Neurotrophic Effects of l-DOPA in Postnatal Midbrain Dopamine Neuron/Cortical Astrocyte Cocultures

Abstract: l -DOPA is toxic to catecholamine neurons in culture, but the toxicity is reduced by exposure to astrocytes. We tested the effect of l -DOPA on dopamine neurons using postnatal ventral midbrain neuron/cortical astrocyte cocultures in serum-free, glia-conditioned medium. l -DOPA (50 µ M ) protected against dopamine neuronal cell death and increased the number and branching of dopamine processes. In contrast to embryonically derived glia-free cultures, where l -DOPA is toxic, postnatal midbrain cultures did not show toxicity at 200 µ M l -DOPA. The stereoisomer d -DOPA (50–400 µ M ) was not neurotrophic. The aromatic amino acid decarboxylase inhibitor carbidopa (25 µ M ) did not block the neurotrophic effect. These data suggest that the neurotrophic effect of l -DOPA is stereospecific but independent of the production of dopamine. However, l -DOPA increased the level of glutathione. Inhibition of glutathione peroxidase by l -buthionine sulfoximine (3 µ M for 24 h) blocked the neurotrophic action of L-DOPA. N -Acetyl- l -cysteine (250 µ M for 48 h), which promotes glutathione synthesis, had a neurotrophic effect similar to that of l -DOPA. These data suggest that the neurotrophic effect of l -DOPA may be mediated, at least in part, by elevation of glutathione content.     

Neurotrophic factors and their receptors in axonal regeneration and functional recovery after peripheral nerve injury

Over a half a century of research has confirmed that neurotrophic factors promote the survival and process outgrowth of isolated neurons in vitro. The mechanisms by which neurotrophic factors mediate these survival-promoting effects have also been well characterized. In vivo, peripheral neurons are critically dependent on limited amounts of neurotrophic factors during development. After peripheral nerve injury, the adult mammalian peripheral nervous system responds by making neurotrophic factors once again available, either by autocrine or paracrine sources. Three families of neurotrophic factors were compared, the neurotrophins, the GDNF family of neurotrophic factors, and the neuropoetic cytokines. Following a general overview of the mechanisms by which these neurotrophic factors mediate their effects, we reviewed the temporal pattern of expression of the neurotrophic factors and their receptors by axotomized motoneurons as well as in the distal nerve stump after peripheral nerve injury. We discussed recent experiments from our lab and others which have examined the role of neurotrophic factors in peripheral nerve injury. Although our understanding of the mechanisms by which neurotrophic factors mediate their effects in vivo are poorly understood, evidence is beginning to emerge that similar phenomena observed in vitro also apply to nerve regeneration in vivo.     

Neurotrophic Factors Attenuate Glutamate-Induced Accumulation of Peroxides, Elevation of Intracellular Ca2+ Concentration, and Neurotoxicity and Increase Antioxidant Enzyme Activities in Hippocampal Neurons

Abstract: Exposure of cultured rat hippocampal neurons to glutamate resulted in accumulation of cellular peroxides (measured using the dye 2,7-dichlorofluorescein). Peroxide accumulation was prevented by an N -methyl- d -aspartate (NMDA) receptor antagonist and by removal of extracellular Ca²⁺, indicating the involvement of NMDA receptor-induced Ca²⁺ influx in peroxide accumulation. Glutamate-induced reactive oxygen species contributed to loss of Ca²⁺ homeostasis and excitotoxic injury because antioxidants (vitamin E, propyl gallate, and N-tert -butyl-α-phenylnitrone) suppressed glutamate-induced elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and cell death. Basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF), but not ciliary neurotrophic factor, each suppressed accumulation of peroxides induced by glutamate and protected neurons against excitotoxicity. bFGF, NGF, and BDNF each increased (to varying degrees) activity levels of superoxide dismutases and glutathione reductase. NGF increased catalase activity, and BDNF increased glutathione peroxidase activity. The ability of the neurotrophic factors to suppress glutamate toxicity and glutamate-induced peroxide accumulation was attenuated by the tyrosine kinase inhibitor genistein, indicating the requirement for tyrosine phosphorylation in the neuroprotective signal transduction mechanism. The data suggest that glutamate toxicity involves peroxide production, which contributes to loss of Ca²⁺ homeostasis, and that induction of antioxidant defense systems is a mechanism underlying the [Ca²⁺]_i-stabilizing and excitoprotective actions of neurotrophic factors.     

重组人睫状神经营养因子的复性研究

利用凝胶层析对人睫状神经营养因子原核基因工程产品进行复性研究,发现此方法的复性率高于稀释透析法.而且在复性的同时也进行了一步纯化工作.该方法简单、迅速、高效、重复性好,可用于规模生产.     

脊髓外科手术术后精神障碍患者发病影响因素及抑制性神经递质水平、神经营养因子表达变化情况分析

摘要 目的：探讨与分析脊髓外科手术术后精神障碍患者发病影响因素及抑制性神经递质水平、神经营养因子表达变化情况。方法：选择2016年9月到2021年5月本院完成脊髓外科手术的患者83例作为研究对象,检测血清抑制性神经递质水平、神经营养因子（NTFs）表达水平。所有患者都给予抑郁自评量表（SDS）调查、执行功能行为评定量表成人版自评问卷（BRIEF-A）评分并进行相关性分析。结果：83例患者术后平均SDS评分为45.10±2.87分,判定为精神障碍23例（精神障碍组）,占比27.7 %。精神障碍组的性别、年龄、手术时间、术中出血量与非精神障碍组对比无差异（P>0.05）,精神障碍组的饮酒、术后清醒时间与非精神障碍组对比有差异（P<0.05）。精神障碍组的BRI自我控制、情感控制、转移、抑制等评分与MI任务启动、任务监督、工作记忆、计划、组织评分都高于非精神障碍组（P<0.05）。精神障碍组的血清NTFs含量低于非精神障碍组,血清HA与5-HT含量高于非精神障碍组（P<0.05）。在83例患者中,Pearson分析显示SDS评分与饮酒、术后清醒时间、血清NTFs、NA、5-HT含量都存在相关性（P<0.05）;二分类logistic逐步回归显示术后清醒时间、血清NTFs、NA、5-HT含量都为导致脊髓外科手术术后精神障碍患者发病的重要因素（P<0.05）。结论：脊髓外科手术术后精神障碍的发生较常见,可导致患者认知与执行功能降低,多伴随有抑制性神经递质水平表达上升与神经营养因子表达下降,血清NTFs、NE、5-HT含量都为导致精神障碍发病的重要因素。     

Tetramethylpyrazine induces the release of BDNF from BM-MSCs through activation of the PI3K/AKT/CREB pathway

Compelling evidences suggest that transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) can be therapeutically effective for central nervous system (CNS) injuries and neurodegenerative diseases. The therapeutic effect of BM-MSCs mainly attributes to their differentiation into neuron-like cells which replace injured and degenerative neurons. Importantly, the neurotrophic factors released from BM-MSCs can also rescue injured and degenerative neurons, which plays a biologically pivotal role in enhancing neuroregeneration and neurological functional recovery. Tetramethylpyrazine (TMP), the main bioactive ingredient extracted from the traditional Chinese medicinal herb Chuanxiong, has been reported to promote the neuronal differentiation of BM-MSCs. This study aimed to investigate whether TMP regulates the release of neurotrophic factors from BM-MSCs. We examined the effect of TMP on brain-derived neurotrophic factor (BDNF) released from BM-MSCs and elucidated the underlying molecular mechanism. Our results demonstrated that TMP at concentrations of lower than 200 μM increased the release of BDNF in a dose-dependent manner. Furthermore, the effect of TMP on increasing the release of BDNF from BM-MSCs was blocked by inhibiting the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT)/cAMP-response element binding protein (CREB) pathway. Therefore, we concluded that TMP could induce the release of BDNF from BM-MSCs through activation of the PI3K/AKT/CREB pathway, leading to the formation of neuroprotective and proneurogenic microenvironment. These findings suggest that TMP possesses novel therapeutic potential to promote neuroprotection and neurogenesis through improving the neurotrophic ability of BM-MSCs, which provides a promising nutritional prevention and treatment strategy for CNS injuries and neurodegenerative diseases via the transplantation of TMP-treated BM-MSCs.     

Long-term changes in neurotrophic factor expression in distal nerve stump following denervation and reinnervation with motor or sensory nerve

Several factors have been proposed to account for poor motor recovery after prolonged denervation, including motor neuron cell death and incomplete or poor regeneration of motor fibers into the muscle. Both may result from failure of the muscle and the distal motor nerve stump to continue expression of neurotrophic factors following delayed muscle reinnervation. This study investigated whether regenerating motor or sensory axons modulate distal nerve neurotrophic factor expression. We found that transected distal tibial nerve up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) mRNA, down-regulated neurotrophin-3 and ciliary neurotrophic factor mRNA, and that although these levels returned to normal with regeneration, the chronically denervated distal nerve stump continued to express these neurotrophic factors for at least 6 months following injury. A sensory nerve (the cutaneous saphenous nerve) sutured to distal tibial nerve lowered injury-induced BDNF and GDNF mRNA levels in distal stump, but repair with a mixed nerve (peroneal, containing muscle and cutaneous axons) was more effective. Repair with sensory or mixed nerves did not affect nerve growth factor or neurotrophin-3 expression. Thus, distal nerve contributed to a neurotrophic environment for nerve regeneration for at least 6 months, and sensory nerve repair helped normalize distal nerve neurotrophic factor mRNA expression following denervation. Furthermore, as BDNF and GDNF levels in distal stump increased following denervation and returned to control levels following reinnervation, their levels serve as markers for the status of regeneration by either motor or sensory nerve.     

Basic Fibroblast Growth Factor Selectively Increases AMPA-Receptor Subunit GluR1 Protein Level and Differentially Modulates Ca2+ Responses to AMPA and NMDA in Hippocampal Neurons

Abstract: The excitatory neurotransmitter glutamate is believed to play important roles in development, synaptic plasticity, and neurodegenerative conditions. Recent studies have shown that neurotrophic factors can modulate neuronal excitability and survival and neurite outgrowth responses to glutamate, but the mechanisms are unknown. The present study tested the hypothesis that neurotrophic factors modulate responses to glutamate by affecting the expression of specific glutamate-receptor proteins. Exposure of cultured embryonic rat hippocampal cells to basic fibroblast growth factor (bFGF) resulted in a concentration-dependent increase in levels of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor subunit GluR1 protein as determined by western blot, dot-blot, and immunocytochemical analyses. In contrast, bFGF did not alter levels of GluP2/3, GluR4, or the NMDA-receptor subunit NR1. Nerve growth factor did not affect GluR1 levels. Calcium-imaging studies revealed that elevation of [Ca²⁺]_i, resulting from selective AMPA-receptor activation, was enhanced in bFGF-pretreated neurons. On the other hand, [Ca²⁺]_i responses to NMDA-receptor activation were suppressed in bFGF-treated neurons, consistent with previous studies showing that bFGF can protect neurons against NMDA toxicity. Moreover, neurons pretreated with bFGF were relatively resistant to the toxicities of glutamate and AMPA, both of which were shown to be mediated by NMDA receptors. These data suggest that differential regulation of the expression of specific glutamate-receptor subunits may be an important mechanism whereby neurotrophic factors modulate activity-dependent neuronal plasticity and vulnerability to excitotoxicity.     

Brain-derived neurotrophic factor promotes neurite growth and survival of antennal lobe neurons in brain from the silk moth, Bombyx mori in vitro

This study was conducted to investigate effects of brain-derived neurotrophic factor on the neurite growth and the survival rate of antennal lobe neurons in vitro, and secretion of brain-derived neurotrophic factor-like neuropeptide from brain into hemolymph in the silk moth, Bombyx mori. In primary culture of antennal lobe neurons with brain-derived neurotrophic factor, it promoted both a neurite extension of putative antennal lobe projection neurons and an outgrowth of branches from principal neurites of putative antennal interneurons with significance (p<0.05). Brain-derived neurotrophic factor also increased significantly a survival rate of antennal lobe neurons (p<0.05). Results from immunolabeling of brain and retrocerebral complex, and ELISA assay of hemolymph showed that brain-derived neurotrophic factor-like neuropeptide was synthesized by both median and lateral neurosecretory cells of brain, then transported to corpora allata for storage, and finally secreted into hemolymph for action. These results will provide valuable information for differentiation of invertebrate brain neurons with brain-derived neurotrophic factor.     

Brain-derived neurotrophic factor levels in the nervous system of wild-type and neurotrophin gene mutant mice

Although brain-derived neurotrophic factor is the most abundant and widely distributed neurotrophin in the nervous system, reproducible determinations of its levels have been hampered by difficulties in raising suitable monoclonal antibodies. Following immunization of mice with recombinant fish and mammalian brain-derived neurotrophic factor, monoclonal antibodies were generated and used in an immunoassay based on the recognition of two different epitopes. Neither antibody crossreacts with neurotrophin homodimers other than brain-derived neurotrophic factor, although reactivity was detected with brain-derived neurotrophic factor/neurotrophin-3 heterodimers. As both nerve growth factor and neurotrophin-3 are known to affect the development of a variety of neurons expressing the brain-derived neurotrophic factor (bdnf) gene, this assay was used to determine levels in tissues isolated from newborn mice carrying a null mutation in the nerve growth factor (ngf) or the neurotrophin-3 (nt3) gene. Marked differences were observed between mutants and wild-type littermates in the PNS, but not in the CNS, suggesting that neither nerve growth factor nor neurotrophin-3 is a unique regulator of brain-derived neurotrophic factor levels in the newborn mouse CNS.     

Inhibition of protein synthesis prevents cell death in sensory and parasympathetic neurons deprived of neurotrophic factor in vitro

Shortly after neurons begin to innervate their targets in the developing vertebrate nervous system they become dependent on the supply of a neurotrophic factor, such as nerve growth factor (NGF) for survival. Recently, Martin et al. (1988) have shown that inhibiting protein synthesis prevents the death of NGF-deprived sympathetic neurons, suggesting that NGF promotes neuronal survival by suppressing an active cell death program. To determine if other neurotrophic factors may regulate neuronal survival by a similar mechanism we examined the effects of inhibiting protein and RNA synthesis in other populations of embryonic neurons that require different neurotrophic factors, namely: 1) trigeminal mesencephalic neurons, a population of proprioceptive neurons that are supported by brain-derived neurotrophic factor; 2) dorsomedial trigeminal ganglion neurons, a population of cutaneous sensory neurons that are supported by NGF; 3) and ciliary ganglion neurons, a population of parasympathetic neurons that are supported by ciliary neuronotrophic factor. Blocking either protein or RNA synthesis rescued all three populations of neurons from cell death induced by neurotrophic factor deprivation in vitro. Thus, at least three different neurotrophic factors appear to promote survival by a similar mechanism that may involve the suppression of an endogenous cell death program.     

The GDNF/RET signaling pathway and human diseases

Glial cell line-derived neurotrophic factor (GDNF) and related molecules, neurturin, artemin and persephin, signal through a unique multicomponent receptor system consisting of RET tyrosine kinase and glycosyl-phosphatidylinositol-anchored coreceptor (GFR1–4). These neurotrophic factors promote the survival of various neurons including peripheral autonomic and sensory neurons as well as central motor and dopamine neurons, and have been expected as therapeutic agents for neurodegenerative diseases. In addition, it turned out that the GDNF/RET signaling plays a crucial role in renal development and regulation of spermatogonia differentiation. RET mutations cause several human diseases such as papillary thyroid carcinoma, multiple endocrine neoplasia types 2A and 2B, and Hirschsprung's disease. The mutations resulted in RET activation or inactivation by various mechanisms and the biological properties of mutant proteins appeared to be correlated with disease phenotypes. The signaling pathways activated by GDNF or mutant RET are being extensively investigated to understand the molecular mechanisms of disease development and the physiological roles of the GDNF family ligands.     

Structural and functional characterization of oversulfated chondroitin sulfate/dermatan sulfate hybrid chains from the notochord of hagfish. Neuritogenic and binding activities for growth factors and neurotrophic factors

Oversulfated chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains were purified from the notochord of hagfish. The chains (previously named CS-H for hagfish) have an average molecular mass of 18 kDa. Composition analysis using various chondroitinases demonstrated a variety of D-glucuronic acid (GlcUA)- and L-iduronic acid (IdoUA)-containing disaccharides variably sulfated with a higher proportion of GlcUA/IdoUA-GalNAc 4,6-O-disulfate, revealing complex CS/DS hybrid features. The hybrid chains showed neurite outgrowth-promoting activity of an axonic nature, which resembled the activity of squid cartilage CS-E and which was abolished fully by chondroitinase ABC digestion and partially by chondroitinase AC-I or B digestion, suggesting the involvement of both GlcUA and IdoUA in neuritogenic activity. Purified CS-H exhibited interactions in a BIAcore system with various heparin-binding proteins and neurotrophic factors (viz. fibroblast growth factor-2, -10, -16, and -18; midkine; pleiotrophin; heparin-binding epidermal growth factor-like growth factor; vascular endothelial growth factor; brain-derived neurotrophic factor; and glial cell line-derived neurotrophic factor), most of which are expressed in the brain, although fibroblast growth factor-1 and ciliary neurotrophic factor showed no binding. Kinetic analysis revealed high affinity binding of these growth factors and, for the first time, of the neurotrophic factors. Competitive inhibition revealed the involvement of both IdoUA and GlcUA in the binding of these growth factors, suggesting the importance of the hybrid nature of CS-H for the efficient binding of these growth factors. These findings, together with those from the recent analysis of brain CS/DS chains from neonatal mouse and embryonic pig (Bao, X., Nishimura, S., Mikami, T., Yamada, S., Itoh, N., and Sugahara, K. (2004) J. Biol. Chem. 279, 9765-9776), suggest physiological roles of the hybrid chains in the development of the brain.     

Qualitative and quantitative re-evaluation of epidermal growth factor-ErbB1 action on developing midbrain dopaminergic neurons in vivo and in vitro: target-derived neurotrophic signaling (Part 1)

Although epidermal growth factor (EGF) receptor (ErbB1) is implicated in Parkinson's disease and schizophrenia, the neurotrophic action of ErbB1 ligands on nigral dopaminergic neurons remains controversial. Here, we ascertained colocalization of ErbB1 and tyrosine hydroxylase (TH) immunoreactivity and then characterized the neurotrophic effects of ErbB1 ligands on this cell population. In mesencephalic culture, EGF and glial-derived neurotrophic factor (GDNF) similarly promoted survival and neurite elongation of dopaminergic neurons and dopamine uptake. The EGF-promoted dopamine uptake was not inhibited by GDNF-neutralizing antibody or TrkB-Fc, whereas EGF-neutralizing antibody fully blocked the neurotrophic activity of the conditioned medium that was prepared from EGF-stimulated mesencephalic cultures. The neurotrophic action of EGF was abolished by ErbB1 inhibitors and genetic disruption of erbB1 in culture. In vivo administration of ErbB1 inhibitors to rat neonates diminished TH and dopamine transporter (DAT) levels in the striatum and globus pallidus but not in the frontal cortex. In parallel, there was a reduction in the density of dopaminergic varicosities exhibiting intense TH immunoreactivity. In agreement, postnatal erbB1-deficient mice exhibited similar decreases in TH levels. Although neurotrophic supports to dopaminergic neurons are redundant, these results confirm that ErbB1 ligands contribute to the phenotypic and functional development of nigral dopaminergic neurons.     

Neurotrophic factors and neurologic disease.

Discovered only 40 years ago, nerve growth factor is the prototypic neurotrophic factor. By binding to specific receptors on certain neurons in the peripheral nervous system and brain, nerve growth factor acts to enhance their survival, differentiation, and maintenance. In recent years, many additional neurotrophic factors have been discovered; some are structurally related to nerve growth factor while others are distinct from it. The robust actions of neurotrophic factors have suggested their use in preventing or lessening the dysfunction and death of neurons in neurologic disorders. We review the progress in defining neurotrophic factors and their receptors and in characterizing their actions. We also discuss some of the uses of neurotrophic factors in animal models of disease. Finally, we discuss how neurotrophic factors could be implicated in the pathogenesis of neurologic disorders.     

Trophic factors in neurodegenerative disorders

Neuronal survival is dependent on continuous trophic stimulation by neurotrophic factors. Anterograde and retrograde transport of neurotrophic factors and their receptors within neurites is essential for the communication of these survival signals. Lack of neurotrophic input has been proposed as a pathomechanism leading to neurodegenerative disease. The present short review provides a summary of some of the recent data on neurotrophic factors in neurodegenerative disorders and describes how disturbances of axonal trafficking might deprive neurons from trophic input.     

Dopamine D2 Receptor-Mediated Regulation of Serotonin Extracellular Concentration in the Dorsal Raphe Nucleus of Freely Moving Rats

Abstract: The neuropeptide-inducing activity of neurotrophic factors was tested in cultured cerebral cortical neurons. Brain-derived neurotrophic factor (BDNF) specifically increased contents of the neuropeptides somatostatin (SOM) and neuropeptide Y (NPY), but its effect on contents of cholecystokinin octapeptide and GABA was much less significant. The maximal induction of NPY content (15-fold increase) was achieved by 20 ng/ml of BDNF. These changes were also reproduced at the mRNA level. In contrast, neurotrophin-3 was much less potent at increasing NPY and SOM contents, and nerve growth factor had no effect on them. The expression of mRNA for NPY and SOM was fully dependent on the presence of BDNF in culture but irrelevant to the survival-promoting activity of BDNF, which has been reported previously. Most of the NPY immunoreactivity induced by BDNF was colocalized with glutamate decarboxylase immunoreactivity in cultured cortical neurons. These results suggest that BDNF regulates the peptidergic expression of GABAergic neurons in the cerebral cortex.     

Effect of Tie-2 conditional deletion of BDNF on atherosclerosis in the ApoE null mutant mouse

The reduced expression (haplodeficiency) of the main brain derived neurotrophic factor receptor, namely TrkB is associated with reduced atherosclerosis, smooth muscle cells accumulation and collagen content in the lesion. These data support the concept that brain derived neurotrophic factor of vascular origin may contribute to atherosclerosis. However, to date, no experimental approach was possible to investigate this issue due to the lethality of brain derived neurotrophic factor null mice. To overcome these limitations, we generated a mouse model with a conditional deletion of brain derived neurotrophic factor in endothelial cells (Tie-2 Cre recombinase) on an atherosclerotic prone background (apolipoprotein E knock out) and investigated the effect of conditional brain derived neurotrophic factor deficiency on atherosclerosis. Despite brain derived neurotrophic factor reduction in the vascular wall, mice with conditional deletion of brain derived neurotrophic factor did not develop larger atherosclerotic lesion compared to controls. Smooth muscle cell content as well as the distribution of total and fibrillar collagen was similar in the atherosclerotic lesions from mice with brain derived neurotrophic factor conditional deficiency compared to controls. Finally an extended gene expression analysis failed to identify pro-atherogenic gene expression patterns among the animal with brain derived neurotrophic factor deficiency. In spite of the reduced brain derived neurotrophic factor expression, similar atherosclerosis development was observed in the brain derived neurotrophic factor conditional deficient mouse compared to controls. These pieces of evidence indicate that endothelial derived-brain derived neurotrophic factor is not a pro-atherogenic factor and would rather suggest to investigate the role of other TrkB activators on atherosclerosis.