1 Block of the human two-pore domain potassium (2-PK) channel TREK-1 by fluoxetine (Prozac R ) and its active metabolite, norfluoxetine, was investigated using whole-cell patch-clamp recording of currents through recombinant channels in tsA 201 cells. 2 Fluoxetine produced a concentration-dependent inhibition of TREK-1 current that was reversible on wash. The IC 50 for block was 19 mM. Block by fluoxetine was voltage-independent. Fluoxetine (100 mM) produced an 84% inhibition of TREK-1 currents, but only a 31% block of currents through a related 2-PK channel, TASK-3. 3 Norfluoxetine was a more potent inhibitor of TREK-1 currents with an IC 50 of 9 mM. Block by norfluoxetine was also voltage-independent. 4 Truncation of the C-terminus of TREK-1 (D89) resulted in a loss of channel function, which could be restored by intracellular acidification or the mutation E306A. The mutation E306A alone increased basal TREK-1 current and resulted in a loss of the slow phase of TREK-1 activation. 5 Progressive deletion of the C-terminus of TREK-1 had no effect on the inhibition of the channel by fluoxetine. The E306A mutation, on the other hand, reduced the magnitude of fluoxetine inhibition, with 100 mM producing only a 40% inhibition. 6 It is concluded that fluoxetine and norfluoxetine are potent inhibitors of TREK-1. Block of TREK-1 by fluoxetine may have important consequences when the drug is used clinically in the treatment of depression.
When associated with the Nogo receptor (NgR), the transmembrane receptor p75NTR signals growth cone collapse. Arrest of CNS axon growth in vivo is mediated by CNS myelin-derived inhibitory ligands through either an unknown pathway after NgR- and Ca2+-dependent activation of the epidermal growth factor receptor (EGFR), and/or sequential Rho-A/ROCK/LIM-kinase/cofilin phosphorylation leading to actin depolymerization. Paradoxically, rat retinal ganglion cell (RGC) axons regenerate through the CNS myelin-rich transected optic nerve after intravitreal sciatic nerve grafting without inhibitory ligand neutralization. Here, we show that optic nerve regeneration in vivo correlates with Schwann cell-derived factor-induced cleavage of NgR and Nogo-A, and inactivation of p75NTR signalling by the induction of regulated intramembranous proteolysis (RIP) and the release of both extracellular (p75ECD) and intracellular (p75ICD) domains. Hence, Schwann cell-derived factors compromise inhibitory signalling by (i) antagonizing ligand/NgR binding with metalloproteinase-cleaved Nogo-A peptides; (ii) RIP of p75NTR; (iii) competitively blocking NgR/p75NTR clustering with soluble p75ECD; and (iv) consequent reduced downstream EGFR phosphorylation and suppression of Rho-A activation. Moreover, in RGC cultures, exogenous tumour necrosis- converting enzyme (TACE) initiates RIP of p75NTR, reduces EGFR phosphorylation, suppresses activation of Rho-A, cleaves the ECD from both NgR and TROY, and disinhibits neurotrophic factor (NTF) stimulated RGC neurite outgrowth in the presence of CNS myelin. Soluble NgRECD binds all CNS myelin-derived ligands and thus has the potential to act as an inhibitory signalling antagonist, but the role of TROY and its shed ectodomain in growth cone mobility is unknown. siRNA knockdown of p75NTR also inactivates Rho-A and disinhibits NTF-stimulated RGC neurite outgrowth in cultures with added CNS myelin. In all the above experimental paradigms, Schwann cell-derived factor/NTF-induced attenuation of NgR/p75NTR signalling suppresses EGFR activation, thereby potentiating axon growth disinhibition.
Xenon neuroprotection against traumatic brain injury can be reversed by increasing the glycine concentration, consistent with inhibition at the N-methyl-D-aspartate receptor glycine site playing a significant role in xenon neuroprotection. Argon and xenon do not act via the same mechanism.
Tissue engineering using a combination of biomaterials and cells represents a new approach to nerve repair. We have investigated the effect that extracellular matrix (ECM) molecules have on Schwann cell (SC) attachment and proliferation on the nerve conduit material poly-3-hydroxybutyrate (PHB), and SC influence on neurite outgrowth in vitro. Initial SC attachment to PHB mats was unaffected by ECM molecules but proliferation increased (laminin > fibronectin > collagen). SCs seeded onto ECM-coated culture inserts suspended above a monolayer of NG108-15 cells determined the effect of released diffusible factors. The effect of direct contact between the two cell types on ECM molecules was also investigated. In both systems SCs enhanced neurite number per cell and percentage of NG108-15 cells sprouting neurites. NG108-15 cells grown in direct contact with SCs had significantly longer neurites than those exposed to diffusible factors when seeded on laminin or fibronectin. Diffusible factors released from SCs cultured on ECM molecules appear to initiate neurite outgrowth, whereas SC-neuron contact promotes neurite elongation. SC proliferation was maximal on poly-D-lysine-coated surfaces, but these cells did not influence neurite outgrowth to the levels of laminin or fibronectin. This suggests that ECM molecules enhance cell number and activate SCs to release neurite promoting factors. Addition of ECM molecules to PHB nerve conduits containing SCs is likely to provide benefits for the treatment of nerve injuries.
Objectives-To determine the neuroprotective efficacy of the inert gas xenon following traumatic brain injury, and to determine whether application of xenon has a clinically relevant therapeutic time window.Design-Controlled animal study. Setting-University research laboratory. Subjects-Male C57BL/6N mice (n=196)Interventions-75% xenon, 50% xenon or 30% xenon, with 25% oxygen (balance nitrogen) treatment following mechanical brain lesion by controlled cortical impact.Measurements & Main Results-Outcome following trauma was measured using: 1) functional neurological outcome score, 2) histological measurement of contusion volume, 3) analysis of locomotor function and gait. Our study shows that xenon-treatment improves outcome following traumatic brain injury. Neurological outcome scores were significantly (p<0.05) better
Background: Xenon is a general anesthetic with neuroprotective properties. Xenon inhibition at the glycine-binding site of the N-Methyl-D-aspartate (NMDA) receptor mediates xenon neuroprotection against ischemic injury in vitro. Here we identify specific amino acids important for xenon binding to the NMDA receptor, with the aim of finding silent mutations that eliminate xenon binding but leave normal receptor function intact. Methods: Site-directed mutagenesis was used to mutate specific amino-acids in the GluN1 subunit of rat NMDA receptors. Mutant GluN1/GluN2A receptors were expressed in HEK 293 cells and were assessed functionally using patchclamp electrophysiology. The responses of the mutant receptors to glycine and anesthetics were determined.Results: Mutation of phenylalanine 758 to an aromatic tryptophan or tyrosine left glycine affinity unchanged, but eliminated xenon binding without affecting the binding of sevoflurane or isoflurane. Conclusions: These findings confirm xenon binds to the glycine site of the GluN1 subunit of the NMDA receptor and indicate that interactions between xenon and the aromatic ring of the phenylalanine 758 residue are important for xenon binding. Our most important finding is that we have identified two mutations, F758W and F758Y, that eliminate xenon binding to the NMDA receptor glycine site without changing the glycine affinity of the receptor or the binding of volatile anesthetics. The identification of these selective mutations will allow knock-in animals to be used to dissect the mechanism(s) of xenon's neuroprotective and anesthetic properties in vivo.
In an attempt to obviate the drawbacks of nerve autograft, ultrathin microporous biodegradable PCL and PCL/PLA films were tested for their compatibility with motor neuron-like NG108-15 cells and primary Schwann cells. Data obtained from MTS colorimetric and DNA fluorimetric assays showed that both cell lines readily attached and proliferated on these materials. Images taken using scanning electron microscope and fluorescence microscope confirmed these observations. Enhanced cell-surface interaction was achieved by pretreating the films in NaOH solution. Importantly, NG108-15 cells could be induced into differentiated phenotype with long, un-branched neurites growing across the surface of the materials. The bipolar spindle-shaped phenotype of Schwann cells was also retained on these scaffolds. Positive immunochemical staining using antibodies against neurofilament for NG108-15 cells and S100 for Schwann cells indicated the expression of these marker proteins. In a small-scaled pilot testing, the performance of PCL conduits in bridging up a 10 mm gap in rat sciatic nerve model was assessed. Immunohistochemical staining showed that regenerated nerve tissue and penetrated Schwann cells have the potential to span the whole length of the conduit in 2 weeks.
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