Tramadol: a Potential Neurotoxic Agent Affecting Prefrontal Cortices in Adult Male Rats and PC-12 Cell Line.

Tramadol is a synthetic analogue of codeine that is often prescribed for the treatment of mild to moderate pains. It has a number of side effects including emotional instability and anxiety. In this study, we focus on the structural and functional changes of prefrontal cortex under chronic exposure to tramadol. At the cellular level, the amounts of ROS and annexin V in PC12 cells were evidently increased upon exposure to tramadol (at a concentration of 600 µM for 48 h). To this end, the rats were daily treated with tramadol at doses of 50 mg/kg for 3 weeks. Our findings reveal that tramadol provokes atrophy and apoptosis by the induction of apoptotic markers such as Caspase 3 and 8, pro-inflammatory markers, and downregulation of GDNF. Moreover, it triggers microgliosis and astrogliosis along with neuronal death in the prefrontal cortex. Behavioral disturbance and cognitive impairment are other side effects of tramadol. Overall, our results indicate tramadol-induced neurodegeneration in the prefrontal cortex mainly through activation of neuroinflammatory response.

FTY720-Mitoxy reduces synucleinopathy and neuroinflammation, restores behavior and mitochondria function, and increases GDNF expression in Multiple System Atrophy mouse models.

Multiple system atrophy (MSA) is a fatal disorder with no effective treatment. MSA pathology is characterized by α-synuclein (aSyn) accumulation in oligodendrocytes, the myelinating glial cells of the central nervous system (CNS). aSyn accumulation in oligodendrocytes forms the pathognomonic glial cytoplasmic inclusions (GCIs) of MSA. MSA aSyn pathology is also associated with motor and autonomic dysfunction, including an impaired ability to sweat. MSA patients have abnormal CNS expression of glial-cell-line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). Our prior studies using the parent compound FTY720, a food and drug administration (FDA) approved immunosuppressive for multiple sclerosis, reveal that FTY720 protects parkinsonian mice by increasing BDNF. Our FTY720-derivative, FTY720-Mitoxy, is known to increase expression of oligodendrocyte BDNF, GDNF, and nerve growth factor (NGF) but does not reduce levels of circulating lymphocytes as it is not phosphorylated so cannot modulate sphingosine 1 phosphate receptors (S1PRs). To preclinically assess FTY720-Mitoxy for MSA, we used mice expressing human aSyn in oligodendrocytes under a 2,' 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter. CNP-aSyn transgenic (Tg) mice develop motor dysfunction between 7 and 9 mo, and progressive GCI pathology. Using liquid chromatography-mass spectrometry (LC-MS/MS) and enzymatic assays, we confirmed that FTY720-Mitoxy was stable and active. Vehicle or FTY720-Mitoxy (1.1 mg/kg/day) was delivered to wild type (WT) or Tg littermates from 8.5-11.5 mo by osmotic pump. We behaviorally assessed their movement by rotarod and sweat production by starch­iodine test. Postmortem tissues were evaluated by qPCR for BDNF, GDNF, NGF and GDNF-receptor RET mRNA and for aSyn, BDNF, GDNF, and Iba1 protein by immunoblot. MicroRNAs (miRNAs) were also assessed by qPCR. FTY720-Mitoxy normalized movement, sweat function and soleus muscle mass in 11.5 mo Tg MSA mice. FTY720-Mitoxy also increased levels of brain GDNF and reduced brain miR-96-5p, a miRNA that acts to decrease GDNF expression. Moreover, FTY720-Mitoxy blocked aSyn pathology measured by sequential protein extraction and immunoblot, and microglial activation assessed by immunohistochemistry and immunoblot. In the 3-nitropropionic acid (3NP) toxin model of MSA, FTY720-Mitoxy protected movement and mitochondria in WT and CNP-aSyn Tg littermates. Our data confirm potent in vivo protection by FTY720-Mitoxy, supporting its further evaluation as a potential therapy for MSA and related synucleinopathies.

Effects of lithium and valproate on behavioral parameters and neurotrophic factor levels in an animal model of mania induced by paradoxical sleep deprivation.

The present study aimed to evaluate the effects of treatment with lithium (Li) and valproate (VPA) on behaviors and brain BDNF, NGF, NT-3, NT-4 and GDNF levels in mice submitted to paradoxical sleep deprivation (PSD), which induces an animal model of mania. Male C57BL/6J mice received an intraperitoneal (i.p.) injection of saline solution (NaCl 0.09%, 1 ml/kg), Li (47.3 mg/kg, 1 ml/kg) or VPA (200 mg/kg, 1 ml/kg) once a day for seven days. Animals were randomly distributed into six groups (n = 10 per group): (1) Control + Sal; (2) Control + Li; (3) Control + VPA; (4) PSD + Sal; (5) PSD + Li; or (6) PSD + VPA. Animals were submitted to 36 h of PSD, and then, they were submitted to the open field test. The frontal cortex and hippocampus were dissected from the brain. The manic-like behaviors in the mice were analyzed. Treatment with Li and VPA reversed the behavioral alterations induced by PSD. PSD decreased BDNF, NGF, and GDNF levels in the frontal cortex and hippocampus of mice. The administration of Li and VPA protected the brain against the damage induced by PSD. However, PSD and the administration of Li and VPA did not affect the levels of NT-3 and NT-4 in either brain structure evaluated. In conclusion, the PSD protocol induced manic-like behavior in rats and induced alterations in neurotrophic factor levels. It seems that neurotrophic factors and sleep are essential targets to treat BD.

FTY720-Mitoxy reduces toxicity associated with MSA-like α-synuclein and oxidative stress by increasing trophic factor expression and myelin protein in OLN-93 oligodendroglia cell cultures.

Multiple system atrophy (MSA) is a fatal demyelinating disorder lacking any disease-modifying therapies. MSA pathology stems from aggregated α-synuclein (aSyn) accumulation in glial cytosolic inclusions of oligodendroglial cell (OLGs), the myelinating cells of brain. In MSA brains and in MSA animal models with aSyn accumulation in OLGs, aberrant expression of brain-derived neurotrophic factor (BDNF) and glial-cell-line-derived neurotrophic factor (GDNF) occur. Nerve growth factor (NGF) expression can also be altered in neurodegenerative diseases. It is unclear if oxidative stress impacts the viability of aSyn-accumulating OLG cells. Here, we show that OLN-93 cells stably expressing human wild type aSyn or the MSA-associated-aSyn-mutants G51D or A53E, are more vulnerable to oxidative stress. In dose response studies we found that OLN-93 cells treated 48 h with 160 nM FTY720 or our new non-immunosuppressive FTY720-C2 or FTY720-Mitoxy derivatives sustained normal viability. Also, FTY720, FTY720-C2, and FTY720-Mitoxy all stimulated NGF expression at 24 h. However only FTY720-Mitoxy also increased BDNF and GDNF mRNA at 24 h, an effect paralleled by increases in histone 3 acetylation and ERK1/2 phosphorylation. Myelin associated glycoprotein (MAG) levels were also increased in OLN-93 cells after 48 h treatment with FTY720-Mitoxy. FTY720, FTY720-C2, and FTY720-Mitoxy all prevented oxidative-stress-associated-cell-death of OLN-93 cells that lack any aSyn expression. However, only FTY720-Mitoxy protected MSA-like aSyn-expressing-OLN-93-cells against oxidative-cell-death. These data identify potent protective effects for FTY720-Mitoxy with regard to trophic factors as well as MAG expression by OLG cells. Testing of FTY720-Mitoxy in mice is thus a judicious next step for neuropharmacological preclinical development.

The role of neurotrophic factors in manic-, anxious- and depressive-like behaviors induced by amphetamine sensitization: Implications to the animal model of bipolar disorder.

BACKGROUND: Bipolar disorder (BD) and substance use disorders share common symptoms, such as behavioral sensitization. Amphetamine-induced behavioral sensitization can serve as an animal model of BD. Neurotrophic factors have an important role in BD pathophysiology. This study evaluated the effects of amphetamine sensitization on behavior and neurotrophic factor levels in the brains of rats. METHODS: Wistar rats received daily intraperitoneal (i.p) injections of dextroamphetamine (d-AMPH) 2 mg/kg or saline for 14 days. After seven days of withdrawal, the animals were challenged with d-AMPH (0.5 mg/kg, i.p) and locomotor behavior was assessed. In a second protocol, rats were similarly treated with d-AMPH (2 mg/kg, i.p) for 14 days. After withdrawal, without d-AMPH challenge, depressive- and anxiety-like behaviors were evaluated through forced swimming test and elevated plus maze. Levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT-3), neurotrophin 4/5 (NT-4/5) and glial-derived neurotrophic factor (GDNF) were evaluated in the frontal cortex, hippocampus, and striatum. RESULTS: D-AMPH for 14 days augmented locomotor sensitization to a lower dose of d-AMPH (0.5 mg/kg) after the withdrawal. d-AMPH withdrawal induced depressive- and anxious-like behaviors. BDNF, NGF, and GDNF levels were decreased, while NT-3 and NT-4 levels were increased in brains after d-AMPH sensitization. LIMITATIONS: Although d-AMPH induces manic-like behavior, the mechanisms underlying these effects can also be related to phenotypes of drug abuse. CONCLUSIONS: Together, vulnerability to mania-like behavior following d-AMPH challenge and extensive neurotrophic alterations, suggest amphetamine-induced behavioral sensitization is a good model of BD pathophysiology.

Astrocyte-Conditioned Medium Protects Prefrontal Cortical Neurons from Glutamate-Induced Cell Death by Inhibiting TNF-α Expression.

OBJECTIVE: Both excitotoxicity and neurotrophin deficiency may contribute to the etiology of depression and neurodegeneration. Astrocytes not only regulate glutamate metabolism and clearance, they also produce neurotrophins in the brain. However, the direct interaction between neurons and astrocytes remains unknown. METHODS: This study evaluated the cellular mechanisms by which astrocyte-conditioned medium (ACM) protects prefrontal cortical neurons from glutamate-induced death by measuring cell viability and morphology as well as mRNA and protein expression of brain-derived neurotrophic factor (BDNF), BDNF receptors, glial cell line-derived neurotrophic factor (GDNF), and the proinflammatory cytokine, tumor necrosis factor (TNF)-α. Neurons and astrocytes were purified from the brains of neonatal 1-day-old Sprague-Dawley rats. ACM was harvested after exposing astrocytes to culture medium containing 100 µM glutamate for 48 h. RESULTS: Glutamate insult (100 µM for 6 h) significantly reduced neuronal cell viability and increased the mRNA expression of BDNF. Glutamate (24 h) decreased neuronal viability and the expression of BDNF, but increased mRNA expression of GFAP, p75 neurotrophin receptor (p75NTR), and TNF-α. ACM pretreatment (2 h) reversed glutamate-decreased cell viability and increased BDNF, but reduced the expression of GDNF, P75NTR, and TNF-α at the mRNA level. Western blotting generally confirmed the mRNA expression following 24 glutamate insults. Furthermore, the glutamate-induced decrease in the protein expression of BDNF and full-length TrkB receptor and increase in pro-BDNF, truncated TrkB isoform 1 receptor, p75NTR, GDNF, and TNF-α were significantly attenuated by ACM pretreatment. CONCLUSIONS: The study demonstrates that ACM exerts neuroprotective effects on cell viability, and this effect is most likely mediated through the modulation of neurotrophin and TNF-α expression.

Citrus Auraptene Induces Glial Cell Line-Derived Neurotrophic Factor in C6 Cells.

We previously demonstrated that auraptene (AUR), a natural coumarin derived from citrus plants, exerts anti-inflammatory effects in the brain, resulting in neuroprotection in some mouse models of brain disorders. The present study showed that treatment with AUR significantly increased the release of glial cell line-derived neurotrophic factor (GDNF), in a dose- and time-dependent manner, by rat C6 glioma cells, which release was associated with increased expression of GDNF mRNA. These results suggest that AUR acted as a neuroprotective agent in the brain via not only its anti-inflammatory action but also its induction of neurotrophic factor. We also showed that (1) the AUR-induced GDNF production was inhibited by U0126, a specific inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2, and by H89, a specific inhibitor of protein kinase A (PKA); and (2) AUR induced the phosphorylation of cAMP response element-binding protein (CREB), a transcription factor located within the nucleus. These results suggest that AUR-stimulated gdnf gene expression was up-regulated through the PKA/ERK/CREB pathway in C6 cells.

Silymarin amplifies apoptosis in ectopic endometrial tissue in rats with endometriosis; implication on growth factor GDNF, ERK1/2 and Bcl-6b expression.

The present prospective study was done to evaluate the effect of silymarin (SMN) on endometriotic-like legions establishment and growth in experimentally-induced endometriosis. For this purpose, the experimental endometriosis was induced in 12 rats and then the animals subdivided into endometriosis-sole and SMN (50 mg kg-1, orally)+endometriosis groups. Following 28 days, the legions establishment, size, Glial cell line-derived neurotrophic factor (GDNF), gfrα1, B Cell Lymphoma 6 (Bcl-6b), Bcl-2, extracellular regulator kinase (ERK1/2) expression ratios, angiogenesis, the apoptosis and fibrosis indices were investigated. The SMN significantly (P < 0.05) decreased the enometriotic-like legions establishment and size, decreased mRNA levels of GDNF, gfrα1, Bcl-6b and Bcl-2 and remarkably diminished GDNF, gfrα1, Bcl-6b and Bcl-2-positive cells distribution/mm2 of tissue versus endometriosis-sole group. The SMN + endometriosis group exhibited a significant (P < 0.05) enhancement in ERK1/2 expression and represented diminished vascularized area and increased apoptosis and fibrosis indices, as well. In conclusion, the SMN by down-regulating GDNF and its receptor gfrα1 expression inhibits GDNF-gfrα1 complex generation and consequently suppresses Bcl-6b expression. Moreover, the SMN by enhancing the ERK1/2 expression and by suppressing the Bcl-2 expression promotes the apoptosis pathway. Finally, the SMN by down-regulating the angiogenesis ratio accelerates apoptosis and consequently induces severe fibrosis in endometriotic-like legions.

BMP6 Downregulates GDNF Expression Through SMAD1/5 and ERK1/2 Signaling Pathways in Human Granulosa-Lutein Cells.

Bone morphogenetic protein (BMP) 6 is a critical regulator of follicular development that is expressed in mammalian oocytes and granulosa cells. Glial cell line‒derived neurotrophic factor (GDNF) is an intraovarian neurotrophic factor that plays an essential role in regulating mammalian oocyte maturation. The aim of this study was to investigate the effect of BMP6 on the regulation of GDNF expression and the potential underlying mechanisms. We used an established immortalized human granulosa cell line (SVOG cells) and primary human granulosa-lutein (hGL) cells as in vitro cell models. Our results showed that BMP6 significantly downregulated the expression of GDNF in both SVOG and primary hGL cells. With dual inhibition approaches (kinase receptor inhibitor and small interfering RNA knockdown), our results showed that both activin receptor kinase-like (ALK) 2 and ALK3 are involved in BMP6-induced downregulation of GDNF. In addition, BMP6 induced the phosphorylation of Sma- and Mad-related protein (SMAD)1/5/8 and ERK1/2 but not AKT or p38. Among three downstream mediators, both SMAD1 and SMAD5 are involved in BMP6-induced downregulation of GDNF. Moreover, concomitant knockdown of endogenous SMAD4 and inhibition of ERK1/2 activity completely reversed BMP6-induced downregulation of GDNF, indicating that both SMAD and ERK1/2 signaling pathways are required for the regulatory effect of BMP6 on GDNF expression. Our findings suggest an additional role for an intrafollicular growth factor in regulating follicular function through paracrine interactions in human granulosa cells.

NODAL secreted by male germ cells regulates the proliferation and function of human Sertoli cells from obstructive azoospermia and nonobstructive azoospermia patients.

This study was designed to explore the regulatory effects of male germ cell secreting factor NODAL on Sertoli cell fate decisions from obstructive azoospermia (OA) and nonobstructive azoospermia (NOA) patients. Human Sertoli cells and male germ cells were isolated using two-step enzymatic digestion and SATPUT from testes of azoospermia patients. Expression of NODAL and its multiple receptors in human Sertoli cells and male germ cells were characterized by reverse transcription-polymerase chain reaction (RT-PCR) and immunochemistry. Human recombinant NODAL and its receptor inhibitor SB431542 were employed to probe their effect on the proliferation of Sertoli cells using the CCK-8 assay. Quantitative PCR and Western blots were utilized to assess the expression of Sertoli cell functional genes and proteins. NODAL was found to be expressed in male germ cells but not in Sertoli cells, whereas its receptors ALK4, ALK7, and ACTR-IIB were detected in Sertoli cells and germ cells, suggesting that NODAL plays a regulatory role in Sertoli cells and germ cells via a paracrine and autocrine pathway, respectively. Human recombinant NODAL could promote the proliferation of human Sertoli cells. The expression of cell cycle regulators, including CYCLIN A, CYCLIN D1 and CYCLIN E, was not remarkably affected by NODAL signaling. NODAL enhanced the expression of essential growth factors, including GDNF, SCF, and BMP4, whereas SB431542 decreased their levels. There was not homogeneity of genes changes by NODAL treatment in Sertoli cells from OA and Sertoli cell-only syndrome (SCO) patients. Collectively, this study demonstrates that NODAL produced by human male germ cells regulates proliferation and numerous gene expression of Sertoli cells.

Ethanol and nicotine interaction within the posterior ventral tegmental area in male and female alcohol-preferring rats: evidence of synergy and differential gene activation in the nucleus accumbens shell.

RATIONALE: Ethanol and nicotine are frequently co-abused. The biological basis for the high co-morbidity rate is not known. Alcohol-preferring (P) rats will self-administer EtOH or nicotine directly into the posterior ventral tegmental area (pVTA). OBJECTIVE: The current experiments examined whether sub-threshold concentrations of EtOH and nicotine would support the development of self-administration behaviors if the drugs were combined. METHODS: Rats were implanted with a guide cannula aimed at the pVTA. Rats were randomly assigned to groups that self-administered sub-threshold concentrations of EtOH (50 mg%) or nicotine (1 µM) or combinations of ethanol (25 or 50 mg%) and nicotine (0.5 or 1.0 µM). Alterations in gene expression downstream projections areas (nucleus accumbens shell, AcbSh) were assessed following a single, acute exposure to EtOH (50 mg%), nicotine (1 µM), or ethanol and nicotine (50 mg% + 1 µM) directly into the pVTA. RESULTS: The results indicated that P rats would co-administer EtOH and nicotine directly into the pVTA at concentrations that did not support individual self-administration. EtOH and nicotine directly administered into the pVTA resulted in alterations in gene expression in the AcbSh (50.8-fold increase in brain-derived neurotrophic factor (BDNF), 2.4-fold decrease in glial cell line-derived neurotrophic factor (GDNF), 10.3-fold increase in vesicular glutamate transporter 1 (Vglut1)) that were not observed following microinjections of equivalent concentrations/doses of ethanol or nicotine. CONCLUSION: The data indicate that ethanol and nicotine act synergistically to produce reinforcement and alter gene expression within the mesolimbic dopamine system. The high rate of co-morbidity of alcoholism and nicotine dependence could be the result of the interactions of EtOH and nicotine within the mesolimbic dopamine system.

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.

Recent advances in the discovery and preclinical testing of novel compounds for the prevention and/or treatment of alcohol use disorders.

Alcohol abuse and dependence have a staggering socioeconomic impact, yet current therapeutic strategies are largely inadequate to treat these disorders. Thus, the development of new strategies that can effectively prevent alcohol use disorders (AUDs) is of paramount importance. Currently approved medications attempt to deter alcohol intake by blocking ethanol metabolism or by targeting the neurochemical systems downstream of the cascades leading to craving and dependence. Unfortunately, these medications have provided only limited success as indicated by the continued high rates of alcohol abuse and alcoholism. The lack of currently available effective treatment strategies is highlighted by the urgent call by the NIAAA to find new and paradigm-changing therapeutics to either prevent or treat alcohol-related problems. This mini-review highlights recent findings from 4 laboratories with a focus on compounds that have the potential to be novel therapeutic agents that can be developed for the prevention and/or treatment of AUDs.

Effects of Panax ginseng on glial cell-derived neurotrophic factor (GDNF) expression and spermatogenesis in rats.

Panax ginseng (PG) is a medicinal herb which has been used to improve male reproduction in traditional Korean medicine. This study investigated the effect of PG on spermatogenesis and the regulation of glial cell-derived neurotrophic factor (GDNF), which has a crucial role in spermatogonial stem cell maintenance. PG was administered to 8-week-old male Wistar rats (1.0 g/kg/day, p.o.) for 56 consecutive days, the sperm formation period of the rat. Sperm analysis, RT-PCR and western blot assays were then carried out. The PG-treated group had significantly enhanced sperm counts, GDNF mRNA level and protein level. These results suggest that PG induces spermatogenesis and GDNF activation in rat testes.

Mechanisms of interleukin-1beta-induced GDNF release from rat glioma cells.

Glial cell line-derived neurotrophic factor (GDNF) is highly expressed both in neurons and astrocytes in injured tissues. Astrocytes support neurons by releasing neurotrophic factors including GDNF. It has been reported that various agents including cytokines such as interleukin (IL)-1beta induce GDNF mRNA expression and the release in astrocytes. However, the mechanism behind the GDNF synthesis and release remains unclear. Herein, we investigated the mechanisms of the IL-1beta-induced GDNF release from rat C6 glioma cells. IL-1beta time dependently stimulated GDNF release from C6 cells. IL-1beta induced the phosphorylation of inhibitor kappa B (IkappaB), p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and signal transducer and activator of transcription (STAT) 3. The IL-1beta-stimulated levels of GDNF were suppressed by wedelolactone, an inhibitor of IkappaB kinase, SB203580, an inhibitor of p38 MAP kinase, PD98059, an inhibitor of MAP kinase kinase 1/2 or Janus family of tyrosine kinase (JAK) inhibitor I, an inhibitor of upstream kinase of STAT3. On the contrary, SP600125, an inhibitor of SAPK/JNK, failed to reduce the IL-1beta-effect. These results strongly suggest that IL-1beta stimulates GDNF release through the pathways of IkappaB-nuclear factor kappa B, p38 MAP kinase, p44/p42 MAP kinase and JAK-STAT3, but not through the SAPK/JNK pathway in glioma cells.

Neuroprotective effects of ginsenoside Rb1 on transient cerebral ischemia in rats.

Previous experiments showed that ginsenoside Rb1 (GRb1) reduced infarct and neuronal deficit in rats followed by transient cerebral ischemia. The mechanism of this neuroprotective function is unclear. Here, we tested whether the effect of GRb1 can be achieved through preventing ischemic neuronal death, modulating apoptotic-related genes and affecting glial-derived neurotrophic factor (GDNF) expression in rats subjected to occlusion of the middle cerebral artery. When GRb1(40 mg/kg, i.p.) was administered immediately after reperfusion, the apoptotic cells in the GRb1 group were decreased significantly from 12 to 72 h of reperfusion compared to the ischemia group by TdT-mediated dUTP-biotin nick-end labeling. Immunostaining and Western blotting analysis showed that the expression of GDNF from 3 to 120 h of the GRb1 group was significantly increased compared to the ischemia group, and GDNF expression peaked at 48 h after reperfusion. The enhanced GDNF mRNA in the GRb1 group was not detected by RT-PCR and in situ hybridization compared to the ischemia group, but GDNF mRNA at 48 h after reperfusion was strongly increased in both the ischemia and GRb1 group when compared to other time points. The number of bcl-2-positive cells was significantly increased from 12 to 120 h of reperfusion compared to the ischemia group. However, the number of bax-positive cells in the GRb1 group was significantly declined compared to the ischemia group. In the GRb1 group, the number of neuronal apoptosis inhibitory protein-positive cells from 12 to 120 h after reperfusion was evidently higher than that in the ischemia group. Therefore, ginsenoside Rb1 prevents ischemic neuronal death induced by transient cerebral ischemia, and this mechanism of which is related to increase the expression of the antiapoptotic genes and modulate the expression of GDNF.

Acidic oligosaccharide sugar chain, a marine-derived oligosaccharide, activates human glial cell line-derived neurotrophic factor signaling.

Gial derived neurotrophic factor (GDNF) modulates neuronal cell differentiation during development and protects against neurodegeneration by preventing apoptosis at maturity. GDNF's role in tissue maintenance has generated interest in the therapeutic potential of GDNF in treating neurological disorders such as Parkinson's disease. Heparan sulfate has been shown to be essential for GDNF signaling and altering the levels of heparan sulfate promotes or inhibits GDNF functional activity. To search for other oligosaccharides capable of modulating GDNF activity as potential therapeutic molecules, we investigated the effect of acidic oligosaccharide sugar chain (AOSC) and its sulfated derivative on GDNF induced neurotrophic events by using Western-blotting, immunofluorescence cell staining, and immunoprecipitation techniques in PC12 cells expressing the GDNF receptors GFR alpha 1-Ret. AOSC significantly improved the neurite outgrowth and activated c-Ret phosphorylation in PC12-GFR alpha 1-Ret cells, but its sulfated derivative inhibited GDNF activity. Studies to understand the opposing biological effects of AOSC and its sulfated derivative on GDNF activity demonstrated that reduced GDNF binding to PC12-GFR alpha 1-Ret cell surface in the presence of the sulfated derivative likely suppressed GDNF activity as both AOSC and its sulfated derivatives had similar binding affinities to GDNF. This study illustrates the importance of oligosaccharide structure and charge on influencing GDNF activity and the potential use of oligosaccharides in modulating GDNF activity for therapeutic purposes.

Toluene inhalation induced neuronal damage in the spinal cord and changes of neurotrophic factors in rat.

We investigated the effects of toluene inhalation on neurons and neurotrophic factors in the spinal cord and the relationship between them. Male Wistar rats were exposed to toluene (1500ppm for 4h per day) for 7 days. To observe damage of the neurons in spinal cord with the toluene, expression of microtubule associated protein 2 (MAP2) and 70kDa heat shock protein (HSP70) in spinal cord were performed by immunohistochemistry. MAP2 was degraded and HSP70-immunoreactivity was enhanced in nerve cell bodies of the gray matter in toluene inhalation group. Immunoreactivity of glial fibrillary acidic protein (GFAP), a marker of astrocytes, was enhanced in the toluene-treated group. Furthermore, glial cell line-derived neurotrophic factor (GDNF)- and brain-derived neurotrophic factor (BDNF)-immunoreactivity in spinal cord were slightly decreased in the treated group. In addition, the concentrations of GDNF and BDNF in the spinal cord were determined using enzyme linked immunosorbent assay (ELISA). Concentration of GDNF was reduced significantly by toluene exposure. BDNF also reduced, but not significantly. The toluene inhalation caused the damage of the neuron in the spinal cord, which was accompanied by the decrease in the neurotrophic factors, such as BDNF and GDNF.

Selective injury to dopaminergic neurons up-regulates GDNF in substantia nigra postnatal cell cultures: role of neuron-glia crosstalk.

The effect of selective injury to dopaminergic neurons on the expression of glial cell line-derived neurotrophic factor (GDNF) was examined in substantia nigra cell cultures. H(2)O(2), mimicking increased oxidative stress, or l-DOPA, the main symptomatic treatment for Parkinson's disease, increased GDNF mRNA and protein levels in a time-dependent mode in neuron-glia mixed cultures. The concentration dependence indicated that mild, but not extensive, injury induced GDNF up-regulation. GDNF neutralization with an antibody decreased dopaminergic cell viability in H(2)O(2)-treated cultures, showing that up-regulation of GDNF was protecting dopaminergic neurons. Neither H(2)O(2) nor l-DOPA directly affected GDNF expression in astrocyte cultures, but conditioned media from challenged mixed cultures increased GDNF mRNA and protein levels in astrocyte cultures, indicating that GDNF up-regulation was mediated by neuronal factors. Since pretreatment with 6-OHDA completely abolished H(2)O(2)-induced GDNF up-regulation, we propose that GDNF up-regulation is triggered by failing dopaminergic neurons that signal astrocytes to increase GDNF expression.

An analog of a dipeptide-like structure of FK506 increases glial cell line-derived neurotrophic factor expression through cAMP response element-binding protein activated by heat shock protein 90/Akt signaling pathway.

Glial cell line-derived neurotrophic factor (GDNF) is an important neurotrophic factor that has therapeutic implications for neurodegenerative disorders. We previously showed that leucine-isoleucine (Leu-Ile), an analog of a dipeptide-like structure of FK506 (tacrolimus), induces GDNF expression both in vivo and in vitro. In this investigation, we sought to clarify the cellular mechanisms underlying the GDNF-inducing effect of this dipeptide. Leu-Ile transport was investigated using fluorescein isothiocyanate-Leu-Ile in cultured neurons, and the results showed the transmembrane mobility of this dipeptide. By liquid chromatography-mass spectrometry and quartz crystal microbalance assay, we identified heat shock cognate protein 70 as a protein binding specifically to Leu-Ile, and molecular modeling showed that the ATPase domain is the predicted binding site. Leu-Ile stimulated Akt phosphorylation, which was attenuated significantly by heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA). Moreover, enhanced interaction between phosphorylated Akt and Hsp90 was detected by immunoprecipitation. Leu-Ile elicited an increase in cAMP response element binding protein (CREB) phosphorylation, which was inhibited by GA, indicating that CREB is a downstream target of Hsp90/Akt signaling. Leu-Ile elevated the levels of GDNF mRNA and protein expression, whereas inhibition of CREB blocked such effects. Leu-Ile promoted the binding activity of phosphorylated CREB with cAMP response element. These findings show that CREB plays a key role in transcriptional regulation of GDNF expression induced by Leu-Ile. In conclusion, Leu-Ile activates Hsp90/Akt/CREB signaling, which contributes to the upregulation of GDNF expression. It may represent a novel lead compound for the treatment of dopaminergic neurons or motoneuron diseases.