Glial Cell Line-derived Neurotrophic Factor Expression Research Articles

MicroRNA-Mediated Suppression of Glial Cell Line-Derived Neurotrophic Factor Expression Is Modulated by a Schizophrenia-Associated Non-Coding Polymorphism.

Plasma levels of glial cell line-derived neurotrophic factor (GDNF), a pivotal regulator of differentiation and survival of dopaminergic neurons, are reportedly decreased in schizophrenia. To explore the involvement of GDNF in the pathogenesis of the disease, a case-control association analysis was performed between five non-coding single nucleotide polymorphisms (SNP) across the GDNF gene and schizophrenia. Of them, the 'G' allele of the rs11111 SNP located in the 3' untranslated region (3'-UTR) of the gene was found to associate with schizophrenia. In silico analysis revealed that the rs11111 'G' allele might create binding sites for three microRNA (miRNA) species. To explore the significance of this polymorphism, transient co-transfection assays were performed in human embryonic kidney 293T (HEK293T) cells with a luciferase reporter construct harboring either the 'A' or 'G' allele of the 3'-UTR of GDNF in combination with the hsa-miR-1185-1-3p pre-miRNA. It was demonstrated that in the presence of the rs11111 'G' (but not the 'A') allele, hsa-miR-1185-2-3p repressed luciferase activity in a dose-dependent manner. Deletion of the miRNA binding site or its substitution with the complementary sequence abrogated the modulatory effect. Our results imply that the rs11111 'G' allele occurring more frequently in patients with schizophrenia might downregulate GDNF expression in a miRNA-dependent fashion.

Glial cell line-derived Neurotrophic factor GDNF Gene Expression Analysis: Unveiling Neuronal Protection Mechanisms in Alzheimer's Disease Management

Background: Alzheimer's disease (AD), accounting for a substantial proportion of dementia cases, presents a formidable challenge to healthcare systems worldwide. The pathogenesis of AD in patients with Type II Diabetes Mellitus (DM) is particularly complex due to the interplay of metabolic and neurodegenerative processes. Understanding the role of Glial Cell Line-Derived Neurotrophic Factor (GDNF) in this context is vital for elucidating potential therapeutic targets. Objective: This study aimed to assess the expression levels of the GDNF gene in patients with Type II DM and dementia and to explore the correlation between GDNF expression and cognitive function. Methods: Blood samples were collected from 88 diabetic patients with dementia and 12 healthy controls. GDNF serum levels were measured using Chemiluminescent Immunoassay (CLIA), and genomic DNA was isolated for expression analysis via RT-qPCR. Cognitive function was evaluated using the Mini Mental State Examination (MMSE). Statistical analysis, including t-tests and one-way ANOVA, was performed using Graphpad Prism version 9.0. Results: Diabetic dementia patients exhibited significantly lower GDNF serum levels (4.37±1.38 µg/mL) compared to healthy controls (11.37±3.64 µg/mL, p=0.014). MMSE scores were also lower in the patient group (12.26±4.56) than in controls (24.1±3.12, p=0.009). RT-qPCR results showed a relative fold decrease in GDNF expression of 4.1 in dementia patients, indicating underexpression of the GDNF gene.1 Conclusion: The reduced expression of GDNF in patients with Type II DM and dementia underscores its potential as a biomarker for cognitive impairment. These findings pave the way for further research into GDNF-targeted therapies, which could prove pivotal in early detection and management of dementia in diabetic populations.

Hepatocyte-derived exosomes deliver the lncRNA CYTOR to hepatic stellate cells and promote liver fibrosis.

Liver fibrosis is characterized by the activation and transformation of hepatic stellate cells (HSCs) induced by various injury factors. The degree of liver fibrosis can be significantly improved, but persistent injury factors present a significant therapeutic challenge. Hepatocytes are the most important parenchymal cell type in the liver. In this study, we explored the molecular mechanisms by which damaged liver cells activate HSCs through extracellular vesicles. We established a coculture model of LO2 and LX2 and validated its exosomal transmission activity. Subsequently, differentially expressed long noncoding RNAs (lncRNAs) were screened through RNA sequencing and their mechanisms of action as competing endogenous RNAs (ceRNAs) further confirmed using biological methods, such as FISH and luciferase assays. Damaged liver cells induced activation of LX2 and upregulation of liver fibrosis-related markers. Exosomes extracted and identified from the supernatant fraction contained differentially expressed lncRNA cytoskeleton regulator RNA (CYTOR) that competed with microRNA-125 (miR-125) for binding to glial cell line-derived neurotrophic factor (GDNF) in HSCs, in turn, promoting LX2 activation. MiR-125 could target and regulate both CYTOR and GDNF and vice versa, as verified using the luciferase assay. In an invivo model, damaged liver extracellular vesicles induced the formation of liver fibrosis. Notably, downregulation of CYTOR within extracellular vesicles effectively inhibited liver fibrosis. The lncRNA CYTOR in exosomes of damaged liver cells is upregulated and modulates the expression of downstream GDNF through activity as a ceRNA, providing an effective mechanism for activation of HSCs.

Interactions between neurotrophins, mood, and physical activity under the conditions of sleep deprivation

Sleep deprivation (DS) is the forced elimination of sleep. While brain-derived neurotrophic factor (BDNF) has been extensively studied in the context of in mood changes following DS, the role of other neurotrophins remains elusive. This study explores the impact of DS on BDNF, glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT3), and neurotrophin-4 (NT4) at mRNA and protein level, considering their potential links to mood disturbances. The study involved 81 participants subjected to polysomnography (PSG) and DS. Blood samples, mood assessments, and actigraphy data were collected twice, after PSG and DS. NT mRNA expression and serum protein concentrations of BDNF, GDNF, NT3, and NT4 were measured. Participants were divided into Responders and Non-Responders based on mood improvement after DS. DS reduced BDNF mRNA expression in all participants, with no change in serum BDNF protein. GDNF protein decreased in Non-Responders, while Responders exhibited reduced GDNF mRNA. NT3 protein increased in both groups, while NT3 mRNA decreased in Respondents. NT4 protein rose universally post-DS, but NT4 mRNA remained unchanged. Physical activity (PA) negatively correlated with mRNA expression of BDNF, GDNF, and NT3 post-DS. The study’s short DS duration and exclusion of immature NT forms limit comprehensive insights. GDNF, together with NT3, might play an important role in mood response to DS. PA during DS seems to impair the mRNA expression of NTs in leukocytes. Future studies on the subject of sleep deprivation might consider investigating the relationship between BDNF and NT4 in the context of their apparent redundancy.

Vitamin D3 improves iminodipropionitrile-induced tic-like behavior in rats through regulation of GDNF/c-Ret signaling activity.

Children with chronic tic disorders (CTD), including Tourette syndrome (TS), have significantly reduced serum 25-hydroxyvitamin D [25(OH)D]. While vitamin D3 supplementation (VDS) may reduce tic symptoms in these children, its mechanism is unclear. The study aim was to investigate the effects and mechanisms of vitamin D deficiency (VDD) and VDS on TS model behavior. Forty 5-week-old male Sprague-Dawley rats were randomly divided into (n = 10 each): control, TS model, TS model with VDD (TS + VDD), or TS model with VDS (TS + VDS; two intramuscular injections of 20,000IU/200g) groups. The VDD model was diet-induced (0IU vitamin D/kg); the TS model was iminodipropionitrile (IDPN)-induced. All groups were tested for behavior, serum and striatal 25(OH)D and dopamine (DA), mRNA expressions of vitamin D receptor (VDR), glial cell line-derived neurotrophic factor (GDNF), protooncogene tyrosine-protein kinase receptor Ret (c-Ret), and DA D1 (DRD1) and D2 (DRD2) receptor genes in the striatum. TS + VDD had higher behavior activity scores throughout, and higher total behavior score at day 21 compared with TS model. In contrast, day 21 TS + VDS stereotyped behavior scores and total scores were lower than TS model. The serum 25(OH)D in TS + VDD was < 20ng/mL, and lower than control. Striatal DA of TS was lower than control. Compared with TS model, striatal DA of TS + VDD was lower, while in TS + VDS it was higher than TS model. Furthermore, mRNA expression of VDR, GDNF, and c-Ret genes decreased in TS model, and GDNF expression decreased more in TS + VDD, while TS + VDS had higher GDNF and c-Ret expressions. VDD aggravates, and VDS ameliorates tic-like behavior in an IDPN-induced model. VDS may upregulate GDNF/c-Retsignaling activity through VDR, reversing the striatal DA decrease and alleviating tic-like behavior.

Utilization of the Rat Tibial Nerve Transection Model to Evaluate Cellular and Molecular Mechanisms Underpinning Denervation-Mediated Muscle Injury.

Peripheral nerve injury denervates muscle, resulting in muscle paralysis and atrophy. This is reversible if timely muscle reinnervation occurs. With delayed reinnervation, the muscle's reparative ability declines, and muscle-resident fibro-adipogenic progenitor cells (FAPs) proliferate and differentiate, inducing fibro-fatty muscle degradation and thereby physical disability. The mechanisms by which the peripheral nerve regulates FAPs expansion and differentiation are incompletely understood. Using the rat tibial neve transection model, we demonstrated an increased FAPs content and a changing FAPs phenotype, with an increased capacity for adipocyte and fibroblast differentiation, in gastrocnemius muscle post-denervation. The FAPs response was inhibited by immediate tibial nerve repair with muscle reinnervation via neuromuscular junctions (NMJs) and sensory organs (e.g., muscle spindles) or the sensory protection of muscle (where a pure sensory nerve is sutured to the distal tibial nerve stump) with reinnervation by muscle spindles alone. We found that both procedures reduced denervation-mediated increases in glial-cell-line-derived neurotrophic factor (GDNF) in muscle and that GDNF promoted FAPs adipogenic and fibrogenic differentiation in vitro. These results suggest that the peripheral nerve controls FAPs recruitment and differentiation via the modulation of muscle GDNF expression through NMJs and muscle spindles. GDNF can serve as a therapeutic target in the management of denervation-induced muscle injury.

Analysis of the effect of adult onset increase of neurotrophic factor GDNF expression in APP‐NL‐G‐F AD mouse model

AbstractBackgroundGradual decline in cholinergic transmission and cognitive function occurs during normal aging, whereas pathological loss of cholinergic function is a hallmark of different types of dementia, including Alzheimer’s disease (AD), Lewy body dementia (LBD), and Parkinson’s disease dementia (PDD). Glial cell line‐derived neurotrophic factor (GDNF) is known to modulate and enhance the dopamine system. However, how endogenous GDNF influences brain cholinergic transmission has remained elusive.MethodRecently we showed that two fold increase in endogenous GDNF expression in Gdnf hypermorphic mice, Gdnf wt/hyper, attenuated age‐associated decline in the cholinergic index and cognitive function observed in Gdnf wt/wt animals. Biochemical analysis revealed that the level of nerve growth factor (NGF), which is important for survival and function of central cholinergic neurons, was significantly increased in several brain areas of old Gdnf wt/hyper miceResultOur results identified endogenous GDNF as a potential enhancer of cholinergic transmission upon aging (11). Here we propose to follow up those results by analyzing the effect of adult onset increase in endogenous GDNF expression on cholinergic index, A‐beta load, NGF expression and cognition in APP‐NLGF mouse model of AD. To accelerate AD onset and to facilitate future studies we have also generated and propose characterization of APP‐NLGF conditional Hypermorph mouse model.ConclusionOur goal is to conclude therapeutic potential of enhanced endogenous GDNF signaling on AD associated cholinergic and cognitive decline to pave way to new dementia treatments based on GDNF mimetics currently under development by several academic and industrial laboratories.

Additional glial cell line-derived neurotrophic factor in vitro promotes the proliferation of undifferentiated spermatogonia from sterile cattleyak

Cattleyak is a typically male sterile species. The meiosis process is blocked and the scarcity of spermatogenic stems cells are both contributing factors to the inability of male cattleyak to produce sperm. While Glial cell line-derived neurotrophic factor (GDNF) is the first discovered growth factor known to promote the proliferation and self-renewal of spermatogenic stem cells, its relationship to the spermatogenesis arrest of cattleyak remains unclear. In this report, we studied the differential expression of GDNF in the testis of yak and cattleyak, and discussed the optimal concentration of GDNF in the culture medium of undifferentiated spermatogonia (UDSPG) of cattleyak in vitro and the effect of GDNF on the proliferation of cattleyak UDSPG. The results indicated that GDNF expression in the testicular tissue of cattleyak was inferior to that of yak. Moreover, the optimum value for the UDSPG in vitro culture was determined to be 20–30 ng/mL for cattleyak. In vitro, the proliferation activity of UDSPG was observed to increase with additional GDNF due to the up-regulation of proliferation-related genes and the down-regulation of differentiation-related genes. We hereby report that the scarcity of cattleyak UDSPG is due to insufficient expression of GDNF, and that the addition of GDNF in vitro can promote the proliferation of cattleyak UDSPG by regulating the expression of genes related to proliferation and differentiation. This work provides a new insight to solve the issue of spermatogenic arrest in cattleyak.

Subthalamic nucleus deep brain stimulation does not alter growth factor expression in a rat model of stable dopaminergic deficiency

BackgroundDeep brain stimulation (DBS) of the subthalamic nucleus (STN) has been a highly effective treatment option for mid-to-late-stage Parkinson’s disease (PD) for decades. Besides direct effects on brain networks, neuroprotective effects of STN-DBS – potentially via alterations of growth factor expression levels – have been proposed as additional mechanisms of action. ObjectiveIn the context of clarifying DBS mechanisms, we analyzed brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) levels in the basal ganglia, motor and parietal cortices, and dentate gyrus in an animal model of stable, severe dopaminergic deficiency. MethodsWe applied one week of continuous unilateral STN-DBS in a group of stable 6-hydroxydopamine (6-OHDA) hemiparkinsonian rats (6-OHDASTIM) in comparison to a 6-OHDA control group (6-OHDASHAM) as well as healthy controls (CTRLSTIM and CTRLSHAM). BDNF and GDNF levels were determined via ELISAs. ResultsThe 6-OHDA lesion did not result in a persistent alteration in either BDNF or GDNF levels in a model of severe dopaminergic deficiency after completion of the dopaminergic degeneration. STN-DBS modestly increased BDNF levels in the entopeduncular nucleus, but even impaired BDNF and GDNF expression in cortical areas. ConclusionsSTN-DBS does not increase growth factor expression when applied to a model of completed, severe dopaminergic deficiency in contrast to other studies in models of modest and ongoing dopaminergic degeneration. In healthy controls, STN-DBS does not influence BDNF or GDNF expression. We consider these findings relevant for clinical purposes since DBS in PD is usually applied late in the course of the disease.

Differential expression of RET and GDNF family receptor, GFR-α1, between striatum and substantia nigra following nigrostriatal lesion: A case for diminished GDNF-signaling

Although glial cell line-derived neurotrophic factor (GDNF) showed efficacy in preclinical and early clinical studies to alleviate parkinsonian signs in Parkinson's disease (PD), later trials did not meet primary endpoints, giving pause to consider further investigation. While GDNF dose and delivery methods may have contributed to diminished efficacy, one crucial aspect of these clinical studies is that GDNF treatment began ∼8 years after PD diagnosis; a time point representing several years after near 100% depletion of nigrostriatal dopamine markers in striatum and at least 50% in substantia nigra (SN), which represents a time point of initiating GDNF treatment later than reported in some preclinical studies. With nigrostriatal terminal loss exceeding 70% at PD diagnosis, we utilized hemiparkinsonian rats to determine if expression of GDNF family receptor, GFR-α1, and receptor tyrosine kinase, RET, differed between striatum and SN at 1 and 4 weeks following a 6-hydroxydopamine (6-OHDA) hemilesion. Whereas GDNF expression changed minimally, GFR-α1 expression decreased progressively in striatum and in tyrosine hydroxylase positive (TH+) cells in SN, correlating with reduced TH cell number. However, in nigral astrocytes, GFR-α1 expression increased. RET expression decreased maximally in striatum by 1 week, whereas in the SN, a transient bilateral increase occurred, returning to control levels by 4 weeks. Expression of brain-derived neurotrophic factor (BDNF) or its receptor, TrkB, were unchanged throughout lesion progression. Together, these results reveal that differential GFR-α1 and RET expression between the striatum and SN, and cell-specific differences in GFR-α1 expression in SN, occur during nigrostriatal neuron loss. Targeting loss of GDNF receptors thus appears critical to enhance GDNF therapeutic efficacy against nigrostriatal neuron loss. Significance statementAlthough preclinical evidence supports that GDNF provides neuroprotection and improves locomotor function in preclinical studies, there is uncertainty if it can alleviate motor impairment in Parkinson's disease patients. Using the established 6-OHDA hemiparkinsonian rat model, we determined whether expression of its cognate receptors, GFR-α1 and RET, were differentially affected between striatum and substantia nigra in a timeline study. In striatum, there was early and significant loss of RET, but a gradual, progressive loss of GFR-α1. In contrast, RET transiently increased in lesioned substantia nigra, but GFR-α1 progressively decreased only in nigrostriatal neurons and correlated with TH cell loss. Our results indicate that direct availability of GFR-α1 may be a critical element that determines GDNF efficacy following striatal delivery.

Cellular Localization and Distribution of TGF-β1, GDNF and PDGF-BB in the Adult Primate Central Nervous System.

The available data on the localization of transforming growth factor beta1 (TGF-β1), glial cell line-derived neurotrophic factor (GDNF), and platelet-derived growth factor-BB (PDGF-BB) in the adult primate and human central nervous system (CNS) are limited and lack comprehensive and systematic information. This study aimed to investigate the cellular localization and distribution of TGF-β1, GDNF, and PDGF-BB in the CNS of adult rhesus macaque (Macaca mulatta). Seven adult rhesus macaques were included in the study. The protein levels of TGF-β1, PDGF-BB, and GDNF in the cerebral cortex, cerebellum, hippocampus, and spinal cord were analyzed by western blotting. The expression and location of TGF-β1, PDGF-BB, and GDNF in the brain and spinal cord was examined by immunohistochemistry and immunofluorescence staining, respectively. The mRNA expression of TGF-β1, PDGF-BB, and GDNF was detected by in situ hybridization. The molecular weight of TGF-β1, PDGF-BB, and GDNF in the homogenate of spinal cord was 25 KDa, 30 KDa, and 34 KDa, respectively. Immunolabeling revealed GDNF was ubiquitously distributed in the cerebral cortex, hippocampal formation, basal nuclei, thalamus, hypothalamus, brainstem, cerebellum, and spinal cord. TGF-β1 was least distributed and found only in the medulla oblongata and spinal cord, and PDGF-BB expression was also limited and present only in the brainstem and spinal cord. Besides, TGF-β1, PDGF-BB, and GDNF were localized in the astrocytes and microglia of spinal cord and hippocampus, and their expression was mainly found in the cytoplasm and primary dendrites. The mRNA of TGF-β1, PDGF-BB, and GDNF was localized to neuronal subpopulations in the spinal cord and cerebellum. These findings suggest that TGF-β1, GDNF and PDGF-BB may be associated with neuronal survival, neural regeneration and functional recovery in the CNS of adult rhesus macaques, providing the potential insights into the development or refinement of therapies based on these factors.

Prenatal BPA exposure disrupts male reproductive functions by interfering with DNA methylation and GDNF expression in the testes of male offspring rats.

BPA is a ubiquitous environmental endocrine-disrupting chemical, and maternal exposure to BPA is associated with impaired male reproductive functions; however, the mechanisms remain to be elucidated. Glial cell line-derived neurotrophic factor (GDNF) plays an important role in maintaining normal spermatogenesis and fertility. However, the effect of prenatal BPA exposure on GDNF expression and its mechanism in the testis has not been reported. In this study, pregnant Sprague-Dawley rats were respectively exposed to 0, 0.05, 0.5, 5, and 50 mg/kg/day BPA via oral gavage from gestational day (GD) 5 to GD 19, with 6 pregnant rats in each group. ELISA, histochemistry, real-time PCR, western blot, and methylation-specific PCR (MSP) were used to detect the sex hormone levels, testicular histopathology, mRNA and protein expression of DNA methyltransferases (DNMTs) and GDNF, and the promoter methylation of Gdnf in the testes of male offspring at postnatal day (PND) 21 and PND 56. Prenatal BPA exposure increased body weight; decreased sperm counts and serum levels of testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH); and induced testicular histological damage, which indicated the damage of male reproductive function. Prenatal BPA exposure also upregulated Dnmt1 in 5 mg/kg group and Dnmt3b in 0.5 mg/kg group, but down-regulated Dnmt1 in 50 mg/kg group at PND 21. At PND 56, Dnmt1 was significantly increased in 0.05 mg/kg group but decreased in 0.5, 5, and 50 mg/kg groups, Dnmt3a was decreased, and Dnmt3b was markedly increased in 0.05 and 0.5 mg/kg groups but decreased in 5 and 50 mg/kg groups. The mRNA and protein expression levels of Gdnf were decreased markedly in 0.5 and 50 mg/kg groups at PND 21. And the methylation level of Gdnf promoter was significantly increased in 0.5 mg/kg group, but decreased in 5 and 50 mg/kg groups at PND 21. In conclusion, our study indicates that prenatal BPA exposure disrupts male reproductive functions, interferes with the expression of DNMTs, and decreases Gdnf expression in the testes of male offspring. Gdnf expression may be regulated by DNA methylation; however, the detailed mechanism needs to be further investigated.

In vivo modulation of endogenous gene expression via CRISPR/Cas9-mediated 3’UTR editing

The 3' untranslated regions (UTRs) modulate gene expression levels by regulating mRNA stability and translation. We previously showed that the replacement of the negative regulatory elements from the 3'UTR of glial cell line-derived neurotrophic factor (GDNF) resulted in increased endogenous GDNF expression while retaining its normal spatiotemporal expression pattern. Here, we have developed a methodology for the generation of in vivo hyper- and hypomorphic alleles via 3'UTR targeting using the CRISPR/Cas9 system. We demonstrate that CRISPR/Cas9-mediated excision of a long inhibitory sequence from Gdnf native 3'UTR in mouse zygotes increases the levels of endogenous GDNF with similar phenotypic alterations in embryonic kidney development as we described in GDNF constitutive and conditional hypermorphic mice. Furthermore, we show that CRISPR/Cas9-mediated targeting of 3’UTRs in vivo allows the modulation of the expression levels of two other morphogens, Gdf11 and Bdnf. Together, our work demonstrates the power of in vivo 3’UTR editing using the CRISPR/Cas9 system to create hyper- and hypomorphic alleles, suggesting wide applicability in studies on gene function and potentially, in gene therapy.

Increased Physiological GDNF Levels Have No Effect on Dopamine Neuron Protection and Restoration in a Proteasome Inhibition Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disease that comprises a range of motor and nonmotor symptoms. Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of dopamine neurons in vitro and in vivo, and intracranial delivery of GDNF has been tested in six clinical trials for treating PD. However, clinical trials with ectopic GDNF have yielded variable results, which could in part result from abnormal expression site and levels caused by ectopic overexpression. Therefore, an important open question is whether an increase in endogenous GDNF expression could be potent in reversing PD progression. Here, we tested the therapeutic potential of endogenous GDNF using mice in which endogenous GDNF can be conditionally upregulated specifically in cells that express GDNF naturally (conditional GDNF hypermorphic mice; GdnfcHyper ). We analyzed the impact of endogenous GDNF upregulation in both neuroprotection and neurorestoration procedures, and for both motor and nonmotor symptoms in the proteasome inhibitor lactacystin (LC) model of PD. Our results showed that upregulation of endogenous GDNF in the adult striatum is not protective in LC-induced PD model in mice. Since age is the largest risk factor for PD, we also analyzed the effect of deletion of endogenous GDNF in aged Gdnf conditional knock-out mice. We found that GDNF deletion does not increase susceptibility to LC-induced damage. We conclude that endogenous GDNF does not impact the outcome in the LC-induced proteasome inhibition mouse model of Parkinson's disease.

Neuroprotective Effects of Human Adipose-Derived Mesenchymal Stem Cells in Oxygen-Induced Retinopathy.

This study was designed to provide evidence of the neuroprotective of human adipose-derived mesenchymal stem cells (hADSCs) in oxygen-induced retinopathy (OIR). In vivo, hADSCs were intravitreally injected into OIR mice. Various assessments, including HE (histological evaluation), TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, electroretinogram (ERG) analysis, and retinal flat-mount examination, were performed separately at postnatal days 15 (P15) and 17 (P17) to evaluate neurological damage and functional changes. Western blot analysis of ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF) was conducted at P17 to elucidate the neuroprotective mechanism. The P17 OIR group exhibited a significant increase in vascular endothelial cell nuclei and neovascularization that breached the ILM (inner limiting membrane) to the P17 control group. In addition, the retinal nonperfusion areas in the P17 OIR group and the number of apoptotic retinal cells in the P15 OIR group were significantly higher than in the corresponding hADSCs treatment group and control group. There was no significant thickness change in the inner nuclear layer (INL) but the outer nuclear layer (ONL) in the P17 OIR treatment group compared with the P17 OIR group. The cell density in the INL and ONL at P17 in the hADSCs treatment group was not significantly different from the OIR group. The amplitude of a-wave and b-wave in scotopic ERG analysis for the P17 OIR group was significantly lower than in the P17 hADSCs treatment group and the P17 control group. Furthermore, the latency of the a-wave and b-wave in the P17 OIR group was significantly longer than in the P17 hADSCs treatment group and the P17 control group. In addition, the expression levels of CNTF and BDNF in the P17 OIR group were statistically higher than those in the P17 control group, whereas the expression of GDNF was statistically lower in the P17 OIR group, compared with the P17 control group. The expression of CNTF and GDNF in the P17 hADSCs treatment group was statistically higher than in the P17 OIR group. However, the expression of BDNF in the P17 hADSCs treatment group was statistically lower than in the P17 OIR group. This study provides evidence for the neuroprotective effects of hADSCs in OIR.

The central glial cell line-derived neurotrophic factor (GDNF) regulates pulmonary function in asthmatic rats.

Asthma is a common chronic inflammatory disease of the airway, but the mechanism is still not fully understood. This study aimed to investigate the effect of glial cell line-derived neurotrophic factor (GDNF) on asthma attacks. An asthmatic rat model was established. GDNF expression in the airway and brain was observed by immunohistochemistry (IHC), and the concentration of GDNF in bronchoalveolar lavage fluid (BALF) was detected by enzyme-linked immunosorbent assay (ELISA). After injection of GDNF and its antibody into the lateral ventricle of asthmatic rats, the pulmonary function was recorded, and the levels of interferon-γ (IFN-γ) and interleukin-4 (IL-4) in BALF were tested. GDNF expressions were increased significantly in the lung tissues of asthmatic rats. In the central nervous system (CNS), GDNF-positive immunoreactive substances were observed in multiple brain regions, including the medial amygdala (MeA), paraventricular nucleus (PVN), cortex, and nucleus of solitary tract (NTS). After injection of GDNF into the lateral ventricles of asthmatic rats, the symptoms of asthma and airway inflammation were significantly aggravated, which could be improved by injection of GDNF antibody into the lateral ventricles. GDNF expression is increased in the lung and brain in asthmatic rats. During an asthma attack, the increased GDNF expressions in the rat brain remarkably aggravate the asthmatic symptoms.

Berberine hydrochloride improves cognitive deficiency through hippocampal up-regulation of neurotrophins following inhalant self-administration of methamphetamine.

Chronic methamphetamine (METH) abuse is recognized as an important risk factor for cognitive impairment. A plant-based isoquinoline alkaloid, Berberine hydrochloride (BER), shows memory and cognition enhancement properties. Due to the aim of the present study which is to investigate the influence of BER administration on METH-induced cognitive deficits, we investigated neurotrophin signaling including brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) as a possible mechanism by which BER exerts its cognitive improvement influences. In this experimental study, thirty-two male Wistar rats were randomly classified into four groups, including non-treated control, intubated control, METH-inhaled, and METH-inhaled + BER-intubated. Rats in the METH-inhaled group underwent METH inhalation for 14 days, and the BER-inhaled and BER-intubated rats were intubated (100mg/kg) for the following three weeks. A novel object recognition task (NORt) was carried out on days 36 and 37. Rats were sacrificed for histological preparations after the behavioral tests. Neurotrophic factors, including GDNF and BDNF, were evaluated by immunofluorescence staining in the hippocampus. This experiment indicated a dramatic improvement in cognitive deficits associated with chronic METH abuse (P<0.001). Furthermore, a significant decrease in the expression of both neurotrophins, GDNF (P<0.001) and BDNF (P<0.001), was observed in the METH-inhaled group compared with the METH-inhaled group treated with BER and non-treated control group. Activation of neurotrophic factors after BER administration resulted in improvement of METH-induced cognitive deficits. Therefore, BER may be considered a promising treatment for METH users who experience cognition deficits.

The Polyunsaturated Fatty Acid EPA, but Not DHA, Enhances Neurotrophic Factor Expression through Epigenetic Mechanisms and Protects against Parkinsonian Neuronal Cell Death.

ω-3 Polyunsaturated fatty acids (PUFAs) have been found to exert many actions, including neuroprotective effects. In this regard, the exact molecular mechanisms are not well understood. Parkinson's disease (PD) is the second most common age-related neurodegenerative disease. Emerging evidence supports the hypothesis that PD is the result of complex interactions between genetic abnormalities, environmental toxins, mitochondrial dysfunction, and other cellular processes, such as DNA methylation. In this context, BDNF (brain-derived neurotrophic factor) and GDNF (glial cell line-derived neurotrophic factor) have a pivotal role because they are both involved in neuron differentiation, survival, and synaptogenesis. In this study, we aimed to elucidate the potential role of two PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and their effects on BDNF and GDNF expression in the SH-SY5Y cell line. Cell viability was determined using the MTT assay, and flow cytometry analysis was used to verify the level of apoptosis. Transmission electron microscopy was performed to observe the cell ultrastructure and mitochondria morphology. BDNF and GDNF protein levels and mRNA were assayed by Western blotting and RT-PCR, respectively. Finally, methylated and hydroxymethylated DNA immunoprecipitation were performed in the BDNF and GDNF promoter regions. EPA, but not DHA, is able (i) to reduce the neurotoxic effect of neurotoxin 6-hydroxydopamine (6-OHDA) in vitro, (ii) to re-establish mitochondrial function, and (iii) to increase BNDF and GDNF expression via epigenetic mechanisms.

Overexpression of microRNA-211 in Functional Dyspepsia via Downregulation of the Glial Cell Line-Derived Neurotrophic Factor (GDNF) by Increasing Phosphorylation of p38 MAPK Pathway.

Overexpression of miRNA-211 suppresses the differentiation of bone marrow stem cells into intestinal ganglion cells via downregulation of GDNF, a regulator of intestine barrier function. The study aimed to investigate the interaction between miR-211 and GDNF on intestinal epithelial cells. The expression levels of miR-211 and GDNF in duodenal biopsy specimens from FD patients and healthy controls were compared. Enteric glia cell (EGCs) cell line transfected with miR-211 mimics and inhibitors were used to clarify the expression levels of GDNF were analyzed by qRT-PCR and ELISA. Intestine epithelial cell (IECs) cell line cultured in medium from ECGs in different transfection conditions were used in wound healing assay, cell proliferation assay, and western blotting for evaluation of p38 MAPK phosphorylation level. MiR-211 expression was significantly upregulated in the duodenal tissue of patients with FD compared to healthy subjects, whereas GDNF expression was significantly downregulated (both p < 0.05). Transfection with miR-211 mimics significantly decreased GDNF mRNA expression and protein secretion (p < 0.001). An inhibited intestinal epithelial cell wound healing (p < 0.05) and increased expression levels of phosphorylated p38 MAPK (p < 0.05) were found in IECs cultured with medium from EGCs transfected with miR-211 mimics. MiR-211 may downregulates GDNF mRNA and protein expression via activation of the pp38 MAPK signaling pathway. Targeting miR-211 or the MAPK pathway may be a potential intervention for FD.

P10.06.A Expression of GDNF and its receptors in glioblastoma

Abstract Background Neurotrophic factors are important for brain development, but their role in glioblastoma, the most aggressive primary brain cancer, is not fully understood. We focused on glial cell line-derived neurotrophic factor (GDNF), which has been shown to play a role in growth and migration in glioblastoma. The aim of this study was to investigate the expression of GDNF and its primary receptor GFRA1 as well as GDNF family receptors GFRA2-4 and RET. Material and Methods Tissue sections from 10 glioblastoma patients were stained with antibodies against GDNF (frozen tissue) and each of the receptors (formalin-fixed paraffin-embedded tissue). Moreover, the sections were stained with an RNA Scope assay for GDNF mRNA. The area fractions of positive receptor staining were quantified with a software-based classifier. Double immunofluorescence stainings were performed for GFRA1 and the astrocytic tumor cell marker GFAP, the microglia and macrophage marker IBA1, and the tumor stem cell markers OLIG2 and SOX2, respectively, for assessment of co-expression. Results GDNF staining in a varying number of cells was seen in 5 of the 10 samples. GDNF mRNA stainings were observed in a varying number of cells in all stained tissues, including in hypoxic areas and in microvascular proliferations. Mean area fractions of the receptors were 31 % (range 7-56) for GFRA1, 3 % (0,2-7) for GFRA2, 0,4 % (0,01-2) for GFRA3, 0,1 % (0,02-0,3) for GFRA4 and 1 % (0,1-2) for RET. Co-expression of GFRA1 and GFAP was widely present. Co-expression of GFRA1 and either OLIG2 or SOX2 was partially present. Co-expression of GFRA1 and IBA1 was limited. Conclusion Our results suggest that GDNF and GFRA1 are widely expressed in glioblastoma tissue, but GFRA2-4 and RET are also expressed and may play a role in glioblastoma tumor biology. Future experimental studies are needed to clarify the functional role of GDNF and its receptors in glioblastoma biology.