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。     

Ciliary neurotrophic factor stimulates neurite outgrowth from spinal cord neurons

Ciliary neurotrophic factor (CNTF) has been shown to promote the survival of motoneurons, but its effects on axonal outgrowth have not been examined in detail. Since nerve growth factor (NGF) promotes the outgrowth of neurites within the same populations of neurons that depend on NGF for survival, we investigated whether CNTF would stimulate neurite outgrowth from motoneurons in addition to enhancing their survival. We found that CNTF is a powerful promoter of neurite outgrowth from cultured chick embryo ventral spinal cord neurons. An effect of CNTF on neurite outgrowth was detectable within 7 hours, and at a concentration of 10 ng/ml, CNTF enhanced neurite length by about 3- to 4-fold within 48 hours. The neurite growth-promoting effect of CNTF does not appear to be a consequence of its survival-promoting effect. To determine whether the effect of CNTF on spinal cord neurons was specific for motoneurons, we analyzed cell survival and neurite outgrowth for motoneurons labeled with diI, as well as for neurons taken from the dorsal half of the spinal cord, which lacks motoneurons. We found that the effect of CNTF was about the same for motoneurons as it was for neurons from the dorsal spinal cord. The responsiveness of a variety of spinal cord neurons to CNTF may broaden the appeal of CNTF as a candidate for the treatment of spinal cord injury or disease. © 1996 Wiley-Liss, Inc.     

Vasoactive intestinal peptide influences neurite outgrowth in cultured rat spinal cord neurons

Abstract

Vasoactive intestinal peptide (VIP) is a neuropeptide which has been shown to exhibit a wide range of neurotrophic effects both in vivo and in vitro . For the purpose of clarifying the effect of VIP on spinal cord neurons, we studied the effect of VIP on neurite outgrowth of fetal rat ventral and dorsal portions of spinal cord in cultures. VIP-treated ventral spinal cord cultures (VSCC), compared with control VSCC, had a significant neurite outgrowth at 10^-8, 10^-6, and 10^-4 M. The effect was considered to be concentration dependent. Morphological changes of the dorsal spinal cord cultures (DSCC) remained unchanged by VIP treatment. Because of their close sequence homology with VIP, PHI-27 (peptide, histidylisoleucine amide) and secretin were also examined with the same experimental conditions as was VIP. Both PHI-27 and secretin had neurite promoting effects in VSCC at 10^-8 and 10^-6 M, respectively. However, there were no neurite promoting effects in DSCC in both of them at any concentrations. VIP had the most potent effect on neurite outgrowth in VSCC, followed by PHI-27, and secretin in their effectiveness concentrations. Our data showing VIP, PHI-27 and secretin have neurotrophic action on VSCC and suggest that a potential therapeutic use of VIP and its related peptides in treating diseases that involve degeneration and death of spinal motor neurons, such as motor neuropathy and amyotrophic lateral sclerosis. [Neurol Res 2001; 23: 851-854]     

Vasoactive intestinal peptide influences neurite outgrowth in cultured rat spinal cord neurons.

Vasoactive intestinal peptide (VIP) is a neuropeptide which has been shown to exhibit a wide range of neurotrophic effects both in vivo and in vitro. For the purpose of clarifying the effect of VIP on spinal cord neurons, we studied the effect of VIP on neurite outgrowth of fetal rat ventral and dorsal portions of spinal cord in cultures. VIP-treated ventral spinal cord cultures (VSCC), compared with control VSCC, had a significant neurite outgrowth at 10(-8), 10(-6), and 10(-4) M. The effect was considered to be concentration dependent. Morphological changes of the dorsal spinal cord cultures (DSCC) remained unchanged by VIP treatment. Because of their close sequence homology with VIP, PHI-27 (peptide, histidylisoleucine amide) and secretin were also examined with the same experimental conditions as was VIP. Both PHI-27 and secretin had neurite promoting effects in VSCC at 10(-8) and 10(-6) M, respectively. However, there were no neurite promoting effects in DSCC in both of them at any concentrations. VIP had the most potent effect on neurite outgrowth in VSCC, followed by PHI-27, and secretin in their effectiveness concentrations. Our data showing VIP, PHI-27 and secretin have neurotrophic action on VSCC and suggest that a potential therapeutic use of VIP and its related peptides in treating diseases that involve degeneration and death of spinal motor neurons, such as motor neuropathy and amyotrophic lateral sclerosis.     

Adipocyte-derived angiopoietin-1 supports neurite outgrowth and synaptogenesis of sensory neurons

Sensory and sympathetic innervation of the white fat tissue (WAT) contributes to lipolysis. In addition, both fiber types adapt in density to weight gain and loss. Because these findings are indicative for a tight control of nerve fiber plasticity by adipokines, we tested whether adipocytes control neurite growth of sensory neurons through angiopoietin-1 (Ang-1). We further considered initial hints that Ang-1-induced neuritogenesis involves transactivation of the high-affinity nerve growth factor (NGF) receptor trkA. Coculturing dorsal root ganglion (DRG) cells with 3T3-L1 adipocytes supported neurite outgrowth. These neurotrophic effects were associated with the increased expression of Ang-1 (presumably in adipocytes) as well as of trkA. The effects were abolished upon inactivating Ang-1 in culture with selective antibodies. Likewise, neurite outgrowth was impaired in the presence of inactivating NGF antibodies as well as upon inhibition of the NGF high-affinity trkA receptor with the antagonist K252a, indicating a tight cooperation of Ang-1 and NGF in the control of neuritogenesis. DRG-adipipocyte cocultures were further used to establish whether sensory neurons would form synaptic contacts with adipocytes. Electron microscopy demonstrated that cultured sensory neurons develop predominantly neuroneuronal synapses but seem to affect adipocytes by synapses en passant. Comparably to the case for neuritogenesis, expression of the presynaptic protein synaptophysin as well of the postsynaptic protein PSD-95 correlated with Ang-1 levels in culture. It is concluded that adipocyte-secreted Ang-1 supports neurite outgrowth, which is involved in synaptogenesis. The novel function of Ang-1 appears to play a physiological role in WAT plasticity.     

Acidic and basic fibroblast growth factors enhance neurite outgrowth in cultured rat spinal cord neurons

Abstract

We have studied neurotrophic effects of acidic fibroblast growth factor (aFCF) and basic fibroblast growth factor (bFGF) on explanted ventral and dorsal spinal cord cultures from 13- and 14-day-old rat embryos. Cultures treated with aFCF and bFGF significantly enhanced neurite outgrowth with cultures of ventral spinal cord, but not with cultures of dorsal spinal cord. Our data suggest that aFCF and bFGF are potent neurotrophic factors on rat ventral spinal cord neurons in vitro. [Neurol Res 1995; 17: 70-72]     

A culture model for neurite regeneration of human spinal cord neurons

Effective therapeutic interventions for injuries of the central nervous system such as spinal cord injury are still unavailable, having a great impact on the quality of life of victims and their families, as well as high costs in medical care. Animal models of spinal cord injury are costly, time-consuming and labor-intensive, making them unsuitable for screening large numbers of experimental conditions. Thus, culture models that recapitulate key aspects of neuronal changes in central nervous system injuries are needed to gain further understanding of the pathological and regenerative mechanisms involved, as well as to accelerate the screening of potential therapeutic agents. In this study we differentiated adherent cultures of dissociated human fetal spinal cord neural precursors into postmitotic neurons which we could then detach from culture plates and successfully freeze down in a viable state. When replated in neuronal medium without neurodifferentiating factors, these ready-to-use human spinal cord neurons remained viable, postmitotic and regenerated neurites in a cell density-dependent manner. Insulin-like growth factor 1 and growth hormone had no effect on neurite regeneration while brain-derived neurotrophic factor increased both the number of cells with neurites as well as the average neurite length. Our model can be applied to investigate factors involved in neuroregeneration of the human spinal cord and since adherent dissociated cell cultures are used, this system has significant potential as a screening platform for therapeutic agents to treat spinal cord injury.     

Developmental patterns of neurite outgrowth from chick embryo spinal cord and retinal neurons on laminin substrates

It has been previously found that neurite outgrowth on collagen substrates decreases with increasing gestational age of chick embryo spinal cord and retinal neurons in tissue culture. In the current study, laminin, polylysine and collagen were compared in their efficacy in promoting neurite extension from chick embryo spinal cord neurons aged 6-16 days or retinal neurons aged 8-16 days in ovo. The percentage of neurons with neurites and the length of the neurites were determined at 1 and 3 days in culture. There was a significant increase in neuritogenesis by laminin and polylysine compared to collagen for both spinal cord and retinal neurons. Further, in spinal cord cultures grown on a laminin substrate, there was no decline in neurite outgrowth with increasing developmental age of the neurons as was seen on collagen and polylysine. Neurite length measurements also demonstrated a significant stimulation of neuritogenesis for spinal cord, but not retinal, neurons by laminin compared to polylysine or collagen in 1-day cultures. The results demonstrate tissue-specific differences in the developmental patterns of neurite outgrowth. Retinal neurons appear to have intrinsic changes in their ability to respond to extracellular promoting factors or substrates, while spinal cord neurite outgrowth can be regulated by these extrinsic factors.     

Laminin supports neurite outgrowth from explants of axotomized adult rat retinal neurons

The influence of laminin on neurite outgrowth from explants of adult rat retina and its distribution in normal and lesioned rat optic nerves were examined. Neurite outgrowth required the presence of laminin in the substratum, and as with a goldfish retinal explant system, was markedly stimulated by prior axotomy. Except for blood vessels and the nerve sheath, normal rat optic nerve was devoid of laminin immunoreactivity. Unlike results seen in the goldfish optic nerve, injury to the rat optic nerve induced no observable increase in laminin content or change in its distribution. The differences in the in vivo regenerative capacities of these two species may in part be related to the differences in their abilities to provide a proper substratum for axon regrowth.     

Effects of calcium ion on neurite outgrowth of rat spinal cord neurons in vitro: the role of non-neuronal cells in regulating neurite sprouting

The interactions of nerve cells with their environment and other cells are specific to different stages of cellular differentiation. Neurite outgrowth was measured from cultured spinal cord neurons under the influence of different Ca²⁺ concentrations. We used fluorodeoxyuridine (FuDr), an antimitotic agent which reduces significantly the proportion of non-neuronal cells in spinal cord cell cultures, to examine the effects of non-neuronal cells on neurite outgrowth. Spinal cord neurons responded to changes in their environment by means of two types of neurite outgrowth: sprouting and elongation. The concurrent presence of non-neuronal cells led to increased sprouting of neurites in certain ionic environments, thus lending support to the idea that non-neuronal cells release diffusible factors which influence sprouting and guide neurite outgrowth.     

Endothelial progenitor cell-conditioned medium promotes angiogenesis and is neuroprotective after spinal cord injury

Endothelial progenitor cells secrete a variety of growth factors that inhibit inflammation, promote angiogenesis and exert neuroprotective effects. Therefore, in this study, we investigated whether endothelial progenitor cell-conditioned medium might have therapeutic effectiveness for the treatment of spinal cord injury using both in vitro and in vivo experiments. After primary culture of bone marrow-derived macrophages, lipopolysaccharide stimulation was used to classically activate macrophages to their proinflammatory phenotype. These cells were then treated with endothelial progenitor cell-conditioned medium or control medium. Polymerase chain reaction was used to determine mR NA expression levels of related inflammatory factors. Afterwards, primary cultures of rat spinal cord neuronal cells were prepared and treated with H_2O_2 and either endothelial progenitor cell-conditioned medium or control medium. Hoechst 33258 and propidium iodide staining were used to calculate the proportion of neurons undergoing apoptosis. Aortic ring assay was performed to assess the effect of endothelial progenitor cell-conditioned medium on angiogenesis. Compared with control medium, endothelial progenitor cell-conditioned medium mitigated the macrophage inflammatory response at the spinal cord injury site, suppressed apoptosis, and promoted angiogenesis. Next, we used a rat model of spinal cord injury to examine the effects of the endothelial progenitor cell-conditioned medium in vivo. The rats were randomly administered intraperitoneal injection of PBS, control medium or endothelial progenitor cell-conditioned medium, once a day, for 6 consecutive weeks. Immunohistochemistry was used to observe neuronal morphology. Terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling assay was performed to detect the proportion of apoptotic neurons in the gray matter. The Basso, Beattie and Bresnahan Locomotor Rating Scale was used to evaluate the recovery of motor function of the bilateral hind limbs after spinal cord injury. Compared with the other two groups, the number of axons was increased, cavities in the spinal cord were decreased, the proportion of apoptotic neurons in the gray matter was reduced, and the Basso, Beattie and Bresnahan score was higher in the endothelial progenitor cell-conditioned medium group. Taken together, the in vivo and in vitro results suggest that endothelial progenitor cell-conditioned medium suppresses inflammation, promotes angiogenesis, provides neuroprotection, and promotes functional recovery after spinal cord injury.     

Cometin is a novel neurotrophic factor that promotes neurite outgrowth and neuroblast migration in vitro and supports survival of spiral ganglion neurons in vivo

Neurotrophic factors are secreted proteins responsible for migration, growth and survival of neurons during development, and for maintenance and plasticity of adult neurons. Here we present a novel secreted protein named Cometin which together with Meteorin defines a new evolutionary conserved protein family. During early mouse development, Cometin is found exclusively in the floor plate and from E13.5 also in dorsal root ganglions and inner ear but apparently not in the adult nervous system. In vitro, Cometin promotes neurite outgrowth from dorsal root ganglion cells which can be blocked by inhibition of the Janus or MEK kinases. In this assay, additive effects of Cometin and Meteorin are observed indicating separate receptors. Furthermore, Cometin supports migration of neuroblasts from subventricular zone explants to the same extend as stromal cell derived factor 1a. Given the neurotrophic properties in vitro, combined with the restricted inner ear expression during development, we further investigated Cometin in relation to deafness. In neomycin deafened guinea pigs, two weeks intracochlear infusion of recombinant Cometin supports spiral ganglion neuron survival and function. In contrast to the control group receiving artificial perilymph, Cometin treated animals retain normal electrically-evoked brainstem response which is maintained several weeks after treatment cessation. Neuroprotection is also evident from stereological analysis of the spiral ganglion. Altogether, these studies show that Cometin is a potent new neurotrophic factor with therapeutic potential.     

Aged median eminence glial cell cultures promote survival and neurite outgrowth of cocultured neurons

We have recently shown that tanycytes, a particular type of glial cell that has morphological and biochemical similarities with radial glial cells, constitute a preferential support for the regeneration of lesioned neurohypophysial axons. The present study was designed to explore the possible neurotrophic role of tanycytes in vitro. Glial cells derived from the median eminence or from the cerebral cortex of 10-day-old rats were cultured for 4–7 weeks. At these times the majority of the cells identified in the median eminence cultures exhibited immunostaining patterns of tanycytes, as detected in the mediobasal hypothalamus of 10-day-old and adult rats, i.e., they were immunoreactive to vimentin (VIM), to DARPP-32 (a dopamine- and adenosine 3′:5′-monophosphate-regulated phosphoprotein), and to a lesser extent to glial fibrillary acidic protein (GFAP) antibodies. On the other hand, the majority of cells in cortex cultures showed immunostaining patterns of astrocytes, i.e., they were intensely immunoreactive to GFAP and VIM antibodies but negative to DARPP-32. Cells obtained from the dissociation of 3-day-old rat mesencephalon, cortex, and hypothalamus were cocultured on these glial monolayers, and the number of surviving neurons and their neurite length were quantified after 8 days. Our data showed that, when compared with astrocytes, tanycytes greatly improved both survival (six- to ten-fold higher) and neurite outgrowth (two- to five-fold longer) of cocultured neurons whatever their origin. Experiments performed by coculturing neurons on millicell inserts placed above the glial monolayers showed that diffusible factors from median eminence glial cells slightly increased survival (1.7-fold higher) of cocultured neurons but had no significant effect on neurite outgrowth. These observations indicate: 1) that aged tanycytes have a capacity to support survival and neurite outgrowth for a variety of postnatal neurons; and 2) that this neurotrophic effect is exerted mainly by means of specific molecules bound to the tanycytic plasmalemma limiting membrane and/or to the extracellular matrix. © 1996 Wiley-Liss, Inc.     

Extracts of muscle biopsies from patients with spinal muscular atrophies inhibit neurite outgrowth from spinal neurons

Preparations derived from embryonic and neonatal chick muscle enhance neurite outgrowth when added to cultures of embryonic chick spinal neurons. In the presence of soluble extracts of biopsied muscle from 15 of 20 patients with spinal muscular atrophy (SMA), the in vitro neurite-promoting activity of neonatal chick muscle was inhibited. There was no comparable inhibition using extracts from 20 age-matched pathologic or morphologically normal controls. The neurite-promoting activity in media conditioned by embryonic myotubes was not inhibited by extracts of the SMA group.     

Effects of calcium ion on neurite outgrowth of rat spinal cord neurons in vitro: The role of non-neuronal cells in regulating neurite sprouting

The interactions of nerve cells with their environment and other cells are specific to different stages of cellular differentiation. Neurite outgrowth was measured from cultured spinal cord neurons under the influence of different Ca²⁺ concentrations. We used fluorodeoxyuridine (FuDr), an antimitotic agent which reduces significantly the proportion of non-neuronal cells in spinal cord cell cultures, to examine the effects of non-neuronal cells on neurite outgrowth. Spinal cord neurons responded to changes in their environment by means of two types of neurite outgrowth: sprouting and elongation. The concurrent presence of non-neuronal cells led to increased sprouting of neurites in certain ionic environments, thus lending support to the idea that non-neuronal cells release diffusible factors which influence sprouting and guide neurite outgrowth.     

Neurite outgrowth through lesions of neonatal opossum spinal cord in culture

The aim of these experiments was to analyze neurite outgrowth during regeneration of opossum spinal cord isolated from Monodelphis domestica and maintained in culture for 3–5 days. Lesions were made by crushing with forceps. In isolated spinal cords of animals aged 3 days, neurites entered the crush and grew along the basal lamina of the pia mater. Growth cones with pleiomorphic appearance containing vesicles, mitochondria and microtubules were abundant in the marginal zone, as were synaptoid contacts with active zones facing basal lamina. In preparations from animals aged 11–12 days, the lesion site was disrupted and contained only degenerating axons, debris and vesicles. Axons and growth cones entered the edge of the lesion but did not extend into it. Lesions in young animals extended over distances of more than 1 mm and contained no radial glia. The damaged area in older preparations was restricted to the crush site with normal astrocytes, oligodendrocytes and neurons immediately adjacent to the lesion. Thus, similar crushes produced more extensive damage in younger spinal cords that were capable of regeneration than in older cords that were not. Dorsal root ganglion fibers labeled with carbocyanine dye (DiI) were observed by video imaging as they grew through lesions. Individual growth cones examined subsequently by electron microscopy had grown again along pial basal lamina. After 5 days in culture dorsal root stimulation gave rise to discharges in ventral roots beyond the lesion indicating that synaptic connections were formed by growing fibers. © 1996 Wiley-Liss, Inc.     

Prion protein as trans-interacting partner for neurons is involved in neurite outgrowth and neuronal survival

Many uncertainties remain regarding the physiological function of the prion protein PrP and the consequences of its conversion into the pathological scrapie isoform in prion diseases. Here, we show for the first time that different signal transduction pathways are involved in neurite outgrowth and neuronal survival elicited by PrP in cell culture of primary neurons. These pathways include the nonreceptor Src-related family member p59(Fyn), PI3 kinase/Akt, cAMP-dependent protein kinase A, and MAP kinase. Regulation of Bcl-2 and Bax expression also correlates with the survival effect elicited by PrP. The combined results, along with our observation that PrP carries the recognition molecule-related HNK-1 carbohydrate, argue strongly for a role of the molecule in neural recognition by interacting with yet unknown heterophilic neuronal receptors, as shown by comparison of neurite outgrowth from neurons of PrP-deficient and wild-type mice.     

Age-dependent patterns and rates of neurite outgrowth from CNS neurons on Schwann cell-derived basal lamina and laminin substrata

In the present study, we have examined the growth characteristics of CNS neurons on type I collagen, detergent-treated collagen (dColl), Schwann cell-derived basal lamina (SC-BL), and purified laminin substrata. Neurons from the cerebral cortex, septal basal forebrain, and lumbosacral spinal cord were obtained from embryonic age (E) 15 and E18 rats and grown in vitro as explants on the test substrata. Neurons from either embryonic age displayed radial neurite outgrowth on collagen and dColl substrata. However, pretreatment of collagen with detergents slightly diminished its ability to support neurite outgrowth, as evidence by the 20-40% decrease in the rate of neurite growth on dColl versus the rate calculated for neurons on collagen. In contrast to the similar growth characteristics of E15 and E18 neurons on collagen and dColl, the pattern of neurite outgrowth for CNS neurons on SC-BL and laminin substrata was age dependent. Most E15 neurons grown on SC-BL extended neurites that grew identically to those observed on dColl; these 'non-orienting' neurites maintained a radial orientation to their outgrowth despite encountering interposing channels of SC-BL and grew at rates equal to that calculated for neurons on dColl. E15 neurons placed on laminin substrata showed similar growth patterns and rates equal to that calculated for neurons on dColl. E15 neurons placed on laminin substrata showed similar growth patterns and rates to neurons on collagen. In contrast, neurons from E18 rats exhibited neurites that preferentially grew in intimate association with SC-BL channels once contact with the channels was established. These 'orienting' neurites faithfully elongated within the SC-BL and demonstrated a 1.4- to 2.0-fold increase in growth rate compared with the sister cultures of neurons grown on dColl. Furthermore, E18 neurons exhibited a 1.4-fold increase in growth on laminin compared with E18 neurons grown on collagen. A minor population of neurites exhibiting similar characteristics to orienting neurites was also observed in E15 cultures. It is hypothesized that orienting and non-orienting neurites reflect the outgrowth of 'regenerating' and 'developing' neurons, respectively, and may indicate an inherent difference in the ability of regenerating and developing neurons to recognize and respond to the same guidance signals.     

Bone mesenchymal stromal cells stimulate neurite outgrowth of spinal neurons by secreting neurotrophic factors

It has been demonstrated that bone mesenchymal stromal cells (BMSCs) stimulate neurite outgrowth from dorsal root ganglion (DRG) neurons. The present in vitro study tested the hypothesis that BMSCs stimulate the neurite outgrowth from spinal neurons by secreting neurotrophic factors. Spinal neurons were cocultured with BMSCs, fibroblasts and control medium in a non-contact system. Neurite outgrowth of spinal neurons cocultured with BMSCs was significantly greater than the neurite outgrowth observed in neurons cultured with control medium or with fibroblasts. In addition, BMSC-conditioned medium increased the length of neurites from spinal neurons compared to those of neurons cultured in the control medium or in the fibroblasts-conditioned medium. BMSCs expressed brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The concentrations of BDNF and GDNF in BMSC-conditioned medium were 132±12 and 70±6 pg ml(-1), respectively. The addition of anti-BDNF and anti-GDNF antibodies to BMSC-conditioned medium partially blocked the neurite-promoting effect of the BMSC-conditioned medium. In conclusion, our results demonstrate that BMSCs promote neurite outgrowth in spinal neurons by secreting soluble factors. The neurite-promoting effect of BMSCs is partially mediated by BDNF and GDNF.