Mesenchymal Stem Cell Treatment Perspectives in Peripheral Nerve Regeneration: Systematic Review.

Traumatic peripheral nerve lesions affect hundreds of thousands of patients every year; their consequences are life-altering and often devastating and cause alterations in movement and sensitivity. Spontaneous peripheral nerve recovery is often inadequate. In this context, nowadays, cell therapy represents one of the most innovative approaches in the field of nerve repair therapies. The purpose of this systematic review is to discuss the features of different types of mesenchymal stem cells (MSCs) relevant for peripheral nerve regeneration after nerve injury. The published literature was reviewed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A combination of the keywords "nerve regeneration", "stem cells", "peripheral nerve injury", "rat", and "human" were used. Additionally, a "MeSH" research was performed in PubMed using the terms "stem cells" and "nerve regeneration". The characteristics of the most widely used MSCs, their paracrine potential, targeted stimulation, and differentiation potentials into Schwann-like and neuronal-like cells are described in this paper. Considering their ability to support and stimulate axonal growth, their remarkable paracrine activity, their presumed differentiation potential, their extremely low immunogenicity, and their high survival rate after transplantation, ADSCs appear to be the most suitable and promising MSCs for the recovery of peripheral nerve lesion. Clinical considerations are finally reported.

Nanofibrous Nerve Conduits with Nerve Growth Factors and Bone Marrow Stromal Cells Pre-Cultured in Bioreactors for Peripheral Nerve Regeneration.

Peripheral nerve injury (PNI) was the leading cause of permanent dysfunction in movement and sensation. Synthesized nerve guide conduits (NGCs) with Schwann Cells (SCs) can help peripheral nerve regeneration. However, poor accessibility of SCs and lack of full coverage of seeded cells on NGCs can lead to failure of nerve regeneration across long gaps and full functional recovery. To overcome these limitations, bone marrow stromal cells (BMSCs) and a novel culture method were proposed in the current study. BMSCs were harvested and seeded on a never growth factor (NGF)-loaded PCL nanofibrous NGCs and cultured with a rotary cell culture system (RCCS) before implantation. The NGCs were tested in vitro with PC-12 cells to validate the bioactivity of released NGF and to access its ability to promote neurite extension. Also, the NGCs were tested in vivo with rat sciatic nerve model to exam its potential in bridging the long gap (15 mm segmental defect). The efficacy of the NGCs was investigated based on the results of the functional test, electrophysiology test, muscle atrophy, and histological analysis. The results of in vitro PC-12 cell study confirmed the bioactivity of released NGF and showed a significant increase in the neurite extension with the help of PEG-diamine and BSA. These results showed that the novel loading method could preserve the bioactivity of growth factors and achieve a sustained release in vitro. Besides, the results of the in vivo study exhibited a significant increase with the combination of all additives. These results showed that with the help of NGF and RCCS, the NGCs with the seeded BMSCs could enhance peripheral nerve regeneration across long nerve injury gaps.

Enhancement of sciatic nerve regeneration with dual delivery of vascular endothelial growth factor and nerve growth factor genes.

BACKGROUND: Peripheral nerve injury is one common clinical disease worldwide, in which sciatic nerve is anatomically the most challenging to regenerate given its length and large cross-sectional area. For the present, autologous nerve grafting remains to be the most ideal strategy when treating with sciatic nerve injury. However, this method sacrifices healthy nerves and requires highly intensive surgery, still calling for other advanced alternatives for nerve grafting. RESULTS: In this study, we utilized previously well-established gene delivery system to dually deliver plasmid DNA (pDNA) encoding vascular endothelial growth factor (VEGF) and nerve growth factor (NGF), exploring therapeutics for sciatic nerve injury. Low-molecular-weight branched polyethylenimine (bPEI) was constructed as the backbone structure of gene vectors, and it was further crosslinked to synthesize degradable polycations via the conjugation of dialdehydes. Potential synergistic effect between VEGF and NGF proteins were observed on rat sciatic nerve crush injury model in this study. CONCLUSIONS: We concluded that dual delivery of plasmid VEGF and NGF as gene therapy could enhance sciatic nerve regeneration.

Exosomes from human adipose-derived stem cells promote sciatic nerve regeneration via optimizing Schwann cell function.

Human adipose-derived stem cells (ASCs) have a potential for the treatment of peripheral nerve injury. Recent studies demonstrated that stem cells can mediate therapeutic effect by secreting exosomes. We aimed to investigate the effect of human ASCs derived exosomes (ASC-Exos) on peripheral nerve regeneration in vitro and in vivo. Our results showed after being internalized by Schwann cells (SCs), ASC-Exos significantly promoted SC proliferation, migration, myelination, and secretion of neurotrophic factors by upregulating corresponding genes in vitro. We next evaluated the efficacy of ASC-Exo therapy in a rat sciatic nerve transection model with a 10-mm gap. Axon regeneration, myelination, and restoration of denervation muscle atrophy in ASC-Exos treated group was significantly improved compared to vehicle control. This study demonstrates that ASC-Exos effectively promote peripheral nerve regeneration via optimizing SC function and thereby represent a novel therapeutic strategy for regenerative medicine and nerve tissue engineering.

The TSC1-mTOR-PLK axis regulates the homeostatic switch from Schwann cell proliferation to myelination in a stage-specific manner.

Proper peripheral myelination depends upon the balance between Schwann cell proliferation and differentiation programs. The serine/threonine kinase mTOR integrates various environmental cues to serve as a central regulator of cell growth, metabolism, and function. We report here that tuberous sclerosis complex 1 (TSC1), a negative regulator of mTOR activity, establishes a stage-dependent program for Schwann cell lineage progression and myelination by controlling cell proliferation and myelin homeostasis. Tsc1 ablation in Schwann cell progenitors in mice resulted in activation of mTOR signaling, and caused over-proliferation of Schwann cells and blocked their differentiation, leading to hypomyelination. Transcriptome profiling analysis revealed that mTOR activation in Tsc1 mutants resulted in upregulation of a polo-like kinase (PLK)-dependent pathway and cell cycle regulators. Attenuation of mTOR or pharmacological inhibition of polo-like kinases partially rescued hypomyelination caused by Tsc1 loss in the developing peripheral nerves. In contrast, deletion of Tsc1 in mature Schwann cells led to redundant and overgrown myelin sheaths in adult mice. Together, our findings indicate stage-specific functions for the TSC1-mTOR-PLK signaling axis in controlling the transition from proliferation to differentiation and myelin homeostasis during Schwann cell development.

Myelin extracellular leaflet compaction requires apolipoprotein D membrane management to optimize lysosomal-dependent recycling and glycocalyx removal.

To compact the extracellular sides of myelin, an important transition must take place: from membrane sliding, while building the wraps, to membrane adhesion and water exclusion. Removal of the negatively charged glycocalyx becomes the limiting factor in such transition. What is required to initiate this membrane-zipping process? Knocking-out the Lipocalin Apolipoprotein D (ApoD), essential for lysosomal functional integrity in glial cells, results in a specific defect in myelin extracellular leaflet compaction in peripheral and central nervous system, which results in reduced conduction velocity and suboptimal behavioral outputs: motor learning is compromised. Myelination initiation, growth, intracellular leaflet compaction, myelin thickness or internodal length remain unaltered. Lack of ApoD specifically modifies Plp and P0 protein expression, but not Mbp or Mag. Late in myelin maturation period, ApoD affects lipogenic and growth-related, but not stress-responsive, signaling pathways. Without ApoD, the sialylated glycocalyx is maintained and ganglioside content remains high. In peripheral nervous system, Neu3 membrane sialidase and lysosomal Neu1 are coordinately expressed with ApoD in subsets of Schwann cells. ApoD-KO myelin becomes depleted of Neu3 and enriched in Fyn, a kinase with pivotal roles in transducing axon-derived signals into myelin properties. In the absence of ApoD, partial permeabilization of lysosomes alters Neu1 location as well. Exogenous ApoD rescues ApoD-KO hypersialylated glycocalyx in astrocytes, demonstrating that ApoD is necessary and sufficient to control glycocalyx composition in glial cells. By ensuring lysosomal functional integrity and adequate subcellular location of effector and regulatory proteins, ApoD guarantees the glycolipid recycling and glycocalyx removal required to complete myelin compaction.

Tissue Inhibitor of Metalloproteinase-3 Promotes Schwann Cell Myelination.

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.

Schwann cell-specific deletion of the endosomal PI 3-kinase Vps34 leads to delayed radial sorting of axons, arrested myelination, and abnormal ErbB2-ErbB3 tyrosine kinase signaling.

The PI 3-kinase Vps34 (Pik3c3) synthesizes phosphatidylinositol 3-phosphate (PI3P), a lipid critical for both endosomal membrane traffic and macroautophagy. Human genetics have implicated PI3P dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating Charcot-Marie-Tooth (CMT) peripheral neuropathy. Here, we investigated the role of Vps34, and PI3P, in mouse Schwann cells by selectively deleting Vps34 in this cell type. Vps34-Schwann cell knockout (Vps34SCKO ) mice show severe hypomyelination in peripheral nerves. Vps34-/- Schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. Upon reaching the promyelinating stage, Vps34-/- Schwann cells are significantly impaired in the elaboration of myelin. Nerves from Vps34SCKO mice contain elevated levels of the LC3 and p62 proteins, indicating impaired autophagy. However, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in Vps34SCKO mice is caused by impaired autophagy. Endosomal trafficking is also disturbed in Vps34-/- Schwann cells. We investigated the activation of the ErbB2/3 receptor tyrosine kinases in Vps34SCKO nerves, as these proteins, which play essential roles in Schwann cell myelination, are known to traffic through endosomes. In Vps34SCKO nerves, ErbB3 was hyperphosphorylated on a tyrosine known to be phosphorylated in response to neuregulin 1 exposure. ErbB2 protein levels were also decreased during myelination. Our findings suggest that the loss of Vps34 alters the trafficking of ErbB2/3 through endosomes. Abnormal ErbB2/3 signaling to downstream targets may contribute to the hypomyelination observed in Vps34SCKO mice.

[Effect of short-term low-frequency electrical stimulation on nerve regeneration of delayed nerve defect during operation].

Objective: To explore the effect of short-term low-frequency electrical stimulation (SLES) during operation on nerve regeneration in delayed peripheral nerve injury with long gap. Methods: Thirty female adult Sprague Dawley rats, weighing 160-180 g, were used to prepare 13-mm defect model by trimming the nerve stumps. Then all rats were randomly divided into 2 groups, 15 rats in each group. After nerve defect was bridged by the contralateral normal sciatic nerve, SLES was applied in the experimental group, but was not in the control group. The spinal cords and dorsal root ganglions (DRGs) were harvested to carry out immunofluorescence histochemistry double staining for growth-associated proteins 43 (GAP-43) and brain-derived neurotrophic factor (BDNF) at 1, 2, and 7 days after repair. Fluorogold (FG) retrograde tracing was performed at 3 months after repair. The mid-portion regenerated segments were harvested to perform Meyer's trichrome staining, immunofluorescence double staining for neurofilament (NF) and soluble protein 100 (S-100) on the transversely or longitudinal sections at 3 months after repair. The segment of the distal sciatic nerve trunk was harvested for electron microscopy and morphometric analyses to measure the diameter of the myelinated axons, thickness of myelin sheaths, the G ratio, and the density of the myelinated nerve fibers. The gastrocnemius muscles of the operated sides were harvested to measure the relative wet weight ratios. Karnovsky-Root cholinesterase staining of the motor endplate was carried out. Results: In the experimental group, the expressions of GAP-43 and BDNF were higher than those in the control group at 1 and 2 days after repair. The number of labeled neurons in the anterior horn of gray matter in the spinal cord and DRGs at the operated side from the experimental group was more than that from the control group. Meyer's trichrome staining, immunofluorescence double staining, and the electron microscopy observation showed that the regenerated nerves were observed to develop better in the experimental group than the control group. The relative wet weight ratio of experimental group was significantly higher than that of the control group ( t=4.633, P=0.000). The size and the shape of the motor endplates in the experimental group were better than those in the control group. Conclusion: SLES can promote the regeneration ability of the short-term (1 month) delayed nerve injury with long gap to a certain extent.

A Long-Gap Peripheral Nerve Injury Therapy Using Human Skeletal Muscle-Derived Stem Cells (Sk-SCs): An Achievement of Significant Morphological, Numerical and Functional Recovery.

Losses in vital functions of the somatic motor and sensory nervous system are induced by severe long-gap peripheral nerve transection injury. In such cases, autologous nerve grafts are the gold standard treatment, despite the unavoidable sacrifice of other healthy functions, whereas the prognosis is not always favorable. Here, we use human skeletal muscle-derived stem cells (Sk-SCs) to reconstitute the function after long nerve-gap injury. Muscles samples were obtained from the amputated legs from 9 patients following unforeseen accidents. The Sk-SCs were isolated using conditioned collagenase solution, and sorted as CD34+/45- (Sk-34) and CD34-/45-/29+ (Sk-DN/29+) cells. Cells were separately cultured/expanded under optimal conditions for 2 weeks, then injected into the athymic nude mice sciatic nerve long-gap model (7-mm) bridging an acellular conduit. After 8-12 weeks, active cell engraftment was observed only in the Sk-34 cell transplanted group, showing preferential differentiation into Schwann cells and perineurial/endoneurial cells, as well as formation of the myelin sheath and perineurium/endoneurium surrounding regenerated axons, resulted in 87% of numerical recovery. Differentiation into vascular cell lineage (pericyte and endothelial cells) were also observed. A significant tetanic tension recovery (over 90%) of downstream muscles following electrical stimulation of the sciatic nerve (at upper portion of the gap) was also achieved. In contrast, Sk-DN/29+ cells were completely eliminated during the first 4 weeks, but relatively higher numerical (83% vs. 41% in axon) and functional (80% vs. 60% in tetanus) recovery than control were observed. Noteworthy, significant increase in the formation of vascular networks in the conduit during the early stage (first 2 weeks) of recovery was observed in both groups with the expression of key factors (mRNA and protein levels), suggesting the paracrine effects to angiogenesis. These results suggested that the human Sk-SCs may be a practical source for autologous stem cell therapy following severe peripheral nerve injury.

Axonal Localization of Integrins in the CNS Is Neuronal Type and Age Dependent.

The regenerative ability of CNS axons decreases with age, however, this ability remains largely intact in PNS axons throughout adulthood. These differences are likely to correspond with age-related silencing of proteins necessary for axon growth and elongation. In previous studies, it has been shown that reintroduction of the α9 integrin subunit (tenascin-C receptor, α9) that is downregulated in adult CNS can improve neurite outgrowth and sensory axon regeneration after a dorsal rhizotomy or a dorsal column crush spinal cord lesion. In the current study, we demonstrate that virally expressed integrins (α9, α6, or ß1 integrin) in the adult rat sensorimotor cortex and adult red nucleus are excluded from axons following neuronal transduction. Attempts to stimulate transport by inclusion of a cervical spinal injury and thus an upregulation of extracellular matrix molecules at the lesion site, or cotransduction with its binding partner, ß1 integrin, did not induce integrin localization within axons. In contrast, virally expressed α9 integrin in developing rat cortex (postnatal day 5 or 10) demonstrated clear localization of integrins in cortical axons revealed by the presence of integrin in the axons of the corpus callosum and internal capsule, as well as in the neuronal cell body. Furthermore, examination of dorsal root ganglia neurons and retinal ganglion cells demonstrated integrin localization both within peripheral nerve as well as dorsal root axons and within optic nerve axons, respectively. Together, our results suggest a differential ability for in vivo axonal transport of transmembrane proteins dependent on neuronal age and subtype.

Gelatin-methacrylamide gel loaded with microspheres to deliver GDNF in bilayer collagen conduit promoting sciatic nerve growth.

In this study, we fabricated glial cell-line derived neurotrophic factor (GDNF)-loaded microspheres, then seeded the microspheres in gelatin-methacrylamide hydrogel, which was finally integrated with the commercial bilayer collagen membrane (Bio-Gide(®)). The novel composite of nerve conduit was employed to bridge a 10 mm long sciatic nerve defect in a rat. GDNF-loaded gelatin microspheres had a smooth surface with an average diameter of 3.9±1.8 µm. Scanning electron microscopy showed that microspheres were uniformly distributed in both the GelMA gel and the layered structure. Using enzyme-linked immunosorbent assay, in vitro release studies (pH 7.4) of GDNF from microspheres exhibited an initial burst release during the first 3 days (18.0%±1.3%), and then, a prolonged-release profile extended to 32 days. However, in an acidic condition (pH 2.5), the initial release percentage of GDNF was up to 91.2%±0.9% within 4 hours and the cumulative release percentage of GDNF was 99.2%±0.2% at 48 hours. Then the composite conduct was implanted in a 10 mm critical defect gap of sciatic nerve in a rat. We found that the nerve was regenerated in both conduit and autograft (AG) groups. A combination of electrophysiological assessment and histomorphometry analysis of regenerated nerves showed that axonal regeneration and functional recovery in collagen tube filled with GDNF-loaded microspheres (GM + CT) group were similar to AG group (P>0.05). Most myelinated nerves were matured and arranged densely with a uniform structure of myelin in a neat pattern along the long axis in the AG and GM + CT groups, however, regenerated nerve was absent in the BLANK group, left the 10 mm gap empty after resection, and the nerve fiber exhibited a disordered arrangement in the collagen tube group. These results indicated that the hybrid system of bilayer collagen conduit and GDNF-loaded gelatin microspheres combined with gelatin-methacrylamide hydrogels could serve as a new biodegradable artificial nerve guide for nerve tissue engineering.

Ageing-induced changes in the redox status of peripheral motor nerves imply an effect on redox signalling rather than oxidative damage.

Ageing is associated with loss of skeletal muscle fibres, atrophy of the remaining fibres and weakness. These changes in muscle are accompanied by disruption of motor neurons and neuromuscular junctions although the direct relationship between the nerve and muscle degeneration is not understood. Oxidative changes have been implicated in the mechanisms leading to age-related loss of muscle mass and in degeneration of the central nervous system, but little is known about age-related changes in oxidation in specific peripheral nerves that supply muscles that are affected by ageing. We have therefore examined the sciatic nerve of old mice at an age when loss of tibialis anterior muscle mass and function is apparent. Sciatic nerve from old mice did not show a gross increase in oxidative damage, but electron paramagnetic resonance (EPR) studies indicated an increase in the activity of superoxide and/or peroxynitrite in the nerves of old mice at rest that was further exacerbated by electrical stimulation of the nerve to activate muscle contractions. Proteomic analyses indicated that specific redox-sensitive proteins are increased in content in the nerves of old mice that may reflect an adaptation to regulate the increased superoxide/peroxynitrite and maintain redox homoeostasis. Analysis of redox active cysteines showed some increase in reversible oxidation in specific proteins in nerves of old mice, but this was not universally seen across all redox-active cysteines. Detailed analysis of the redox-active cysteine in one protein in the nerve of old mice that is key to redox signalling (Peroxiredoxin 6, Cys 47) showed a minor increase in reversible oxidation that would be compatible with a change in its redox signalling function. In conclusion, the data presented indicate that sciatic nerve from old mice does not show a gross increase in oxidative damage similar to that seen in the TA and other muscles that it innervates. Our results indicate an adaptation to increased oxidation with minor changes in the oxidation of key cysteines that may contribute to defective redox signalling in the nerve.

Tissue-engineered conduit promotes sciatic nerve regeneration following radiation-induced injury as monitored by magnetic resonance imaging.

PURPOSE: To observe the longitudinal changes in peripheral nerve repaired with chitosan conduits in a rat model of radiation-induced neuropathy. MATERIALS AND METHODS: Four months after 40 Gy radiation to the right lower limbs, forty-two rats were divided randomly into three groups. Chitosan conduits were implanted with (group A, n=12) or without (group B, n=12) mesenchymal stem cells (MSCs), and untreated controls (group C, n=12). Following sciatic nerve MR imaging (including T2WI and Gd-DTPA enhanced T1WI), functional evaluation and electrophysiological exam were performed two-monthly, final histological assessments were done at the end of one year. The differences among the experimental and control groups were statistically analysed with Fisher's PLSD or t-test. RESULTS: The compound muscle action potentials (CMAPs) and sciatic function index (SFI) had declined since 4 months after radiation injury. The focal nerve enlargement and hyperintensity, the perineurium and connecting muscle enhancement were demonstrated by MR neurography images. After chitosan tube implantation, the normalized signal intensities (SIs) in group A were declined more rapidly than SIs in other groups. The histological assessments indicated that group A had better remyelination, combined with higher CMAPs amplitude and SFI score than other groups. CONCLUSION: A single fraction dose of 40 Gy can be used to establish a rat model of sciatic nerve injury. Longitudinal electrophysiological examination and MR neurography are useful to evaluate the post-irradiation sciatic neuropathy. The rats with tissue-engineered conduits implantation showed some improvement of lower limb function, accompanied by a normalization of (T1W/T2W) MR signal.

Neuronal Regulation of Schwann Cell Mitochondrial Ca(2+) Signaling during Myelination.

Schwann cells (SCs) myelinate peripheral neurons to promote the rapid conduction of action potentials, and the process of myelination is known to be regulated by signals from axons to SCs. Given that SC mitochondria are one of the potential regulators of myelination, we investigated whether SC mitochondria are regulated by axonal signaling. Here, we show a purinergic mechanism that sends information from neurons to SC mitochondria during myelination. Our results show that electrical stimulation of rat sciatic nerve increases extracellular ATP levels enough to activate purinergic receptors. Indeed, electrical stimulation of sciatic nerves induces Ca(2+) increases in the cytosol and the mitochondrial matrix of surrounding SCs via purinergic receptor activation. Chronic suppression of this pathway during active myelination suppressed the longitudinal and radial development of myelinating SCs and caused hypomyelination. These results demonstrate a neuron-to-SC mitochondria signaling, which is likely to have an important role in proper myelination.

E-cadherin enhances neuregulin signaling and promotes Schwann cell myelination.

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.

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.

Giant scaffolding protein AHNAK1 interacts with ß-dystroglycan and controls motility and mechanical properties of Schwann cells.

The profound morphofunctional changes that Schwann cells (SCs) undergo during their migration and elongation on axons, as well as during axon sorting, ensheathment, and myelination, require their close interaction with the surrounding laminin-rich basal lamina. In contrast to myelinating central nervous system glia, SCs strongly and constitutively express the giant scaffolding protein AHNAK1, localized essentially underneath the outer, abaxonal plasma membrane. Using electron microscopy, we show here that in the sciatic nerve of ahnak1(-) (/) (-) mice the ultrastructure of myelinated, and unmyelinated (Remak) fibers is affected. The major SC laminin receptor ß-dystroglycan co-immunoprecipitates with AHNAK1 shows reduced expression in ahnak1(-) (/) (-) SCs, and is no longer detectable in Cajal bands on myelinated fibers in ahnak1(-) (/) (-) sciatic nerve. Reduced migration velocity in a scratch wound assay of purified ahnak1(-) (/) (-) primary SCs cultured on a laminin substrate indicated a function of AHNAK1 in SC motility. This was corroborated by atomic force microscopy measurements, which revealed a greater mechanical rigidity of shaft and leading tip of ahnak1(-) (/) (-) SC processes. Internodal lengths of large fibers are decreased in ahnak1(-) (/) (-) sciatic nerve, and longitudinal extension of myelin segments is even more strongly reduced after acute knockdown of AHNAK1 in SCs of developing sciatic nerve. Together, our results suggest that by interfering in the cross-talk between the transmembrane form of the laminin receptor dystroglycan and F-actin, AHNAK1 influences the cytoskeleton organization of SCs, and thus plays a role in the regulation of their morphology and motility and lastly, the myelination process.

Effect of electrical stimulation of sciatic nerve on synaptic plasticity of spinal dorsal horn and spinal c-fos expression in neonatal, juvenile and adult rats.

To explore the response to nociceptive stimuli in spinal cord of immature rat and observe the electrical stimulation of the sciatic nerve on synaptic plasticity of the spinal dorsal horn and spinal c-fos expression in rats of different ages, MK-801 was added to the spinal cord of rats, and the resulting changes in field potential as well as c-fos expression were recorded. LTP in neonatal rats was mainly evoked by A-type nerve fibers, whereas LTP in juvenile and adult rats was mainly evoked by C-type nerve fibers. C-fos expression was significantly increased in the superficial and deep layers of the spinal dorsal horn and in the ventral horn in neonatal rats indicating that the pain signal changed with age.

Hyccin, the molecule mutated in the leukodystrophy hypomyelination and congenital cataract (HCC), is a neuronal protein.

"Hypomyelination and Congenital Cataract", HCC (MIM #610532), is an autosomal recessive disorder characterized by congenital cataract and diffuse cerebral and peripheral hypomyelination. HCC is caused by deficiency of Hyccin, a protein whose biological role has not been clarified yet. Since the identification of the cell types expressing a protein of unknown function can contribute to define the physiological context in which the molecule is explicating its function, we analyzed the pattern of Hyccin expression in the central and peripheral nervous system (CNS and PNS). Using heterozygous mice expressing the b-galactosidase (LacZ) gene under control of the Hyccin gene regulatory elements, we show that the gene is primarily expressed in neuronal cells. Indeed, Hyccin-LacZ signal was identified in CA1 hippocampal pyramidal neurons, olfactory bulb, and cortical pyramidal neurons, while it did not colocalize with oligodendroglial or astrocytic markers. In the PNS, Hyccin was detectable only in axons isolated from newborn mice. In the brain, Hyccin transcript levels were higher in early postnatal development (postnatal days 2 and 10) and then declined in adult mice. In a model of active myelinogenesis, organotypic cultures of rat Schwann cells (SC)/Dorsal Root Ganglion (DRG) sensory neurons, Hyccin was detected along the neurites, while it was absent from SC. Intriguingly, the abundance of the molecule was upregulated at postnatal days 10 and 15, in the initial steps of myelinogenesis and then declined at 30 days when the process is complete. As Hyccin is primarily expressed in neurons and its mutation leads to hypomyelination in human patients, we suggest that the protein is involved in neuron-to-glia signalling to initiate or maintain myelination.