Glial Cell Line-derived Neurotrophic Factor Secretion Research Articles

The role of Sertoli cells-secreted factors in different stages of germ cells development in mice exposed to BDE-209

Decabromodiphenyl ether (BDE-209), a frequently used brominated flame retardant, readily enters the environment and is difficult to degrade with bioaccumulation. BDE-209 could cause male reproductive toxicity, but the regulatory functions of Sertoli cells-secreted factors remain uncertain. In present study, male mice were treated with 75 mg/kg BDE-209 and then stopped exposure for 50 days. Exogenous Glial cell line-derived neurotrophic factor (GDNF), a Sertoli cell-secreted factor, was injected into testes of mice treated with BDE-209 for 50 days to explore the role of GDNF in BDE-209-induced reproductive toxicity. The mouse spermatogonia cell line GC-1 spg was used in vitro to further verify regulatory effects of Sertoli cells-secreted factors on meiotic initiation. The results showed that BDE-209 inhibited expressions of the self-renewal pathway GFRα-1/RAS/ERK1/2 in spermatogonial stem cells (SSCs), and reduced expressions of spermatogonia proliferation-related pathway NRG3/ERBB4 and meiosis initiation factor Stra8. Furthermore, BDE-209 decreased the levels of both GDNF and retinoic acid (RA) secreted by Sertoli cells in testes. Importantly, the alterations of above indicators induced by BDE-209 did not recover after 50-day recovery period. After exogenous GDNF injection, the decreased expression of GFRα-1/RAS/ERK in SSCs was reversed. However, the level of RA and expressions of NRG3/ERBB4/Stra8 were not restored. The in vitro experimental results showed that exogenous RA reversed the reductions in NRG3/ERBB4/Stra8 and ameliorated inhibition of GC-1 spg cells proliferation induced by BDE-209. These results suggested that Sertoli cells-secreted factors play roles in regulating various stages of germ cell development. Specifically, BDE-209 affected the self-renewal of SSCs by decreasing GDNF secretion resulting in the inhibition of GFRα-1/RAS/ERK pathway; BDE-209 hindered the proliferation of spermatogonia and initiation of meiosis by inhibiting the secretion of RA and preventing RA from binding to RARα, resulting in the suppression of NRG3/ERBB4/Stra8 pathway. As a consequence, spermatogenesis was compromised, leading to persistent male reproductive toxicity.

Glucan Coated Iron Oxide Nanoparticles for Regulating Survival, Inflammation and Oxidative Stress in Parkinsons Disease

This study assessed the mechanism of Iron Oxide Nanoparticles (IONP) coated with glucan in regulating survival, inflammation and oxidative stress in Parkinson’s disease (PD). IONP coated with glucan (NPs) was established and identified. BV-2 cells were randomly divided into the following three sets; control set, PD set, and NPs set. Apoptotic activity of nerve cells, expression of TNF-α, IL-6 and IL-1β, and content of reactive oxygen species (ROS) and malondialdehyde (MDA) were was also analyzed. The expression of NF-κB and Inductible Nitric Oxide Synthase (iNOS) was detected with Western Blot, while content of glialcellline-derivedneurotrophicfactor (GDNF) was analyzed by Proliferation of nerve cells was prompted and apoptosis was restrained in the NPs set. The levels of TNF-α, IL-6 and IL-1β were reduced while contents of ROS and MDA were reduced. The expression of NF-κB and iNOS were restrained. The levels of inflammatory factors were reduced, while secretion of GDNF was reduced. The survival of nerve cells in the PD was prompted by NPs, while inflammation and oxidative stress were restrained.

Sleeping Beauty transposon system for GDNF overexpression of entrapped stem cells in fibrin hydrogel in a rat model of Parkinson’s disease

There is currently no causal treatment available for Parkinson’s disease (PD). However, the use of glial cell line–derived neurotrophic factor (GDNF) to provide regenerative effects for neurons is promising. Such approaches require translational delivery systems that are functional in diseased tissue. To do so, we used a non-viral Sleeping Beauty (SB) transposon system to overexpress GDNF in adipose tissue–derived mesenchymal stromal cells (adMSCs). Entrapment of cells in fibrin hydrogel was used to boost potential neurorestorative effects. Functional GDNF-adMSCs were able to secrete 1066.8 ± 169.4 ng GDNF/120,000 cells in vitro. The GDNF-adMSCs were detectable for up to 1 month after transplantation in a mild 6-hydroxydopamine (6-OHDA) hemiparkinson male rat model. Entrapment of GDNF-adMSCs enabled GDNF secretion in surrounding tissue in a more concentrated manner, also tending to prolong GDNF secretion relatively. GDNF-adMSCs entrapped in hydrogel also led to positive immunomodulatory effects via an 83% reduction of regional IL-1β levels compared to the non-entrapped GDNF-adMSC group after 1 month. Furthermore, GDNF-adMSC-treated groups showed higher recovery of tyrosine hydroxylase (TH)-expressing cells, indicating a neuroprotective function, although this was not strong enough to show significant improvement in motor performance. Our findings establish a promising GDNF treatment system in a PD model. Entrapment of GDNF-adMSCs mediated positive immunomodulatory effects. Although the durability of the hydrogel needs to be extended to unlock its full potential for motor improvements, the neuroprotective effects of GDNF were evident and safe. Further motor behavioral tests and other disease models are necessary to evaluate this treatment option adequately.Graphical

Implantation of glial cell line-derived neurotrophic factor-expressing adipose tissue-derived stromal cells in a rat Parkinson’s disease model

ABSTRACT In previous studies, the effects of glial cell line-derived neurotrophic factor (GDNF) expressing adipose tissue-derived stromal cells (ADSCs) on Parkinson’s disease (PD) models have been studied but have not been elucidated. The present study aims to investigate this phenomenon and trace their differentiation in vivo. In our study, ADSCs were harvested from adult Sprague-Dawley rats, then genetically modified into GDNF-expressing system by lentivirus. The secretion of GDNF from the transduced cells was titrated by enzyme-linked immunosorbent assay (ELISA). Cellular differentiation in vitro was observed after induction. To examine survival and differentiation in vivo, they were injected into the striatum of 6-hydroxydopamine-lesioned rats, whose apomorphine-induced rotations were examined 2, 7, 14 and 21d after grafting. It’s found that GDNF-expressing ADSCs can differentiate into neuron-like cells in vitro. Moreover, engrafted GDNF-expressing ADSCs survived at least 90 days post-grafting and differentiated into dopaminergic neuron-like cells. Most importantly, these cells drastically improved the clinical symptoms of PD rats. In conclusion, ADSCs can be efficiently engineered by lentivirus system and deliver a therapeutic level of the transgene to target tissues. GDNF-ADSCs can improve behavior phenotype in the rat PD model. Moreover, ADSCs is a more readily available source of dopaminergic neurons, though a more effective procedure needs to be developed to enrich the number of differentiation.

Effect of brefelamide on proliferation of 1321N1 human astrocytoma cells induced by glial cell line-derived neurotrophic factor.

Malignant gliomas are highly resistant to chemotherapy and radiation and more effective options for treatment are urgently needed. We reported previously that the aromatic amide brefelamide, which is isolated from methanolic extracts of the cellular slime molds Dictyostelium giganteum and D. brefeldianum, hinders cellular proliferation in a glioma model utilizing 1321N1 human astrocytoma cells. Herein, we examined the mechanisms underlying the inhibition of 1321N1 cell proliferation by brefelamide. Glial cell line-derived neurotrophic factor (GDNF) was found to enhance the rate of proliferation of serum-free cultured 1321N1 cells, but did not affect proliferation in PC12 cells. Brefelamide pretreatment inhibited GDNF-induced cell proliferation and expression of rearranged during transfection (RET). GDNF enhanced the phosphorylation of extracellular signal-regulated kinase (ERK), AKT, and c-jun-N-terminal kinase (JNK); however, brefelamide pretreatment inhibited these effects. Brefelamide also reduced the expression of GDNF mRNA and GDNF secretion. Together, the findings from this study indicate that brefelamide inhibits the proliferation of 1321N1 cell via several mechanisms including reduced GDNF receptor expression and GDNF secretion, and reduced phosphorylation of ERK, AKT, and JNK.

Encapsulated cell device approach for combined electrical stimulation and neurotrophic treatment of the deaf cochlea

Profound hearing impairment can be overcome by electrical stimulation (ES) of spiral ganglion neurons (SGNs) via a cochlear implant (CI). Thus, SGN survival is critical for CI efficacy. Application of glial cell line-derived neurotrophic factor (GDNF) has been shown to reduce SGN degeneration following deafness. We tested a novel method for local, continuous GDNF-delivery in combination with ES via a CI. The encapsulated cell (EC) device contained a human ARPE-19 cell-line, genetically engineered for secretion of GDNF. In vitro, GDNF delivery was stable during ES delivered via a CI. In the chronic in vivo part, cats were systemically deafened and unilaterally implanted into the scala tympani with a CI and an EC device, which they wore for six months. The implantation of control devices (same cell-line not producing GDNF) had no negative effect on SGN survival. GDNF application without ES led to an unexpected reduction in SGN survival, however, the combination of GDNF with initial, short-term ES resulted in a significant protection of SGNs. A tight fibrous tissue formation in the scala tympani of the GDNF-only group is thought to be responsible for the increased SGN degeneration, due to mechanisms related to an aggravated foreign body response. Furthermore, the fibrotic encapsulation of the EC device led to cell death or cessation of GDNF release within the EC device during the six months in vivo. In both in vitro and in vivo, fibrosis was reduced by CI stimulation, enabling the neuroprotective effect of the combined treatment. Thus, fibrous tissue growth limits treatment possibilities with an EC device. For a stable and successful long-term neurotrophic treatment of the SGN via EC devices in human CI users, it would be necessary to make changes in the treatment approach (provision of anti-inflammatories), the EC device surface (reduced cell adhesion) and the ES (initiation prior to fibrosis formation).

Atypical antipsychotic treatment increases glial cell line-derived neurotrophic factor serum levels in drug-free schizophrenic patients along with improvement of psychotic symptoms and therapeutic effects.

Glial cell line-derived neurotrophic factor (GDNF) plays an increasingly vital role in the pathogenesis of neuropsychiatric illnesses. Antipsychotic medications were shown to stimulate GDNF secretion from C6 glioma cells. The aims of this study were to investigate the serum concentration of GDNF, to monitor the therapeutic effect of atypical antipsychotics related to GDNF levels in drug-free schizophrenia patients, and to examine these levels in relation to psychotic symptoms. We recruited 138 drug-free schizophrenic patients and compared them with 77 matched healthy subjects. All patients were treated with atypical antipsychotic monotherapy. GDNF serum levels and psychiatric symptoms were assessed at baseline and after 2, 4, 6 and 8 weeks. GDNF levels gradually increased accompanied by a reduction in psychiatric symptoms during antipsychotic therapy. The levels of GDNF in responders were significantly increased after 8 weeks of treatment, however, no significant change was found in non-responders. Furthermore, a negative association between GDNF levels following pharmacotherapy and disease duration in schizophrenic subjects could be observed. The present study suggests that GDNF may be involved in the etiology of schizophrenia and pharmacological treatment.

Glial Cell Line Derived Neurotrophic Factor Protein Secretion by Skeletal Muscles is Altered by Activity of L‐Type Calcium Channels

Glial cell line‐derived neurotrophic factor (GDNF) is an important signaling molecule throughout the peripheral nervous system. GDNF protein is produced and secreted by skeletal muscle cells and helps maintain motor neuron innervation at the neuromuscular junction. Treatment with exogenous GDNF prevents denervation which occurs with aging and neurodegenerative disease. The therapeutic potential of GDNF cannot be fully explored without understanding the mechanisms by which GDNF protein production is regulated. Although the specific cellular events necessary for regulation of GDNF secretion remains to be elucidated, previous studies from our laboratory have shown that increased skeletal muscle activity significantly alters GDNF protein levels in vivo and in vitro. The primary objective of this study was to determine the role of calcium in regulating GDNF protein secretion by skeletal muscle cells. Skeletal muscle cells (C2C12) were grown in culture and allowed to differentiate into myotubes which display a contractile phenotype. The following treatments were given, Nifedipine to block the voltage‐gated L‐type calcium channels, and Bay K8644 to enhance calcium influx through L‐type calcium channels. Both Western Blot and ELISA were employed for protein identification and GDNF protein quantification respectively. Preliminary results indicate that treatment of myotubes with Bay K8644 (100μM) caused an increase in GDNF secretion to 170% of that in untreated controls. Treatment of cells with Nifedipine blocked the stimulatory effect of Bay K8644, and treatment with Nifedipine alone (10μM and 100μM) inhibited GDNF secretion to 77% of that in control cells. The results suggest that secretion of GDNF protein by C2C12 myotubes is regulated by intracellular calcium levels. Acquiring a greater understanding of the role that calcium plays in regulating GDNF production may help to identify potential sites for therapeutic intervention to increase or decrease GDNF production.Support or Funding InformationNSF Grant DBI‐1062883 and NIH Grant 1R15AG022908‐01A2

Tetrahydroxystilbene Glucoside Improves Neurotrophic Factors Release in Cultured Astroglia.

Tetrahydroxystilbene glucoside (TSG), one of the main ingredients of Polygonum multiflorum, has a great number of beneficial effects for health including anti-oxidant, free radicalscavenging and anti-inflammatory properties. However, the potential effects of TSG on neurotrophic factors release remain unclear. In this study, rat primary astroglia cultures were applied to investigate TSG-mediated neurotrophic effects. The protein levels and production of glial cell-line derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in astroglia and the culture medium were determined by western blotting assay and ELISA, respectively. Results indicated that TSG increased the production of neurotrophic factors in a concentration-dependent manner. At different time points of TSG treatment, the BDNF and NGF production in the culture medium was increased 48 h after treatment, while GDNF secretion was initially induced 24 h after TSG treatment. Consistent with the neurotrophic factors release, TSG significantly increased the BDNF, GDNF and NGF protein expressions in astroglia. Furthermore, TSG significantly induced the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and a specific ERK inhibitor-U0126 inhibited TSG-mediated secretion of BDNF, GDNF and NGF. Overall, this study demonstrated that TSG induces astroglia-derived neurotrophic factors release, suggesting TSG might hold a therapeutic potential for neurological disorders.

DNA damage induces GDNF secretion in the tumor microenvironment with paracrine effects promoting prostate cancer treatment resistance

Though metastatic cancers often initially respond to genotoxic therapeutics, acquired resistance is common. In addition to cytotoxic effects on tumor cells, DNA damaging agents such as ionizing radiation and chemotherapy induce injury in benign cells of the tumor microenvironment resulting in the production of paracrine-acting factors capable of promoting tumor resistance phenotypes. In studies designed to characterize the responses of prostate and bone stromal cells to genotoxic stress, we found that transcripts encoding glial cell line-derived neurotrophic factor (GDNF) increased several fold following exposures to cytotoxic agents including radiation, the topoisomerase inhibitor mitoxantrone and the microtubule poison docetaxel. Fibroblast GDNF exerted paracrine effects toward prostate cancer cells resulting in enhanced tumor cell proliferation and invasion, and these effects were concordant with the expression of known GDNF receptors GFRA1 and RET. Exposure to GDNF also induced tumor cell resistance to mitoxantrone and docetaxel chemotherapy. Together, these findings support an important role for tumor microenvironment damage responses in modulating treatment resistance and identify the GDNF signaling pathway as a potential target for improving responses to conventional genotoxic therapeutics.

Zirconium oxide ceramic foam: a promising supporting biomaterial for massive production of glial cell line-derived neurotrophic factor.

This study investigated the potential application of a zirconium oxide (ZrO2) ceramic foam culturing system to the production of glial cell line-derived neurotrophic factor (GDNF). Three sets of ZrO2 ceramic foams with different pore densities of 10, 20, and 30 pores per linear inch (PPI) were prepared to support a 3D culturing system. After primary astrocytes were cultured in these systems, production yields of GDNF were evaluated. The biomaterial biocompatibility, cell proliferation and activation of cellular signaling pathways in GDNF synthesis and secretion in the culturing systems were also assessed and compared with a conventional culturing system. In this study, we found that the ZrO2 ceramic foam culturing system was biocompatible, using which the GDNF yields were elevated and sustained by stimulated cell proliferation and activation of signaling pathways in astrocytes cultured in the system. In conclusion, the ZrO2 ceramic foam is promising for the development of a GDNF mass production device for Parkinson's disease treatment.

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.

Effects of acetylcholine and electrical stimulation on glial cell line-derived neurotrophic factor production in skeletal muscle cells

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor required for survival of neurons in the central and peripheral nervous system. Specifically, GDNF has been characterized as a survival factor for spinal motor neurons. GDNF is synthesized and secreted by neuronal target tissues, including skeletal muscle in the peripheral nervous system; however, the mechanisms by which GDNF is synthesized and released by skeletal muscle are not fully understood. Previous results suggested that cholinergic neurons regulate secretion of GDNF by skeletal muscle. In the current study, GDNF production by skeletal muscle myotubes following treatment with acetylcholine was examined. Acetylcholine receptors on myotubes were identified with labeled alpha-bungarotoxin and were blocked using unlabeled alpha-bungarotoxin. The question of whether electrical stimulation has a similar effect to that of acetylcholine was also investigated. Cells were stimulated with voltage pulses; at 1 and 5Hz frequencies for times ranging from 30min to 48h. GDNF content in myotubes and GDNF in conditioned culture medium were quantified by enzyme-linked immunosorbant assay. Results suggest that acetylcholine and short-term electrical stimulation reduce GDNF secretion, while treatment with carbachol or long-term electrical stimulation enhances GDNF production by skeletal muscle.

Non-viral human proGDNF gene delivery to rat bone marrow stromal cells under ex vivo conditions

Glial cell line-derived neurotrophic factor (GDNF) is one of the most important proteins playing a pivotal role in growing and repairing of the nervous system. GDNF therapy is one of the suggested options in the treatment of neurodegenerative diseases. Limitations in the viral gene delivery and its side effects after therapy have encouraged us to use a non-viral method one for this purpose. We transfected rat bone marrow stromal cells (BMSCs) in ex vivo conditions using Lipofectamine 2000 reagent with pEGFP-C1 and a constructed vector carrying the human proGDNF (pSecTag2/HygroB-human proGDNF), transiently and stably, respectively. The rate of transient transfection of rat BMSCs was eight percent and transfected rat BMSCs with pSecTag2/HygroB-human proGDNF stabilized by adding Hygromycin B in cell culture medium at 200μg/ml. Semi-quantitative data analysis from Western-blot technique showed that stable transfected cells secrete GDNF at higher level in comparison with control cells (6.530fold in the supernatant). The present study supports the utility of liposome-mediated transfection for overexpressing human GDNF in rat BMSCs. For this purpose and in order to get more yield of human GDNF secretion from the stable transfected rat BMSCs, we used a vector containing another signal sequence instead of its own pre-segment of proGDNF protein. This is the first report in this regard and the data presented will be potentially useful for human gene transfer therapies in a variety of neurodegenerative diseases.

Electroacupuncture enhances hippocampal NSCs proliferation in cerebral ischemia-reperfusion injured rats via activation of notch signaling pathway

The purpose of this study was to investigate the mechanisms by which electroacupuncture (EA) enhances hippocampal neural stem cells (NSCs) proliferation in cerebral ischemia-reperfusion (I/R) injured rats. A total of 72 male adult Sprague-Dawley rats were randomly divided into the sham operation control group (SC), the ischemia control group (IC) and the EA group. Middle cerebral artery occlusion (MCAO) was performed to establish the focal cerebral I/R injury model. Proliferation of hippocampal NSCs in cerebral I/R injured rats was determined by the Nestin immunohistochemical staining. Activation of the notch signaling pathway was detected by Western blotting and reverse transcription polymerase chain reaction analysis. The serum level of neurotrophic factors, e.g., the brain-derived neurotrophic factor (BDNF) and the Glial cell line-derived neurotrophic factor (GDNF), was measured using enzyme-linked immunosorbent assay (ELISA). The results showed that EA at Quchi (LI11) and Zusanli (ST36) acupoints significantly alleviated neurological deficits, reduced infarct volumes and promoted the proliferation of hippocampal NSCs in cerebral I/R injured rats. The crucial signaling molecules in the notch signaling pathway were activated and the secretion of BDNF and GDNF was increased upon EA. The protein and mRNA levels of Cyclin D1, Cdk4 and p-Rb were increased, while p21 and p27 transcripts were suppressed by notch signaling. These results suggest that the up-regulatory effect of EA on the notch signaling pathway and neurotrophic factor secretion may result in the promotion of NSCs proliferation and consequently a therapeutic effect on cerebral ischemia.

The reduction in immunogenicity of neurotrophin overexpressing stem cells after intra-striatal transplantation by encapsulation in an in situ gelling collagen hydrogel

Delivery of neurotrophic factors to the brain via genetically modified bone marrow-derived mesenchymal stem cells (MSCs) offers a promising neuroprotective strategy for neurodegenerative diseases. However, MSCs delivered to the CNS typically show poor survival post-transplantation, which is accompanied by microglial activation and astrocyte recruitment at the graft site. Recent studies have shown the potential of biomaterials to provide a supportive matrix for transplanted cells which may assist in the grafting process. In this study, an in situ gelling type I collagen hydrogel was evaluated as an intracerebral transplantation matrix for delivery of glial cell line-derived neurotrophic factor (GDNF)-overexpressing MSCs to the rat brain (GDNF-MSCs). In vitro analyses demonstrated that this collagen hydrogel did not affect the viability of the GDNF-MSCs nor did it prevent GDNF secretion into the surrounding medium. In vivo analyses also confirmed that the collagen hydrogel did not negatively impact on the survival of the cells and permitted GDNF secretion into the striatal parenchyma. Importantly, this study also revealed that transplanting GDNF-MSCs in a collagen hydrogel significantly diminished the host brain's response to the cells by reducing the recruitment of both microglia and astrocytes at the site of delivery. In conclusion, this hydrogel, which is composed of the natural extracellular matrix, collagen, was shown to be a well-tolerated cell delivery platform technology which could be functionalised to further aid cell support and graft integration.

Repeated Systemic Administration of Human Adipose-Derived Stem Cells Attenuates Overt Diabetic Nephropathy in Rats

Adipose-derived stem cells (ASCs) can alleviate acute kidney injury and promote kidney cell regeneration and repair. To investigate the role of ASCs in diabetic nephropathy (DN), Sprague-Dawley rats were made diabetic by intraperitoneal injection of streptozotocin (STZ) after uninephrectomy. After 12 weeks, proteinuria was well established. Five times of 5×10(6) human ASCs repeatedly injected through a tail vein at 4 weekly intervals. A reduction in proteinuria was not observed in diabetic rats until 24 weeks. However, urinary protein excretion was significantly suppressed at 28 weeks and persisted up to 32 weeks after STZ treatment. ASC treatment significantly attenuated glomerulus hypertrophy and tubular interstitial injury, and led to the downregulation of WT-1 and synaptopodin expression. CFSE labeled ASCs were injected into DN rats via the tail vein. Within 24 h after injection, the cells were detected in lung, spleen, and peritubular regions, but rarely in pancreas. Human Alu gene expression was detected in lung and spleen up to 4 weeks after ASCs injection. ASC treatment did not improve hyperglycemia or pancreatic damage. In vitro, recombinant human glial cell line-derived neurotrophic factor (GDNF) prevented podocyte injury by high glucose similarly to ASC-conditioned medium. After blocking GDNF in ASC-CM with neutralizing antibody, the therapeutic effect of ASC-CM was significantly decreased. ASCs cocultured with podocytes restored the downregulation of synaptopodin expression, which was weakened by GDNF-RNA interfering. These findings indicate that repeated intravenous ASC can reduce diabetic kidney damage in rats even at the progressive stage, and promote podocyte recovery via GDNF secretion.

Effect of platelet-rich plasma (PRP) concentration on proliferation, neurotrophic function and migration of Schwann cellsin vitro

Platelet-rich plasma (PRP) contains various growth factors and appears to have the potential to promote peripheral nerve regeneration, but evidence is lacking regarding its biological effect on Schwann cells (SCs). The present study was designed to investigate the effect of PRP concentration on SCs in order to determine the plausibility of using this plasma-derived therapy for peripheral nerve injury. PRP was obtained from rats by double-step centrifugation and was characterized by determining platelet numbers and growth factor concentrations. Primary cultures of rat SCs were exposed to various concentrations of PRP (40%, 20%, 10%, 5% and 2.5%). Cell proliferation assays and flow cytometry were performed to study to assess SC proliferation. Quantitative real-time PCR and ELISA analysis were performed to determine the ability of PRP to induce SCs to produce nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF). Microchemotaxis assay was used to analyse the cell migration capacity. The results obtained indicated that the platelet concentration and growth factors in our PRP preparations were significantly higher than in whole blood. Cell culture experiments showed that 2.5-20% PRP significantly stimulated SC proliferation and migration compared to untreated controls in a dose-dependent manner. In addition, the expression and secretion of NGF and GDNF were significantly increased. However, the above effects of SCs were suppressed by high PRP concentrations (40%). In conclusion, the appropriate concentration of PRP had the potency to stimulate cell proliferation, induced the synthesis of neurotrophic factors and significantly increased migration of SCs dose-dependently. Copyright © 2013 John Wiley & Sons, Ltd.

The Effects of Progesterone on Glial Cell Line-derived Neurotrophic Factor Secretion from C6 Glioma Cells.

Progesterone is a steroid hormone whose biology has been greatly studied within the confines of reproductive function. In recent years, the neuroprotective effects of progesterone have attracted growing interest. Glial cell line-derived neurotrophic factor (GDNF), is a neurotrophic factor which plays a crucial role in the development and maintenance of distinct sets of central and peripheral neurons. In the present study, we investigated the potential implication of GDNF in the neuroprotective action of progesterone. Cultured rat C6 glioma cells were treated with progesterone (100 nm, 1 µM, and 10 µM) or its vehicle. After 24, 36, 48 and 72 hr, GDNF protein levels were measured in the cell-conditioned media and cell lysates using a GDNF ELISA kit. Cell numbers were determined by a cell-counting assay kit. Forty-eight hr treatment with progesterone (10 µM) resulted in a significant elevation of GDNF secretion from C6 glioma cells that remained elevated up to 72 hr. The intracellular content of GDNF and cell numbers were not affected by progesterone treatment. Stimulation of GDNF release from glial cells appears as a potential mechanism through which progesterone exerts its neuroprotective effects.

Non-viral gene therapy for GDNF production in RCS rat: the crucial role of the plasmid dose

Glial cell line-derived neurotrophic factor (GDNF) is one of the candidate molecules among neurotrophic factors proposed for a potential treatment of retinitis pigmentosa (RP). It must be administered repeatedly or through sustained releasing systems to exert prolonged neuroprotective effects. In the dystrophic Royal College of Surgeon's (RCS) rat model of RP, we found that endogenous GDNF levels dropped during retinal degeneration time course, opening a therapeutic window for GDNF supplementation. We showed that after a single electrotransfer of 30 μg of GDNF-encoding plasmid in the rat ciliary muscle, GDNF was produced for at least 7 months. Morphometric, electroretinographic and optokinetic analyses highlighted that this continuous release of GDNF delayed photoreceptors (PRs) as well as retinal functions loss until at least 70 days of age in RCS rats. Unexpectedly, increasing the GDNF secretion level accelerated PR degeneration and the loss of electrophysiological responses. This is the first report: (i) demonstrating the efficacy of GDNF delivery through non-viral gene therapy in RP; (ii) establishing the efficacy of intravitreal administration of GDNF in RP associated with a mutation in the retinal pigment epithelium; and (iii) warning against potential toxic effects of GDNF within the eye/retina.