Fibroblast growth factor treatment produces differential effects on survival and neurite outgrowth from identified bulbospinal neurons in vitro

The in vivo application of appropriate trophic factors may enhance regeneration of bulbospinal projections after spinal cord injury. Currently, little is known about the sensitivities of specific bulbospinal neuron populations to the many identified trophic factors. We devised novel in vitro assays to study trophic effects on the survival and neurite outgrowth of identified bulbospinal neurons. Carbocyanine dye crystals implanted into the cervical spinal cord of embryonic day (E)5 chick embryos retrogradely labeled developing bulbospinal neurons. On E8, dissociated cultures containing labeled bulbospinal neurons were prepared. Fibroblast growth factor (FGF)-2 (but not FGF-1) promoted the survival of bulbospinal neurons. FGF receptor expression was widespread in the E8 brainstem, but not detected in young bulbospinal neurons, suggesting that nonneuronal cells mediated the FGF-stimulated survival response. Astrocytes synthesize a variety of trophic factors, and astrocyte-conditioned medium (ACM) also promoted the survival of bulbospinal neurons. As might be expected, FGF-2 function blocking antibodies did not suppress ACM-promoted survival, nor did an ELISA detect FGF-2 in ACM. This suggests that nonneuronal cells synthesize other factors in response to exogenous FGF-2 which promote the survival of bulbospinal neurons. Focusing on vestibulospinal neurons, dissociated (survival assay) or explant (neurite outgrowth assay) cultures were prepared. FGF-2 promoted both survival and neurite outgrowth of identified vestibulospinal neurons. Interestingly, FGF-1 promoted neurite outgrowth but not survival; the converse was true of FGF-9. Thus, differential effects of specific growth factors on survival or neurite outgrowth of bulbospinal neurons were distinguished.     

IGF-1 and BDNF promote chick bulbospinal neurite outgrowth in vitro

Injured neurons in the CNS do not experience significant functional regeneration and so spinal cord insult often results in permanently compromised locomotor ability. The capability of a severed axon to re-grow is thought to depend on numerous factors, one of which is the decreased availability of neurotrophic factors. Application of trophic factors to axotomized neurons has been shown to enhance survival and neurite outgrowth. Although brainstem-spinal connections play a pivotal role in motor dysfunction after spinal cord injury, relatively little is known about the trophic sensitivity of these populations. This study explores the response of bulbospinal populations to various trophic factors. Several growth factors were initially examined for potential trophic effects on the projection neurons of the brainstem. Brain derived neurotrophic factor (BDNF) and insulin-like growth factor (IGF-1) significantly enhance mean process length in both the vestibulospinal neurons and spinal projection neurons from the raphe nuclei. Nerve growth factor (NGF), neurotrophin-4 (NT-4) and glial derived neurotrophic factor (GDNF) did not effect process outgrowth in vestibulospinal neurons. At the developmental stages used in this study, it was determined that receptors for BDNF and IGF-1 were present both on bulbospinal neurons and on surrounding cells with a non-neuronal morphology.     

Triiodothyronine Exerts a Trophic Action on Rat Sensory Neuron Survival and Neurite Outgrowth through Different Pathways

Apart from several growth factors which play a crucial role in the survival and development of the central and peripheral nervous systems, thyroid hormones can affect different processes involved in the differentiation and maturation of neurons. The present study was initiated to determine whether triiodothyronine (T₃) affects the survival and neurite outgrowth of primary sensory neurons in vitro. Dorsal root ganglia (DRG) from 19-day-old embryos or newborn rats were plated in explant or dissociated cell cultures. The effect of T₃ on neuron survival was tested, either in mixed DRG cell cultures, where neurons grow with non-neuronal cells, or in neuron-enriched cultures where non-neuronal cells were eliminated at the outset. T₃, in physiological concentrations, promoted the growth of neurons in mixed DRG cell cultures as well as in neuron-enriched cultures without added nerve growth factor (NGF). Since neuron survival in neuron-enriched cultures cannot be promoted by endogenous neurotrophic factors synthesized by non-neuronal cells, the increased number of surviving neurons was due to a direct trophic action of T₃. Another trophic effect was revealed in this study: T₃ sustained the neurite outgrowth of sensory neurons in DRG explants. The stimulatory effect of T₃ on nerve fibre outgrowth was considerably reduced when non-neuronal cell proliferation was inhibited by the antimitotic agent cytosine arabinoside, and was completely suppressed when the great majority of non-neuronal cells were eliminated in neuron-enriched cultures. These results indicate that the stimulatory effect of T₃ on neurite outgrowth is mediated through non-neuronal cells. It is conceivable that T₃ up-regulates Schwann cell expression of a neurotrophic factor, which in turn stimulates axon growth of sensory neurons. Together, these results demonstrate that T₃ promotes both survival and neurite outgrowth of primary sensory neurons in DRG cell cultures. The trophic actions of T₃ on neuron survival and neurite outgrowth operate under two different pathways.     

Fibroblast growth factors (FGF-1, FGF-2) promote migration and neurite growth of mouse cochlear ganglion cells in vitro: immunohistochemistry and antibody perturbation

To study the effect of FGF in the early development of the sensory neurons of the auditory system, we established a culture preparation of ganglionic neuroblasts engaged in migration and process outgrowth. The presumed anlage of the cochlear ganglion was dissected from E11 otocysts, just as the neuronal precursors were migrating. The cultures were divided into 4 groups and supplemented for 7-9 days with either hrFGF-1 or hrFGF-2 or both or with defined medium only (control group). Measurements of the increase in explant growth, neuroblast migration, and neurite outgrowth were made by time-lapse imaging techniques in living cultures. Either FGF-1 or FGF-2 alone stimulated early migration and outgrowth of the ganglion cells by 5-10x. The effect of combining FGF-1 and FGF-2 was greater than either alone, but less than additive, consistent with a shared receptor. BrdU labeling confirmed that the effect was on migration, not on proliferation. Adding a neutralizing antibody for FGF-2 to the cultures inhibited migration and neurite outgrowth, suggesting an endogenous FGF-2 activity in these functions. Immunocytochemical observations in vitro and in situ with antibodies to FGF-1, FGF-2, or FGF receptor (R1) demonstrated immunopositive staining of the migrating ganglionic neuroblasts, their processes, and growth cones at corresponding stages (E13). Also non-neuronal cells, hair cells, and Schwann cells (in situ) expressed FGF-1 and FGF-2. Evidently both FGF-1 and FGF-2 play important roles in the migration and initial differentiation of cochlear ganglion neurons in the mouse.     

The physiological and pharmacological role of basic fibroblast growth factor in the dopaminergic nigrostriatal system

Basic fibroblast growth factor (FGF-2) is a physiological relevant neurotrophic factor in the nigrostriatal system and hence a promising candidate for the establishment of alternative therapeutic strategies in Parkinson's disease. FGF-2 and its high-affinity receptors (FGFR) display an expression in the developing, postnatal, and adult substantia nigra (SN) and in the striatum. Exogenous application promoted survival, neurite outgrowth and protection from neurotoxin-induced death of dopaminergic (DA) neurons both in vitro and in vivo. In animal models of Parkinson's disease, co-transplantation of fetal DA cells with FGF-2 expressing cells increased survival and functional integration of the grafted DA neurons resulting in improved behavioral performance. Analyzing the physiological function of the endogenous FGF-2 system during development and after neurotoxin-induced lesion revealed for the DA neurons of the SNpc a dependence on FGFR3 signaling during development. In addition, in the absence of FGF-2 an increased number of DA neurons was found, whereas enhanced levels of FGF-2 resulted in a reduced DA cell density. Following neurotoxin-induced lesion of DA neurons, FGF-2-deleted mice displayed a higher extent of DA neuron death whereas in FGF-2 overexpressing mice more DA neurons were protected. According to the data, FGF-2 seems to promote DA neuron survival via FGFR3 during development, whereas absence of this ligand could be compensated by other members of the FGF family. In contrast, in the adult organism, FGF-2 cannot be compensated by other factors under lesion conditions suggesting a central role for this molecule in the nigrostriatal system.     

Physiological function and putative therapeutic impact of the FGF-2 system in peripheral nerve regeneration--lessons from in vivo studies in mice and rats

Diffusible and substratum-bound molecules regulate development and regeneration of the peripheral nervous system. The understanding of physiological function of these factors could have an impact on the development of new therapeutic strategies to stimulate nerve regeneration across long gaps. Within the group of trophic factors, basic fibroblast growth factor (FGF-2) and its high-affinity receptors are expressed in the intact peripheral nervous system and regulated following nerve injury. After exogenous application, FGF-2 promotes neuronal survival and neurite outgrowth in vitro and in vivo. In this review, animal studies on the physiological role of the endogenous FGF-2 system and the regenerative capacity after exogenous FGF-2 administration are summarized. The concept of FGF-2 function is discussed in context with other growth factors that are also physiologically relevant in the peripheral nervous system. Studies of sciatic nerve axotomy in FGF-2- and FGF receptor (R) 3-deleted mice, respectively, strongly suggested that FGF-2 binding to FGFR3 is involved in injury-induced neuronal apoptosis. At the lesion site, inhibition of myelination and stimulation of Schwann cell proliferation by FGF-2 via FGFR1/2 is suggested from rat and mouse studies, whereas neurite formation is very likely enhanced via FGFR3 activation. Additionally to these demonstrated physiological functions of endogenous FGF-2, administration of FGF-2 isoforms in the rat model of nerve regeneration across long gaps revealed a role of the high molecular weight isoforms of FGF-2 on sensory recovery. Within the group of physiologically relevant trophic factors, the FGF-2 system seems to be crucially involved in the scenario of peripheral nerve development and regeneration.     

Estrogen increases sensory nociceptor neuritogenesis in vitro by a direct, nerve growth factor-independent mechanism

Estrogen affects many aspects of the nervous system, including pain sensitivity and neural regulation of vascular function. We have shown that estrogen elevation increases sensory nociceptor innervation of arterioles in Sprague-Dawley rat mammary gland, external ear and mesentery, suggesting widespread effects on sensory vasodilatory innervation. However, it is unclear whether estrogen elicits nociceptor hyperinnervation by promoting target release of neurotrophic factors, or by direct effects on sensory neurons. To determine if estrogen may promote axon sprouting by increasing release of target-derived diffusible factors, dorsal root ganglia explants were co-cultured with mesenteric arterioles for 36 h in the absence or presence of 17beta-estradiol (E2). Mesenteric arteriolar target substantially increased neurite outgrowth from explanted ganglia, but estrogen had no effect on outgrowth, suggesting that estrogen does not increase the availability of trophic proteins responsible for target-induced neurite outgrowth. To assess the direct effects of estrogen, dissociated neonatal dorsal root ganglion neurons were cultured for 3 days in the absence or presence of E2 and nerve growth factor (NGF; 1-10 ng/mL), and immunostained for the nociceptor markers peripherin or calcitonin gene-related peptide. NGF increased neuron size, survival and numbers of neurons with neurites, but did not affect neurite area per neuron. Estrogen did not affect neuron survival, size or numbers of neurons with neurites, but did increase neurite area per neuron. The effects of these agents were not synergistic. We conclude that estrogen exerts direct effects on nociceptor neurons to promote axon outgrowth, and this occurs through an NGF-independent mechanism.     

Transforming growth factor-β mediates the neurotrophic effect of fibroblast growth factor-2 on midbrain dopaminergic neurons

Fibroblast growth factor (FGF)-2 is an established neurotrophic factor for dopaminergic (DAergic) neurons in the ventral midbrain. Its survival and differentiation-promoting effects on DAergic neurons in vitro and in vivo are crucially dependent on the presence, numerical expansion and maturation of astroglial cells. We show now that transforming growth factor (TGF)-β, an established trophic factor for DAergic neurons and product of astroglial cells, mediates the trophic effect of FGF-2 on DAergic neurons cultured from the embryonic rat midbrain floor. Antibodies to TGF-β that neutralize the isoforms -β1, -β2 and -β3 abolish the trophic effect of FGF-2. FGF-2 increases TGF-β3 mRNA and amounts of biologically active TGF-β determined in a mink lung epithelial cell assay in a time-dependent manner. FGF-2 also induces levels of active TGF-β in neonatal rat astrocytes cultured from midbrain, striatum and cortex. We conclude that TGF-β is required for mediating the survival promoting effect of FGF-2 on DAergic and, possibly, cortical and striatal neurons grown in the presence of glial cells.     

Choroid coat extract and ciliary neurotrophic factor strongly promote neurite outgrowth in the embryonic chick retina

Previous studies have shown that extracts from the target optic tectum stimulate neurite outgrowth from retinal explants. The present study indicates that the choroid coat is an even richer source of retinotrophic activity. We thus studied the effects of recombinant rat ciliary neurotrophic factor (CNTF) on primary cultures of dissociated chick ciliary ganglion neurons and retinal explants for a comparison with choroid coat extract from the E 18 chick. For our assays, E9 ciliary neurons were incubated in collagen gels and retinal explants were cultured on collagen gels with the addition of the trophic factors and maintained for two or four days. Survival of ciliary neurons per area as well as maximal neurite length in retinal cultures were determined. Growth responses occurred in a dose-dependent manner both to CNTF and choroid extract. Immunofluorescence examination of cells and developing processes showed 200 kdal neurofilament positivity demonstrating that the cells studied were neurons with neurites. It is concluded that a trophic activity of the choroid as well as the recombinant CNTF stimulate retinal neuron survival and neurite extension. The results suggest that CNTF may have developmental functions in the establishment of the visual pathways.     

Nerve growth factor and a fibroblast growth factor-like neurotrophic activity in cerebrospinal fluid of brain injured human patients

We report here the presence of nerve growth factor (NGF) in the cerebrospinal fluid (CSF) of some brain-injured human patients soon after injury. The NGF was quantified against a recombinant human NGF standard in a two-site enzyme-linked immunoabsorbant assay using antibodies against murine B NGF. None of the samples collected more than 2 days after injury contained detectable levels of NGF. When the CSF was assayed for the ability to promote neurite outgrowth from PC12 cells, neurite outgrowth was reduced, but not completely blocked, by antibodies to B NGF, suggesting that there were other biologically active factors present. Fibroblast growth factor (FGF) also promotes neurite outgrowth in PC12 cells. In an initial screening for the presence of FGF, we employed PC12 cells and NR119 cells, PC12 variants in which recombinant human B NGF, but not recombinant human basic FGF, promotes neurite outgrowth. CSF from brain injury patients promoted greater neurite outgrowth from PC12 cells than from NR119 cells, suggesting that some of the biological activity associated with the injury CSF may be due a FGF. This possibility is further supported by the observation that the biological activity of the injury CSF significantly reduced by batch absorption with heparin Sepharose, suggesting the presence of a heparin binding neurotrophic factor. Neurotrophic factors appear in CSF as a consequence of diverse types of brain injury, including head trauma, intracerebral hemorrhage and subarachnoid hemorrhage. The appearance of these factors may reflect important common elements in the complex series of cellular changes occuring in response to acute brain injury.     

Motor neuron survival and neuritic extension from spinal cord explants induced by factors released from denervated muscle

Extracts prepared from denervated adult skeletal muscle contain increased amounts of neurotrophic activity which promotes both survival of dissociated motor neurons and the outgrowth of neurites from explants of spinal cord maintained in serum-free defined media. The trophic activity is specific for motor neurons and reaches a peak within the first week post-denervation. In these most potent extracts the neurite outgrowth enhancement is a linearly increasing function of protein concentration at low concentrations; at higher concentrations the neurite activity-concentration relationship saturates and in the milligram range the relationship becomes inhibitory. When media containing active denervated muscle extract was preincubated over polycationic substrata, it lost the ability to promote neuritic growth; this could be restored if fresh extract was added to the cultures. Thus it was demonstrated that within the denervated muscle extract there are physically separable agents responsible for neuron survival and neurite expression. It is possible that the release of neurotrophic factors may be in part responsible for the in vivo phenomenon of nerve sprouting.     

Growth responses of different subpopulations of adult sensory neurons to neurotrophic factors in vitro

Different subpopulations of adult primary sensory neurons in the dorsal root ganglia express receptors for different trophic factors, and are therefore potentially responsive to distinct trophic signals. We have compared the effect of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and NT-3, and of glial cell line-derived neurotrophic factor (GDNF) on neurite outgrowth in dissociated cultures of sensory neurons from the lumbar ganglia of young adult rats, and attempted to establish subset-specific effects of these trophic factors. We analysed three parameters of neurite growth (percentage of process-bearing neurons, length of longest neurite and total neurite length), which may correlate with particular types of axon growth in vivo, and may therefore respond differently to trophic factor presence. Our results showed that percentage of process-bearing neurons and total neurite length were influenced by trophic factors, whilst the length of the longest neurite was trophic factor independent. Only NGF and GDNF were found to enhance significantly the proportion of process-bearing neurons in vitro. GDNF was more effective than NGF on small, IB4- neurons, which are known to develop GDNF responsiveness early in postnatal development. NGF, and to a much lesser extent GDNF, enhanced the total length of the neurites produced by neurons in culture. BDNF exerted an inhibitory effect on growth, and both BDNF and NT-3 could partially block some of the growth-promoting effects of NGF on specific neuronal subpopulations.     

Critical Periods of Basic Fibroblast Growth Factor and Brain-Derived Neurotrophic Factor in the Development of the Chicken Cochleovestibular Ganglionin Vitro

The temporal roles of brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2 (FGF-2) in the development of sensory neurons have been studied in a cell culture preparation which models normal embryonic inner ear development (normocytic). Previous studies showed that FGF-2 stimulated migration and differentiation of ganglion cells for the first 2 daysin vitro,but after 5 days led to degeneration, implicating other factors in their later development. To see if BDNF could be such a factor, otocysts were explanted from white leghorn embryos at the time when ganglion cell precursors normally start migrating from the otic epithelium. Cultures were grown in a defined medium, either with or without human recombinant FGF-2 for 2 days or with BDNF. On Day 3, FGF-2 was replaced either with BDNF in defined medium or with defined medium only. Measurements of neuroblast migration and neurite outgrowth were made by time-lapse imaging in living cultures. In cultures receiving BDNF on Day 3, cell migration and neurite outgrowth from the explant increased for more than 3 weeks but not in cultures receiving only defined medium from Day 3. Cultures did not survive more than 3–4 days when receiving either BDNF in defined medium or defined medium alone from the first day. A neutralizing antibody to BDNF inhibited neuronal migration and neurite outgrowth, and it also blocked the effects of exogenous BDNF. BDNF did not enhance the effects of FGF-2 by interacting with it. These experiments defined a temporal sequence in which FGF-2 acts early in development, while BDNF affects a later stage.     

Schwann cells transduced with a lentiviral vector encoding Fgf‐2 promote motor neuron regeneration following sciatic nerve injury

Fibroblast growth factor 2 (FGF‐2) is a trophic factor expressed by glial cells and different neuronal populations. Addition of FGF‐2 to spinal cord and dorsal root ganglia (DRG) explants demonstrated that FGF‐2 specifically increases motor neuron axonal growth. To further explore the potential capability of FGF‐2 to promote axon regeneration, we produced a lentiviral vector (LV) to overexpress FGF‐2 (LV‐FGF2) in the injured rat peripheral nerve. Cultured Schwann cells transduced with FGF‐2 and added to collagen matrix embedding spinal cord or DRG explants significantly increased motor but not sensory neurite outgrowth. LV‐FGF2 was as effective as direct addition of the trophic factor to promote motor axon growth in vitro. Direct injection of LV‐FGF2 into the rat sciatic nerve resulted in increased expression of FGF‐2, which was localized in the basal lamina of Schwann cells. To investigate the in vivo effect of FGF‐2 overexpression on axonal regeneration after nerve injury, Schwann cells transduced with LV‐FGF2 were grafted in a silicone tube used to repair the resected rat sciatic nerve. Electrophysiological tests conducted for up to 2 months after injury revealed accelerated and more marked reinnervation of hindlimb muscles in the animals treated with LV‐FGF2, with an increase in the number of motor and sensory neurons that reached the distal tibial nerve at the end of follow‐up. GLIA 2014;62:1736–1746     

Cooperation between nerve growth factor and laminin or fibronectin in promoting sensory neuron survival and neurite outgrowth

Chick embryo dorsal root ganglion (DRG) neurons were purified by differential adhesion to plastic. The purified neurons were used to study the cooperation between nerve growth factor (NGF) and laminin or fibronectin in promoting neuron survival and neurite outgrowth. NGF alone supported the survival of only 20% embryonic day 10 (E10) cells, of which only 40-50% had neurites. Treatment of the substrate with fibronectin or laminin increased survival in the presence of NGF up to 80% of the seeded neurons, all of which showed extensive neurite outgrowth. Survival and neurite outgrowth were also enhanced by the combined effects of elevated potassium and laminin. In contrast to E8-10 cells, 85% of E16 neurons survived in the basal culture conditions, i.e. without additional NGF, fibronectin or laminin, although neurite outgrowth was enhanced by all 3 proteins. Antisera to NGF, laminin and fibronectin, each independently decreased survival and neurite outgrowth of DRG neurons, totally with E9 and partially with E16 cells. The results suggest that the cooperative actions of extracellular matrix proteins and NGF are essential for survival and neurite outgrowth of embryonic DRG neurons and that these neuronal requirements change during development.     

Olfactory ensheathing cells represent an optimal substrate for hippocampal neurons: an in vitro study

Olfactory ensheathing cells (OECs) are cells that display Schwann cell or astrocyte-like properties. They are a source of growth factors and adhesion molecules which play a very important role as neuronal support enhancing cellular survival. Over the past 10 years, OECs have emerged as a leading reparative candidate, when transplanted into the injured spinal cord, having shown significant promise in the regeneration of spinal cord lesions. In this study we assessed the efficacy of OECs on the survival and neurite outgrowth of hippocampal neurons in vitro. Co-cultures of OECs and hippocampal of postnatal rats were successfully established and cells were immunocytochemically characterized. Some hippocampal cultures were added with growth factors, as bFGF, NGF and GDNF. Furthermore, conditioned medium from OECs cultures was used to feed some hippocampal neurons coverslips. Our results show that in co-cultures of hippocampal neurons and OECs the number of neurons and their neurite outgrowth were significantly increased in comparison with controls. Moreover, we showed that NGF and GDNF promoted a more positive effect in both neuronal survival and neurite outgrowth than bFGF. OEC-conditioned media stimulated both the neuronal survival and dense neurite outgrowth. These data indicate that OECs, as a source of growth factors, can promote the survival and the neurite outgrowth of hippocampal neurons in vitro and that bFGF, NGF and GDNF support them differently. Therefore, as OECs and their secreted growth factors appear to exert a neuroprotective effect for functional restoration and for neural plasticity in neurodegenerative disorders, they might be considered an approach for functional recovery.     

Serotonin and nialamide differentially regulate survival and growth of cultured serotonin and catecholamine neurons.

In this morphometric analysis of immunoreactive serotonin (5-HT) and tyrosine hydroxylase (TH) neurons in culture, 5-HT and the MAO inhibitor nialamide influenced the survival, cell body size and neurite outgrowth of embryonic day 14 (E14) 5-HT neurons after treatment from 1-3 days in vitro (DIV), but did not significantly affect E14 or E15 TH neurons of either the noradrenergic or dopaminergic phenotype. These treatments had minimal effects on 5-HT neurons derived from E15 embryos. The stimulatory effects of 5-HT on survival and somal growth of E14 5-HT neurons was in contrast to its inhibitory effects on neurite outgrowth, suggesting trophic and inhibitory autoregulation of different cellular compartments of developing 5-HT neurons. The decreased sensitivity of E15 5-HT neurons to these treatments, despite similar viability and growth of these neurons in control cultures, suggests the existence of a critical period for this regulation during the initial period of serotonergic neurogenesis when these neurons are forming the bilateral B4-9 raphe complex. The lack of significant effects of 5-HT on TH neurons suggests differential sensitivities of 5-HT and TH neurons to developmental regulation by this neurotransmitter.     

蛋白激酶C调节脊髓神经元突起的生长(英文)

目的关于蛋白激酶C(PKC)在神经元突起生长和神经再生中的作用,目前仍存有争议。本研究主要观察PKC对离体培养的脊髓神经元生长的调节作用,旨在阐明PKC对突起生长的调节作用。方法分离纯化胎龄14天(E14)的SD胎鼠的脊髓前角神经元,进行原代培养,并检测不同时相点膜/浆PKC活性(m/c-PKCactivity)的比值。结果神经元培养3-11d期间,神经元内m/c-PKC比值以及PKC-βII在突起中的表达水平均与突起生长呈显著相关关系(r=0.95,P<0.01;r=0.73,P<0.01)。此外,PKC激动剂PMA能显著提高m/c-PKC比值,且与神经突起的生长一致(r=0.99,P<0.01)。而PKC抑制剂GF109203X则能显著抑制突起生长,且不被PMA作用所逆转。结论PKC的活性在脊髓神经元突起生长调节中具有重要作用,其中βII亚型可能扮演重要角色。     

Neurite growth promotion by nerve growth factor and insulin-like growth factor-1 in cultured adult sensory neurons: role of phosphoinositide 3-kinase and mitogen activated protein kinase

Although neurons of the PNS no longer require neurotrophins such as Nerve Growth Factor (NGF) for their survival, such factors are involved in regulating axonal sprouting and regeneration after injury. In addition to the neurotrophin receptors, sensory neurons are reported to express IGF-1, EGF and FGF receptors. To investigate the influence of growth factors in addition to NGF, we examined the effects of IGF-1 EGF and FGF on neurite growth from adult rat dorsal root ganglion sensory neurons in both dissociated cultures and in compartmented cultures. As expected, NGF elicited robust neuritic growth in both the dissociated and compartmented cultures. The growth response to IGF-1 was similar, although there was minimal neurite growth in response to EGF or FGF. In addition, IGF-1 (but neither FGF nor EGF), when applied to cell bodies in compartmented cultures, potentiated the distal neurite growth into NGF-containing side compartments. This potentiation was not seen when these factors were provided along with NGF in the side compartments of compartmented cultures, or in the dissociated cultures. To determine the contribution of signaling intermediates downstream of receptor activation, we used inhibitors of the potential effectors and Western blotting. The PI 3-kinase inhibitor, LY294002 attenuated neurite growth evoked by NGF, IGF and EGF in dissociated cultures, although the MAP kinase kinase (MEK) inhibitor PD098059 diminished the growth in only IGF. Immunoprecipitation and Western blotting results demonstrated differential activation of MAPK, PI 3-kinase, PLCgamma1 and SNT by the different factors. Activation of PI 3-kinase and SNT by both NGF and IGF-1 correlated with their effects on neurite growth. These results support the hypothesis that the PI 3-kinase pathway plays an important role in neuritogenesis.