Cell Line-derived Neurotrophic Factor mRNA Research Articles

Antidepressant amitriptyline-induced matrix metalloproteinase-9 activation is mediated by Src family tyrosine kinase, which leads to glial cell line-derived neurotrophic factor mRNA expression in rat astroglial cells.

BackgroundAstrocytes have been implicated in the pathophysiology of mood disorders and in the mechanism of the pharmacological effects of antidepressant drugs by the production of neurotrophic/growth factors. Previous studies have identified astrocyte‐expressed Gαi/o‐coupled lysophosphatidic acid receptor 1 (LPAR1), as being involved in antidepressant‐induced production of glial cell line‐derived neurotrophic factor (GDNF) and matrix metalloproteinase‐9 (MMP‐9) activation, an important step in the production of GNDF. However, the precise mechanism of MMP‐9 activation by antidepressants has yet to be identified, in particular the intracellular signaling pathway between LPAR1/Gαi/o and MMP‐9.Methods and ResultsTreatment of rat C6 astroglial cells (C6 cells) with amitriptyline increased Src family tyrosine kinase phosphorylation in a time and concentration‐dependent manner. Amitriptyline‐induced GDNF mRNA expression was blocked by Src family tyrosine kinase inhibitors. In addition, inhibiting Src family tyrosine kinase blocked amitriptyline‐induced zymographic MMP‐9 activation in C6 cells. The amitriptyline‐induced zymographic MMP‐9 activity was completely blocked by selective inhibition of Gαi/o protein and LPAR1. Furthermore, the amitriptyline‐induced Src family tyrosine kinase phosphorylation was blocked by LPAR1, but not MMP‐9 inhibition, indicating that Src family tyrosine kinase involvement is downstream of LPAR1.ConclusionsThe current findings suggest that the pharmacological effect of antidepressant such as amitriptyline is mediated through an intracellular signaling pathway via the LPAR1/Gαi/o/Src family tyrosine kinase, which leads to MMP‐9 activation and GDNF production.

Lactobacillus plantarum YS-3 Prevents Activated Carbon-Induced Constipation in Mice.

The aim of this study was to determine the effects of Lactobacillus plantarum YS-3 (LP-YS3) on activated carbon-induced constipation in Kunming mice. The results of the experiment show that the antigastric acid activity and bile salt tolerance of LP-YS3 were stronger than those of Lactobacillus bulgaricus (LB). LP-YS3 inhibited loss of body weight caused by constipation and further reductions in fecal weight, particle number, and water content in mice. Moreover, LP-YS3 elevated the gastrointestinal transit rate and reduced the time required for initial black stool defecation. LP-YS3 also elevated motilin (MTL), endothelin (ET), acetylcholinesterase (AChE), substance P (SP), and VIP serum levels and reduced somatostatin (SS) levels in constipated mice. Hematoxylin-eosin (H&E) staining revealed that high concentration of LP-YS3 reduced the incidence of injuries to small intestine villi and the intestinal wall compared to carbon-induced constipation groups. Reverse transcription-polymerase chain reaction and western blot experiments demonstrated that LP-YS3 upregulated c-Kit, stem cell factor, and glial cell line-derived neurotrophic factor mRNA and protein expression and downregulated transient receptor potential vanilloid 1 and nitric oxide synthase expression in small intestine tissue from constipated mice. In conclusion, high concentrations of LP-YS3 had stronger and more beneficial effects than LB. Based on these results, we conclude that LP-YS3 can effectively inhibit constipation.

The expression of glial cell line-derived neurotrophic factor mRNA by antidepressants involves matrix metalloproteinase-9 activation in rat astroglial cells

Neurotrophic/growth factors derived from glial cells, especially astrocytes, have been implicated in mood disorders and the pharmacological effects of antidepressant drugs. Previous studies demonstrated that the release of glial cell line-derived neurotrophic factor (GDNF) induced by the tricyclic antidepressant amitriptyline was significantly inhibited by a broad-spectrum matrix metalloproteinase (MMP) inhibitor in rat C6 astroglial cells (C6 cells). However, it is unknown whether amitriptyline affects MMP enzymatic activity or expression, and the MMP subtype has yet to be identified. The current study measured the effect of antidepressants on MMP activity with gelatin zymography, an in vitro assay for enzymatic activity, in C6 cells and primary cultured rat astrocytes (primary astrocytes). Treatment with amitriptyline increased zymographic MMP-9 activity without changing MMP-9 mRNA expression in C6 cells. Several different classes of antidepressants significantly increased zymographic MMP-9 activity in C6 cells and primary astrocytes, whereas antipsychotic drugs without antidepressant pharmacological activity did not. The amitriptyline-induced expression of GDNF mRNA was completely blocked by selective inhibition of MMP-9 in C6 cells. Treatment of C6 cells and primary astrocytes with exogenous recombinant MMP-9 increased GDNF mRNA expression, similar to that observed with amitriptyline. Inhibiting MMP-3 blocked amitriptyline-induced zymographic MMP-9 activation in C6 cells and primary astrocytes, indicating that MMP-3 is necessary for MMP-9 activity. The current study suggests that MMP-9 activation is indispensable in the amitriptyline-induced expression of GDNF mRNA in astrocytes and further supports a role of astrocytic neurotrophic/growth factors in the pharmacological effect of antidepressants.

Muscular mechanical hyperalgesia after lengthening contractions in rats depends on stretch velocity and range of motion.

The current study investigated stretch variables and mechanical factors of lengthening contractions (LC) in the processes leading to muscular mechanical hyperalgesia in rats to understand mechanisms underpinning delayed onset muscle soreness (DOMS). Under isoflurane anaesthesia, ankle extensor muscles were loaded with repetitive LC with angular stretch velocities (50°, 100°, 200° and 400°/s) at a fixed range of motion (ROM) of 90°, and with ROMs (30°, 60°, 90° and 120°) at a fixed velocity of 200°/s. Mechanical hyperalgesia was observed in a velocity- and ROM-dependent manner. Under the fixed ROM, integrated torque generated during LC (iTq[max] ) was inversely correlated with the velocity, but the rate of torque increase during LC (rTq[max] ) was positively and significantly correlated with the velocity, and the magnitude of hyperalgesia was correlated with rTq[max] (p<0.001). When the velocity was fixed, iTq[max] was significantly correlated with ROM, and the magnitude of hyperalgesia was correlated with iTq[max] (p<0.01). Necrotic myofibres were observed only sparsely (<0.8%) after any of the LC protocols tested. Up-regulation of nerve growth factor and glial cell line-derived neurotrophic factor mRNA in the muscle was positively correlated with the increases in the LC velocity and ROM (p<0.05~0.001). Both velocity and ROM are pivotal variables determining the initiation of mechanical hyperalgesia. Neurotrophic factor-mediated peripheral mechanisms, but apparently not inflammatory changes caused by myofibre damage, are responsible for the mechanical hyperalgesia. Mechanical hyperalgesia appears after LC in a stretch velocity- and range of motion-dependent manner. The rate of torque increase and integrated torque are the crucial factors. Neurotrophic factor-mediated peripheral pain mechanisms without robust inflammatory changes caused by myofibre damage were required for this mechanical hyperalgesia.

Induction of Neurotrophic and Differentiation Factors in Neural Stem Cells by Valproic Acid

Valproic acid (VPA), a short-chain fatty acid, is used clinically as an anticonvulsant and mood stabilizer. Valproic acid also inhibits histone deacetylase activity, which is associated with histone hyperacetylation and changes in gene expression. In this study, we examined the effects of VPA on the expression of selected neurotrophic and differentiation factors in C17.2 neural stem cells. Reverse transcription-polymerase chain reaction analysis revealed a significant increase in conserved dopamine neurotrophic factor (CDNF) and glial cell line-derived neurotrophic factor mRNA expression, after treatment with clinically relevant concentrations of VPA (0.5 or 1.0 mM) for 24 hr. Increases in the protein expression of CDNF and mesencephalic astrocyte-derived neurotrophic factor were also observed, after similar treatment with VPA. In addition, significant increases in the mRNA levels of the early dopaminergic neuron marker, nuclear receptor-related protein 1(Nurr1), and the transcriptional regulator, early growth response protein 1 (Egr1), were observed after treatment with VPA for 24 hr. C17.2 neural stem cells exhibited high basal mRNA expression of brain-derived neurotrophic factor and SRY (sex determining region Y)-box 2 (Sox2), which was not altered by VPA treatment. Western analysis revealed hyperacetylation of histone H3 proteins in C17.2 cells after VPA treatment for 24 hr or 48 hr, suggesting involvement of an epigenetic mechanism in the observed gene induction by this drug. These findings support a role for VPA in modulating neurotrophic and differentiation factor expression, in keeping with its reported neuroprotective and neurodevelopmental effects.

Electroacupuncture-regulated neurotrophic factor mRNA expression in the substantia nigra of Parkinson's disease rats.

Acupuncture for the treatment of Parkinson's disease has a precise clinical outcome. This study investigated the effect of electroacupuncture at Fengfu (GV16) and Taichong (LR3) acupoints in rat models of Parkinson's disease induced by subcutaneous injection of rotenone into rat neck and back. Reverse transcription-PCR demonstrated that brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression was significantly increased in the substantia nigra of rat models of Parkinson's disease, and that abnormal behavior of rats was significantly improved following electroacupuncture treatment. These results indicated that electroacupuncture treatment upregulated brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor mRNA expression in the substantia nigra of rat models of Parkinson's disease. Thus, electroacupuncture may be useful in the treatment of Parkinson's disease.

Antidepressant Acts on Astrocytes Leading to an Increase in the Expression of Neurotrophic/Growth Factors: Differential Regulation of FGF-2 by Noradrenaline

Recently, multiple neurotrophic/growth factors have been proposed to play an important role in the therapeutic action of antidepressants. In this study, we prepared astrocyte- and neuron-enriched cultures from the neonatal rat cortex, and examined the changes in neurotrophic/growth factor expression by antidepressant treatment using real-time PCR. Treatment with amitriptyline (a tricyclic antidepressant) significantly increased the expression of fibroblast growth factor-2 (FGF-2), brain-derived neurotrophic factor, vascular endothelial growth factor and glial cell line-derived neurotrophic factor mRNA with a different time course in astrocyte cultures, but not in neuron-enriched cultures. Only the expression of FGF-2 was higher in astrocyte cultures than in neuron-enriched cultures. We focused on the FGF-2 production in astrocytes. Several different classes of antidepressants, but not non-antidepressants, also induced FGF-2 mRNA expression. Noradrenaline (NA) is known to induce FGF-2 expression in astrocyte cultures, as with antidepressants. Therefore, we also assessed the mechanism of NA-induced FGF-2 expression, in comparison to amitriptyline. NA increased the FGF-2 mRNA expression via α1 and β-adrenergic receptors; however, the amitriptyline-induced FGF-2 mRNA expression was not mediated via these adrenergic receptors. Furthermore, the amitriptyline-induced FGF-2 mRNA expression was completely blocked by cycloheximide (an inhibitor of protein synthesis), while the NA-induced FGF-2 mRNA was not. These data suggest that the regulation of FGF-2 mRNA expression by amitriptyline was distinct from that by NA. Taken together, antidepressant-stimulated astrocytes may therefore be important mediators that produce several neurotrophic/growth factors, especially FGF-2, through a monoamine-independent and a de novo protein synthesis-dependent mechanism.

FTY720 ameliorates MOG‐induced experimental autoimmune encephalomyelitis by suppressing both cellular and humoral immune responses

FTY720, an oral sphingosine 1-phosphate (S1P) receptor modulator, has shown efficacy in phase II trials in patients with relapsing-remitting multiple sclerosis (MS). Although this molecule is thought to immunosuppress by inhibiting lymphocyte egress from the lymph nodes, the full spectrum of FTY720's actions has not yet been uncovered. In this study, we investigated the effects of FTY720 treatment on disease severity and histopathology of MOG-induced experimental autoimmune encephalomyelitis (EAE) in the dark agouti (DA) rat, a model that closely mimics several features of MS. The effects of FTY720 on T-cell subsets, anti-MOG antibody production, and mRNA expression of a number of cytokines and other genes were also examined. Commencement of treatment before disease onset prevented the appearance of clinical disease. Therapeutic treatment after established disease reduced clinical scores and substantially attenuated inflammation, demyelination, and axon loss. EAE suppression was associated with a reduction in all measured T-cell subsets in blood and spleen and a significant decrease in serum IgG(2a) levels. However, in the lymph nodes, all T-cell subsets except for naïve T cells and recent thymic emigrants remained unaffected. In addition, FTY720 treatment led to a significant inhibition in interferon-gamma, inducible nitric oxide synthase, and glial cell line-derived neurotrophic factor mRNA expression in the MOG-EAE spinal cord. In conclusion, our findings indicate that FTY720-mediated S1P receptor modulation ameliorates chronic relapsing MOG-EAE by suppressing both cellular and humoral immune responses.

NGF, BDNF, NT-3, and GDNF mRNA Expression in Rat Skeletal Muscle following Denervation and Sensory Protection

Poor muscle and nerve functional recovery after nerve damage is a serious clinical problem, particularly if there is prolonged delay before nerve-muscle contact is reestablished. Our previous studies showed that sensory nerve cross-anastomosis (sensory protection) provides support to the denervated muscle. In the present study, we analyzed neurotrophic factor mRNA expression by RT-PCR in denervated rat gastrocnemius muscle receiving sensory protection with the saphenous nerve, compared to normal innervated muscle, to denervated muscle, and to denervated muscle repaired immediately with the peroneal (motor) nerve, after periods of 3 days to 3 months. No significant differences in mRNA levels of beta-actin, nerve growth factor, brain-derived neurotrophic factor or neurotrophin-3 were found between the sensory protection treatment and the denervated or the motor repair groups. However, sensory protection resulted in levels of muscle glial cell line-derived neurotrophic factor mRNA expression that were lower than in denervated muscle and higher than in muscle given immediate motor repair. These results demonstrate that glial cell line-derived neurotrophic factor mRNA is elevated following denervation but is partially down-regulated by sensory protection. Our study suggests that sensory protection provides a modified trophic environment by modulating neurotrophic factor synthesis in muscle.

Expression of glial cell line-derived neurotrophic factor mRNA in the human newborn and adult hippocampal formation

Glial cell line-derived neurotrophic factor (GDNF) mRNA-containing neurons were found in normal neonate and adult human hippocampus with a localization pattern consistently similar among different ages. They were numerous in proximal CA1 to CA3 pyramidal layer, granular layer and hilus, and sparse in oriens and molecular layers. The present data provide a map of GDNF-producing neurons in the human archicortex and suggest a role for GDNF in neuronal function throughout life.

Activated Macrophages and Microglia Induce Dopaminergic Sprouting in the Injured Striatum and Express Brain-Derived Neurotrophic Factor and Glial Cell Line-Derived Neurotrophic Factor

Nigrostriatal dopaminergic neurons undergo sprouting around the margins of a striatal wound. The mechanism of this periwound sprouting has been unclear. In this study, we have examined the role played by the macrophage and microglial response that follows striatal injury. Macrophages and activated microglia quickly accumulate after injury and reach their greatest numbers in the first week. Subsequently, the number of both cell types declines rapidly in the first month and thereafter more slowly. Macrophage numbers eventually cease to decline, and a sizable group of these cells remains at the wound site and forms a long-term, highly activated resident population. This population of macrophages expresses increasing amounts of glial cell line-derived neurotrophic factor mRNA with time. Brain-derived neurotrophic factor mRNA is also expressed in and around the wound site. Production of this factor is by both activated microglia and, to a lesser extent, macrophages. The production of these potent dopaminergic neurotrophic factors occurs in a similar spatial distribution to sprouting dopaminergic fibers. Moreover, dopamine transporter-positive dopaminergic neurites can be seen growing toward and embracing hemosiderin-filled wound macrophages. The dopaminergic sprouting that accompanies striatal injury thus appears to result from neurotrophic factor secretion by activated macrophages and microglia at the wound site.

Increased expression of glial cell line-derived neurotrophic factor mRNA in muscle biopsies from patients with amyotrophic lateral sclerosis.

The expression of glial cell line-derived neurotrophic factor (GDNF) mRNA and brain-derived neurotrophic factor (BDNF) mRNA were studied in muscle biopsies from five patients with amyotrophic lateral sclerosis (ALS), six patients with other neuromuscular diseases and eight healthy control persons. All five patients with ALS had higher GDNF mRNA expressions in their biopsies than the healthy control group (almost a three fold increase). Among the other patients only one, who had a rapidly progressing toxic polyneuropathy, showed a GDNF mRNA expression above those of the controls. The BDNF mRNA expressions in the biopsies from the ALS patients were in the same range as those from the healthy controls, although the mean value of the ALS patients was higher. The only biopsy that showed a markedly higher BDNF mRNA expression was taken from one patient with progressive muscular atrophy. These results suggest that increased GDNF mRNA expression in muscle is an unspecific response to ongoing denervation and that this response is maintained in ALS, at least temporarily. If increased GDNF mRNA in muscle proves to be a constant finding in ALS the rationale for the use of GDNF as a therapeutic agent in ALS must be questioned.

Distribution of glial cell line-derived neurotrophic factor mRNA in human colon suggests roles for muscularis mucosae in innervation

Background/Purpose: Glial cell line-derived neurotrophic factor (GDNF) is a ligand for the receptor complex of GDNF family receptor alphas (GFRαs) and Ret receptor tyrosine kinase, the product of a known Hirschsrung's disease gene. The aim of this study was to analyze the mRNA distribution of these genes in the developing human intestine to understand their roles in enteric innervation. Methods: Cryosections of fetal and newborn stomach, ileum, and colon were hybridized in situ with S 35-labeled cRNA probes to GDNF, Ret, GFRα-1 or GFRα-2. GDNF mRNA levels in fetal ileum and colon were compared by reverse transcription-polymerase chain reaction (PCR). Results: GDNF mRNA expression was abundant in the muscularis mucosae of both fetal and newborn colon but was found neither in the neural plexuses nor in other regions of the intestine. Accordingly, by reverse transcription-PCR, GDNF mRNA level was many times higher in colon than ileum. Ret, GFRα-1 and GFRα-2 mRNA were expressed in the ganglionic cells of both myenteric and submucosal plexuses throughout the intestine. Conclusions: The highly restricted distribution of GNDF mRNA suggests an important role for muscularis mucosae in the development of human enteric nervous system. Ret, GFRα-1, and GFRα-2 most likely act as GDNF receptors in colon but may have alternative ligands in other enteric segments.

Enhanced glial cell line-derived neurotrophic factor mRNA expression upon (-)-deprenyl and melatonin treatments.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to be a preferentially selective neurotrophic factor for dopamine (DA) neurons. In the present study, we have examined the distribution of GDNF mRNA expression in several major DA-containing cell body and terminal areas and the regulation of GDNF mRNA expression upon various pharmacological treatments. Results indicated that there is a relatively higher GDNF mRNA level in neurons of the nigrostriatal and mesolimbic dopaminergic pathways. Upon chronic 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) treatment (30 mg/ kg, i.p., for 7 days), DA level was decreased, whereas GDNF mRNA expression was increased in the striatum, suggesting that more GDNF is synthesized and expressed to cope with the neurotoxin insult. Furthermore, among several DA neuron protective and/or therapeutic agents examined, both intrastriatal injections of (-)-deprenyl (1.25 microg and 2.5 microg) and melatonin (30 microg, 60 microg, and 120 microg) significantly enhanced GDNF mRNA expression in the striatum, whereas the same concentrations of (-)-deprenyl did not affect monoamine oxidase B (MAOB) activity, although it increased glutathione peroxidase (GPx) and/or superoxide dismutase (SOD) activities. Similarly, the same concentrations of melatonin did not alter SOD or GPx activities, except that the highest dose of melatonin (120 microg) increased lipid peroxidation in the striatum. Conversely, GM1 ganglioside injection (45 microg) lacked of an effect on GDNF mRNA expression. Together, these results suggest that both (-)-deprenyl and melatonin up-regulate GDNF gene expression at threshold doses lower than that needed for altering MAOB activity and/or the antioxidant enzyme systems, respectively. These results provide new information on the neuroprotective and therapeutic mechanisms of (-)-deprenyl and melatonin on DA neurons.

Induction of Interleukin-1 Associated with Compensatory Dopaminergic Sprouting in the Denervated Striatum of Young Mice: Model of Aging and Neurodegenerative Disease

Young mice challenged with the neurotoxin 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP), which selectively destroys the substantia nigra dopaminergic neurons in the midbrain, exhibit spontaneous recovery of dopaminergic nerve terminals. However, such recovery becomes attenuated with age. Here we report that newly sprouted fibers originate from spared dopaminergic neurons in the ventral tegmental area. We found that interleukin-1 (IL-1), an immune response-generated cytokine that can enhance dopaminergic sprouting when exogenously applied, increased dramatically in the denervated striatum of young mice (2 months) compared with middle-aged mice (8 months) after MPTP treatment. Young mice displayed a maximal 500% induction of IL-1alpha synthesis that remained elevated for several weeks in the dorsal and ventral striatum, whereas middle-aged mice exhibited a modest 135% induction exclusively in the dorsal striatum for a week. IL-1alpha immunoreactivity was localized in GFAP-immunoreactive hypertrophied astrocytes and neurons within the denervated striatum of young mice. However, no induction of IL-1alpha mRNA was seen in the midbrain in either age group despite glial activation. Because we have reported that IL-1 can regulate astroglia-derived dopaminergic neurotrophic factors, it was surprising that no changes were observed in acidic and basic fibroblast growth factor or glial cell line-derived neurotrophic factor mRNA levels associated with MPTP-induced plasticity of dopaminergic neurons in the striatum of young mice. Interestingly, we found that dopaminergic neurons express IL-1 receptors, thus suggesting that IL-1alpha could directly act as a target-derived dopaminergic neurotrophic factor to initiate or enhance the sprouting of dopaminergic axonal terminals. These findings strongly suggest that IL-1alpha could play an important role in MPTP-induced plasticity of dopaminergic neurons.