Glial Cell Line-derived Neurotrophic Factor mRNA Levels Research Articles

Intestinal flora imbalance affects the prognosis of Parkinson's disease by regulating the expression of fecal microRNA-146a and GDNF: A Preliminary Study

IntroductionThere is mounting evidence showing the association between the gut microbiome and Parkinson’s disease (PD). In this study, we aimed to explore the role of intestinal flora imbalance in the progression of PD and the underlying molecular mechanism involving the regulatory relationship between miR-146a and glial cell line–derived neurotrophic factor (GDNF).Material and methodsA total of 42 PD patients were recruited in this study and grouped according to their prognosis. Real-time PCR, computational analysis and luciferase assay were carried out to explore the role of miR-146a and GDNF in PD patients as well as the regulatory relationship between the expression of miR-146a and GDNF. A rat model was used to study the effect of intestinal flora imbalance by treating the rats with Lactobacillus rhamnosus NCDC17.ResultsA lower count of total lactobaccilus and B. fragilis may predict the progression of PD. GDNF was identified as a virtual target gene of miR-146a, whose expression in fecal, plasma and cerebral tissue samples was significantly reduced in PD patients. In addition, the presence of miR-146a precursors and GDNF siRNA significantly reduced the mRNA and protein levels of GDNF in SH-SY5Y and U251 cells, and the inhibitory effect of miR-146a precursors on GDNF expression was similar to that of GDNF siRNA.ConclusionsTaken together, the findings of this study demonstrated that GDNF is a key factor involved in the prognosis of PD. we suggested that the administration of Lactobacillus rhamnosus NCDC17 promoted the pathogenesis of PD by regulating the expression of miR-146a and GDNF in the brain.

GDNF to the rescue: GDNF delivery effects on motor neurons and nerves, and muscle re-innervation after peripheral nerve injuries.

Peripheral nerve injuries commonly occur due to trauma, like a traffic accident. Peripheral nerves get severed, causing motor neuron death and potential muscle atrophy. The current golden standard to treat peripheral nerve lesions, especially lesions with large (≥ 3 cm) nerve gaps, is the use of a nerve autograft or reimplantation in cases where nerve root avulsions occur. If not tended early, degeneration of motor neurons and loss of axon regeneration can occur, leading to loss of function. Although surgical procedures exist, patients often do not fully recover, and quality of life deteriorates. Peripheral nerves have limited regeneration, and it is usually mediated by Schwann cells and neurotrophic factors, like glial cell line-derived neurotrophic factor, as seen in Wallerian degeneration. Glial cell line-derived neurotrophic factor is a neurotrophic factor known to promote motor neuron survival and neurite outgrowth. Glial cell line-derived neurotrophic factor is upregulated in different forms of nerve injuries like axotomy, sciatic nerve crush, and compression, thus creating great interest to explore this protein as a potential treatment for peripheral nerve injuries. Exogenous glial cell line-derived neurotrophic factor has shown positive effects in regeneration and functional recovery when applied in experimental models of peripheral nerve injuries. In this review, we discuss the mechanism of repair provided by Schwann cells and upregulation of glial cell line-derived neurotrophic factor, the latest findings on the effects of glial cell line-derived neurotrophic factor in different types of peripheral nerve injuries, delivery systems, and complementary treatments (electrical muscle stimulation and exercise). Understanding and overcoming the challenges of proper timing and glial cell line-derived neurotrophic factor delivery is paramount to creating novel treatments to tend to peripheral nerve injuries to improve patients’ quality of life.

Experimental diabetes negatively affects the spermatogonial stem cells' self-renewal by suppressing GDNF network interactions.

The present study was done to analyse the time-dependent effects of diabetes on Sertoli cells-spermatogonial stem cells' (SSCs) network interaction by focusing on glial cell line-derived neurotrophic factor (GDNF) and its special receptors, gfrα1 and c-RET as well as the Bcl-6b. In total, 40 Wistar rats were considered in; control, 20, 45 and 60days diabetes-induced groups. An experimental diabetes was induced by STZ. The GDNF, gfrα1, c-RET and Bcl-6b expressions were evaluated. The serum level of testosterone, tubular repopulation (RI) and spermiogenesis (SPI) indices, general histological alterations, germ cells, mRNA damage, sperm count and viability were assessed. The diabetes, in a time-dependent manner, diminished mRNA and protein levels of GDNF, gfrα1, c-RET and Bcl-6b versus control group (p<.05), enhanced percentage of seminiferous tubules with negative RI, SPI, and diminished Leydig and Sertoli cells distribution, serum levels of testosterone, sperm count and viability. Finally, the number, percentage of cells and seminiferous tubules with normal mRNA content were significantly (p<.05) diminished. In conclusion, as a new data, we showed that the diabetes by inducing severe mRNA damage and suppressing GDNF, gfrα1, c-RET and Bcl-6b expressions, potentially affects the Sertoli-SSCs' network and consequently inhibits the SSCs' self-renewal process.

The Effect of Atypical Antipsychotic Drugs on the Neurotrophic Factors Gene Expression in the MPTP Model of Parkinson’s Disease

Atypical antipsychotics (AAP) are used in the therapy of Parkinson’s disease (PD) for elimination of psychotic symptoms. As the brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and cerebral dopamine neurotrophic factor (CDNF) play a crucial role in the PD treatment, the aim of our study was the investigation of the effects of chronic treatment with commonly used AAP, clozapine and quetiapine, on the motor behavior and the BDNF, GDNF and CDNF genes expression in the mouse brain in the PD model produced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Clozapine and quetiapine (1 mg/kg, i.p.) were administered 48 hours after the last MPTP injection, and treatment continued for the following 16 days. Then animals were euthanized, substantia nigra (SN), striatum (St) and hippocampus (Hc) were extracted for RT-PCR and tyrosine hydroxylase (TH) western blot assessment. MPTP treatment led to 50% depletion in the TH protein level in the St. MPTP caused significant decrease in both BDNF and GDNF mRNA level in the Hc and St, respectively. At the same time, increase in the GDNF expression in the MPTP + clozapine group in the SN was found. MPTP caused dramatic decrease in the CDNF mRNA level in the SN with simultaneous increase in the St. Both clozapine and quetiapine decreased it to a normal level in the St. The effect of AAP clozapine and quetiapine on the GDNF and CDNF genes expression in the pharmacological model of PD has been shown for the first time.

Smilagenin Protects Dopaminergic Neurons in Chronic MPTP/Probenecid-Lesioned Parkinson's Disease Models.

Current therapies for Parkinson’s disease (PD) only offer limited symptomatic alleviation but fail to hamper the progress of the disease. Thus, it is imperative to establish new approaches aiming at protecting or reversing neurodegeneration in PD. Recent work elucidates whether smilagenin (abbreviated SMI), a steroidal sapogenin from traditional Chinese medicinal herbs, can take neuroprotective effect on dopaminergic neurons in a chronic model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) conjuncted with probenecid mice. We reported for the first time that SMI significantly improved the locomotor ability of chronic MPTP/probenecid–lesioned mice. SMI increased the tyrosine hydroxylase (TH) positive and Nissl positive neuron number in the substantia nigra pars compacta (SNpc), augmented striatal DA and its metabolites concentration and elevated striatal dopamine transporter density (DAT). In addition, dopamine receptor D2R not D1R was down-regulated by MPTP/probenecid and slightly raised by SMI prevention. What’s more, we discovered that SMI markedly elevated striatal glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) protein levels in SMI prevented mice. And we found that SMI increased GDNF and BDNF mRNA level by promoting CREB phosphorylation in 1-methyl-4-phenylpyridimium (MPP+) treated SH-SY5Y cells. The results illustrated that SMI could prevent the impairment of dopaminergic neurons in chronic MPTP/probenecid-induced mouse model.

Alterations of DNA Methylation at GDNF Gene Promoter in the Ventral Tegmental Area of Adult Depression-Like Rats Induced by Maternal Deprivation

Objective: To study the expression and DNA methylation of the Glial cell line-derived neurotrophic factor (GDNF) gene in the development of depression-like behaviors in rats experiencing maternal deprivation stress in early life.Methods: Newborn SD rats were randomly assigned to a normal control group (NOR) or maternal deprivation group (MD). An open field test (OPT), sucrose preference test (SPT), and a forced swimming test (FST) were used to evaluate rats' behaviors. Protein, mRNA, and methylation levels were measured by ELISA/Western blot, real-time PCR, and BiSulfte Amplicon sequencing PCR, respectively.Results: MD rats had significantly shorter total distance and more fecal pellets in OPT, a lower sucrose preference rate in SPT, and a longer immobility time in FST than NOR rats. Compared with NOR rats, MD rats showed a significantly higher plasma corticosterone (CORT) level. The levels of plasma dopamine (DA) and the GDNF were significantly lower in the MD rats than in NOR rats. In the ventral tegmental area (VTA) tissues, MD rats had a significantly higher level of methylation at the GDNF gene promoter than NOR rats. The expression of the GDNF mRNA and protein were significantly lower in MD rats than in NOR rats. The total distance was significantly correlated with plasma DA and GDNF, the DNA methylation level at the GDNF promoter and the GDNF mRNA level in the VTA. Fecal pellets showed a significant correlation with plasma CORT. The sucrose preference rate was significantly correlated with plasma DA, the DNA methylation level at the GDNF promoter and the GDNF mRNA level in the VTA. Immobility time showed a significant correlation with the plasma DA, the plasma GDNF and the GDNF mRNA level in the VTA.Conclusion: up-regulation of DNA methylation at the GDNF gene promotor and the subsequent down-regulation of the GDNF gene expression in the VTA, may be involved in the development of depression-like behaviors in rats experiencing MD in early life.

The effect of atypical antipsychotic drugs on the neurotrophic factors gene expression in the MPTP model of Parkinson's disease

Atypical antipsychotics (AAP) are used in the therapy of Parkinson’s disease (PD) for elimination of psychotic symptoms. As the brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and cerebral dopamine neurotrophic factor (CDNF) play a crucial role in the PD treatment, the aim of our study was the investigation of the effects of chronic treatment with commonly used AAP, clozapine and quetiapine, on the motor behavior and the BDNF, GDNF and CDNF genes expression in the mouse brain in the PD model produced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Clozapine and quetiapine (1 mg/kg, i.p.) were administered 48 hours after the last MPTP injection, and treatment continued for the following 16 days. Then animals were euthanized, substantia nigra (SN), striatum (St) and hippocampus (Hc) were extracted for RT-PCR and tyrosine hydroxylase (TH) western blot assessment. MPTP treatment led to 50% depletion in the TH protein level in the St. MPTP caused significant decrease in both BDNF and GDNF mRNA level in the Hc and St, respectively. At the same time, increase in the GDNF expression in the MPTP + clozapine group in the SN was found. MPTP caused dramatic decrease in the CDNF mRNA level in the SN with simultaneous increase in the St. Both clozapine and quetiapine decreased it to a normal level in the St. The effect of AAP clozapine and quetiapine on the GDNF and CDNF genes expression in the pharmacological model of PD has been shown for the first time.

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.

Ultrasound-Triggered Effects of the Microbubbles Coupled to GDNF Plasmid-Loaded PEGylated Liposomes in a Rat Model of Parkinson's Disease.

Background: The purpose of this study was to investigate ultrasound-triggered effects of PEGylated liposomes-coupled microbubbles mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF) plasmid (PLs-GDNF-MBs) on behavioral deficits and neuron loss in a rat model of Parkinson's disease (PD).Methods: The unloaded PLs-MBs were characterized for particle size, concentration and zeta potential. PD rat model was established by a unilateral 6-hydroxydopamine (6-OHDA) lesion. Rotational, climbing pole, and suspension tests were used to evaluate behavioral deficits. The immunohistochemical staining of tyrosine hydroxylase (TH) and dopamine transporter (DAT) was used to assess the neuron loss. The expression levels of GDNF and nuclear receptor-related factor 1 (Nurr1) were determined by western blot and qRT-PCR analysis.Results: The particle size of PLs-MBs was gradually increased, while the concentration and absolute zeta potential were gradually decreased in a time-dependent manner after injection. 6-OHDA elevated amphetamine-induced rotations and decreased the TH and DAT immunoreactivity compared to sham group. However, these effects were blocked by the PLs-GDNF-MBs. In addition, the mRNA and protein expression levels of GDNF and Nurr1 were increased after PLs-GDNF-MBs treatment.Conclusions: The delivery of PLs-GDNF-MBs into the brains using MRI-guided focused ultrasound alleviates the behavioral deficits and neuron loss in the rat model of PD.

Pulsed Radiofrequency Improves Neuropathic Pain in Chronic Constriction Injury Rats through the Upregulation of the Transcription and Translation Levels of Glial Cell Line-Derived Neurotrophic Factor

Background: Several studies in recent years have confirmed that the direct application of pulsed radiofrequency (PRF) on peripheral nerve compression points can alleviate hyperalgesia in animal models of neuropathic pain (NP). However, the mechanism underlying the treatment of peripheral nerves by PRF is unclear. Objectives: We aim to observe changes in pain behavior after the application of PRF on the ligation site of the sciatic nerves (SNs) of rats with chronic constriction injury (CCI) and to investigate the effects of PRF on the transcription and translation levels of glial cell line-derived neurotrophic factor (GDNF) in nerve tissues at the treatment site. Study Design: A randomized, experimental trial. Setting: Experimental Animal Center, Beijing Tiantan Hospital. Methods: Ninety-six adult male Sprague-Dawley rats were randomly divided into 4 groups: sham-sham (SS) group, sham-PRF (SP) group, CCI-sham (CS) group, and CCI-PRF (CP) group. The right SNs of rats in the CS and CP group were ligated to establish the CCI model. The right SNs in the SS and SP groups were isolated and exposed but without being ligated. On the fourteenth day after CCI/sham operation, PRF treatment was performed on the midpoint of the ligation sites of the SN in the CP group and the corresponding sites in the SP group. The electrode was only placed at the ligation sites of the SN in the CS group and the corresponding sites in the SS group without current being applied. The 50% paw withdrawal threshold (50%PWT) and thermal withdrawal latency (TWL) of rats in all of the groups were measured. The transcription and translation levels of GDNF of the PRF/sham treatment sites were measured before and after treatment by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assays (ELISAs). Results: The 50%PWT value of the hind paws of rats in the CP group gradually increased on day 6 after the PRF treatment and was significantly higher than that in the CS group (6 days after treatment P &lt; 0.05; 14 days after treatment P &lt; 0.01). The TWL value in the CP group was higher than that in the CS group 2 days after treatment (P &lt; 0.05) and was significantly higher (P &lt; 0.01) from day 6 until the end of the experiment. On the day 6 and 14 after PRF treatment, the mRNA and protein expression levels of GDNF at the ligation sites of the SNs of rats in the CP group were higher than both the levels before treatment and those in the CS group (P &lt; 0.01). Limitations: The efficacy of PRF treatment in the CCI model was only tested within 14 days, and the changes in GDNF levels were only tested at 3 time-points before and after treatment. Conclusions: The direct application of PRF on SN ligation sites in the CCI model can safely and effectively alleviate NP. One of the mechanisms of this effect could be the upregulation of the transcription and translation of GDNF in compressed SNs. Key words: Pulsed radiofrequency, chronic constriction injury, sciatic nerve, 50% paw withdrawal threshold, thermal withdrawal latency, glia cell line-derived neurotrophic factor, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay

Suppression of MicroRNA-383 Enhances Therapeutic Potential of Human Bone-Marrow-Derived Mesenchymal Stem Cells in Treating Spinal Cord Injury via GDNF

Background/Aims: Transplantation of bone-marrow-derived mesenchymal stem cells (MSCs) has been used to treat spinal cord injury (SCI) to enhance tissue repair and neural cell regeneration. Glial cell line derived neurotrophic factor (GDNF) is an identified neural growth and survival factor. Here, we examined whether modification of GDNF levels in MSCs may further increase the potential of MSCs in promoting neural cell regeneration and subsequently the therapeutic outcome. Methods: We examined the mRNA and protein levels of GDNF in human MSCs by RT-qPCR and Western blot, respectively. Bioinformatics analyses were done to predict microRNAs (miRNAs) that target GDNF in MSCs. The functional binding of miRNAs to GDNF mRNA was examined by a dual luciferase reporter assay. MSCs were transduced with adeno-associated virus (AAV) carrying null or antisense for miR-383 (as-miR-383), which were transplanted into nude rats that underwent SCI. The intact tissue, cavity volume, and recovery of locomotor activity were assessed. Results: MSCs expressed very low GDNF protein, but surprisingly high levels of GDNF mRNA. Bioinformatics analyses showed that miR-383 inhibited protein translation of GDNF, through binding to the 3’-UTR of the GDNF mRNA. MSCs transduced with AAV-as-miR-383 further increased the intact tissue percentage, decreased cavity volume, and enhanced the recovery of locomotor activity in nude rats that underwent SCI, compared to MSCs. Conclusions: Suppression of miR-383 may increase the therapeutic potential of human bone-marrow-derived MSCs in treating SCI via augmentation of GDNF protein levels.

Glial cell line-derived neurotrophic factor in genetically defined fear-induced aggression.

Glial cell line-derived neurotrophic factor (GDNF) plays an important role in maintenance of neuronal system throughout life. However, there is a lack of data on the involvement of GDNF in the regulation of different kinds of behavior. In this study, GDNF, its precursor (proGDNF) and GDNF mRNA levels were investigated in the brain of rats selectively bred for 85 generations for either high level or for the lack of affective aggressiveness toward human. It was found that GDNF mRNA level was decreased in the frontal cortex, increased in the raphe nuclei area of the midbrain of aggressive rats compared to tame animals and was not detected in the amygdala and hypothalamus. The level of proGDNF was reduced in the raphe nuclei area of the midbrain of highly aggressive rats and was not detected in the striatum, nucleus accumbens of investigated animals. Two forms of mature GDNF - monomer and dimer - were revealed. GDNF monomer level was increased in the raphe nuclei area, substantia nigra and amygdala of aggressive rats and it was not found in the frontal cortex and nucleus accumbens of investigated rats. Dimer GDNF level was found in all investigated brain structures. It was reduced in the hippocampus and increased in amygdala of highly aggressive rats. Thus, considerable structure-specific differences in GDNF expression between highly aggressive and nonaggressive rats were shown. The data suggested the implication of both mature GDNF monomer and dimer as well as proGDNF in the mechanism underlying genetically defined aggressiveness.

Acute and chronic effects of lithium on BDNF and GDNF mRNA and protein levels in rat primary neuronal, astroglial and neuroastroglia cultures.

The neuroprotective effect of lithium has been attributed to its therapeutic action. However, the role of glial cells particularly astrocytes, and the possible interactions between neurons and astrocytes in neuroprotective effects of lithium have been disregarded. Thus, the aim of this study was to evaluate the direct effects of lithium on brain derived neurotrophic factor (BDNF) and glial cell line derived neurotrophic factor (GDNF) in rat primary neuronal, astrocytes, and mixed neuro-astroglial cultures to assess the possible effects of lithium on astrocytes and neuro-astroglia interactions. Rat primary astrocyte, neuronal and mixed neuro-astrocyte cultures were prepared from cortices of 18-day embryos. Cell cultures were exposed to lithium (1 mM) or vehicle for 1 day (acute) or 7 days (chronic). BDNF and GDNF mRNA and protein levels were determined by RT-PCR and ELISA, respectively. Chronic but not acute lithium treatment increased intracellular BDNF and GDNF protein levels in rat primary neuronal and astrocyte cultures, respectively (P<0.05). However, chronic lithium treatment had no significant effect on intracellular BDNF protein level in astrocyte and mixed neuron-astrocyte cultures or GDNF protein levels in mixed neuron-astrocyte culture. Furthermore, acute and chronic lithium treatment had no significant effect on mRNA and extracellular BDNF and GDNF protein levels in three studied cultures. Present study showed that chronic lithium treatment affected neurotrophins both in neurons and astrocytes in a cell-type specific manner with no effect on neuron-astrocyte interactions. The findings of this study also highlighted the importance of astrocytes as drug targets involved in the neuroprotective action of lithium.

Calcitonin gene-related peptide regulation of glial cell-line derived neurotrophic factor in differentiated rat myotubes.

Glial cell-line derived neurotrophic factor (GDNF) is the most potent trophic factor for motoneuron survival and neuromuscular junction formation. GDNF is upregulated in injured or denervated skeletal muscle and returns to normal levels following reinnervation. However, the mechanism by which GDNF is regulated in denervated muscle is not well understood. The nerve-derived neurotransmitter calcitonin gene-related peptide (CGRP) is upregulated following neuromuscular injury and is subsequently released from motoneurons at the neuromuscular junction. CGRP also promotes nerve regeneration, but the mechanism is not well understood. The current study investigates whether this increase in CGRP regulates GDNF, thus playing a key role in promoting regeneration of injured nerves. This study demonstrates that CGRP increases GDNF secretion without affecting its transcription or translation. Rat L6 myoblasts were differentiated into myotubes and subsequently treated with CGRP. GDNF mRNA expression levels were quantified by quantitative real-time reverse transcription-polymerase chain reaction, and secreted GDNF was quantified in the conditioned medium by ELISA. CGRP treatment increased secreted GDNF protein without altering GDNF mRNA levels. The translational inhibitor cycloheximide did not affect CGRP-induced GDNF secreted protein levels, whereas the secretional inhibitor brefeldin A blocked the CGRP-induced increase in GDNF. This study highlights the importance of injury-induced upregulation of CGRP by exposing its ability to increase GDNF levels and demonstrates a secretional mechanism for regulation of this key regeneration-promoting neurotrophic factor.

6-Hydroxydopamine induces distinct alterations in GDF5 and GDNF mRNA expression in the rat nigrostriatal system in vivo

Growth/differentiation factor (GDF)5 and glial cell line-derived neurotrophic factor (GDNF) are neurotrophic factors that promote the survival of midbrain dopaminergic neurons in vitro and in vivo. Both factors have potent neurotrophic and neuroprotective effects in rat models of Parkinson's disease (PD) and represent promising new therapies for PD. The aim of this study was to investigate the expression of GDF5, GDNF and their receptors in the nigrostriatal dopaminergic system in rat models of PD. It found that endogenous GDF5, GDNF and their receptors are differentially expressed in two 6-hydroxydopamine lesion models of PD. In both striatal and medial forebrain bundle (MFB) lesion models, striatal levels of GDF5 mRNA increased at 10 days post-lesion, while GDNF mRNA levels in the nigrostriatal system decreased after 10 and 28 days. Midbrain mRNA levels for both GDF5 receptors transiently increased after striatal lesion, whereas those of two GDNF receptors decreased at later time-points in both models. Despite the fact that exogenous GDF5 and GDNF have comparable effects on dopaminergic neurons in vitro and in vivo, their endogenous responses to neurotoxic injury are different. This highlights the importance of studying neurotrophic factor expression at distinct disease stages and in various animal models of PD.

Selegiline rescues gait deficits and the loss of dopaminergic neurons in a subacute MPTP mouse model of Parkinson’s disease

The monoamine oxidase type-B (MAO-B) inhibitor, selegiline, is often recommended as a first-line treatment for Parkinson's disease (PD) and has been shwon to possess neuroprotective effects. The aim of the present study was to determine whether selegiline increases the levels of the neurotrophic factors (NTFs), glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), and whether it rescues motor dysfunction and the loss of dopaminergic neurons in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced lesions. We found that the oral administration of selegiline (1.0 mg/kg/day for 14 days) successfully suppressed the MPTP-induced reduction of nigral dopaminergic neurons and striatal fibers (192.68 and 162.76% of MPTP-exposed animals, respectively; both P<0.001). Moreover, improvements in gait dysfunction were observed after 7 and 14 days of a low dose of selegiline that is reported not to inhibit MAO‑B. Furthermore, there was a significant increase in GDNF and BDNF mRNA (2.10 and 2.75-fold) and protein levels (143.53 and 157.05%) in the selegiline-treated mice compared with the saline-treated MPTP-exposed mice. In addition, the Bax/Bcl-2 gene and protein expression ratios were significantly increased in the MPTP-exposed mice, and this effect was reversed by selegiline. Correlation analysis revealed that gait measurement and GDNF/BDNF levels positively correlated with the number of dopaminergic neurons. These findings demonstrate that selegiline has neurorescue effects that are possibly associated with the induction of NTFs and anti-apoptotic genes.

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 neurotrophicfactors. Res and its derivative may be effective for the treatment of neuropathic diseases induced by EtOH.

Positive autoregulation of GDNF levels in the ventral tegmental area mediates long-lasting inhibition of excessive alcohol consumption

Glial cell line-derived neurotrophic factor (GDNF) is an essential growth factor for the survival and maintenance of the midbrain dopaminergic (DA-ergic) neurons. Activation of the GDNF pathway in the ventral tegmental area (VTA), where the GDNF receptors are expressed, produces a long-lasting suppression of excessive alcohol consumption in rats. Previous studies conducted in the DA-ergic-like cells, SHSY5Y, revealed that GDNF positively regulates its own expression, leading to a long-lasting activation of the GDNF signaling pathway. Here we determined whether GDNF activates a positive autoregulatory feedback loop in vivo within the VTA, and if so, whether this mechanism underlies the long-lasting suppressive effects of the growth factor on excessive alcohol consumption. We found that a single infusion of recombinant GDNF (rGDNF; 10 μg) into the VTA induces a long-lasting local increase in GDNF mRNA and protein levels, which depends upon de novo transcription and translation of the polypeptide. Importantly, we report that the GDNF-mediated positive autoregulatory feedback loop accounts for the long-lasting inhibitory actions of GDNF in the VTA on excessive alcohol consumption. Specifically, the long-lasting suppressive effects of a single rGDNF infusion into the VTA on excessive alcohol consumption were prevented when protein synthesis was inhibited, as well as when the upregulation of GDNF expression was prevented using short hairpin RNA to focally knock down GDNF mRNA in the VTA. Our results could have implications for the development of long-lasting treatments for disorders in which GDNF has a beneficial role, including drug addiction, chronic stress and Parkinson's disease.

The efficiency of male fertility restoration is dependent on the recovery kinetics of spermatogonial stem cells after cytotoxic treatment with busulfan in mice

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and represent a crucial resource for male fertility restoration. It has not been well documented, however, whether the recovery of SSC population size after cytotoxic damage associates with the kinetics of male fertility restoration. We addressed this issue using the mouse as a model. Following single injections of busulfan at 15, 30 or 45 mg/kg into male mice, we examined their ability to sire offspring at different times by natural mating and determined SSC numbers using spermatogonial transplantation. We measured testis physiological parameters (testis weights, sperm counts, serum and intratesticular testosterone levels, and histological assessments of spermatogenic recovery) and quantified the expression of glial-cell-line-derived neurotrophic factor (GDNF) transcripts. Regardless of busulfan doses, fertility was lost within 4 weeks after treatment, while more than 95% of SSCs were lost within 3 days. Fertility and SSC numbers gradually recovered with time, but the recoveries were delayed at higher busulfan doses. Interestingly, SSC numbers reached ∼30% of before-treatment levels by 4 weeks prior to the time of fertility restoration, across the dose groups. Sperm counts were ∼20% of before-treatment levels at the onset of fertility restoration, regardless of busulfan doses. We detected a significant increase in total GDNF mRNA per testis immediately after busulfan treatment. The loss and restoration of fertility after busulfan treatment are direct consequences of SSC loss and expansion. Our data suggest that there is a threshold in SSC numbers that allows for male fertility restoration and that the testicular somatic environment responds rapidly and temporarily to the loss of spermatogonia, including SSCs, by altering GDNF mRNA levels. This study provides fundamental information to clinically apply SSCs for male fertility restoration in the future.

Noribogaine, but not 18‐MC, exhibits similar actions as ibogaine on GDNF expression and ethanol self‐administration

Ibogaine is a naturally occurring alkaloid that has been reported to decrease various adverse phenotypes associated with exposure to drugs of abuse and alcohol in human and rodent models. Unfortunately, ibogaine cannot be used as a medication to treat addiction because of severe side effects. Previously, we reported that the desirable actions of ibogaine to reduce self-administration of, and relapse to, alcohol consumption are mediated via the upregulation of the expression of the glial cell line-derived neurotrophic factor (GDNF) in the midbrain ventral tegmental area (VTA), and the consequent activation of the GDNF pathway. The ibogaine metabolite, noribogaine, and a synthetic derivative of ibogaine, 18-Methoxycoronaridine (18-MC), possess a similar anti-addictive profile as ibogaine in rodent models, but without some of its adverse side effects. Here, we determined whether noribogaine and/or 18-MC, like ibogaine, increase GDNF expression, and whether their site of action to reduce alcohol consumption is the VTA. We used SH-SY5Y cells as a cell culture model and found that noribogaine, like ibogaine, but not 18-MC, induces a robust increase in GDNF mRNA levels. Next, we tested the effect of intra-VTA infusion of noribogaine and 18-MC on rat operant alcohol self-administration and found that noribogaine, but not 18-MC, in the VTA decreases responding for alcohol. Together, our results suggest that noribogaine and 18-MC have different mechanisms and sites of action.