E19, aggrecan is seen throughout the cerebral cortex, whereas the distribution of versican is considerably more limited, being confined essentially to the marginal zone and subplate. At 1 week postnatal, both aggrecan and versican are present in the prospective white matter and in the molecular and granule cell layers of the cerebellum, but neither proteoglycan is seen in the external granule cell layer. alpha- but not beta-versican staining is seen in Purkinje cells, and aggrecan staining of Purkinje cells is also rather minimal. In the spinal cord at E13, aggrecan is present in the dorsal root entry zone, ventral funiculus, mantle layer, and floor plate, as well as in the dorsal root ganglia and ventral roots. However, alpha-versican is confined to the dorsal root entry zone and the ependyma surrounding the spinal canal, and beta-versican is not present in spinal cord parenchyma at this developmental stage, being limited to the surrounding connective tissue. By E19, there are significant amounts of all three proteoglycans in the spinal cord. Aggrecan staining is most intense in the lateral funiculus and the fasciculi gracilis and cuneatus, where alpha-versican staining is also strong. In contrast, beta-versican is seen predominantly in the motor columns. Differences in the localization and temporal expression patterns of these chondroitin sulfate proteoglycans suggest that, like neurocan and phosphacan, they have partially complementary roles during central nervous system development.