Neuronal age influences the response to neurite outgrowth inhibitory activity in the central and peripheral nervous systems.

Axonal regeneration is abortive in the central nervous system (CNS) of adult mammals, but readily occurs in the injured peripheral nervous system (PNS). Recent experiments indicate an important role for both intrinsic neuronal features and extrinsic substrate properties in determining the propensity for axonal regrowth. In particular, certain components of adult mammalian CNS myelin have been shown to exert a strong inhibitory influence on neurite outgrowth. To determine whether the potent neurite outgrowth inhibitory activity found in CNS myelin may also be present in PNS myelin and to study the influence of neuronal age on neurite outgrowth, we used a cryoculture assay in which dissociated rat dorsal root ganglion (DRG) neurons of different ages were challenged to extend neurites on fractionated myelin and cryostat sections from the PNS (sciatic nerve and myelin-free degenerated sciatic nerve) and CNS (optic nerve) of adult rats. The CNS environment of the optic nerve did not support E17 to P8 DRG neurite adhesion or outgrowth. E17 DRG neurons, unlike their older counterparts, however, were able to attach and extend neurites onto normal sciatic nerve and onto purified PNS myelin. In contrast, a vigorous neurite outgrowth response from all the ages tested was observed on the myelin-free degenerated sciatic nerve. These results indicate that PNS myelin is a potent inhibitor of neurite outgrowth and that DRG neuronal age plays an important role in determining the propensity for neurite outgrowth and regenerative response on inhibitory PNS and CNS substrata.     

L1 neural cell adhesion molecule is a survival factor for fetal dopaminergic neurons

Cell adhesion molecules play a central role in neural development and are also critically involved in axonal regeneration and synaptic plasticity in the adult nervous system. We investigated whether the neural cell adhesion molecule L1 was capable of stimulating survival and differentiation in the mid-brain dopaminergic neurons which degenerate in Parkinson's disease. Monoclonal L1 antibodies, known to enhance neurite outgrowth, were substrate-coated or added at the time of plating to medium of cultures containing mid-brain dopaminergic neurons from 14-day-old fetal rats. Tritiated dopamine uptake per well and the number of tyrosine hydroxylase-immunopositive neurons increased in a dose-dependent manner with increasing concentrations of L1 antibody, suggesting that L1 acts directly or indirectly as a growth factor for dopaminergic neurons. A monoclonal L1 antibody not enhancing neurite outgrowth was ineffective. The growth-promoting effects of L1 antibodies on dopaminergic neurons in culture did not appear to be mediated by the cAMP-activated protein kinase A pathway, since combined treatment with a phosphodiesterase inhibitor had only additive effects on the L1-induced increase of dopamine uptake, and in addition, antibodies against L1 failed to protect cultures of dopaminergic neurons against the neurotoxin MPP⁺, whereas pretreatment with forskolin and phosphodiesterase type-IV inhibitors was strongly protective. J. Neurosci. Res. 53:129–134, 1998. © 1998 Wiley-Liss, Inc.     

Neurite Outgrowth on Non-permissive Substrates In Vitro is Enhanced by Ectopic Expression of the Neural Adhesion Molecule L1 by Mouse Astrocytes

Axonal regrowth in the lesioned central nervous system (CNS) of adult mammals is, in part, prevented by non-permissive properties of glial cells and myelin. To test if ectopic expression of the neurite outgrowth promoting recognition molecule L1 will overcome these non-permissive influences and promote neurite outgrowth, L1 was expressed in astrocytes of transgenic mice using regulatory sequences of the glial fibrillary acidic protein (GFAP) gene. Northern blot analysis of different transgenic lines revealed different levels of transgenically expressed L1. Cultured astrocytes derived from transgenic animals displayed L1 immunoreactivity at the cell surface and in situ hybridization and immunocytochemical analysis of optic nerves from adult transgenic mice localized L1 expression to astrocytes. Expression of L1 protein by transgenic astrocytes was significantly upregulated in lesioned optic nerves. When mouse small cerebellar neurons or chick dorsal root ganglion neurons were cultured on cryosections of lesioned optic nerves or astrocyte monolayers from transgenic mice, respectively, neurite outgrowth was increased up to 400% on tissue sections and 50% on astrocytes compared with similar preparations from non-transgenic mice. The increase in neurite outgrowth on tissue sections or astrocyte monolayers from different transgenic lines was proportional to the different levels of L1 expression. Moreover, increased neurite outgrowth on these substrates was specifically inhibited by polyclonal L1 antibodies. In vivo , rescue of severed axons was enhanced in transgenic versus wild type animals, while regrowth of axons was slightly, but not significantly, increased. Together, our observations demonstrate that L1 promotes neurite outgrowth when expressed ectopically by astrocytes and that L1 is able to overcome, at least partially, the non-permissive substrate properties of differentiated CNS glial cells in vitro.     

FLRT3 is expressed in sensory neurons after peripheral nerve injury and regulates neurite outgrowth

We used a molecular screen to identify genes upregulated in regenerating adult rat dorsal root ganglion cells. FLRT3 mRNA and protein characterized by a fibronectin type III domain and a leucine-rich repeat motif was upregulated in damaged sensory neurons. The protein was then transported into their peripheral and central processes where the FLRT3 protein was localized to presynaptic axon terminals. In vitro, the FLRT3 protein was expressed at the cell surface, regulated neurite outgrowth in sensory neurons, but did not exhibit homophilic binding. FLRT3 was widely expressed in the developing embryo, particularly in the central nervous system and somites. However, in the adult, we found no evidence for accumulation or reexpression of the FLRT3 protein in damaged axons of the central nervous system. We conclude that FLRT3 codes for a putative cell surface receptor implicated in both the development of the nervous system and in the regeneration of the peripheral nervous system (PNS).     

Galanin and galanin-like peptide modulate neurite outgrowth via protein kinase C-mediated activation of extracellular signal-related kinase

The neuropeptide galanin is widely distributed in the central nervous system and plays a role in a number of processes in the adult brain. Galanin also has neurotrophic effects in the developing nervous system and after nerve injury. The current study investigated the mechanism by which galanin promotes neurite outgrowth in the neuronal cell line PC12 and in neurospheres derived from adult hippocampal progenitor cells. We demonstrated that galanin can induce extracellular signal-related kinase (ERK) phosphorylation transiently in a concentration-dependent manner in neurons. Galanin-like peptide, which is thought to signal primarily through the GalR2 receptor subtype, induced ERK phosphorylation with similar kinetics to galanin. In functional studies, the ability of galanin and galanin-like peptide to induce neurite outgrowth was dependent on activation of both protein kinase C and ERK. This study identified a novel physiological role for galanin-induced ERK phosphorylation and identified ERK and protein kinase C as important signaling components in the galanin-mediated modulation of neurite outgrowth.     

Neurotrophic activity of S-100 beta in cultures of dorsal root ganglia from embryonic chick and fetal rat

We report here that S-100 beta, a protein with neurotrophic activity on central nervous system neurons, stimulates neuritic outgrowth from cultures of dorsal root ganglia (DRG). S-100 beta elicited neurites from explant and dissociated cell cultures of embryonic chick DRG, and the extent of the response varied with the age of the embryo. Specificity was demonstrated by the observation that incubation of S-100 beta with antibodies directed against S-100 beta reduced the neurite outgrowth, whereas incubation of S-100 beta with normal rabbit serum had little effect. S-100 beta also stimulated the area of neuritic outgrowth from organotypic cultures of fetal rat DRG, showing that the activity of the protein is not restricted to a particular species or culture condition. A mutant S-100 beta lacking neurotrophic activity on cerebral cortex neurons was unable to effectively stimulate neurite outgrowth from DRG cultures. These studies suggest that S-100 beta may play a role in neuronal growth and/or maintenance in the peripheral nervous system.     

Activation of c-Jun N-terminal kinase is required for neurite outgrowth of dopaminergic neuronal cells

Recent studies indicate that activation of stress-activated protein kinases may be implicated in a broad range of biological activities including differentiation. To directly examine whether stress-activated protein kinases are involved in neuronal differentiation, we utilized retinoic acid-induced and spontaneous models of neurite outgrowth in dopaminergic neurons. Here, we show that retinoic acid-induced neurite outgrowth in MN9D dopaminergic neuronal cells was accompanied by activation of c-Jun N-terminal kinase but not p38. Consequently, cotreatment with a specific inhibitor of c-Jun N-terminal kinase or overexpression of c-Jun N-terminal kinase-binding domain of c-Jun N-terminal kinase-interacting protein-1 blocked retinoic acid-induced neurite outgrowth. In primary cultures of dopaminergic neurons, the extent of neurite outgrowth increased spontaneously in a time-dependent manner. When these cultures were treated with a specific inhibitor of c-Jun N-terminal kinase, the total extent of neurites, the primary neurite length and the number of neurites per cell were suppressed significantly. Thus, our data indicate that the c-Jun N-terminal kinase signal seems to play an important role during morphological differentiation in cultured dopaminergic neurons.     

The molluscan RING-finger protein L-TRIM is essential for neuronal outgrowth

The tripartite motif proteins TRIM-2 and TRIM-3 have been put forward as putative organizers of neuronal outgrowth and structural plasticity. Here, we identified a molluscan orthologue of TRIM-2/3, named L-TRIM, which is up-regulated during in vitro neurite outgrowth of central neurons. In adult animals, L-Trim mRNA is ubiquitously expressed at low levels in the central nervous system and in peripheral tissues. Central nervous system expression of L-Trim mRNA is increased during postnatal brain development and during in vitro and in vivo neuronal regeneration. In vitro double-stranded RNA knock-down of L-Trim mRNA resulted in a >70% inhibition of neurite outgrowth. Together, our data establish a crucial role for L-TRIM in developmental neurite outgrowth and functional neuronal regeneration and indicate that TRIM-2/3 family members may have evolutionary conserved functions in neuronal differentiation.     

pp60(c-src) is a negative regulator of laminin-1-mediated neurite outgrowth in chick sensory neurons

Multiple protein tyrosine kinases regulate neurite outgrowth in the developing nervous system. To begin to unravel the complexity of this regulation, we addressed the role of one specific kinase, pp60(c-src), in chick dorsal root ganglion (DRG) neurons grown on laminin-1, a well-characterized system to study neurite outgrowth. Pharmacological inhibition of all tyrosine kinases by genestein treatment of chick DRG neurons significantly increased neurite number and length by approximately 50%. Similar increases in these parameters occurred when src-family kinases were inhibited using PP2. To implicate pp60(c-src) directly in neurite outgrowth, we inactivated it in DRG neuronal growth cones using Chromophore-Assisted Laser Inactivation (CALI). CALI of pp60(c-src) resulted in an 85% inactivation of its kinase activity and a 63% reduction in phosphotyrosine immunofluorescence in neurons. Microscale CALI of pp60(c-src) in DRG growth cones caused a significant and acute two-fold increase in neurite extension rate during irradiation. These findings demonstrate that pp60(c-src) is a negative regulator of laminin-1-mediated neurite outgrowth in chick sensory neurons.     

Suppressor of cytokine signalling-2 and epidermal growth factor regulate neurite outgrowth of cortical neurons

Factors that regulate neurite outgrowth are important in determining the wiring of the central nervous system. Here we describe that the intracellular regulator of cytokine signalling, suppressor of cytokine signalling-2 (SOCS2) and epidermal growth factor (EGF), both of which are expressed in the cortical plate during neural development, promote neurite outgrowth of cortical neurons. Cortical neurons derived from transgenic mice that over-express SOCS2 had an increased rate of neurite outgrowth and an increased length and number of primary neurites compared with wild-type neurons. EGF produced a similar effect in wild-type cortical neurons and further enhanced the SOCS2-induced neurite outgrowth. The mechanism of neurite outgrowth induction by SOCS2 and EGF at least partially overlapped as phosphorylation of the EGF receptor in SOCS2 over-expressing or EGF-stimulated neurons was increased on Tyrosine845, the Src binding site and neurite outgrowth in both protocols was blocked by inhibitors of the EGF receptor kinase and Src kinase.     

Epac mediates cyclic AMP-dependent axon growth, guidance and regeneration

A decline in developing neuronal cAMP levels appears to render mammalian axons susceptible to growth inhibitory factors in the damaged CNS. cAMP elevation enhances axon regeneration, but the cellular mechanisms involved have yet to be fully elucidated. Epac has been identified as a signaling protein that can be activated by cAMP independently of PKA, but little is known of its expression or role in the nervous system. We report that Epac expression is developmentally regulated in the rat nervous system, and that activation of Epac promotes DRG neurite outgrowth and is as effective as cAMP elevation in promoting neurite regeneration on spinal cord tissue. Additionally, siRNA mediated knockdown of Epac reduces DRG neurite outgrowth, prevents the increased growth promoted by cAMP elevation and also diminishes the ability of embryonic neurons to grow processes on spinal cord tissue. Furthermore, we show that asymmetric activation of Epac promotes attractive growth cone turning in a similar manner to cAMP activation. We propose that Epac plays a role in mediating cAMP-dependent axon growth and guidance, and may provide an important target for inducing axon regeneration in vivo.     

Predegenerated peripheral nerve grafts rescue retinal ganglion cells from axotomy-induced death

The inability of axons to grow across damaged central nervous system tissue is a well-known consequence of injury to the brain and spinal cord of adult mammals. Our previous studies showed that predegenerated peripheral nerve grafts facilitate neurite outgrowth from the injured hippocampus and that this effect was particularly distinct when 7-, 28-, and 35-day-predegenerated nerve grafts were used. The purpose of the present study was to use the above method to induce and support the regrowth of injured nerve fibers as well as the survival of retinal ganglion cells (RGCs). Adult Sprague-Dawley rats were assigned to three groups. In the experimental groups transected optic nerve was grafted with peripheral nerve (predegenerated for 7 days (PD) or nonpredegenerated). In the control group, the optic nerve was totally transected. RGCs and growing fibers labeled with fluorescent tracers were examined. They were counted and the results were subjected to statistical analysis. Retinal ganglion cells survived in the groups treated with predegenerated as well as nonpredegenerated grafts; however, the number of surviving retinal ganglion cells was significantly higher in the first one. In both groups the regrowth of the transected optic nerve was observed but the distance covered by regenerating fibers was longer in the PD group. No fibers inside grafts and no labeled cells in retinas were present in the control animals. On the basis of the obtained results we can state that the predegeneration of grafts enhance their neurotrophic influence upon the injured retinal ganglion cells.     

Multimodal signaling by the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) promotes neurite extension

Aggregating proteoglycans (PG) bearing chondroitin sulfate (CS) side chains associate with hyaluronan and various secreted proteins to form a complex of extracellular matrix (ECM) that inhibits neural plasticity in the central nervous system (CNS). Chondroitinase treatment depletes PGs of their CS side chains and enhances neurite extension. Increasing evidence from in vivo models indicates that proteolytic cleavage of the PG core protein by members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of glutamyl-endopeptidases also promotes neural plasticity. The purpose of this study was to determine whether proteolytic action of the ADAMTSs influences neurite outgrowth in cultured neurons. Transfection of primary rat neurons with ADAMTS4 cDNA induced longer neurites, whether the neurons were grown on a monolayer of astrocytes that secrete inhibitory PGs or on laminin/poly-L-lysine substrate alone. Similar results were found when neurons were transfected with a construct encoding a proteolytically inactive, point mutant of ADAMTS4. Addition of recombinant ADAMTS4 or ADAMTS5 protein to immature neuronal cultures also enhanced neurite extension in a dose-dependent manner, an effect demonstrated to be dependent on the activation of MAP ERK1/2 kinase. These results suggest that ADAMTS4 enhances neurite outgrowth via a mechanism that does not require proteolysis but is dependent on activation of the MAP kinase cascade. Thus a model to illustrate multimodal ADAMTS activity would entail proteolysis of CS-bearing PGs to create a loosened matrix environment more favorable for neurite outgrowth, and enhanced neurite outgrowth directly stimulated by ADAMTS signaling at the cell surface.     

ROCK inhibition promotes adult retinal ganglion cell neurite outgrowth only in the presence of growth promoting factors

Lesioned central nervous system (CNS) axons fail to regenerate because of limited availability of neurotrophic factors (NTF) to promote neuron survival and drive axon regeneration through an environment rich in multiple myelin- and non myelin-derived axon growth inhibitory ligands that initiate growth cone collapse through the Rho/Rho kinase (ROCK) signalling pathway. However, pharmacological inhibition of Rho and ROCK promotes neurite outgrowth in PC12, Ntera-2 cells and embryonic/early postnatal neurons in culture. We have used our well-characterised CNS myelin-inhibited adult rat retinal culture model to show that Y27632 only promotes disinhibited neurite outgrowth if RGC are co-stimulated with ciliary neurotrophic factor (CNTF). Y27632 in CNTF-stimulated retinal cultures promotes optimal RGC neurite outgrowth at 10 μM concentrations, while higher concentrations negatively correlate with RGC neurite outgrowth and survival. Raising the levels of cAMP in Y27632-treated retinal cultures also promotes significant RGC neurite outgrowth, an effect that is potentiated by the further inclusion of CNTF. Our results suggest that Y27632-induced ROCK inhibition promotes robust disinhibited axon regeneration of adult neurons only when growth promoting factors are added and/or cAMP levels are raised.     

Interleukin-17A increases neurite outgrowth from adult postganglionic sympathetic neurons

Inflammation can profoundly alter the structure and function of the nervous system. Interleukin (IL)-17 has been implicated in the pathogenesis of several inflammatory diseases associated with nervous system plasticity. However, the effects of IL-17 on the nervous system remain unexplored. Cell and explant culture techniques, immunohistochemistry, electrophysiology, and Ca2+ imaging were used to examine the impact of IL-17 on adult mouse sympathetic neurons. Receptors for IL-17 were present on postganglionic neurons from superior mesenteric ganglia (SMG). Supernatant from activated splenic T lymphocytes, which was abundant in IL-17, dramatically enhanced axonal length of SMG neurons. Importantly, IL-17-neutralizing antiserum abrogated the neurotrophic effect of splenocyte supernatant, and incubation of SMG neurons in IL-17 (1 ng/ml) significantly potentiated neurite outgrowth. The neurotrophic effect of IL-17 was accompanied by inhibition of voltage-dependent Ca2+ influx and was recapitulated by incubation of neurons in a blocker of N-type Ca2+ channels (ω-conotoxin GVIA; 30 nM). IL-17-induced neurite outgrowth in vitro appeared to be independent of glia, as treatment with a glial toxin (AraC; 5 μM) did not affect the outgrowth response to IL-17. Moreover, application of the cytokine to distal axons devoid of glial processes enhanced neurite extension. An inhibitor of the NF-κB pathway (SC-514; 20 μM) blocked the effects of IL-17. These data represent the first evidence that IL-17 can act on sympathetic somata and distal neurites to enhance neurite outgrowth, and identify a novel potential role for IL-17 in the neuroanatomical plasticity that accompanies inflammation.     

Lysophosphatidic acid influences the morphology and motility of young,postmitotic cortical neurons

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that produces process retraction and cell rounding through its cognate receptors in neuroblastoma cell lines. Although the expression profile of LPA receptors in developing brains suggests a role for LPA in central nervous system (CNS) development, how LPA influences the morphology of postmitotic CNS neurons remains to be determined. Here we have investigated the effects of exogenous LPA on the morphology of young, postmitotic neurons in primary culture. When treated with LPA, these neurons responded by not only retracting processes but also producing retraction fiber "caps" characterized by fine actin filaments emanating from a dense core. Retraction fiber caps gradually vanished due to the outward spread of regrowing membranes along the fibers, suggesting a role for caps as scaffolds for regrowth of retracted processes. Furthermore, LPA also affects neuronal migration in vitro and in vivo. Taken together, these results implicate LPA as an extracellular lipid signal affecting process outgrowth and migration of early postmitotic neurons during development.     

Microglia enhance dorsal root ganglion outgrowth in Schwann cell cultures

Transplantation of cellular populations to facilitate regrowth of damaged axons is a common experimental therapy for spinal cord injury. Schwann cells (SC) or microglia grafted into injury sites can promote axonal regrowth of central projections of dorsal root ganglion (DRG) sensory neurons. We sought to determine whether the addition of microglia or microglia-derived secretory products alters DRG axon regrowth upon cultures of SC. Rat DRG explants were grown on monolayers consisting of either SC, microglia, SC exposed to microglia-conditioned medium (MCM), or co-cultures with different relative concentrations of microglia. Image analysis revealed that, compared to SC alone, the extent of neurite outgrowth was significantly greater on SC-microglia co-cultures. Immunocytochemistry for extracellular matrix molecules showed that microglial cells stained positively for growth-promoting thrombospondin, whereas laminin and the inhibitory chondroitin sulfate proteoglycans (CSPGs) were localized primarily to SC. Notably, immunoreactivity for CSPGs appeared reduced in areas associated with DRG outgrowth in co-cultures and SC exposed to MCM. These results show that microglia or their secreted products can augment SC-mediated DRG regrowth in vitro, indicating that co-grafting SC with microglia provides a novel approach to augment sensory fiber regeneration after spinal cord injury.     

A quantitative method for analysis of in vitro neurite outgrowth

The adult mammalian CNS is extremely limited in its ability to regenerate axons following injury. Glial scar, neuroinflammatory processes and molecules released from myelin impair axonal regrowth and contribute to the lack of neural regeneration. An in vitro assay that quantitates neurite outgrowth from cultured neurons as a model of neuronal regenerative potential is described. Specifically, the neurite outgrowth from primary neurons (rat cerebellar granule neurons; CGNs) and a neuronal cell line (NG108-15) were quantitatively measured after optimization of culture conditions. After cultures were fixed and immunostained to label neurons and nuclei, microscope images were captured and an image analysis algorithm was developed using Image-Pro Plus software to allow quantitative analysis. The algorithm allowed the determination of total neurite length, number of neurons, and number of neurons without neurites. The algorithm also allows for end-user control of thresholds for staining intensity and cell/nuclei size. This assay represents a useful tool for quantification of neurite outgrowth from a variety of neuronal sources with applications that include: (1) assessment of neurite outgrowth potential; (2) identification of molecules that can block or stimulate neurite outgrowth in conventional culture media; and (3) identification of agents that can overcome neurite outgrowth inhibition by inhibitory substrates.