Robo3.1A suppresses Slit‐mediated repulsion by triggering degradation of Robo2

Slits and Robos control the midline crossing of commissural axons, which are not sensitive to the midline repellent Slit before crossing but gain Slit responsiveness to exit the midline and avoid recrossing. Robo3.1A promotes midline crossing of commissural axons by suppressing the axonal responsiveness to the midline repellent Slit, but the underlying mechanism remains unclear. By using a cell surface binding assay and immunoprecipitation, we observed that Robo3.1A did not bind Slit on its own but prevented the specific binding of Slit to the cell surface when it was coexpressed with its close homologue Robo1 or Robo2 (Robo1/2), which are known to mediate the Slit repulsion. Cotransfection with Robo3.1A significantly reduced the protein level of Robo2 in HEK293 cells, and overexpression of Robo3.1A also significantly decreased Robo2 protein level in cerebellar granule cells. Downregulation of endogenous Robo3 by specific small interference RNA (siRNA) significantly increased Robo1 protein level, Slit binding to the cell surface was significantly elevated, and Slit‐triggered growth cone collapse appeared after downregulation of Robo3 in cultured cortical neurons. Immunocytochemical staining showed that Robo2 and Robo3 colocalized in intracellular vesicles positive for the marker of late endosomes and lysosomes, but not trans‐Golgi apparatus and early endosomes. Thus Robo3.1A may prevent the Slit responsiveness by recruiting Robo1/2 into a late endosome‐ and lysosome‐dependent degradation pathway. © 2014 Wiley Periodicals, Inc.     

Slit2/Robo1 signaling in glioma migration and invasion

Slit2/Robo1 is a conserved ligand-receptor system, which greatly affects the distribution, migration, axon guidance and branching of neuron cells. Slit2 and its transmembrane receptor Robo1 have different distribution patterns in gliomas. The expression of Slit2 is at very low levels in pilocytic astrocytoma, fibrillary astrocytoma and glioblastoma, while Robo1 is highly expressed in different grades of gliomas at both mRNA and protein levels. Acquisition of insidious invasiveness by malignant glioma cells involves multiple genetic alterations in signaling pathways. Although the specific mechanisms of tumor-suppressive effect of Slit2/Robo1 have not been elucidated, it has been proved that Slit2/Robo1 signaling inhibits glioma cell migration and invasion by inactivation of Cdc42-GTP. With the research development on the molecular mechanisms of Slit2/Robo1 signaling in glioma invasion and migration, Slit2/Robo1 signaling may become a potential target for glioma prevention and treatment.     

Dynamic expression of Slit1–3 and Robo1–2 in the mouse peripheral nervous system after injury

The Slit family of axon guidance cues act as repulsive molecules for precise axon pathfinding and neuronal migration during nervous system development through interactions with specific Robo receptors.Although we previously reported that Slit1–3 and their receptors Robo1 and Robo2 are highly expressed in the adult mouse peripheral nervous system,how this expression changes after injury has not been well studied.Herein,we constructed a peripheral nerve injury mouse model by transecting the right sciatic nerve.At 14 days after injury,quantitative real-time polymerase chain reaction was used to detect mRNA expression of Slit1–3 and Robo1–2 in L4–5 spinal cord and dorsal root ganglia,as well as the sciatic nerve.Immunohistochemical analysis was performed to examine Slit1–3,Robo1–2,neurofilament heavy chain,F4/80,and vimentin in L4–5 spinal cord,L4 dorsal root ganglia,and the sciatic nerve.Co-expression of Slit1–3 and Robo1–2 in L4 dorsal root ganglia was detected by in situ hybridization.In addition,Slit1–3 and Robo1–2 protein expression in L4–5 spinal cord,L4 dorsal root ganglia,and sciatic nerve were detected by western blot assay.The results showed no significant changes of Slit1–3 or Robo1–2 mRNA expression in the spinal cord within 14 days after injury.In the dorsal root ganglion,Slit1–3 and Robo1–2 mRNA expression were initially downregulated within 4 days after injury;however,Robo1–2 mRNA expression returned to the control level,while Slit1–3 mRNA expression remained upregulated during regeneration from 4–14 days after injury.In the sciatic nerve,Slit1–3 and their receptors Robo1–2 were all expressed in the proximal nerve stump;however,Slit1,Slit2,and Robo2 were barely detectable in the nerve bridge and distal nerve stump within 14 days after injury.Slit3 was highly ex-pressed in macrophages surrounding the nerve bridge and slightly downregulated in the distal nerve stump within 14 days after injury.Robo1 was upregulated in vimentin-positive cells and migrating Schwann cells inside the nerve bridge.Robo1 was also upregulated in Schwann cells of the distal nerve stump within 14 days after injury.Our findings indicate that Slit3 is the major ligand expressed in the nerve bridge and distal nerve stump during peripheral nerve regeneration,and Slit3/Robo signaling could play a key role in peripheral nerve repair after injury.This study was approved by Plymouth University Animal Welfare Ethical Review Board (approval No.30/3203) on April 12,2014.     

Classic axon guidance molecules control correct nerve bridge tissue formation and precise axon regeneration

The peripheral nervous system has an astonishing ability to regenerate following a compression or crush injury;however,the potential for full repair following a transection injury is much less.Currently,the major clinical challenge for peripheral nerve repair come from long gaps between the proximal and distal nerve stumps,which prevent regenerating axons reaching the distal nerve.Precise axon targeting during nervous system development is controlled by families of axon guidance molecules including Netrins,Slits,Ephrins and Semaphorins.Several recent studies have indicated key roles of Netrin1,Slit3 and EphrinB2 signalling in controlling the formation of new nerve bridge tissue and precise axon regeneration after peripheral nerve transection injury.Inside the nerve bridge,nerve fibroblasts express EphrinB2 while migrating Schwann cells express the receptor EphB2.EphrinB2/EphB2 signalling between nerve fibroblasts and migrating Schwann cells is required for Sox2 upregulation in Schwann cells and the formation of Schwann cell cords within the nerve bridge to allow directional axon growth to the distal nerve stump.Macrophages in the outermost layer of the nerve bridge express Slit3 while migrating Schwann cells and regenerating axons express the receptor Robo1;within Schwann cells,Robo1 expression is also Sox2-dependent.Slit3/Robo1 signalling is required to keep migrating Schwann cells and regenerating axons inside the nerve bridge.In addition to the Slit3/Robo1 signalling system,migrating Schwann cells also express Netrin1 and regenerating axons express the DCC receptor.It appears that migrating Schwann cells could also use Netrin1 as a guidance cue to direct regenerating axons across the peripheral nerve gap.Engineered neural tissues have been suggested as promising alternatives for the repair of large peripheral nerve gaps.Therefore,understanding the function of classic axon guidance molecules in nerve bridge formation and their roles in axon regeneration could be highly beneficial in developing engineered neural tissue for more effective peripheral nerve repair.     

Schwann cell‐derived exosomes enhance axonal regeneration in the peripheral nervous system

Axonal regeneration in the peripheral nervous system is greatly supported by Schwann cells (SCs). After nerve injury, SCs dedifferentiate to a progenitor‐like state and efficiently guide axons to their original target tissues. Contact and soluble factors participate in the crosstalk between SCs and axons during axonal regeneration. Here we show that dedifferentiated SCs secrete nano‐vesicles known as exosomes which are specifically internalized by axons. Surprisingly, SC‐derived exosomes markedly increase axonal regeneration in vitro and enhance regeneration after sciatic nerve injury in vivo. Exosomes shift the growth cone morphology to a pro‐regenerating phenotype and decrease the activity of the GTPase RhoA, involved in growth cone collapse and axon retraction. Altogether, our work identifies a novel mechanism by which SCs communicate with neighboring axons during regenerative processes. We propose that SC exosomes represent an important mechanism by which these cells locally support axonal maintenance and regeneration after nerve damage. GLIA 2013;61:1795–1806     

Sphingosine‐1‐phosphate induces Ca2+ signaling and CXCL1 release via TRPC6 channel in astrocytes

A biologically active lipid, sphingosine‐1‐phosphate (S1P) is highly abundant in blood, and plays an important role in regulating the growth, survival, and migration of many cells. Binding of the endogenous ligand S1P results in activation of various signaling pathways via G protein‐coupled receptors, some of which generates Ca²⁺ mobilization. In astrocytes, S1P is reported to evoke Ca²⁺ signaling, proliferation, and migration; however, the precise mechanisms underlying such responses in astrocytes remain to be elucidated. Transient receptor potential canonical (TRPC) channels are Ca²⁺‐permeable cation channels expressed in astrocytes and involved in Ca²⁺ influx after receptor stimulation. In this study, we investigated the involvement of TRPC channels in S1P‐induced cellular responses. In Ca²⁺ imaging experiments, S1P at 1 μM elicited a transient increase in intracellular Ca²⁺ in astrocytes, followed by sustained elevation. The sustained Ca²⁺ response was markedly suppressed by S1P₂ receptor antagonist JTE013, S1P₃ receptor antagonist CAY10444, or non‐selective TRPC channel inhibitor Pyr2. Additionally, S1P increased chemokine CXCL1 mRNA expression and release, which were suppressed by TRPC inhibitor, inhibition of Ca²⁺ mobilization, MAPK pathway inhibitors, or knockdown of the TRPC channel isoform TRPC6. Taken together, these results demonstrate that S1P induces Ca²⁺ signaling in astrocytes via G_q‐coupled receptors S1P₂ and S1P₃, followed by Ca²⁺ influx through TRPC6 that could activate MAPK signaling, which leads to increased secretion of the proinflammatory or neuroprotective chemokine CXCL1.     

STIM1, STIM2, and Orai1 regulate store‐operated calcium entry and purinergic activation of microglia

Activation of microglia is the first and main immune response to brain injury. Release of the nucleotides ATP, ADP, and UDP from damaged cells regulate microglial migration and phagocytosis via purinergic P2Y receptors. We hypothesized that store‐operated Ca²⁺ entry (SOCE), the prevalent Ca²⁺ influx mechanism in non‐excitable cells, is a potent mediator of microglial responses to extracellular nucleotides. Expression analyses of STIM Ca²⁺ sensors and Orai Ca²⁺ channel subunits, that comprise the molecular machinery of SOCE, showed relevant levels of STIM1, STIM2, and Orai1 in cultured mouse microglia. STIM1 expression and SOCE were down‐regulated by treatment of microglia with lipopolysaccharide, suggesting that inflammation limits SOCE by lower STIM1 abundance. Ca²⁺ entry induced by cyclopiazonic acid, ATP, the P2Y₆ receptor agonist UDP, or the P2Y₁₂ receptor agonist 2‐methylthio‐ADP (2‐MeSADP) was clearly affected in microglia from Stim1^–/–, Stim2^–/–, and Orai1^–/– mice. SOCE blockers or ablation of STIM1, STIM2, or Orai1 severely impaired nucleotide‐induced migration and phagocytosis in microglia. Thus, this study assigns SOCE, regulated by STIM1, STIM2, and Orai1 an essential role in purinergic signaling and activation of microglia. GLIA 2015;63:652–663     

Functional ionotropic glutamate receptors on peripheral axons and myelin

Introduction: Neurotransmitter‐dependent signaling is traditionally restricted to axon terminals. However, receptors are present on myelinating glia, suggesting that chemical transmission may also occur along axons. Methods: Confocal microscopy and Ca²⁺‐imaging using an axonally expressed FRET‐based reporter was used to measure Ca²⁺ changes and morphological alterations in myelin in response to stimulation of glutamate receptors. Results: Activation of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) or N‐methyl‐D‐aspartate (NMDA) receptors induced a Ca²⁺ increase in axon cylinders. However, only the latter caused structural alterations in axons, despite similar Ca²⁺ increases. Myelin morphology was significantly altered by NMDA receptor activation, but not by AMPA receptors. Cu²⁺ ions influenced the NMDA receptor‐dependent response, suggesting that this metal modulates axonal receptors. Glutamate increased ribosomal signal in Schwann cell cytoplasm. Conclusions: Axon cylinders and myelin of peripheral nervous system axons respond to glutamate, with a consequence being an increase in Schwann cell ribosomes. This may have implications for nerve pathology and regeneration. Muscle Nerve 54 : 451–459, 2016     

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. GLIA 2015;63:1522–1536     

Erythropoietin promotes Schwann cell migration and assembly of the provisional extracellular matrix by recruiting β1 integrin to the cell surface

In peripheral nerve injury, Schwann cells undergo profound phenotypic modulation, adopting a migratory phenotype and remodeling the extracellular matrix so that it is permissive for axonal regrowth. Erythropoietin (Epo) and its receptor (EpoR) are expressed by Schwann cells after nerve injury, regulating inflammatory cytokine expression and minimizing the duration of neuropathic pain. The mechanism of Epo activity in the injured peripheral nerve remains incompletely understood. Herein, we demonstrate that Epo promotes Schwann cell migration in vitro on fibronectin (FN)‐coated surfaces. Epo also rapidly recruits β1 integrin subunit to the Schwann cell surface by a JAK‐2‐dependent pathway. Although β1 integrin subunit‐containing integrins were not principally responsible for Schwann cell adhesion or migration on FN under basal conditions, β1 gene‐silencing blocked the ability of Epo to promote cell migration. Epo also induced Schwann cell FN expression in vitro and in vivo. The FN was organized into insoluble fibrils by Epo‐treated Schwann cells in vitro and into an extensive matrix surrounding Schwann cells in vivo. Our results support a model in which Epo promotes Schwann cell migration and assembly of the provisional extracellular matrix in the injured peripheral nerve by its effects on integrin recruitment to the cell surface and local FN production. © 2009 Wiley‐Liss, Inc.     

The Neuregulin‐Rac‐MKK7 pathway regulates antagonistic c‐jun/Krox20 expression in Schwann cell dedifferentiation

Schwann cells respond to nerve injury by dedifferentiating into immature states and producing neurotrophic factors, two actions that facilitate successful regeneration of axons. Previous reports have implicated the Raf‐ERK cascade and the expression of c‐jun in these Schwann cell responses. Here we used cultured primary Schwann cells to demonstrate that active Rac1 GTPase (Rac) functions as a negative regulator of Schwann cell differentiation by upregulating c‐jun and downregulating Krox20 through the MKK7‐JNK pathway, but not through the Raf‐ERK pathway. The activation of MKK7 and induction of c‐jun in sciatic nerves after axotomy was blocked by Rac inhibition. Microarray experiments revealed that the expression of regeneration‐associated genes, such as glial cell line‐derived neurotrophic factor and p75 neurotrophin receptor, after nerve injury was dependent on Rac but not on ERK. Finally, the inhibition of ErbB2 signaling prevented MKK7 activation, c‐jun induction, and Rac‐dependent gene expression in sciatic nerve explant cultures. Taken together, our results indicate that the neuregulin‐Rac‐MKK7‐JNK/c‐jun pathway regulates Schwann cell dedifferentiation following nerve injury.     

β2 Adrenergic receptor activation induces microglial NADPH oxidase activation and dopaminergic neurotoxicity through an ERK‐dependent/protein kinase A‐independent pathway

Activation of the β2 adrenergic receptor (β2AR) on immune cells has been reported to possess anti‐inflammatory properties, however, the pro‐inflammatory properties of β2AR activation remain unclear. In this study, using rat primary mesencephalic neuron‐glia cultures, we report that salmeterol, a long‐acting β2AR agonist, selectively induces dopaminergic (DA) neurotoxicity through its ability to activate microglia. Salmeterol selectively increased the production of reactive oxygen species (ROS) by NADPH oxidase (PHOX), the major superoxide‐producing enzyme in microglia. A key role of PHOX in mediating salmeterol‐induced neurotoxicity was demonstrated by the inhibition of DA neurotoxicity in cultures pretreated with diphenylene‐iodonium (DPI), an inhibitor of PHOX activity. Mechanistic studies revealed the activation of microglia by salmeterol results in the selective phosphorylation of ERK, a signaling pathway required for the translocation of the PHOX cytosolic subunit p47^phox to the cell membrane. Furthermore, we found ERK inhibition, but not protein kinase A (PKA) inhibition, significantly abolished salmeterol‐induced superoxide production, p47^phox translocation, and its ability to mediate neurotoxicity. Together, these findings indicate that β2AR activation induces microglial PHOX activation and DA neurotoxicity through an ERK‐dependent/PKA‐independent pathway. © 2009 Wiley‐Liss, Inc.     

Intracellular Ca2+ release‐dependent inactivation of Ca2+ currents in thalamocortical relay neurons

Neuronal Ca²⁺ channels are rapidly inactivated by a mechanism that is termed Ca²⁺‐dependent inactivation (CDI). In this study we investigated the influence of intracellular Ca²⁺ release on CDI of high‐voltage‐activated Ca²⁺ channels in rat thalamocortical relay neurons by combining voltage‐clamp, Ca²⁺ imaging and immunological techniques. Double‐pulse protocols revealed CDI, which depended on the length of the conditioning pulses. Caffeine caused a concentration‐dependent increase in CDI that was accompanied by an increase in the duration of Ca²⁺ transients. Inhibition of ryanodine receptors and endoplasmic Ca²⁺ pumps (by thapsigargin or cyclopiazonic acid) resulted in a reduction of CDI. In contrast, inhibition of inositol 1,4,5‐tris‐phosphate receptors by intracellular application of 2‐aminoethoxy diphenyl borate or heparin did not influence CDI. The block of transient receptor potential channels by extracellular application of 2‐aminoethoxy diphenyl borate, however, resulted in a significant reduction of CDI. The central role of L‐type Ca²⁺ channels was emphasized by the near‐complete block of CDI by nifedipine, an effect only surpassed when Ca²⁺ was replaced by Ba²⁺ and chelated by 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′,‐tetraacetic acid (BAPTA). Trains of action potential‐like stimuli induced a strong reduction in high‐voltage‐activated Ca²⁺ current amplitude, which was significantly reduced when intracellular Ca²⁺ stores were made inoperative by thapsigargin or Ba²⁺/BAPTA. Western blotting revealed expression of L‐type Ca²⁺ channels in thalamic and hippocampal tissue but not liver tissue. In summary, these results suggest a cross‐signalling between L‐type Ca²⁺ channels and ryanodine receptors that controls the amount of Ca²⁺ influx during neuronal activity.     

Canonical transient receptor potential channel subtype 3‐mediated hair cell Ca2+ entry regulates sound transduction and auditory neurotransmission

The physiological significance of canonical transient receptor potential (TRPC) ion channels in sensory systems is rapidly emerging. Heterologous expression studies show that TRPC3 is a significant Ca²⁺ entry pathway, with dual activation via G protein‐coupled receptor (GPCR)–phospholipase C–diacylglycerol second messenger signaling, and through negative feedback, whereby a fall in cytosolic Ca²⁺ releases Ca²⁺–calmodulin channel block. We hypothesised that the latter process contributes to cochlear hair cell cytosolic Ca²⁺ homeostasis. Confocal microfluorimetry with the Ca²⁺ indicator Fluo‐4 acetoxymethylester showed that, when cytosolic Ca²⁺ was depleted, Ca²⁺ re‐entry was significantly impaired in mature TRPC3^?/? inner and outer hair cells. The impact of this disrupted Ca²⁺ homeostasis on sound transduction was assessed with the use of distortion product otoacoustic emissions (DPOAEs), which constitute a direct measure of the outer hair cell transduction that underlies hearing sensitivity and frequency selectivity. TRPC3^?/? mice showed significantly stronger DPOAE (2f₁ ? f₂) growth functions than wild‐type (WT) littermates within the frequency range of best hearing acuity. This translated to hyperacusis (decreased threshold) measured by the auditory brainstem response (ABR). TRPC3^?/? and WT mice did not differ in the levels of temporary and permanent threshold shift arising from noise exposure, indicating that potential GPCR signaling via TRPC3 is not pronounced. Overall, these data suggest that the Ca²⁺ set‐point in the hair cell, and hence membrane conductance, is modulated by TRPC3s through their function as a negative feedback‐regulated Ca²⁺ entry pathway. This TPRC3‐regulated Ca²⁺ homeostasis shapes the sound transduction input–output function and auditory neurotransmission.     

Plasmalemmal and mitochondrial Na+‐Ca2+ exchange in neuroglia

In the absence of the electrical signaling for which neurons are so highly specialized, GLIA rely on the slow propagation of ionic signals to mediate network events such as Ca²⁺ and Na⁺ waves. Glia differ from neurons in another important way, they are replete with a high density of ionic‐transport proteins that are essential for them to fulfil their basic functions as guardians of the intra and extra‐cellular milieux. Both the signaling and the homeostatic properties of glial cells are therefore particularly dependent upon the regulation of the two principle physiological metal cations, Ca²⁺ and Na⁺. For both ions, glia express high‐affinity/low capacity ATP‐fuelled pumps that can rapidly move small numbers of ions against an electro‐chemical gradient. For both Ca²⁺ and Na⁺ regulation, a single transporter family, the Na⁺‐Ca²⁺ exchanger (NCX), is used to maintain cellular ion homeostasis over the longer term and under conditions of prolonged or acute ionic dysregulation in astrocytes, oligodendroglia and microglia. Our understanding of glial NCX, both plasmalemmal and mitochondrial, is undergoing the kind of transformation that our understanding of glial cells, in general, has undergone in recent decades. These exchange proteins are becoming increasingly recognized for their essential roles in intracellular homeostasis while their signaling functions are starting to come to light. This review summarizes these key aspects and highlights the many areas where work has yet to begin in this rapidly evolving field. GLIA 2016;64:1646–1654     

Dopamine and full‐field illumination activate D1 and D2‐D5‐type receptors in adult rat retinal ganglion cells

Dopamine can regulate signal generation and transmission by activating multiple receptors and signaling cascades, especially in striatum, hippocampus, and cerebral cortex. Dopamine modulates an even larger variety of cellular properties in retina, yet has been reported to do so by only D1 receptor‐driven cyclic adenosine monophosphate (cAMP) increases or D2 receptor‐driven cAMP decreases. Here, we test the possibility that dopamine operates differently on retinal ganglion cells, because the ganglion cell layer binds D1 and D2 receptor ligands, and displays changes in signaling components other than cAMP under illumination that should release dopamine. In adult rat retinal ganglion cells, based on patch‐clamp recordings, Ca²⁺ imaging, and immunohistochemistry, we find that 1) spike firing is inhibited by dopamine and SKF 83959 (an agonist that does not activate homomeric D1 receptors or alter cAMP levels in other systems); 2) D1 and D2 receptor antagonists (SCH 23390, eticlopride, raclopride) counteract these effects; 3) these antagonists also block light‐induced rises in cAMP, light‐induced activation of Ca²⁺/calmodulin‐dependent protein kinase II, and dopamine‐induced Ca²⁺ influx; and 4) the Ca²⁺ rise is markedly reduced by removing extracellular Ca²⁺ and by an IP3 receptor antagonist (2‐APB). These results provide the first evidence that dopamine activates a receptor in adult mammalian retinal neurons that is distinct from classical D1 and D2 receptors, and that dopamine can activate mechanisms in addition to cAMP and cAMP‐dependent protein kinase to modulate retinal ganglion cell excitability. J. Comp. Neurol. 520:4032–4049, 2012. © 2012 Wiley Periodicals, Inc.