Cell Line-derived Neurotrophic Factor Gene Research Articles

Environmental enrichment accentuates glucose-induced feeding suppression and glial cell line-derived neurotrophic factor gene expression in the hypothalamus of mice

ABSTRACT The hypothalamus controls food intake by integrating nutrient signals, of which one of the most important is glucose. Consequently, impairments in hypothalamic glucose-sensing mechanisms are associated with hyperphagia and obesity. Environmental enrichment (EE) is an animal housing protocol that provides complex sensory, motor, and social stimulations and has been proven to reduce adiposity in laboratory mice. However, the mechanism by which EE promotes adiposity-suppressing effect remains incompletely understood. Neurotrophic factors play an important role in the development and maintenance of the nervous system, but they are also involved in the hypothalamic regulation of feeding. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) are expressed in the hypothalamus and their expression is stimulated by glucose. EE is associated with increased expression of Bdnf mRNA in the hypothalamus. Therefore, we hypothesized that EE potentiates the anorectic action of glucose by altering the expression of neurotrophic factor genes in the hypothalamus. Male C57BL/6 mice were maintained under standard or EE conditions to investigate the feeding response to glucose and the associated expression of feeding-related neurotrophic factor genes in the hypothalamus. Intraperitoneal glucose injection reduced food intake in both control and EE mice with a significantly greater reduction in the EE group compared to the control group. EE caused a significantly enhanced response of Gdnf mRNA expression to glucose without altering basal Gdnf mRNA expression and Bdnf mRNA response to glucose. These findings suggest that EE enhances glucose-induced feeding suppression, at least partly, by enhancing hypothalamic glucose-sensing ability that involves GDNF.

Tobacco and betel quid chewing is associated with glial cell line-derived neurotrophic factor (GDNF) gene variants in three Indian populations

Tobacco and betel quid chewing is associated with glial cell line-derived neurotrophic factor (GDNF) gene variants in three Indian populations

Glucose Stimulates Glial Cell Line-Derived Neurotrophic Factor Gene Expression in Microglia through a GLUT5-Independent Mechanism.

Feeding-regulating neurotrophic factors are expressed in both neurons and glial cells. However, nutritional regulation of anorexigenic glial cell line-derived neurotrophic factor (GDNF) and orexigenic mesencephalic astrocyte-derived neurotrophic factor (MANF) expression in specific cell types remains poorly understood. Hypothalamic glucose sensing plays a critical role in the regulation of food intake. It has been theorized that local glucose concentration modulates microglial activity partially via glucose transporter 5 (GLUT5). We hypothesized that an increased local glucose concentration stimulates GDNF expression while inhibiting MANF expression in the hypothalamus and microglia via GLUT5. The present study investigated the effect of glucose on Gdnf and Manf mRNA expression in the mouse hypothalamus and murine microglial cell line SIM-A9. Intracerebroventricular glucose treatment significantly increased Gdnf mRNA levels in the hypothalamus without altering Manf mRNA levels. Exposure to high glucose caused a significant increase in Gdnf mRNA expression and a time-dependent change in Manf mRNA expression in SIM-A9 cells. GLUT5 inhibitor treatment did not block glucose-induced Gdnf mRNA expression in these cells. These findings suggest that microglia are responsive to changes in the local glucose concentration and increased local glucose availability stimulates the expression of microglial GNDF through a GLUT5-independent mechanism, contributing to glucose-induced feeding suppression.

Role of Glial Cell Line-Derived Neurotrophic Factor in the Maintenance of Adult Mesencephalic Catecholaminergic Neurons.

The glial cell line-derived neurotrophic factor has a potent neuroprotective action on mesencephalic dopamine neurons, which are progressively lost in Parkinson's disease. Intrastriatal administration of this factor is a promising therapy for Parkinson's disease. Glial cell line-derived neurotrophic factor is naturally produced in restricted cerebral regions, such as the striatum, septum, and thalamus; however, its effects in the adult brain remain under debate. We sought to clarify the physiologic role of endogenous glial cell line-derived neurotrophic factor in the survival of catecholaminergic neurons of the substantia nigra pars compacta and the locus coeruleus in adult mice. We used 2 new Cre recombinase-based mouse models to delete a floxed-glial cell line-derived neurotrophic factor gene. The first model had Cre expression in the parvalbumin expressing interneurons, as these cells represent the major source of striatal glial cell line-derived neurotrophic factor. The second model was an estrogen receptor 2-based inducible Cre triggered by tamoxifen at 2 months of age. We found that the floxed-glial cell line-derived neurotrophic factor gene was resilient to ablation by Cre-induced recombination and that parvalbumin-driven Cre was particularly inefficient to do so. The inducible-Cre model allowed an average 70% to 80% reduction in glial cell line-derived neurotrophic factor messenger ribonucleic acid and protein in striatum and septum with moderate significant loss of catecholamine neurons in the nigrostriatal pathway and, more markedly, in the locus coeruleus. This was accompanied with mild locomotor decline. Our data support qualitatively the view that brain glial cell line-derived neurotrophic factor is needed for the maintenance of adult central catecholaminergic neurons. © 2020 International Parkinson and Movement Disorder Society.

GDNFOS1 knockdown decreases the invasion and viability of glioblastoma cells.

Glioblastoma multiforme is the most aggressive primary brain cancer in adults. Therefore, it is important to investigate the mechanisms associated with cell viability and invasion ability of the cells in glioblastoma multiforme. The opposite strand of the glial cell line-derived neurotrophic factor (GDNF) gene is used to transcribe the cis-antisense GDNF opposite strand (GDNFOS) gene, which belongs to the long noncoding RNAs. The current study assessed the effects of GDNFOS1 overexpression and interference on GDNF expression, cell viability and invasion ability in U87 and U251 MG glioblastoma cells. Overexpression and interference were performed using constructed lentiviral vectors, including long non-coding RNA GDNFOS1 overexpression vector, pL-short hairpin RNA (shRNA)-GDNFOS1-9, pL-shRNA-GDNFOS1-49, pL-shRNA-GDNFOS1-248, pL-shRNA-GDNFOS1-9+49, pL-shRNA-GDNFOS1-9+248 and pL-shRNA-GDNFOS1-49+248. Reverse transcription-quantitative PCR was used to determine the efficiency of interference and overexpression of GDNFOS1 in U87 and U251 MG cells. GDNF protein expression in U87 and U251 MG cells was detected using western blot analysis. In addition, cell viability was detected using a cell counting kit-8 assay at 24, 48 and 72 h after GDNFOS1 overexpression or interference. A transwell invasion assay was used to detect invasion ability. Different shRNA sequences were tested and the results revealed that a combination (pL-shRNA-GDNFOS1-49+248) was most effective in the knock-down GDNFOS1. Compared with the control group, GDNF expression in U87 MG cells was significantly increased in the GDNFOS1 overexpression group and decreased in the shRNA-GDNFOS1-248 group. U87 MG cell viability was significantly increased in the GDNFOS1 overexpression group at 24, 48 and 72 h compared with the negative control group. The viability of U87 MG cells was decreased in the GDNFOS1 interference group at 72 h when compared with the control group. The relative invasive ability was significantly increased in the GDNFOS1 overexpression group when compared with the negative control group. The invasive ability was significantly decreased in the GDNFOS1 interference group when compared with the negative control group. Similar results were exhibited by the U251 MG cells. Overall, GDNF expression, cell viability and invasion ability of glioblastoma cells significantly increased with GDNFOS1 overexpression and decreased with GDNFOS1 interference.

Timed GDNF gene therapy using an immune-evasive gene switch promotes long distance axon regeneration.

Neurosurgical repair in patients with proximal nerve lesions results in unsatisfactory recovery of function. Gene therapy for neurotrophic factors is a powerful strategy to promote axon regeneration. Glial cell line-derived neurotrophic factor (GDNF) gene therapy promotes motor neuron survival and axon outgrowth; however, uncontrolled delivery of GDNF results in axon entrapment. We report that time-restricted GDNF expression (1 month) using an immune-evasive doxycycline-inducible gene switch attenuated local axon entrapment in avulsed reimplanted ventral spinal roots, was sufficient to promote long-term motor neuron survival (24 weeks) and facilitated the recovery of compound muscle action potentials by 8 weeks. These improvements were associated with an increase in long-distance regeneration of motor axons. In contrast, persistent GDNF expression impaired axon regeneration by inducing axon entrapment. These findings demonstrate that timed expression can resolve the deleterious effect of uncontrolled growth factor delivery and shows that inducible growth factor gene therapy can be employed to enhance the efficacy of axon regeneration after neurosurgical repair of a proximal nerve lesion in rats. This preclinical study is an important step in the ongoing development of a neurotrophic factor gene therapy for patients with severe proximal nerve lesions.

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.

Transplantation of Amniotic Fluid-Derived Stem Cells Preconditioned with Glial Cell Line-Derived Neurotrophic Factor Gene Alleviates Renal Fibrosis

Amniotic fluid-derived stem cells (AFSCs), which exhibit both embryonic and mesenchymal stem cell characteristics, have been shown to mitigate the degree of renal interstitial fibrosis. The aim of the present study was to determine whether transplantation of glial cell line-derived neurotrophic factor (GDNF)–modified AFSCs is more useful than transplantation of unmodified AFSCs for the treatment of renal interstitial fibrosis. Mice were randomly assigned to a sham-operation group (sham), a unilateral ureteral obstruction (UUO)-saline solution group (UUO), an AFSC transplantation group (AFSC) and a GDNF-modified AFSC transplantation group (GDNF-AFSC) and sacrificed at days 3 and 7 post-surgery (six in each group). We showed that GDNF-AFSCs noticeably suppressed oxidative stress and inflammation; additionally, GDNF-AFSCs positively regulated peritubular capillaries (PTCs), vascular endothelial growth factor (VEGF), hypoxia inducible factor-1α (HIF-1α), and transforming growth factor-β1 (TGF-β1) protein levels. Transmission electron microscopy (TEM) revealed that mitochondrial injury induced by the UUO model was significantly ameliorated after the mice were treated with GDNF-AFSCs. Therefore, we determined that GDNF gene promotes the abilities of AFSCs to inhibit inflammatory and oxidative stress effects, repair renal microvessels, relieve tissue hypoxia and mitochondrial damage, and, ultimately, alleviate renal interstitial fibrosis.

Association of medullary sponge kidney and hyperparathyroidism with RET G691S/S904S polymorphism: a case report

BackgroundMedullary sponge kidney is a rare renal malformation, which usually manifests as nephrocalcinosis, renal tubular acidosis, and recurrent urinary tract infections. Medullary sponge kidney is often associated with renal developmental anomalies and tumors, and its exact pathogenesis is not yet clearly explained. Given the key role of the interaction of glial cell line-derived neurotrophic factor gene, GDNF, and the “rearranged during transfection” proto-oncogene, RET, in kidney and urinary tract development, variations in these genes are proposed to be candidates for medullary sponge kidney. Hyperparathyroidism is observed in a few patients with medullary sponge kidney, but the exact pathogenesis of this association is unknown. This case report highlights the coexistence of these two conditions associated with RET polymorphism, which contributes toward the understanding of the pathogenesis of medullary sponge kidney.Case presentationA 52-year-old Chinese woman with recurrent renal stones presented to our hospital. Subsequently she was diagnosed as having medullary sponge kidney and tertiary hyperparathyroidism and underwent parathyroidectomy. Genomic DNA was isolated from lymphocytes and the GDNF and RET genes were determined by Sanger sequencing. Two RET polymorphisms were found in our patient, one was nonsynonymous c.2071G>A (G691S; rs1799939) located in exon 11, the other was synonymous c.2712C>G. (p.S904S; rs1800863) located in exon 15.ConclusionsWe demonstrated a case of medullary sponge kidney combined with tertiary hyperparathyroidism, which contributes to further understanding of the pathogenesis of this disease. Besides, we also found RET G691S/S904S polymorphism in this patient, but additional studies are required to explore the role of the RET gene in medullary sponge kidney with hyperparathyroidism.

Intranasal Delivery of pGDNF DNA Nanoparticles Provides Neuroprotection in the Rat 6-Hydroxydopamine Model of Parkinson's Disease.

Glial cell line-derived neurotrophic factor (GDNF) gene therapy could offer a disease-modifying treatment for Parkinson's disease (PD). Here, we report that plasmid DNA nanoparticles (NPs) encoding human GDNF administered intranasally to rats induce transgene expression in the brain and protect dopamine neurons in a model of PD. To first test whether intranasal administration could transfect cells in the brain, rats were sacrificed 1week after intranasal pGDNF NPs or the naked plasmid. GDNF ELISA revealed significant increases in GDNF expression throughout the brain for both treatments. To assess whether expression was sufficient to protect dopamine neurons, naked pGDNF and pGDNF DNA NPs were given intranasally 1week before a unilateral 6-hydroxydopamine lesion in a rat model of PD. Three to four weeks after the lesion, amphetamine-induced rotational behavior was reduced, and dopaminergic fiber density and cell counts in the lesioned substantia nigra and nerve terminal density in the lesioned striatum were significantly preserved in rats given intranasal pGDNF. The NPs afforded a greater level of neuroprotection than the naked plasmid. These results provide proof-of-principle that intranasal administration of pGDNF DNA NPs can offer a non-invasive, non-viral gene therapy approach for early-stage PD.

Glial Cell Line-Derived Neurotrophic Factor Gene Delivery in Parkinson's Disease: A Delicate Balance between Neuroprotection, Trophic Effects, and Unwanted Compensatory Mechanisms.

Glial cell line-derived neurotrophic factor (GDNF) and Neurturin (NRTN) bind to a receptor complex consisting of a member of the GDNF family receptor (GFR)-α and the Ret tyrosine kinase. Both factors were shown to protect nigro-striatal dopaminergic neurons and reduce motor symptoms when applied terminally in toxin-induced Parkinson's disease (PD) models. However, clinical trials based on intraputaminal GDNF protein administration or recombinant adeno-associated virus (rAAV)-mediated NRTN gene delivery have been disappointing. In this review, several factors that could have limited the clinical benefits are discussed. Retrograde transport of GDNF/NRTN to the dopaminergic neurons soma is thought to be necessary for NRTN/GFR-α/Ret signaling mediating the pro-survival effect. Therefore, the feasibility of treating advanced patients with neurotrophic factors is questioned by recent data showing that: (i) tyrosine hydroxylase-positive putaminal innervation has almost completely disappeared at 5 years post-diagnosis and (ii) in patients enrolled in the rAAV-NRTN trial more than 5 years post-diagnosis, NRTN was almost not transported to the substantia nigra pars compacta. In addition to its anti-apoptotic and neurotrophic properties, GDNF also interferes with dopamine homeostasis via time and dose-dependent effects such as: stimulation of dopamine neuron excitability, inhibition of dopamine transporter activity, tyrosine hydroxylase phosphorylation, and inhibition of tyrosine hydroxylase transcription. Depending on the delivery parameters, the net result of this intricate network of regulations could be either beneficial or deleterious. In conclusion, further unraveling of the mechanism of action of GDNF gene delivery in relevant animal models is still needed to optimize the clinical benefits of this new therapeutic approach. Recent developments in the design of regulated viral vectors will allow to finely adjust the GDNF dose and period of administration. Finally, new clinical studies in less advanced patients are warranted to evaluate the potential of AAV-mediated neurotrophic factors gene delivery in PD. These will be facilitated by the demonstration of the safety of rAAV administration into the human brain.

Egr-1 and RNA POL II facilitate glioma cell GDNF transcription induced by histone hyperacetylation in promoter II.

The specific mechanisms for epigenetic regulation of gene transcription remain to be elucidated. We previously demonstrated that hyperacetylation of histone H3K9 in promoter II of glioma cells promotes high transcription of the glial cell line-derived neurotrophic factor (GDNF) gene. This hyperacetylation significantly enhanced Egr-1 binding and increased the recruitment of RNA polymerase II (RNA POL II) to that region (P < 0.05). Egr-1 expression was abnormally increased in C6 glioma cells. Further overexpression of Egr-1 significantly increased Egr-1 binding to GDNF promoter II, while increasing RNA POL II recruitment, thus increasing GDNF transcription (P < 0.01). When the acetylation of H3K9 in the Egr-1 binding site was significantly reduced by the histone acetyltransferase (HAT) inhibitor curcumin, binding of Egr-1 to GDNF promoter II, RNA POL II recruitment, and GDNF mRNA expression were significantly downregulated (P < 0.01). Moreover, curcumin attenuated the effects of Egr-1 overexpression on Egr-1 binding, RNA POL II recruitment, and GDNF transcription (P < 0.01). Egr-1 and RNA POL II co-existed in the nucleus of C6 glioma cells, with overlapping regions, but they were not bound to each other. In conclusion, highly expressed Egr-1 may be involved in the recruitment of RNA POL II in GDNF promoter II in a non-binding manner, and thereby involved in regulating GDNF transcription in high-grade glioma cells. This regulation is dependent on histone hyperacetylation in GDNF promoter II.

New non-renal congenital disorders associated with medullary sponge kidney (MSK) support the pathogenic role of GDNF and point to the diagnosis of MSK in recurrent stone formers.

Medullary sponge kidney (MSK) is a congenital renal disorder. Its association with several developmental abnormalities in other organs hints at the likelihood of some shared step(s) in the embryogenesis of the kidney and other organs. It has been suggested that the REarranged during Transfection (RET) proto-oncogene and the Glial cell line-Derived Neurotrophic Factor (GDNF) gene are defective in patients with MSK, and both RET and GDNF are known to have a role in the development of the central nervous system, heart, and craniofacial skeleton. Among a cohort of 143 MSK patients being followed up for nephrolithiasis and chronic kidney disease at our institution, we found six with one or more associated non-renal anomalies: one patient probably has congenital hemihyperplasia and hypertrophic cardiomyopathy with adipose metaplasia and mitral valve prolapse; one has Marfan syndrome; and the other four have novel associations between MSK and nerve and skeleton abnormalities described here for the first time. The discovery of disorders involving the central nervous system, cardiovascular system and craniofacial skeleton in MSK patients supports the hypothesis of a genetic alteration on the RET-GDNF axis having a pivotal role in the pathogenesis of MSK, in a subset of patients at least. MSK seems more and more to be a systemic disease, and the identification of extrarenal developmental defects could be important in arousing the suspicion of MSK in recurrent stone formers.

Non-invasive, neuron-specific gene therapy by focused ultrasound-induced blood-brain barrier opening in Parkinson's disease mouse model

Focused ultrasound (FUS)-induced with microbubbles (MBs) is a promising technique for noninvasive opening of the blood-brain barrier (BBB) to allow targeted delivery of therapeutic substances into the brain and thus the noninvasive delivery of gene vectors for CNS treatment. We have previously demonstrated that a separated gene-carrying liposome and MBs administration plus FUS exposure can deliver genes into the brain, with the successful expression of the reporter gene and glial cell line-derived neurotrophic factor (GDNF) gene. In this study, we further modify the delivery system by conjugating gene-carrying liposomes with MBs to improve the GDNF gene-delivery efficiency, and to verify the possibility of using this system to perform treatment in the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced animal disease model. FUS-BBB opening was verified by contrast-enhanced MRI, and GFP gene expression was verified via in vivo imaging system (IVIS). Western blots as well as enzyme-linked immunosorbent assay (ELISA) were conducted to measure protein expression, and immunohistochemistry (IHC) was conducted to test the Tyrosine hydroxylase (TH)-neuron distribution. Dopamine (DA) and its metabolites as well as dopamine active transporter (DAT) were quantitatively analyzed to show dopaminergic neuronal dopamine secretion/activity/metabolism. Motor performance was evaluated by rotarod test weekly. Results demonstrated that the LpDNA-MBs (gene-liposome-MBs) complexes successfully serve as gene carrier and BBB-opening catalyst, and outperformed the separated LpDNA/MBs administration both in terms of gene delivery and expression. TH-positive IHC and measurement of DA and its metabolites DOPAC and HVA confirmed improved neuronal function, and the proposed system also provided the best neuroprotective effect to retard the progression of motor-related behavioral abnormalities. Immunoblotting and histological staining further confirmed the expression of reporter genes in neuronal cells. This study suggests that FUS exposures with the administration of LpDNA-MBs complexes synergistically can serve as an effective gene therapy strategy for MPTP-animal treatment, and may have potential for further application to perform gene therapy for neurodegenerative disease.

Relationship Between Chronic Tinnitus and Glial Cell Line-Derived Neurotrophic Factor Gene rs3812047, rs1110149, and rs884344 Polymorphisms in a Turkish Population.

Glial cell line-derived neurotrophic factor (GDNF) plays a key role in early development of central auditory pathway and the inner ear. However, the auditory pathway studies of GDNF gene polymorphisms are scarce in the literature, and the studies especially associated with tinnitus are limited. Our study aimed to identify whether GDNF gene polymorphisms play any roles in the pathophysiology of tinnitus by investigating the relationship between tinnitus and GDNF polymorphisms. A total of 52 patients with chronic tinnitus and ages ranging from 18 to 55 were admitted to the Ear-Nose-Throat outpatient clinic of Celal Bayar University Medical Faculty Hospital of Manisa, Turkey and constituted the study group. Another 42 patients of the same age range, without tinnitus symptoms and lacking any systemic disease, were also admitted to the clinic and formed the control group. The tympanometric, audiological, and psychoacoustic assessments of the subjects were performed. Deoxyribonucleic acid samples obtained using venous blood taken for routine inspections were used to investigate GDNF gene polymorphisms (rs884344, rs3812047, and rs1110149) by polymerase chain reaction-based restriction fragment length polymorphism method. No correlation could be detected between GDNF rs884344 and rs3812047 polymorphisms and subjects with tinnitus (p>0.05). Heterozygosity was significantly lower for GDNF rs1110149 polymorphism in tinnitus subjects compared to the controls (p<0.05). However, the allele frequencies for all 3 polymorphisms were not significantly different between tinnitus and control groups (p>0.05). Failure to detect correlations between tinnitus and GDNF gene polymorphisms suggests this may be due to the fact that the GDNF gene has a variable expression pattern in different tissues and pathologies. Therefore, the study should be improved and its scope should be expanded by including a larger group of patients and different tissues to investigate the expression pattern of GDNF.

APP-dependent glial cell line-derived neurotrophic factor gene expression drives neuromuscular junction formation.

Besides its crucial role in the pathogenesis of Alzheimer's disease, the knowledge of amyloid precursor protein (APP) physiologic functions remains surprisingly scarce. Here, we show that APP regulates the transcription of the glial cell line-derived neurotrophic factor (GDNF). APP-dependent regulation of GDNF expression affects muscle strength, muscular trophy, and both neuronal and muscular differentiation fundamental for neuromuscular junction (NMJ) maturation in vivo In a nerve-muscle coculture model set up to modelize NMJ formation in vitro, silencing of muscular APP induces a 30% decrease in secreted GDNF levels and a 40% decrease in the total number of NMJs together with a significant reduction in the density of acetylcholine vesicles at the presynaptic site and in neuronal maturation. These defects are rescued by GDNF expression in muscle cells in the conditions where muscular APP has been previously silenced. Expression of GDNF in muscles of amyloid precursor protein null mice corrected the aberrant synaptic morphology of NMJs. Our findings highlight for the first time that APP-dependent GDNF expression drives the process of NMJ formation, providing new insights into the link between APP gene regulatory network and physiologic functions.-Stanga, S., Zanou, N., Audouard, E., Tasiaux, B., Contino, S., Vandermeulen, G., René, F., Loeffler, J.-P., Clotman, F., Gailly, P., Dewachter, I., Octave, J.-N., Kienlen-Campard, P. APP-dependent glial cell line-derived neurotrophic factor gene expression drives neuromuscular junction formation.

Expression of GDNF Throughout Rat Brain After Intranasal Delivery of hGDNF Plasmid Nanoparticles

Glial cell line‐derived neurotrophic factor (GDNF) gene therapy is a promising approach for CNS disorders such as Parkinson's disease (PD). We are examining intranasal administration of PEG‐CK30 DNA nanoparticles (NPs) encoding hGDNF, developed by Copernicus Therapeutics, Inc. We have shown that intranasal NPs incorporating hGDNF DNA (pGDNF), or a hGDNF‐eGFP fusion plasmid (pUGG), transfect brain cells in vivo, and that intranasal pGDNF NPs protect substantia nigra (SN) dopamine neurons in a rat model of PD.Here, we examined transfection in rat brain 7 days after intranasal delivery of these NPs. ELISA showed increased GDNF levels throughout the brain of rats given pGDNF relative to saline controls. Immunohistochemistry (IHC) revealed that the number of eGFP positive cells was significantly higher in rats given intranasal pUGG NPs compared to background fluorescence in controls, with the highest numbers in midbrain. Double label IHC showed that most eGFP positive cells were immediately adjacent to capillary endothelial cells staining for RECA‐1, and they were often contiguous to GFAP positive astrocytic endfeet enwrapping capillaries. When eGFP cells were observed adjacent to dopamine neurons in SN, or NeuN labeled cells in any region, these neurons were also found close to a capillary. This pattern suggests that pericytes are transfected after intranasal administration of DNA NPs, consistent with distribution by perivascular transport. These results confirm delivery and transfection of cells in rat brain after intranasal administration of Copernicus' DNA NPs and support use of intranasal hGDNF NPs as a non‐invasive gene therapy for PD.

Association of 2 Neurotrophic Factor Polymorphisms With Efficacy of Paroxetine in Patients With Major Depressive Disorder in a Chinese Population

To evaluate the influence of the single nucleotide polymorphism (SNP) rs 6265 in the brain-derived neurotrophic factor (BDNF) gene and 21 SNPs of the glial cell line-derived neurotrophic factor (GDNF) gene on the efficacy of paroxetine in patients with major depressive disorder (MDD). Genotyping for BDNF and GDNF polymorphisms was performed in 298 patients with MDD who started 20 mg paroxetine per day and had their plasma concentrations measured after 6 weeks. The SNPs were selected from the HapMap Chinese ethnic group and literature reports. Changes in the severity of MDD were assessed with the Hamilton Depression Rating Scale (HAM-D) at baseline and at a 6-week follow-up. Paroxetine plasma concentration was measured using high-performance liquid chromatography with fluorescence detection. The Sequenom MassArray system was used for genotyping. At the 6-week follow-up, 219 of the 298 patients (73.5%) were responders and 79 patients (26.5%) were nonresponders to paroxetine treatment. The lower threshold concentration of paroxetine for response was 50 ng/mL, and a linear relationship was found between paroxetine plasma concentration and clinical response. The allele types for the SNPs rs 6265 (P < 0.001), rs 2973049 (P = 0.005), and rs 2216711 (P = 0.006) demonstrated significant associations with paroxetine treatment remission at week 6. Genetic variants in the BDNF and GDNF regions may be indicators of treatment response to paroxetine in patients with MDD.

Glial cell line-derived neurotrophic factor gene delivery via a polyethylene imine grafted chitosan carrier

Parkinson’s disease is known to result from the loss of dopaminergic neurons. Direct intracerebral injections of high doses of recombinant glial cell line-derived neurotrophic factor (GDNF) have been shown to protect adult nigral dopaminergic neurons. Because GDNF does not cross the blood–brain barrier, intracerebral gene transfer is an ideal option. Chitosan (CHI) is a naturally derived material that has been used for gene transfer. However, the low water solubility often leads to decreased transfection efficiency. Grafting of highly water-soluble polyethylene imines (PEI) and polyethylene glycol onto polymers can increase their solubility. The purpose of this study was to design a non-viral gene carrier with improved water solubility as well as enhanced transfection efficiency for treating Parkinsonism. Two molecular weights (Mw =600 and 1,800 g/mol) of PEI were grafted onto CHI (PEI600-g-CHI and PEI1800-g-CHI, respectively) by opening the epoxide ring of ethylene glycol diglycidyl ether (EX-810). This modification resulted in a non-viral gene carrier with less cytotoxicity. The transfection efficiency of PEI600-g-CHI/deoxyribonucleic acid (DNA) polyplexes was significantly higher than either PEI1800-g-CHI/DNA or CHI/DNA polyplexes. The maximal GDNF expression of PEI600-g-CHI/DNA was at the polymer:DNA weight ratio of 10:1, which was 1.7-fold higher than the maximal GDNF expression of PEI1800-g-CHI/DNA. The low toxicity and high transfection efficiency of PEI600-g-CHI make it ideal for application to GDNF gene therapy, which has potential for the treatment of Parkinson’s disease.

Analgesic effect of lentivirus-mediated RNA interference of the expression of glial cell line-derived neurotrophic factor gene on bone cancer pain

Objective To observe the analgesic effects of lentivirus-mediated RNA interference of the expression of glial cell line-derived neurotrophic factor (GDNF) on bone cancer pain.Methods (1)Lvs-siGDNF was constructed and bone cancer pain models were established by intra-tibial injection of MRMT-1 cells.(2) The rats were randomly divided into 4 groups according to different drugs which were infused into subarachnoid space through the intrathecal tubes:psiHIV-U6,psiHIV-GDNF-mU6,mor-phine,and normal saline.(3) The pain threshold at different time points (normal rats,7 days and 14 days after dosing) was measured.(4) The changes of pain threshold were observed from preoperative day to 7 days and 14 days after administration.The levels of GDNF,glial fibrillary acidic protein (GFAP),and Fos were detected by immunofluorescent staining technique and Western blotting.Results (1) As compared with control group (0.117 ±0.060) and transfection group (0.113 ±0.072),GDNF siRNA sequence 3 (0.045 ± 0.034) was the most effective interference segment confirmed by Q-PCR (P ＜ 0.05).The percentage of fluorescent cells to the brightfield was 0.87.TLvs-siGDNF was 3.48 × 108 pfu/ml.(2)Thermal pain threshold at 7th day (3.80 ±0.69) and 14th day (3.71 ± 1.10),and mechanical pain thresh-old at 14th day (2.58 ± 1.93) were all increased in siGDNF group than normal saline group (1.38 ± 1.01,1.11 ± 1.02,and 1.53 ±0.69) and psiHIV-U6 group (1.39 ± 1.18,0.67 ±0.27,and 1.21 ± 1.06)(P ＜ 0.05),while there was no significant difference in mechanical pain threshold at 7 day after administration (P ＞ 0.05).(3) RNA interference effectively decreased the expression level of GDNF in siGDNF group (P ＜0.05).(4) As compared with other groups,the expression levels of Fos and GFAP in the spinal dorsal horn were decreased in morphine group and siGDNF group (P ＜ 0.05).Conclusion The analgesic effect of siGDNF is similar to that of morphine in bone cancer rats,and siGDNF worked effectively more than 2 weeks.SiGDNF realized analgesic effect probably by inhibiting the astrocyte activation. Key words: Bone cancer pain; Glial cell line-derived neurotrophic factor; RNA interference; Lentiviral