Amelioration of retarded neurite outgrowth of dorsal root ganglion neurons by overexpression of PKCdelta in diabetic rats

To examine which isoform of protein kinase C (PKC) may be associated with impaired nerve regeneration in diabetes, we compared neurite outgrowth of isolated dorsal root ganglion (DRG) neurons in streptozocin (STZ)-induced diabetic and control rats. Neurite outgrowth was significantly retarded in diabetic neurons. Rottlerin, a PKCdelta specific inhibitor, significantly retracted neurite outgrowth whereas G?6976, an inhibitor specific for classical PKCs, had no effect, suggesting a significant role of PKCdelta in neurite outgrowth of DRG neurons. The expression of phosphorylated PKCdelta, but not total PKCdelta, in DRGs was decreased in diabetic rats. When this reduced expression was restored by overexpressing the PKCdelta in isolated DRG neurons, retardation of neurite outgrowth was significantly reversed in diabetic rats. These results suggest that a decrease in phosphorylated PKCdelta is at least in part responsible for impaired neurite outgrowth in diabetes, and that PKCdelta plays a significant role in the pathogenesis of diabetic neuropathy. This observation provides a useful clue for the treatment of diabetic neuropathy.     

Pituitary adenylate cyclase-activating peptide (PACAP) induces differentiation in the neuronal F11 cell line through a PKA-dependent pathway

PACAP is a peptide with neuroprotective activity, which induces adenylate cyclase and protein kinase A (PKA) activity. PACAP has also been shown to induce neurite outgrowth in PC12 cells and dorsal root ganglion (DRG) neurons. Here, we report that exogenous PACAP38 promotes neurite outgrowth in the F11 neuroblastoma/dorsal DRG hybrid cell line. Using an automated microscopy system, we show that PACAP38 induces a 170-fold increase in neurite length, with an EC50 of 3.1 nM, compared to 3.7 microM for forskolin and 143.4 microM for dibutyril cyclic AMP (dbcAMP). PACAP38 induced a 4-fold increase in the level of phosphorylation of cAMP-responsive element binding protein (CREB) in F11 cells with an EC50 of 130 pM. In contrast a peptide related to PACAP, vasoactive intestinal peptide (VIP) failed to induce CREB phosphorylation or neurite outgrowth in F11 cells. Addition of the nonselective phosphodiesterase inhibitor, isobutyl methylxanthine (IBMX) increased the potency of PACAP at inducing neurite outgrowth by ten-fold. The PKA inhibitor, H89, was a potent inhibitor of PACAP38-induced neurite outgrowth. The delta-opioid receptor agonist, SNC 80, did not inhibit PACAP-induced neurogenesis even though it did reduce CREB phosphorylation. In contrast to previous studies in PC12 cells, PACAP38 failed to show MEK1 activation in F11 cells. PACAP is upregulated in DRG neurons as a result of injury, and F11 cells provide an easily accessible in vitro model for understanding mechanisms underlying PACAP differentiation and neurogenesis.     

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.     

NGF对鸡胚背根神经节神经突起生长作用的机制研究

目的探讨神经生长因子(nerve growth factor,NGF)促进鸡胚背根神经节(dorsal root ganglion,DRG)神经突起生长的作用机制。方法实验采用9 d的鸡胚分离背根神经节,原代培养法,观察鸡胚DRG的体外生长情况。通过半定量PCR检测诱导型一氧化氮合酶(iNOS)mRNA表达,采用NO检测试剂盒检测NO释放水平。结果 NGF能明显促进鸡胚背根神经节神经突起生长,同时可见NGF抑制iNOS mRNA表达,NO检测结果显示,添加NGF培养的背根神经节上清NO分泌水平明显降低,与阴性对照组比较差异显著(P0.05)。结论 NGF可促进鸡胚背根神经节神经突起生长,其作用与其下调iNOS mRNA表达及抑制神经损伤因子NO释放有关。     

体外低频电刺激促进背根神经元突起的生长：上调钙离子介导的脑源性神经营养因子表达可为其途径

摘要 背景：短时低频电刺激已被证明可显著促进周围神经系统损伤后轴突的再生,目前对电刺激是如何促进其突起生长还有待证实。 目的：体外培养背根神经元,观察短时低频电刺激对神经元突起生长的影响,探讨电刺激发挥作用可能的细胞信号分子。 设计、时间及地点：体外培养背根神经元及离体电刺激处理,于2007-05/2008-10在上海交通大学医学院完成。 材料：新生48h Sprague-Dawley大鼠20只(中科院上海生命科学研究所动科所)。 方法：体外培养背根神经元,随机分为两组,正常对照组(n = 6)及电刺激组(n = 8)。电刺激组施予离体电刺激(20Hz, 100μs, 3V),持续作用1h。为探讨电刺激发挥作用经由的细胞信号分子,在施予电刺激前预先加入钙离子通道阻滞剂Nifedipine孵育4小时,再给予电刺激,再次检测各组神经元突起的生长情况。 主要观察指标：β-tubulin染神经元,测量各组神经元突起的长度。RT-PCR、 western blot和ELISA分别检测神经元BDNF的表达和分泌。 结果：短时低频电刺激促进神经元突起的生长,增强其表达和分泌BDNF (P < 0.05)。Nifedipine的使用削弱了电刺激对神经元突起生长及BDNF合成的促进作用 (P < 0.05)。 结论：短时低频电刺激促进体外培养的背根神经元突起的生长及BDNF的合成,初步认为电刺激对神经元突起生长的促进作用,至少通过促发钙内流所致BDNF表达和分泌增多所致。 关键词：电刺激;背根神经元;突起生长;BDNF;Ca2+     

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.     

Rat astrocytes and Schwann cells in culture synthesize nerve growth factor-like neurite-promoting factors

Neurite-promoting activity in feeding medium conditioned by rat astrocytes and Schwann cells in culture was examined. The conditioned medium (CM) from both types of glial cultures stimulated extensive neurite outgrowth from embryonic chick dorsal root ganglia (DRG) as well as pheochromocytoma (PC12) cells. Both the DRG and PC12 cells also produce neurite outgrowth in the presence of nerve growth factor (NGF). With the DRG, the neurite growth rates observed with the glial cell CM were identical to growth rates seen with NGF. Although anti-NGF antibody did not inhibit the neurite outgrowth produced by either of the glial CM, a nerve growth factor radioreceptor assay did detect an NGF-like molecule in both CM. Since the extensive neurite outgrowth stimulated by the glial CM was not mimicked by pure laminin alone, we conclude that the glial neurite promoting factors are distinct from laminin.     

Soluble myelin-associated glycoprotein released from damaged white matter inhibits axonal regeneration

The adult, mammalian CNS does not regenerate after injury largely because of a glial scar and inhibitors of regeneration in myelin. To date, two myelin inhibitors, myelin-associated glycoprotein (MAG) and Nogo, both transmembrane proteins, have been identified. No secreted inhibitors of regeneration have been described. However, a proteolytic fragment of MAG (dMAG), consisting of the entire extracellular domain, is readily released from myelin and is found in vivo. Here, we show, first, that a soluble, chimeric form of MAG (MAG-Fc), when secreted from CHO cells in a collagen gel and hence in the absence of a fixed substrate, inhibits/deflects neurite outgrowth from P6 dorsal root ganglion (DRG) neurons. This inhibition was blocked when a MAG monoclonal antibody was included in the gel and a control chimera sialoadhesin-Fc (Sn-Fc), which, like MAG, binds neurons in a sialic acid-dependent manner but does not inhibit axonal growth, had no effect. Using the same assay system we showed that factors secreted from damaged white matter inhibited/deflected neurite outgrowth. This inhibition was neutralized when a MAG monoclonal antibody was included in the gel and there was no inhibition when white matter from a MAG knockout mouse was used. Factors secreted from damaged white matter from wild-type mice had no effect on neurite outgrowth from E18 DRG neurons. These results show that factors secreted from damaged white matter inhibit axonal regeneration and that the majority of inhibitory activity can be accounted for by dMAG. Thus, released dMAG is likely to play an important role in preventing regeneration, immediately after injury before the glial scar forms.     

Riluzole promotes cell survival and neurite outgrowth in rat sensory neurones in vitro

This study explored the effects of riluzole administration on cell survival and neurite growth in adult and neonatal rat dorsal root ganglion (DRG) neurones in vitro. Neuronal survival was assessed by comparing numbers of remaining neurones in vehicle- and riluzole-treated cultures. A single dose of 0.1 microm riluzole was sufficient to promote neuronal survival in neonatal DRG cultures, whereas repeated riluzole administration was necessary in adult cultures. However, a single administration of riluzole was sufficient to induce neuritogenesis, promote neurite branching and enhance neurite outgrowth in both neonatal and adult DRG cultures. The effects of a single dose of riluzole on adult DRG neurones after peripheral nerve or dorsal root injury were also studied in vitro at 48 h. For both types of injury, riluzole enhanced neurite outgrowth in terms of number, length and branch pattern significantly more on the injured side as compared with the contralateral side. No effect was seen on cell survival. The results suggest that, in addition to its cell survival effects, riluzole has novel growth-promoting effects on sensory neurones in vitro and that riluzole may offer a new way to promote sensory afferent regeneration following peripheral injury.     

Acetylcholinesterase antibody treatment results in neurite detachment and reduced outgrowth from cultured neurons: Further evidence for a cell adhesive role for neuronal acetylcholinesterase

Data from our laboratory and others demonstrate that acetylcholinesterase (AChE) is expressed transiently by neurons during periods of neurite outgrowth preceding synaptogenesis, suggesting an extrasynaptic function for this molecule. These findings, along with reports that AChE shares amino acid sequence homology and structural similarities with known cell adhesion molecules, have led to the theory that, during development, AChE may exert a morphogenic effect through cell adhesion. To further test this hypothesis, we have examined the effects of an AChE monoclonal antibody (MAB304) on neurite outgrowth in primary cultures of rat dorsal root ganglion (DRG) neurons. Short-term, high-concentration antibody treatment produced a rapid detachment of established DRG neurites, which was followed by regrowth upon removal of the antibody from the culture medium. This effect appeared to be site-specific, because other AChE antibodies that were able to detect AChE immunocytochemically failed to produce this disadhesion. Long-term, low-concentration antibody exposure produced a 50% reduction in total area of outgrowth, in which neurites were more densely packed and interlaced compared with the neurites in control cultures. These results extend our previous observations on the outgrowth perturbing effects of AChE inhibitor treatment and provide further evidence that AChE may support neurite outgrowth through a cell adhesive role. J. Neurosci. Res. 53:454–464, 1998. © 1998 Wiley-Liss, Inc.     

Enhanced neurogenesis from neural progenitor cells with G1/S-phase cell cycle arrest is mediated by transforming growth factor beta1

We have previously demonstrated that a G1/S-phase cell cycle blocker, deferoxamine (DFO), increased the number of new neurons from rat neurosphere cultures, which correlated with prolonged expression of cyclin-dependent kinase (cdk) inhibitor p27(kip1) [H. J. Kim et al. (2006)Brain Research, 1092, 1-15]. The present study focuses on neuronal differentiation mechanisms following treatment of neural stem/progenitor cells (NPCs) with a G1/S-phase cell cycle blocker. The addition of DFO (0.5 mm) or aphidicolin (Aph) (1.5 microm) to neurospheres for 8 h, followed by 3 days of differentiation, resulted in an increased number of neurons and neurite outgrowth. DFO induced enhanced expression of transforming growth factor (TGF)-beta1 and cdk5 at 24 h after differentiation, whereas Aph only increased TGF-beta1 expression. DFO-induced neurogenesis and neurite outgrowth were attenuated by administration of a cdk5 inhibitor, roscovitine, suggesting that the neurogenic mechanisms differ between DFO and Aph. TGF-beta1 (10 ng/mL) did not increase neurite outgrowth but rather the number of beta-tubulin III-positive cells, which was accompanied by enhanced p27(kip1) mRNA expression. In addition, TGF-beta receptor type II expression was observed in nestin-positive NPCs. Results indicated that DFO-induced TGF-beta1 signaling activated smad3 translocation from the cytoplasm to the nucleus. In contrast, TGF-beta1 signaling inhibition, via a TGF-beta receptor type I inhibitor (SB-505124), resulted in decreased DFO-induced neurogenesis, in conjunction with decreased p27(kip1) protein expression and smad3 translocation to the nucleus. These results suggest that cell cycle arrest during G1/S-phase induces TGF-beta1 expression. This, in turn, prompts enhanced neuronal differentiation via smad3 translocation to the nucleus and subsequent p27(kip1) activation in NPCs.     

Role for myelin-associated glycoprotein as a functional tenascin-R receptor

The expression of the immunoglobulin superfamily member myelin-associated glycoprotein (MAG) and the extracellular matrix glycoprotein tenascin-R (TN-R) by oligodendrocytes overlaps in time and space. The two molecules can be neurite outgrowth-inhibitory or -promoting depending on the neuronal cell type and the environment in which they are presented. Here we show that the two molecules directly bind to each other in vitro and that binding sites on TN-R localize to two domains, the fibrinogen domain and the epidermal growth factor-like repeat domain with the N-terminal cysteine-rich stretch. We further show by a functional assay, namely the repulsion of MAG-transfected Chinese hamster ovary cells (CHO) cells from a TN-R substrate, that MAG is part of the signalling pathway of TN-R for cell repulsion. When coated as a uniform substrate, MAG was inhibitory for neurite outgrowth of hippocampal and cerebellar neurons in vitro, when compared to poly-L-lysine, while TN-R enhanced neurite outgrowth. When added to MAG, TN-R neutralized the neurite outgrowth-inhibitory effects of MAG, presumably by blocking the neurite outgrowth-inhibitory domain of MAG.     

Neurite outgrowth and GAP-43 mRNA expression in cultured adult rat dorsal root ganglion neurons: Effects of NGF or prior peripheral axotomy

Adult dorsal root ganglion (DRG) cells are capable of neurite outgrowth in vivo and in vitro after axotomy. We have investigated, in cultured adult rat DRG cells, the relative influence of nerve growth factor (NGF) or a prior peripheral nerve lesion on the capacity of these neurons to produce neurites. Since there is evidence suggesting that the growth-associated protein GAP-43 may play a crucial role in axon elongation during development and regeneration, we have also compared the effect of these treatments on GAP-43 mRNA expression. NGF increased the early neurite outgrowth in a subpopulation of DRG cells. This effect was substantially less, however, than that resulting from preaxotomy, which initiated an early and profuse neurite outgrowth in almost all cells. No difference in the expression of GAP-43 mRNA was found between neurons grown in the presence or absence of NGF over 1 week of culture, in spite of the increased growth produced by NGF. In contrast, cultures of neurons that had been preaxotomized showed substantial increase in GAP-43 mRNA and NGF had, as expected, a significant effect on substance P mRNA levels. Two forms of growth may be present in adult DRG neurons: an NGF-independent, peripheral nerve injury-provoked growth associated with substantial GAP-43 upregulation, and an NGF-dependent growth that may underlie branching or sprouting of NGF-sensitive neurons, but which is not associated with increased levels of GAP-43 mRNA. © 1994 Wiley-Liss, Inc.     

Evidence for the direct role of acetylcholinesterase in neurite outgrowth in primary dorsal root ganglion neurons

Dorsal root ganglion (DRG) neurons show a transient peak expression of acetylcholinesterase (AChE) during periods of axonal outgrowth prior to synaptogenesis, suggesting that AChE has a non-enzymatic role during development. We have previously shown that perturbation of cell surface AChE in cultured embryonic rat DRG neurons results in decreased neurite outgrowth and neurite detachment. In this report, we demonstrate a direct correlation between endogenous AChE content and neurite outgrowth in primary DRG neurons. Adenoviral vectors were constructed using full-length rat AChE(T) cDNA in either the sense or antisense orientations to overexpress or knock down AChE expression, respectively. Treatment with the sense-expressing vector produced a 2.5-fold increase in AChE expression and a 2-fold increase in neurite length compared with either untreated or null virus-treated control cells. Conversely, treatment with the antisense-expressing vector reduced AChE expression by 40% and resulted in a reduction in neurite length of similar magnitude. We also observed that overexpression of AChE resulted in greater branching at the distal tips of each primary neurite as well as an increase in cell body size. These findings further indicate that AChE expressed on the axonal surface of developing DRG neurons may modulate their adhesive properties and thereby support axonal development.     

The microenvironment in chronic pancreatitis and pancreatic cancer induces neuronal plasticity

Background Pancreatic neuropathy in chronic pancreatitis (CP) and pancreatic cancer (PCa) is characterized by pancreatic neuropathy, i.e. increased neural density and hypertrophy, which are associated with neuropathic pain. To better understand the mechanism of these neuropathic alterations, we aimed at achieving an in‐vitro simulation of the intrapancreatic neuroplasticity. Methods Dissociated myenteric plexus (MP) and dorsal root ganglia (DRG) neurons of newborn rats were treated with normal human pancreas (NP), CP or PCa tissue extracts. Furthermore, MP and DRG neurons were cultured in supernatants from different pancreatic cancer cell lines (PCC) and human pancreatic stellate cells (hPSC) obtained from either CP or PCa tissues. For analysis, the neurite density, outgrowth, neuronal branching capacity and perikaryonal size were quantified. Key Results Myenteric plexus and DRG neurons grown in CP and PCa tissue extracts built denser networks than in NP extracts. Both neuronal types showed a strong neurite outgrowth, more complex branching pattern and a somatic hypertrophy in CP and PCa extracts. Pancreatic cancer cell supernatants induced a prominent neurite outgrowth, increased neurite density and perikaryonal hypertrophy in MP and DRG neurons. Supernatants of CP‐derived hPSC strongly stimulated neurite outgrowth. Glial density in MP cultures was strikingly increased by PCa tissue extracts. Conclusions & Inferences Intrapancreatic microenvironment in CP and PCa induces neuroplastic alterations under in‐vitro conditions, leading to increased neural density and hypertrophy. Thus, due to its neurotrophic attributes, the intrapancreatic microenviroment in CP and PCa seems to be a key player in the generation of pancreatic neuropathy and neuroplasticity.     

Synthesis of a growth-associated protein by embryonic rat cerebrocortical neurons in vitro

Proteins synthesized by embryonic rat cortical cultures were studied under conditions that were either permissive or nonpermissive to neurite outgrowth. Freshly dissected cortex from embryonic day 17 rat pups was mechanically dissociated and plated on poly(L-lysine) substrate in the presence of (1) serum-free media, which allowed neuronal survival but no outgrowth; (2) serum, which allowed survival of both neurons and glia as well as neurite outgrowth; or (3) a hormone-supplemented defined media, which allowed preferential survival and outgrowth of neurons. In addition, postnatal tissue was cultured as a source of glia. Cultures were pulse-labeled with 35S-methionine 48 hr after plating and the protein synthesis patterns examined by 2-dimensional gel electrophoresis followed by fluorography. The expression of an acidic 50 kDa protein, associated with the particulate fraction of cells, was found to be a prominent correlate of neurite outgrowth. This protein was synthesized in serum- or hormone-treated embryonic cultures showing neurite outgrowth but was undetectable in embryonic cultures without outgrowth or in postnatal glial cultures. By virtue of its migration position on 2-dimensional gels, its presence in a light membrane fraction, and its cleavage products after Staphylococcus aureus protease treatment, the 50 kDa protein appears to be identical to an acidic 43-49 kDa protein that has been identified in several developing and regenerating neural pathways, as well as to the B-50 phosphoprotein. These findings lend support for a critical role of this protein in neural development and demonstrate the feasibility of using primary CNS cell cultures to study its biosynthesis and function.     

Uridine induces differentiation in human neuroblastoma cells via protein kinase C epsilon

Uridine metabolism has an important role in the physiopathology of the nervous system. In this paper, we have explored the effects of exogenous uridine on LAN-5 human neuroblastoma cells. Cells were exposed to uridine for 4 days and cell proliferation, neurite outgrowth, and 160 kDa neurofilament (NF) expression were the parameters measured. Our results showed that 10 microg/ml uridine decreased cell proliferation, this effect being associated with an increase in cell differentiation, as evidenced by neurite outgrowth and NF expression. These effects can be prevented by dipyridamole (10 microM), an inhibitor of nucleotides and nucleosides uptake. In the literature, neuroblastoma cells differentiation has been demonstrated to involve Protein Kinase C epsilon (PKCepsilon). After treatment with uridine, we observed in LAN-5 cells an increase in PKCepsilon protein level. This increase was inhibited by dipyridamole. Moreover, the increase of neurite outgrowth induced by uridine was inhibited by treatment with bisindolylmaleimide I (GF109203X), an inhibitor of PKC. Our data suggest that PKCepsilon is involved in uridine-induced cell differentiation in human neuroblastoma cells.     

Low Density, but not High Density, C6 Glioma Cells Support Dorsal Root Ganglion and Sympathetic Ganglion Neurite Growth

Accumulating evidence suggests that an inhibitory influence of the environment on growth cones plays a crucial role in development and regeneration of neuronal projections. Oligodendrocyte-associated neurite growth inhibiting substance is one of the most extensively studied molecules. Molecular biological studies, however, remain slow in progress. Although finding clonal cells that express such factors would facilitate the analysis of inhibitory influences on neurite growth, few cell lines have been reported to express neurite growth inhibitor. We therefore investigated the possibility of a clonal glial cell line to differentiate and express inhibitory or non-permissive features for neurite outgrowth in culture. We chose the C6 glioblastoma cell line and examined neurite extension from chick dorsal root ganglion (DRG) explants. Neurites from embryonic day 9 DRG extensively grew on C6 cells that were cultured at low cell density, while they failed to grow on C6 cells cultured at high density, even in the presence of nerve growth factor in high concentrations. Membrane extract from high density C6 cells, when used as culture substratum, was less permissive for neurite outgrowth compared to extract from low density cells. Treatment of the membrane extract derived from high density C6 cells with trypsin made it less non-permissive for neurite growth. These results suggest that C6 cells are induced to express a non-permissive property for neurite outgrowth by culturing them at high density.