Triptolide Upregulates NGF Synthesis in Rat Astrocyte Cultures

Triptolide (T10), an extract from the traditional Chinese herb, Tripterygium wilfordii Hook F (TWHF), has been shown to attenuate the rotational behavior induced by d-amphetamine and prevent the loss of dopaminergic neurons in the substantia nigra in rat models of Parkinson’s disease. To examine if the neuroprotective effect is mediated by its stimulation of production of neurotrophic factors from astrocytes, we investigated the effect of T10 on synthesis and release of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in rat astrocyte cultures. T10 did not affect the synthesis and release of either BDNF or GDNF. However, it significantly increased NGF mRNA expression. It also increased both intracellular NGF and NGF level in culture medium. These results indicate that the neuroprotective effect of T10 might be mediated, at least in part, via a stimulation of the production and release of NGF in astrocytes. Authors Bing Xue and Jian Jiao contributed equally to this work.     

The protective effects of resveratrol on Schwann cells with toxicity induced by ethanol in vitro

Schwann cells (SCs) are the myelin forming cells in the peripheral nervous system, they play a key role in the pathology of various polyneuropathies and provide trophic support to axons via expression of various neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). Ethanol (EtOH) adversely affected both SCs proliferation and myelin formation in culture. Resveratrol (Res) has been shown to regulate many cellular processes and to display multiple protective and therapeutic effects. Whether Res has protective effects on SCs with EtOH-induced toxicity is still unclear. The protective efficacy of Res on EtOH-treated SCs in vitro was investigated in the present study. Res improved cell viability of the EtOH-treated SCs. Hoechst 33342 staining and terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling analysis showed that the EtOH-induced apoptosis was inhibited by Res. The effects of Res were blocked by the 5′-adenosine monophosphate-activated protein kinase inhibitor Compound C and the silencing information regulator T1 inhibitor nicotinamide. Res could increase the mRNA and protein levels of BDNF and GDNF in the EtOH-treated SCs. However, the EtOH-induced increase of NGF in the SCs is inhibited by Res. The data from the present study indicate that Res protects SCs from EtOH-induced cell death and regulates the expression of neurotrophic factors. Res and its derivative may be effective for the treatment of neuropathic diseases induced by EtOH.     

Intrastriatal administration of human immunodeficiency virus‐1 glycoprotein 120 reduces glial cell‐line derived neurotrophic factor levels and causes apoptosis in the substantia nigra

Uninfected neurons of the substantia nigra (SN) degenerate in human immunodeficiency virus (HIV)‐positive patients through an unknown etiology. The HIV envelope glycoprotein 120 (gp120) causes apoptotic neuronal cell death in the rodent striatum, but its primary neurotoxic mechanism is still under investigation. Previous studies have shown that gp120 causes neurotoxicity in the rat striatum by reducing brain‐derived neurotrophic factor (BDNF). Because glial cell line‐derived neurotrophic factor (GDNF) and BDNF are neurotrophic factors crucial for the survival of dopaminergic neurons of the SN, we investigated whether gp120 reduces GDNF and BDNF levels concomitantly to induce apoptosis. Rats received a microinjection of gp120 or vehicle into the striatum and were sacrificed at various time intervals. GDNF but not BDNF immunoreactivity was decreased in the SN by 4 days in gp120‐treated rats. In these animals, a significant increase in the number of caspase‐3‐ positive neurons, both tyrosine hydroxylase (TH)‐positive and ‐negative, was observed. Analysis of TH immunoreactivity revealed fewer TH‐positive neurons and fibers in a medial and lateral portion of cell group A9 of the SN, an area that projects to the striatum, suggesting that gp120 induces retrograde degeneration of nigrostriatal neurons. We propose that dysfunction of the nigrostriatal dopaminergic system associated with HIV may be caused by a reduction of neurotrophic factor expression by gp120. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of axotomized retinal ganglion cells in adult rats: comparison to and combination with brain-derived neurotrophic factor (BDNF)

Adult rat retinal ganglion cells (RGC) undergo degeneration after optic nerve transection. Studies have shown that exogenously applied neurotrophic factors such as brain-derived neurotrophic factor (BDNF) can attenuate axotomy-induced as well as developmental RGC death. Here, we examined whether glial cell line-derived neurotrophic factor (GDNF), a known neurotrophic factor for dopaminergic neurons and motor neurons, could provide neurotrophic support to RGC in adult rats. We determined whether RGC could retrogradely transport GDNF from their target tissue. After injection into the superior colliculus of adult rats, 125I-GDNF was retrogradely transported to contralateral eyes but not to ipsilateral eyes. The transport of 125I-GDNF could be blocked by coinjection of excess unlabeled GDNF, indicating that it was receptor mediated. We tested whether intravitreally applied GDNF could prevent axotomy-induced RGC degeneration. The RGC were prelabeled with Fluorogold (FG) and axotomized by intraorbital optic nerve transection. GDNF, BDNF (positive control), cytochrome c (negative control), or a GDNF/BDNF combination was injected intravitreally on days 0 and 7. On day 14, FG-labeled RGC were counted from whole-mount retinas. We found that, similar to BDNF, GDNF could significantly attenuate the degeneration of RGC in a dose-dependent fashion. Furthermore, the combination treatment of GDNF and BDNF showed better protection than either factor used individually. Our data indicate that GDNF is a neurotrophic factor for the adult rat RGC. GDNF, like BDNF, may be useful for the treatment of human RGC degenerative diseases.     

Postnatal roles of glial cell line-derived neurotrophic factor family members in nociceptors plasticity

The neurotrophin and glial cell line-derived neurotrophic factor (GDNF) family of growth factors have been extensively studied because of their proven ability to regulate development of the peripheral nervous system. The neurotrophin family,which includes nerve growth factor (NGF), NT-3, NT4/5 and BDNF, is also known for its ability to regulate the function of adult sensory neurons. Until recently, little was known concerning the role of the GNDF-family (that includes GDNF, artemin, neurturin and persephin) in adult sensory neuron function. Here we describe recent data that indicates that the GDNF family can regulate sensory neuron function, that some of its members are elevated in inflammatory pain models and that application of these growth factors produces pain in vivo. Finally we discuss how these two families of growth factors may converge on a single membrane receptor, TRPV 1, to produce long-lasting hyperalgesia.     

A detoxified extract of Rhus verniciflua Stokes upregulated the expression of BDNF and GDNF in the rat brain and the human dopaminergic cell line SH-SY5Y

Rhus verniciflua Stokes (RVS) has traditionally been used as a food supplement and a traditional herbal medicine for centuries in Korea. This study attempted to evaluate the effects of RVS on the expression of Brain derived neurotrophic factor (BDNF) and Glial cell line-derived neurotrophic factor (GDNF) in SH-SY5Y cells and the rat brain. The results indicated that RVS is a potent inducer of Neurotrophic factor (NTF) production both in vitro and in vivo. Treatment with 10 μg/ml and 10 mg/kg RVS for 4 h of SH-SY5Y cells and rats yielded significant increases in BDNF and GDNF protein levels. We also detected BDNF and GDNF immunoreactive neurons in the rat brain. Both BDNF and GDNF-immunohistochemical staining was markedly enhanced in the animals treated with RVS. These results suggest that RVS serves as an ideal adjuvant in regard to regulating NTF expression, and can contribute to neuroprotection in neurodegenerative diseases.     

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.     

Long-term glial cell line-derived neurotrophic factor overexpression in the intact nigrostriatal system in rats leads to a decrease of dopamine and increase of tetrahydrobiopterin production

Parkinson's disease (PD) is characterized by the progressive degeneration of the nigrostriatal dopaminergic system. Brain delivery of glial cell line-derived neurotrophic factor (GDNF) has been shown to protect and restore the dopaminergic pathway in various animal models of PD. However, GDNF overexpression in the dopaminergic pathway leads to a time-dependent down-regulation of tyrosine hydroxylase (TH), a key enzyme in dopamine synthesis. In order to elucidate GDNF-mediated biochemical effects on dopaminergic neurons, we overexpressed GDNF in the intact rat striatum using a lentiviral vector-mediated gene transfer technique. Long-term GDNF overexpression led to increased GTP cyclohydrolase I (GTPCH I) activity and tetrahydrobiopterin (BH4) levels. Further, we observed a down-regulation of TH enzyme activity in morphologically intact striatal dopaminergic nerve terminals, as well as a significant decrease of dopamine levels in striatal tissue samples. These results indicate that long-term GDNF delivery is a major factor affecting dopamine biosynthesis via a direct or indirect modulation of TH and GTPCH I and further underscore the importance of assessing both GDNF dose and delivery duration prior to clinical application in order to circumvent potentially adverse pharmacological effects on the biosynthesis of dopamine.     

Neural Stem-Like Cells Derived from Human Amnion Tissue are Effective in Treating Traumatic Brain Injury in Rat

Although human amnion derived mesenchymal stem cells (AMSC) are a promising source of stem cells, their therapeutic potential for traumatic brain injury (TBI) has not been widely investigated. In this study, we evaluated the therapeutic potential of AMSC using a rat TBI model. AMSC were isolated from human amniotic membrane and characterized by flow cytometry. After induction, AMSC differentiated in vitro into neural stem-like cells (AM-NSC) that expressed higher levels of the neural stem cell markers, nestin, sox2 and musashi, in comparison to undifferentiated AMSC. Interestingly, the neurotrophic factors, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT-3), glial cell derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) were markedly upregulated after neural stem cell induction. Following transplantation in a rat TBI model, significant improvements in neurological function, brain tissue morphology, and higher levels of BDNF, NGF, NT-3, GDNF and CNTF, were observed in the AM-NSC group compared with the AMSC and Matrigel groups. However, few grafted cells survived with minimal differentiation into neural-like cells. Together, our results suggest that transplantation of AM-NSC promotes functional rehabilitation of rats with TBI, with enhanced expression of neurotrophic factors a likely mechanistic pathway.     

Neurotrophic factors (BDNF and GDNF) and the serotonergic system of the brain

Neurotrophic factors play a key role in development, differentiation, synaptogenesis, and survival of neurons in the brain as well as in the process of their adaptation to external influences. The serotonergic (5-HT) system is another major factor in the development and neuroplasticity of the brain. In the present review, the results of our own research as well as data provided in the corresponding literature on the interaction of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) with the 5-HT-system of the brain are considered. Attention is given to comparison of BDNF and GDNF, the latter belonging to a different family of neurotrophic factors and being mainly considered as a dopaminergic system controller. Data cited in this review show that: (i) BDNF and GDNF interact with the 5-HT-system of the brain through feedback mechanisms engaged in autoregulation of the complex involving 5-HT-system and neurotrophic factors; (ii) GDNF, as well as BDNF, stimulates the growth of 5-HT neurons and affects the expression of key genes of the brain 5-HT-system–those coding tryptophan hydroxylase-2 and 5-HT_1A and 5-HT_2A receptors. In turn, 5-HT affects the expression of genes that control BDNF and GDNF in brain structures; (iii) the difference between BDNF and GDNF is manifested in different levels and relative distribution of expression of these factors in brain structures (BDNF expression is highest in hippocampus and cortex, GDNF expression in the striatum), in varying reaction of 5-HT2A receptors on BDNF and GDNF administration, and in different effects on certain types of behavior.     

GDNF对精原干细胞增殖及其分化的调控机制

阐述了胶质细胞源性神经营养因子(GDNF)及其受体与精原干细胞增殖和分化的关系。GDNF能够促进未分化的精原细胞增长,并且可以调节精原干细胞自我更新与分化的微环境,参与其分化的第一步,是精原干细胞存活的重要营养因子。     

Distribution of Brain-Derived Neurotrophic Factor in Rats and Its Changes with Development in the Brain

Abstract: A newly established, sensitive, two-site enzyme-immunoassay system for brain-derived neurotrophic factor (BDNF) is described. Using this system, we investigated the tissue distribution of BDNF and developmental changes in tissue levels of BDNF in rats. The minimal limit of detection of the assay was 3 pg/0.2 ml of assay mixture. BDNF was successfully solubilized from tissues in the presence of guanidine hydrochloride but not in any of the other buffers examined. In the rat brain at 1 month of age, the highest level of BDNF was detected in the hippocampus (5.41 ng/g of wet weight), followed by the hypothalamus (4.23 ng/g) and the septum (1.68 ng/g). In other regions, levels of BDNF ranged between 0.9 and 1.7 ng/g. The level of BDNF in the posterior lobes of the cerebellum from rats at 30 days of age was slightly higher than that in the anterior lobes. The concentration of BDNF increased in all regions of the brain with postnatal development. In peripheral tissues, BDNF was found at very low concentrations (0.65 ng/g in the spleen, 0.21 ng/g in the thymus, and 0.06 ng/g in the liver). The subfractionation of the hippocampal homogenate indicated that ∼50% of BDNF was contained in the crude nuclear fraction. Immunoblots of BDNF-immunoreactive proteins extracted from the hippocampus, hypothalamus, and cerebellum contained doublet bands of protein of ∼14 kDa, a value close to the molecular mass of recombinant human BDNF. Immunocytochemical investigations showed that, in the hippocampus, BDNF was localized in the nucleus of the granule cells in the dentate gyrus and of the cells in the pyramidal cell layer. The frequency of cells that were stained in the dentate gyrus was greater than that of cells in the pyramidal cell layer.     

Chronic tinnitus and BDNF/GDNF CpG promoter methylations: a case–control study

Brain-derived neurotrophic factor (BDNF) and Glial-derived neurotrophic factor (GDNF) are neurotrophic factors that play key roles in the auditory pathway. While the relationship between serum levels and polymorphisms of BDNF/GDNF and chronic tinnitus is emphasized in the literature, there is no study showing the link between the promoter methylations of these genes and tinnitus. For this purpose, the relationship between chronic tinnitus and peripheral blood derived BDNF/GDNF promoter methylations was investigated to identify their role in the pathophysiology of tinnitus. In this case–control study, we examined the possible effects of BDNF/GDNF methylations in the blood samples of patients with tinnitus complaints for more than 3 months. Sixty tinnitus subjects between the ages of 18–55 and 50 healthy control subjects in the same age group who were free of any otorhinolaryngology and systemic disease were selected for examination. Methylation of total 12 CpG sites in BDNF and GDNF promoter regions were determined by the bisulfite-pyrosequencing method. Statistically significant differences were detected between BDNF CpG6 and GDNF CpG3-5-6 methylation ratios in the comparison of control group and the chronic tinnitus patients (P?=?0.002, 0.0005, 0.00003, and 0.0029, respectively). To our knowledge, this is the first study in the literature investigating the relationship between chronic tinnitus and peripheral blood derived BDNF/GDNF promoter methylations. It is believed that the current results might be supported by investigating the relationships between BDNF/GDNF methylations and genotypes in future research using higher sample sizes.

     

Production and Characterization of Recombinant Mouse Brain-Derived Neurotrophic Factor and Rat Neurotrophin-3 Expressed in Insect Cells

Abstract: Bioactive brain-derived neurotrophic factor (BDNF) and neurotrophin-3 were produced using the baculovirus expression system and purified to homogeneity using ion-exchange and reversed-phase chromatography. Yields of purified neurotrophin-3 (300–500 μg/L) were similar to levels reported for baculovirus-expressed nerve growth factor (NGF), whereas initial yields of BDNF were significantly lower (20–50 μg/L). Improved production of BDNF (150–200 μg/L) was achieved by expressing BDNF from a chimeric prepro-NGF/mature BDNF construct using the Trichoplusia ni insect cell line, Tn-5B1-4. Examination of the distribution of BDNF protein from both the nonchimeric prepro-BDNF and the chimeric prepro-NGF/mature BDNF viruses in Sf-21-and Tn-5B1-4-infected cells suggests a specific deficiency in the Tn-5B1-4 cells in processing the nonchimeric precursor. In addition, the vast majority of the BDNF protein at 2 days after infection was intracellular and insoluble. N-terminal amino acid sequencing of purified recombinant BDNF and neurotrophin-3 demonstrated that the insect cells processed their precursors to the correct N-terminus expected for the mature protein. Bioactivity was characterized in vitro on primary neuronal cultures from the CNS and PNS.     

bFGF与BDNF对损伤后成年小鼠离体培养嗅觉上皮细胞发育的影响

在成熟神经系统中,嗅觉上皮（ＯＥ）很特殊,它能不断产生新的神经元。本文用细胞培养、组织化学和免疫细胞化学技术对成年小鼠的ＯＥ进行了研究。实验显示：双极细胞ＮＦ、ＮＳＥ、ＭＡＰ２、ＯＭＰ和ｔａｕ蛋白免疫染色为阳性,但ｋｅｒａｔｉｎ免疫染色为阴性,说明双极细胞是神经元。用不同浓度血清的培养基。离体培养成年小鼠的ＯＥ,观察碱性成纤维细胞生长因子（ｂＦＧＦ）和脑源神经营养因子（ＢＤＮＦ）对ＯＥ细胞数量和突     

Activation of RET tyrosine kinase regulates interleukin-8 production by multiple signaling pathways

Interleukin-8 (IL-8) is known to contribute to human cancer progression through its potential function as a mitogenic, angiogenic, or motogenic factor. We found a high level of IL-8 production in SK-N-MC human primitive neuroectodermal tumor cells transfected with the human RET gene (SK-N-MC (RET) cells) in response to glial cell line-derived neurotrophic factor (GDNF) stimulation. IL-8 was also produced at high levels in TT human medullary thyroid carcinoma and TPC-1 human papillary thyroid carcinoma cell lines both of which express activated RET tyrosine kinase. To investigate which signaling pathways are responsible for IL-8 expression, we treated SK-N-MC (RET) cells with several kinase inhibitors before GDNF stimulation. The results showed that a MEK1 inhibitor, PD98059, a p38MAPK inhibitor, SB202190, and a protein kinase C (PKC) inhibitor, Calphostin C, markedly decreased the IL-8 secretion from SK-N-MC (RET) cells at 24 h after GDNF stimulation. In contrast, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, increased its secretion. These results thus suggested that IL-8 production by RET tyrosine kinase is regulated by multiple signaling pathways.