Neurite Inhibition Research Articles

Growth-inhibiting extracellular matrix proteins also inhibit electrical activity by reducing calcium and increasing potassium conductances

Inhibitionof neurite sprouting and electrical activity by extracellular matrix (ECM) glycoproteins was studied during neurite regeneration by using anterior pagoda (AP) neurons of the leech. Adult isolated neurons were plated in culture inside ganglion capsules, which among many ECM proteins, contain a group of inhibitory peanut lectin- (PNA) binding glycoproteins. These proteins inhibit neurite production and contribute to the formation of a bipolar outgrowth pattern by AP neurons. Addition of PNA lectin to the culture medium to block the inhibitory effects of ECM glycoproteins induced an increase of neurite sprouting, the loss of the bipolar pattern, and also an increase in the amplitude and duration of action potentials evoked by intracellular current injection. PNA lectin had independent effects on neurite sprouting and electrical activity, since there was no correlation between the total neurite length and the amplitude of the action potentials. Moreover, action potentials were increased by the presence of PNA lectin even in neurons that did not grow. The changes induced by PNA lectin on the active conductances underlying the action potentials were estimated by quantitative model simulations. We predict that the increases in the amplitude and duration of the action potential induced by PNA lectin were due to an increase in a calcium conductance and a reduction in the delayed rectifier potassium conductance. Our results suggest that inhibitory ECM glycoproteins may use independent signaling pathways to inhibit neurite sprouting and electrical activity. These proteins affect the action potential by changing the proportion of inward and outward active conductances.

The Role of Kalirin9 in p75/Nogo Receptor-mediated RhoA Activation in Cerebellar Granule Neurons

p75 and the Nogo receptor form a signaling unit for myelin inhibitory molecules, with p75 being responsible for RhoA activation. Because p75 lacks the GDP/GTP exchange factor domain, it has remained unclear how p75 activates RhoA. Here, we report that Kalirin9, a dual RhoGEF, binds p75 directly and regulates p75-Nogo receptor-dependent RhoA activation and neurite inhibition in response to myelin-associated glycoprotein. The region of p75 that Kalirin9 binds includes its mastoparan-like fifth helix, which was shown to recruit RhoGDI-RhoA. As predicted from the presence of a shared binding site, we found that Kalirin9 competes with RhoGDI for p75 binding in a dose-dependent manner in vitro. In line with these data, myelin-associated glycoprotein addition to cerebellar granule neurons resulted in a reduction in the association of Kalirin9 with p75, and a simultaneous increase in the binding of RhoGDI to p75. These results reveal a mechanism by which the fifth helix of p75 regulates RhoA activation.

Aggrecan components differentially modulate nerve growth factor–responsive and neurotrophin‐3‐responsive dorsal root ganglion neurite growth

Aggrecan is one of the major chondroitin sulfate proteoglycans (CSPGs) expressed in the central nervous system. The signaling pathways activated downstream of cell interaction with aggrecan and with CSPGs in general and the importance of chondroitin sulfate-glycosaminoglycan side chains in their inhibition are unclear. Therefore, to analyze the effect of different components of aggrecan in inhibiting neurite growth, neurite outgrowth was quantified in an in vitro model in which chick dorsal root ganglion (DRG) explants were grown on substrates containing aggrecan bound to hyaluronan and link protein as a macromolecular aggregate, aggrecan monomers, hyaluronan, or ChABC-treated aggrecan. Aggrecan aggregate, aggrecan monomer, and hyaluronan inhibited neurite outgrowth from nerve growth factor (NGF)- and neurotrophin-3 (NT3)-responsive DRG neurons. Aggrecan inhibition was dependent on its chondroitin sulfate-glycosaminoglycans, as ChABC digestion alleviated neurite inhibition because of aggrecan. Growth cones displayed full or partial collapse on aggrecan aggregate, hyaluronan, and ChABC-treated aggrecan. Inhibition of Rho kinase (ROCK) with Y27632 increased neurite growth on some but not all of the aggrecan components tested. With NGF in the culture medium, Y27632 increased neurite outgrowth on aggrecan aggregate, monomers, and ChABC-treated aggrecan, but not on hyaluronan. The ROCK inhibitor also increased NT3-responsive outgrowth on aggrecan aggregate and hyaluronan, but not on ChABC-treated aggrecan. This study showed that the matrix proteoglycan aggrecan and its components have multiple effects on neurite outgrowth and that some of these effects involve the Rho/ROCK pathway.

Phenoxodiol protects against Cisplatin induced neurite toxicity in a PC-12 cell model

BackgroundMany commonly used chemotherapeutic agents, such as Cisplatin, are restricted in their potential anti-neoplastic effectiveness by their side effects, with one of the most problematic being induction of peripheral neuropathy. Although a number of different neurotrophic, neuroprotective or anti-oxidant treatments have been tried in order to prevent or treat the neuropathies, to date they have met with limited success. Phenoxodiol is a new chemotherapeutic agent that has anti-proliferative and apoptotic effects on a range of cancer cells. PC12 cells are a commonly used neuronal cell model for examination of neurite outgrowth. In this study we examined whether phenoxodiol could protect against Cisplatin induced neurite inhibition in PC12 cells as an indication of the potential to protect against neuropathy.ResultsUsing the PC12 neuronal cell line, concentrations of Cisplatin were chosen that induced moderate or strong neurite toxicity within 24 hrs but were not cytotoxic. The effect of Phenoxodiol on Cisplatin induced neurite toxicity was assessed by measurement of neurite outgrowth. Addition of phenoxodiol at 100 nM or 1 μM showed no cytotoxicity and blocked the Cisplatin induced neurite toxicity, while phenoxodiol at 10 μM was cytotoxic and enhanced neurite toxicity of Cisplatin. When Cisplatin was added for 24 hrs, then washed out and the cells allowed to recover for 48 hrs, neurite outgrowth was not restored and addition of phenoxodiol did not further promote recovery or restore the Cisplatin treated cells.ConclusionIn addition to its potential as a chemotherapeutic agent Phenoxodiol may thus also have the potential to be used in conjunction with Cisplatin chemotherapy to prevent induction of neuropathy.

NOGO-A induction and localization during chick brain development indicate a role disparate from neurite outgrowth inhibition

BackgroundNogo-A, a myelin-associated protein, inhibits neurite outgrowth and abates regeneration in the adult vertebrate central nervous system (CNS) and may play a role in maintaining neural pathways once established. However, the presence of Nogo-A during early CNS development is counterintuitive and hints at an additional role for Nogo-A beyond neurite inhibition.ResultsWe isolated chicken NOGO-A and determined its sequence. A multiple alignment of the amino acid sequence across divergent species, identified five previously undescribed, Nogo-A specific conserved regions that may be relevant for development. NOGO gene transcripts (NOGO-A, NOGO-B and NOGO-C) were differentially expressed in the CNS during development and a second NOGO-A splice variant was identified. We further localized NOGO-A expression during key phases of CNS development by in situ hybridization. CNS-associated NOGO-A was induced coincident with neural plate formation and up-regulated by FGF in the transformation of non-neural ectoderm into neural precursors. NOGO-A expression was diffuse in the neuroectoderm during the early proliferative phase of development, and migration, but localized to large projection neurons of the optic tectum and tectal-associated nuclei during architectural differentiation, lamination and network establishment.ConclusionThese data suggest Nogo-A plays a functional role in the determination of neural identity and/or differentiation and also appears to play a later role in the networking of large projection neurons during neurite formation and synaptogenesis. These data indicate that Nogo-A is a multifunctional protein with additional roles during CNS development that are disparate from its later role of neurite outgrowth inhibition in the adult CNS.

Synergistic Interactions between Commonly Used Food Additives in a Developmental Neurotoxicity Test

Exposure to non-nutritional food additives during the critical development window has been implicated in the induction and severity of behavioral disorders such as attention deficit hyperactivity disorder (ADHD). Although the use of single food additives at their regulated concentrations is believed to be relatively safe in terms of neuronal development, their combined effects remain unclear. We therefore examined the neurotoxic effects of four common food additives in combinations of two (Brilliant Blue and L-glutamic acid, Quinoline Yellow and aspartame) to assess potential interactions. Mouse NB2a neuroblastoma cells were induced to differentiate and grow neurites in the presence of additives. After 24 h, cells were fixed and stained and neurite length measured by light microscopy with computerized image analysis. Neurotoxicity was measured as an inhibition of neurite outgrowth. Two independent models were used to analyze combination effects: effect additivity and dose additivity. Significant synergy was observed between combinations of Brilliant Blue with L-glutamic acid, and Quinoline Yellow with aspartame, in both models. Involvement of N-methyl-D-aspartate (NMDA) receptors in food additive-induced neurite inhibition was assessed with a NMDA antagonist, CNS-1102. L-glutamic acid- and aspartame-induced neurotoxicity was reduced in the presence of CNS-1102; however, the antagonist did not prevent food color-induced neurotoxicity. Theoretical exposure to additives was calculated based on analysis of content in foodstuff, and estimated percentage absorption from the gut. Inhibition of neurite outgrowth was found at concentrations of additives theoretically achievable in plasma by ingestion of a typical snack and drink. In addition, Trypan Blue dye exclusion was used to evaluate the cellular toxicity of food additives on cell viability of NB2a cells; both combinations had a straightforward additive effect on cytotoxicity. These data have implications for the cellular effects of common chemical entities ingested individually and in combination.

From Axon Guidance to Neurite Inhibitor to Viral Receptor

Two groups report activities for ephrins beyond axon guidance and cell migration. Bonaparte et al . performed a microarray screen comparing cells that were permissive and nonpermissive for Nipah virus (NiV)- or Hendra virus (HeV)-mediated cell fusion (a surrogate measure of infection) and identified ephrin B2 (EFNB2), whose mRNA was abundant in fusogenic cells. When expressed in nonfusogenic cells, EFNB2 rendered those cells competent for HeV-mediated fusion. A soluble Fc fusion protein (EFNB2/Fc) blocked HeV- and NiV-stimulated cell fusion of EFNB2-transfected cells, and this fusion protein coimmunoprecipitated and interacted with soluble forms of the attachment glycoprotein (sG) from the two viruses in surface plasmon resonance assays. Viral infection was also assayed; EFNB2/Fc prevented HeV and NiV infection in permissive cells, and EFNB2 was sufficient to allow infection by HeV and NiV when transfected into nonpermissive cells. Benson et al . report that ephrin B3 is expressed in postnatal myelinating oligodendrocytes of the central nervous system and is a constituent of purified myelin. Neurite outgrowth of postnatal cortical neurons, which express EphA4 (the receptor for ephrin B3), was inhibited when the cells were cultured in the presence of soluble ephrin B3/Fc fusion protein or a soluble fusion of the known myelin-based outgrowth inhibitor, myelin-associated glycoprotein (MAG). Indeed, the ephrin B3/Fc was more potent than MAG/Fc. Cortical neurons plated on myelin deficient for ephrin B3 showed more outgrowth compared with that of neurons whose growth was inhibited by plating on wild-type myelin. Cortical neurons deficient in p75, the receptor for several myelin-based inhibitors of neurite outgrowth, including MAG, also exhibited decreased neurite growth inhibition. However, neurite extension was restored to that of cells cultured in the absence of myelin when p75 –/– neurons were plated on ephrin B3-deficient myelin. (Thus, the effects of ephrin B3 and those of the other myelin-based inhibitors appear to be additive). Inhibition of neurite outgrowth by myelin was alleviated in both cortical neurons and cerebellar granule neurons when these cells were cultured in the presence of soluble fusion proteins for EphA4 or p75. These results suggest that ephrin-Eph signaling may be another mediator of myelin-based neurite inhibition, which has implications for treatment of spinal cord injury. M. I. Bonaparte, A. S. Dimitrov, K. N. Bossart, G. Crameri, B. A. Mungall, K. A. Bishop, V. Choudhry, D. S. Dimitrov, L.-F. Wang, B. T. Eaton, C. C. Broder, Ephrin-B2 ligand is a functional receptor for Hendra virus and Nipah virus. Proc. Natl. Acad. Sci. U.S.A. 102 , 10652-10657 (2005). [Abstract] [Full Text] M. D. Benson, M. I. Romero, M. E. Lush, Q. R. Lu, M. Henkemeyer, L. R. Parada, Ephrin-B3 is a myelin-based inhibitor of neurite outgrowth. Proc. Natl. Acad. Sci. U.S.A. 102 , 10694-10699 (2005). [Abstract] [Full Text]

MAG Induces Regulated Intramembrane Proteolysis of the p75 Neurotrophin Receptor to Inhibit Neurite Outgrowth

The three known inhibitors of axonal regeneration present in myelin--MAG, Nogo, and OMgp--all interact with the same receptor complex to effect inhibition via protein kinase C (PKC)-dependent activation of the small GTPase Rho. The transducing component of this receptor complex is the p75 neurotrophin receptor. Here we show that MAG binding to cerebellar neurons induces alpha- and then gamma-secretase proteolytic cleavage of p75, in a protein kinase C-dependent manner, and that this cleavage is necessary for both activation of Rho and inhibition of neurite outgrowth.

Neuroprotection and Regeneration Strategies for Spinal Cord Repair

The journey toward a cure for spinal cord injury (SCI) has taken many paths. In this article, we review these paths, and highlight the clinical applications of these experimental repair strategies. Initial strategies involved attempts at neuroprotection with steroids and other anti-inflammatory drugs. Other anti-ischemia treatments, agents to eliminate the damage from excitotoxicity, and anti-apoptotic agents were also tried. Another avenue involved enhancing the function of the remaining uninjured axons by measures to produce remyelination and medications to improve axonal conduction. In the last two decades there has been a major effort to enhance spinal cord axonal regeneration through a variety of techniques including neutralization of neurite inhibition, administration of neurotrophic factors, implantation of synthetic channels, and transplantation of a variety of cell types. Indeed, several of these strategies have been so promising in animals that clinicians have been stimulated to explore their potential human application. We also examine the different experimental models of SCI used to assess repair, and discuss how the injury model impacts on the assessment of axonal regeneration and functional recovery after SCI. The mechanisms of recovery that may be involved after SCI will be analyzed, and their relevance toward finding a cure for human SCI. Unfortunately, the goal of producing significant functional regeneration of the human spinal cord has not yet been achieved despite the many strategies that have been developed. It is our hope that improved understanding of the mechanisms underlying functional recovery will lead to successful therapeutic strategies in humans.

Genetic deletion of the Nogo receptor does not reduce neurite inhibition in vitro or promote corticospinal tract regeneration in vivo

Axon regeneration failure in the adult mammalian CNS is attributed in part to the inhibitory nature of CNS myelin. Three myelin-associated, structurally distinct proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, have been implicated in this inhibition. Neuronal Nogo receptor (NgR) binds to each of the three inhibitors and has been proposed to mediate their inhibitory signals by complexing with a signal-transducing coreceptor, the neurotrophin receptor p75(NTR). To assess the contribution of NgR to mediating myelin inhibitory signals and regeneration failure in vivo, we generated and characterized NgR-deficient mice. Nogo transcripts are up-regulated in NgR mutants, indicating that NgR regulates Nogo in vivo. However, neurite outgrowth from NgR-deficient postnatal dorsal root ganglion or cerebellar granule neurons is inhibited by myelin and by a Nogo-66 substrate to the same extent as is from wild-type neurons, whereas p75(NTR)-deficient neurons are less inhibited. The NgR ligand-binding domain promotes neurite outgrowth on Nogo-66, regardless of the genotype of the neurons, indicating that the NgR ligand-binding domain can act independent of NgR. Thus, NgR is not essential for mediating inhibitory signals from CNS myelin, at least in the neurons tested, whereas p75(NTR) plays a central role in this response. Neither NgR-nor p75(NTR)-deficient mice showed enhanced regeneration of corticospinal tract axons in comparison with wild-type controls after spinal dorsal hemisection. Our results thus fail to support a central role for NgR in axonal growth inhibition in vitro or in corticospinal tract regeneration block in vivo.

Molecular analysis of Nogo expression in the hippocampus during development and following lesion and seizure.

The Nogo gene encodes an integral membrane protein mainly responsible for the neurite inhibition properties of myelin. Here, we analyzed the expression pattern of Nogo-A, Nogo-B, and Nogo-C and Nogo-66 receptor (Ng66R) mRNA during hippocampal development and lesion-induced axonal sprouting. Nogo-A and Nogo-B and Ng66R transcripts preceded the progress of myelination and were highly expressed at postnatal day zero (P0) in all principal hippocampal cell layers, with the exception of dentate granule cells. Only a slight Nogo-C expression was found at P0 in the principal cell layers of the hippocampus. During adulthood, all Nogo splice variants and their receptor were expressed in the neuronal cell layers of the hippocampus, in contrast to the myelin basic protein mRNA expression pattern, which revealed a neuronal source of Nogo gene expression in addition to oligodendrocytes. After hippocampal denervation, the Nogo genes showed an isoform-specific temporal regulation. All Nogo genes were strongly regulated in the hippocampal cell layers, whereas the Ng66R transcripts showed a significant increase in the contralateral cortex. These data could be confirmed on protein levels. Furthermore, Nogo-A expression was up-regulated after kainate-induced seizures. Our data show that neurons express Nogo genes with a clearly distinguishable pattern during development. This expression is further dynamically and isoform-specifically altered after lesioning during the early phase of structural rearrengements. Thus, our results indicate a role for Nogo-A, -B, and -C during development and during the stabilization phase of hippocampal reorganization. Taken together with these data, the finding that neurons in a highly plastic brain region express Nogo genes supports the hypothesis that Nogo may function beyond its known neuronal growth inhibition activity in shaping neuronal circuits.

Metallothionein-III Inhibits Initial Neurite Formation in Developing Neurons as Well as Postinjury, Regenerative Neurite Sprouting

Human metallothionein-III (MT-III) is an inhibitory factor deficient in the Alzheimer's disease (AD) brain. MT-III has been identified as an inhibitor of neurite sprouting, and its deficiency has been proposed to be involved in the formation of neurofibrillary tangles (NFT) in the neuropathology of AD. However, there has been limited investigation of the proposed neurite growth inhibitory properties of MT-III. We have applied recombinant human MT-III to both single cell embryonic cortical neurons (to investigate initial neurite formation), as well as mature (21 days postplating) clusters of cortical neurons (to investigate the regenerative sprouting response following injury). We report that MT-III inhibited the initial formation of neurites by rat embryonic (E18) cortical neurons. This was based on both the percentage of neurite positive neurons and the number of neurites per neuron (45 and 30% inhibition, respectively). Neurite inhibition was only observed in the presence of adult rat brain extract, and was also reversible following replacement of MT-III-containing medium. MT-III inhibited the formation and growth of both axons and dendrites. Of more physiological significance, MT-III also inhibited the regenerative neurite sprouting response following axonal transection. The morphology of sprouting neurites was also altered, with the distal tip often ending in bulb-like structures. Based on these results, we propose that MT-III, in the presence of brain extract, is a potent inhibitor of neurite sprouting, and may be involved in abnormal sprouting potentially underlying both AD and epilepsy.

Axon Guidance in the Mouse Optic Chiasm: Retinal Neurite Inhibition by Ephrin “A”-Expressing Hypothalamic Cells in Vitro

In the mammalian visual system, retinal axons undergo temporal and spatial rearrangements as they project bilaterally to targets on the brain. Retinal axons cross the neuraxis to form the optic chiasm on the hypothalamus in a position defined by overlapping domains of regulatory gene expression. However, the downstream molecules that direct these processes remain largely unknown. Here we use a novel in vitro paradigm to study possible roles of the Eph family of receptor tyrosine kinases in chiasm formation. In vivo, Eph receptors and their ligands distribute in complex patterns in the retina and hypothalamus. In vitro, retinal axons are inhibited by reaggregates of isolated hypothalamic, but not dorsal diencephalic or cerebellar cells. Furthermore, temporal retinal neurites are more inhibited than nasal neurites by hypothalamic cells. Addition of soluble EphA5-Fc to block Eph “A” subclass interactions decreases both the inhibition and the differential response of retinal neurites by hypothalamic reaggregates. These data show that isolated hypothalamic cells elicit specific, position-dependent inhibitory responses from retinal neurites in culture. Moreover, these responses are mediated, in part, by Eph interactions. Together with the in vivo distributions, these data suggest possible roles for Eph family members in directing retinal axon growth and/or reorganization during optic chiasm formation.

Myelin-associated glycoprotein interacts with neurons via a sialic acid binding site at ARG118 and a distinct neurite inhibition site.

Inhibitory components in myelin are largely responsible for the lack of regeneration in the mammalian CNS. Myelin-associated glycoprotein (MAG), a sialic acid binding protein and a component of myelin, is a potent inhibitor of neurite outgrowth from a variety of neurons both in vitro and in vivo. Here, we show that MAG's sialic acid binding site is distinct from its neurite inhibitory activity. Alone, sialic acid-dependent binding of MAG to neurons is insufficient to effect inhibition of axonal growth. Thus, while soluble MAG-Fc (MAG extracellular domain fused to Fc), a truncated form of MAG-Fc missing Ig-domains 4 and 5, MAG(d1-3)-Fc, and another sialic acid binding protein, sialoadhesin, each bind to neurons in a sialic acid- dependent manner, only full-length MAG-Fc inhibits neurite outgrowth. These results suggest that a second site must exist on MAG which elicits this response. Consistent with this model, mutation of arginine 118 (R118) in MAG to either alanine or aspartate abolishes its sialic acid-dependent binding. However, when expressed at the surface of either CHO or Schwann cells, R118-mutated MAG retains the ability to inhibit axonal outgrowth. Hence, MAG has two recognition sites for neurons, the sialic acid binding site at R118 and a distinct inhibition site which is absent from the first three Ig domains.

Why 103 women asked for reversal of sterilisation.

Between 1975 and 1976 103 women requesting sterilisation reversal were interviewed at Hammersmith Hospital. Their average age at sterilisation was 26-7 years; 65 (63-1%) had been sterilised immediately after pregnancy, and many patients had previously used contraception inadequately when they were sterilised. When they had been sterilised 78 (75-7%) patients were unhappily married and remarriage was the chief reason for the request for reversal. Sexual dissatisfaction after sterilisation was common, but there was no obvious increase in menstrual disturbance. Thirty-nine (37-8%) patients had been sterilised by irreversible methods, and in only half the cases sterilised by tubal ligation were conditions technically suitable for reversal surgery. It therefore seems unwise to sterilise women under 30 particularly immediately after pregnancy or if their marriage is in jeopardy.