Glial Cell-derived Neurotrophic Factor Gene Research Articles

Effects of Aerobic Interval Training Along with Ginkgo biloba Supplementation on Passive Avoidance Memory and GDNF Gene Expression in Alzheimer Rats

Background: Alzheimer's disease (AD) represents the most prevalent form of dementia, characterized by memory loss, and is a complex, progressive neurodegenerative condition. Objectives: This study aimed to explore the impact of aerobic interval training (AIT) combined with Ginkgo biloba (GB) supplementation on passive avoidance memory function and the expression of the glial cell-derived neurotrophic factor (GDNF) gene in rats afflicted with AD. Methods: A total of forty-four male Wistar rats, each weighing approximately 250 ± 20 g, were maintained in a controlled environment at a specified temperature. Over 8 weeks (5 days per week), they underwent AIT and received GB supplements at a dosage of 100 mg/kg. The rats were randomly assigned into six groups: Healthy control (HC) with 7 rats, AD control (AC) with 8 rats, amyloid-β (Aβ) injection plus GB supplement (Aβ + GB) with 8 rats, Aβ injection plus GB supplement plus AIT (Aβ + GB + AIT) with 8 rats, Aβ injection plus AIT (Aβ + AIT) with 8 rats, and sham surgery (SS) with 5 rats. The shuttlebox apparatus was utilized to assess the effect of Aβ on passive avoidance memory and to confirm disease induction. GDNF gene expression was measured using the qRT-PCR method. Results: The study found significant improvements in passive avoidance memory in the Aβ + AIT and Aβ + GB + AIT groups compared to the AC group after 8 weeks of AIT and GB supplementation (P = 0.02 and P = 0.000, respectively). Long-term memory performance was notably enhanced in the Aβ + GB + AIT group (P = 0.000). Additionally, GDNF expression significantly increased in the Aβ + GB + AIT and Aβ + GB groups in comparison to the AC group (P = 0.000 and P = 0.01, respectively). Conclusions: A synergistic effect of AIT and GB supplementation was observed, leading to an increase in GDNF gene expression and improvements in both passive avoidance memory and long-term memory in male rats with AD.

Effect of electroacupuncture on gastrointestinal motility in rats with slow transit constipation based on GDNF methylation modification

To observe the effect of electroacupuncture (EA) of "Tianshu"(ST25) and "Dachangshu"(BL25) on the intestinal transit function， expression level of glial cell-derived neurotrophic factor (GDNF) and methylation level of GDNF gene promoter region in colon tissue of rats with slow transit constipation (STC)， so as to explore its mechanisms underlying improvement of STC. Male Sprague-Dawley (SD) rats were randomized into control， saline， model and EA groups (n=16 in each group). The STC model was replicated by gavage of compound diphenoxylate suspension (10 mL· kg-1· d-1) for 28 days. Rats of the saline group received the same dose of normal saline via gavage. EA (2 Hz/15 Hz， 0.1-1 mA) was applied to bila-teral ST25 and BL25 for 15 min， once daily for 14 days. The intestinal transmission function (the intestinal propulsion rate) was assessed by recording the first black grain stool discharge time and the number and weight of the discharged stool grains in 30 min after gavage of the activated carbon suspension (1 mL/100 g， 150 g/L). The score of fecal trait and the weight of stool within 24 h were recorded. The ultrastructural changes of Cajal interstitial cells in the colon tissue were observed by transmission electron microscope. The expression levels of GDNF protein and mRNA in the colon tissue were detected by using Western blot and real-time fluorescent quantitative PCR， separately， and changes of methylation level in the promoter region of GDNF gene detected by using Bisulfite sequencing method. Compared with the control group， the time of the 1st black stool grain discharging was obviously prolonged， and the number and weight of the discharged black stool grains were significantly decreased in the mo-del group (P<0.05)， suggesting a success of STC. The weight and trait score of stool in 24 h， intestinal propulsive rate， and the expression levels of GDNF protein and mRNA were significantly lower in the model group than in the control group (P<0.01， P<0.05). After EA， the weight and trait score of stool within 24 h， intestinal propulsive rate，and the expression levels of GDNF protein and mRNA were significantly increased in the EA group in contrast to the model group (P<0.01，P<0.05). The total CpGs methylation level of GDNF gene in colon tissue was considerably higher in the model group than in the control group (P<0.05)， and markedly lower in the EA group than in the model group (P<0.05). No significant differences were found between the control and saline groups in all the above-mentioned indexes (P>0.05). EA of back-shu and front-mu acupoints can effectively improve symptoms of constipation and intestinal transport function in STC rats， which may be related to its function in up-regulating the expression of GDNF and down-regulating the methylation level in the promoter region of GDNF gene in colon tissue.

Treatment of Parkinson’s disease using focused ultrasound with GDNF retrovirus-loaded microbubbles to open the blood–brain barrier

AbstractThis study aims to prepare ultrasound-targeted glial cell-derived neurotrophic factor (GDNF) retrovirus-loaded microbubbles (M pLXSN-GDNF) to verify the properties of the microbubbles and to study the therapeutic effect of the GDNF retrovirus-loaded microbubbles combined with ultrasound (U) to open the blood–brain barrier (BBB) in a Parkinson’s disease (PD) model in rats, allowing the retrovirus to pass through the BBB and transfect neurons in the substantia nigra of the midbrain, thereby increasing the expression of GDNF. The results of western blot analysis revealed significant differences between U + MpLXSN-EGFP, U + M + pLXSN-GDNF, and M pLXSN-GDNF (P &lt; 0.05) groups. After 8 weeks of treatment, the evaluation of the effect of increased GDNF expression on behavioral deficits in PD model rats was conducted. The rotation symptom was significantly improved in the U + MpLXSN-GDNF group, and the difference before and after treatment was significant (P &lt; 0.05). Also, the content of dopamine and the number of tyrosine hydroxylase-positive (dopaminergic) neurons were found to be higher in the brain of PD rats in the U + M pLXSN-GDNF group than in the control groups. Ultrasound combined with GDNF retrovirus-loaded microbubbles can enhance the transfection efficiency of neurons in vivo and highly express the exogenous GDNF gene to play a therapeutic role in PD model rats.

The role of Glial cell derived neurotrophic factor in head and neck cancer.

Glial cell-derived neurotrophic factor (GDNF) is reported to promote the survival of neurons and salivary gland regeneration after radiation damage. This study investigated the effect of GDNF on cell migration, growth, and response to radiation in preclinical models of head and neck squamous cell carcinoma (HNSCC) and correlated GDNF expression to treatment outcomes in HNSCC patients. Our ultimate goal is to determine whether systemic administration of GDNF at high dose is safe for the management of hyposalivation or xerostomia in HNSCC patients. Three HPV-positive and three HPV-negative cell lines were examined for cell migration, growth, and clonogenic survival in vitro and tumor growth assay in vivo. Immunohistochemical staining of GDNF, its receptors GFRα1 and its co-receptor RET was performed on two independent HNSCC tissue microarrays (TMA) and correlated to treatment outcomes. Results showed that GDNF only enhanced cell migration in two HPV-positive cells at supra-physiologic doses, but not in HPV-negative cells. GDNF did not increase cell survival in the tested cell lines post-irradiation. Likewise, GDNF treatment affected neither tumor growth in vitro nor response to radiation in xenografts in two HPV-positive and two HPV-negative HNSCC models. High stromal expression of GDNF protein was associated with worse overall survival in HPV-negative HNSCC on multivariate analysis in a combined cohort of patients from Stanford University (n = 82) and Washington University (n = 189); however, the association between GDNF gene expression and worse survival was not confirmed in a separate group of HPV-negative HNSCC patients identified from the Cancer Genome Atlas (TCGA) database. Based on these data, we do not believe that GNDF is a safe systemic treatment to prevent or treat xerostomia in HNSCC and a local delivery approach such as intraglandular injection needs to be explored.

Human Neural Stem Cells with GDNF Site-Specific Integration at AAVS1 by Using AAV Vectors Retained Their Stemness.

The neural stem cells (NSCs) have the ability to self-renew, and to migrate to pathologically altered regions of the central nervous system. Glial cell derived neurotrophic factor (GDNF) could protect dopamine neurons and rescue motor neurons in vivo, which has been proposed as a promising candidate for the treatments of degenerative neurological diseases. In order to combine the advantages of neurotrophic factors and stem cells in clinical therapy, we established the modified hNSCs that has site-specific integration of GDNF gene by using recombinant adeno-associated virus (rAAV) vectors. The hNSCs were co-infected by rAAV2-EGFP-GDNF and rAAV2-SVAV2 which provide integrase to specifically integrate GDNF gene into AAVS1 site. The GDNF-hNSCs maintained their original stem cell characteristics and the ability to differentiate into neurons in vitro. In the animal model, the GDNF-hNSCs were specifically transplanted into CA1 area of hippocampi and could migrate to the dentate gyrus region and differentiate into neuronal cells while maintaining GDNF expression. hNSCs with GDNF gene site-specific integration at AAVS1 by using AAV vectors retained their stemness and effectively expressed GDNF, which indicates the potential of employing transplanted hNPCs for treatment of brain injuries and degenerative neurological diseases.

Glial cell-derived neurotrophic factor gene polymorpisms affect severity and functionality of bipolar disorder.

Glial cell-derived neurotrophic factor and other neurotrophins have important role in the development of mental disorders. Here, we aimed to assess the effects of Single nucleotide polymorphisms at potentially regulated regions of GDNF on severity and functionality of bipolar disorder and GDNF serum levels in bipolar disorder patients and healthy volunteers. Severity and functionality of bipolar disorder were evaluated using the Clinical Global Impression and Global Assessment of Functioning scales in sixty-six bipolar disorder patients. The GDNF serum levels obtained from bipolar disorder patients and healthy volunteers who had been already reported SNPs information by our group. GAF scales were lower and GDNF serum levels were higher in Bipolar disorder patients with T/A genotype at 5:37812784 and 5:37812782 compared to patients with T/T genotype. There were significant difference in severity and functionality scores, but not in GDNF serum levels, between patients with G/G and G/A genotype of rs62360370 G>A SNP.rs2075680 C>A and rs79669773 T>C SNPs had no effect on bipolar disorder severity and functionality scores and GDNF serum levels. The results suggest that some SNPs of GDNF have potential association with severity and functionality of bipolar disorder. In addition, except two SNPs, none of GDNF SNPs had association with GDNF serum levels.

Schwann cells promote the capability of neural stem cells to differentiate into neurons and secret neurotrophic factors.

The present study investigated whether co-culturing Schwann cells (SCs) with neural stem cells (NSCs) improves viability, direction of differentiation and secretion of brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) in NSCs. The three groups assessed were as follows: SCs, NSCs, and a co-culture of SCs and NSCs. Cellular morphological changes were observed under an inverted phase contrast microscope and quantified. Cells were identified by immunofluorescence staining: S100 for SCs, Nestin for NSCs, microtubule associated protein (Map) 2 and NeuN for neurons and glial fibrillary acidic protein for astrocytes. Cell viability was evaluated by MTT assay. Secretion of BDNF and GDNF was quantified; mRNA expression was quantified by reverse transcription-quantitative polymerase chain reaction. The majority of NSCs in the co-cultured group differentiated into neurons. The cell survival rate of the co-culture group was significantly higher than the other groups on days 3, 5 and 10 (P<0.01). The secretion of BDNF in the co-culture group was significantly higher than NSCs on days 3, 5 and 7 (P<0.05), while the amount of GDNF in co-culture was significantly higher than both NSCs and SCs on day 1 (P<0.05). BDNF and GDNF gene expression in the co-culture group was significantly higher than SCs (P<0.01). Gene expression of Map2 in co-culture group was also significantly higher than both NSC and SC groups (P<0.01). Therefore, co-cultured SCs and NSCs promote differentiation of NSCs into neurons and secrete higher levels of neurotropic factors including BDNF and GDNF.

Exposure to Early Life Stress Results in Epigenetic Changes in Neurotrophic Factor Gene Expression in a Parkinsonian Rat Model

Early life adversity increases the risk of mental disorders later in life. Chronic early life stress may alter neurotrophic factor gene expression including those for brain derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF) that are important in neuronal growth, survival, and maintenance. Maternal separation was used in this study to model early life stress. Following unilateral injection of a mild dose of 6-hydroxydopamine (6-OHDA), we measured corticosterone (CORT) in the blood and striatum of stressed and nonstressed rats; we also measured DNA methylation and BDNF and GDNF gene expression in the striatum using real time PCR. In the presence of stress, we found that there was increased corticosterone concentration in both blood and striatal tissue. Further to this, we found higher DNA methylation and decreased neurotrophic factor gene expression. 6-OHDA lesion increased neurotrophic factor gene expression in both stressed and nonstressed rats but this increase was higher in the nonstressed rats. Our results suggest that exposure to early postnatal stress increases corticosterone concentration which leads to increased DNA methylation. This effect results in decreased BDNF and GDNF gene expression in the striatum leading to decreased protection against subsequent insults later in life.

Adenoviral vector carrying glial cell-derived neurotrophic factor for direct gene therapy in comparison with human umbilical cord blood cell-mediated therapy of spinal cord injury in rat.

Experimental study. To evaluate the treatment of spinal cord injury with glial cell-derived neurotrophic factor (GDNF) delivered using an adenoviral vector (AdV-GDNF group) in comparison with treatment performed using human umbilical cord blood mononuclear cells (UCB-MCs)-transduced with an adenoviral vector carrying the GDNF gene (UCB-MCs+AdV-GDNF group) in rat. Kazan, Russian Federation. We examined the efficacy of AdV-GDNF and UCB-MCs+AdV-GDNF therapy by conducting behavioral tests on the animals and morphometric studies on the spinal cord, performing immunofluorescence analyses on glial cells, investigating the survival and migration potential of UCB-MCs, and evaluating the expression of the recombinant GDNF gene. At the 30th postoperative day, equal positive locomotor recovery was observed after both direct and cell-based GDNF therapy. However, after UCB-MCs-mediated GDNF therapy, the area of preserved tissue and the number of spared myelinated fibers were higher than those measured after direct GDNF gene therapy. Moreover, we observed distinct changes in the populations of glial cells; expression patterns of the specific markers for astrocytes (GFAP, S100B and AQP4), oligodendrocytes (PDGFαR and Cx47) and Schwann cells (P0) differed in various areas of the spinal cord of rats treated with AdV-GDNF and UCB-MCs+AdV-GDNF. The differences detected in the AdV-GDNF and UCB-MCs+AdV-GDNF groups could be partially explained by the action of UCB-MCs. We discuss the insufficiency and the advantages of these two methods of GDNF gene delivery into the spinal cord after traumatic injury.

Lenti-GDNF gene therapy protects against Alzheimer's disease-like neuropathology in 3xTg-AD mice and MC65 cells.

Glial cell-derived neurotrophic factor (GDNF) is emerging as a potent neurotrophic factor with therapeutic potential against a range of neurodegenerative conditions including Alzheimer's disease (AD). We assayed the effects of GDNF treatment in AD experimental models through gene-therapy procedures. Recombinant lentiviral vectors were used to overexpress GDNF gene in hippocampal astrocytes of 3xTg-AD mice in vivo, and also in the MC65 human neuroblastoma that conditionally overexpresses the 99-residue carboxyl-terminal (C99) fragment of the amyloid precursor protein. After 6months of overexpressing GDNF, 10-month-old 3xTg-AD mice showed preserved learning and memory, while their counterparts transduced with a green fluorescent protein vector showed cognitive loss. GDNF therapy did not significantly reduce amyloid and tau pathology, but rather, induced a potent upregulation of brain-derived neurotrophic factor that may act in concert with GDNF to protect neurons from atrophy and degeneration. MC65 cells overexpressing GDNF showed an abolishment of oxidative stress and cell death that was at least partially mediated by a reduced presence of intracellular C99 and derived amyloid β oligomers. GDNF induced neuroprotection in the AD experimental models used. Lentiviral vectors engineered to overexpress GDNF showed to be safe and effective, both as a potential gene therapy and as a tool to uncover the mechanisms of GDNF neuroprotection, including cross talk between astrocytes and neurons in the injured brain.

Effect of exogenous glial cell-derived neurotrophic factor on development of the enteric nervous system in the rectal end of fetal rats with anorectal malformations

The objective of this study is to analyze the effect of exogenous glial cell-derived neurotrophic factor (GDNF) in the development of the enteric nervous system (ENS) in the rectal end of fetal rats. Thirty pregnant Sprague-Dawley rats were categorized randomly into three groups: ethylene thiourea (ETU), ETU+GDNF, and control. On day 10 of gestation, ETU was injected via a gastric tube in the ETU group and ETU+GDNF group. On day 11 of gestation, GDNF was administered through the tail vein in the ETU+GDNF group. On day 20 of gestation, fetal rats were harvested by cesarean section. The prevalence of anorectal malformations (ARMs) in the fetal rats was observed. GDNF expression in the rectal end of fetal rats was detected by immunohistochemical and Western blotting analyses. The prevalence of ARMs in the ETU group and ETU+GDNF group was 51.4 and 52.5%, respectively, but the difference between the two groups was not significant (P>0.05). In the rectal end of fetal rats with an anus, GDNF expressions in the three groups were not significantly different (P>0.05). In the rectal end of fetal rats without an anus: GDNF expression in the ETU+GDNF group was significantly higher than that in the ETU group (P=0.036); GDNF expression in the rectal end of fetal rats without an anus from the ETU group and ETU+GDNF group was significantly lower than that of fetal rats with an anus (ETU group P=0.001; ETU+GDNF group P=0.028). There was a significant difference in the gray level ratio of GDNF and actin between the ETU group and ETU+GDNF group (P<0.0001), and the expression in the ETU+GDNF group was significantly up-regulated. GDNF could not totally prevent the occurrence of ETU-induced ARMs, but it up-regulated expression of the GDNF gene in the wall of the rectal end, thereby promoting the growth of a hypogenetic ENS.

GDNF and BDNF gene interplay in chronic tinnitus.

Glial cell-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) play key roles in the early development of the central auditory pathway and the inner ear. Both have been successfully employed to treat experimental forms of hearing loss and are likely to operate in a broad spectrum of auditory phenotypes, including phantom perceptions of sound. We conducted a genetic association study addressing five biallelic candidate variants in 240 Caucasian subjects who had been diagnosed with tinnitus for more than 6 months. Allele frequencies were determined for three GDNF and two BDNF markers, including a functional missense substitution (V66M). When data were compared to previously examined control populations, no significant allelic associations were noted after corrections for multiple testing. However, using a multiple regression approach and scores from a validated self-report questionnaire, GDNF and BDNF genotypes jointly predicted tinnitus severity in women (N=69, uncorrected p=0.04) but not in men (N=171, n.s.). The present findings serve as an incentive for further explorations of neurotrophic factors' role in predicting clinical features of tinnitus. Possible implications of sexually dimorphic at-risk genotypes are discussed with regard to hearing and neural plasticity.

Neuroprotection by Gene Therapy Targeting Mutant SOD1 in Individual Pools of Motor Neurons Does not Translate Into Therapeutic Benefit in fALS Mice

A major challenge in neurological gene therapy is delivery of the transgene to sufficient cell numbers in an atraumatic manner. This is particularly difficult for motor neuron (MN) diseases that have cells located across the entire spinal cord, brain stem, and cortex. We have used the familial mouse model of amyotrophic lateral sclerosis (ALS) to examine the feasibility of body-wide intramuscular injections of adeno-associated virus serotype 6 (AAV6), a vector capable of axonal retrograde transport, to deliver therapeutic genetic information across the lower MN axis. Neonatal muscle delivery of AAV expressing small hairpin RNAs (shRNAs) against the toxic transgene in this model, human mutant superoxide dismutase 1 (mSOD1), led to significant mSOD1 knockdown in the muscle as well as innervating MNs. This knockdown conferred neuroprotection and halted muscle atrophy in individually targeted MN pools. However, despite the vector being targeted to MNs that innervate muscle groups controlling eating, breathing, and locomotion, this approach was unable to therapeutically impact on disease progression in the ALS mouse model. These results stress the complexity of gene delivery for mSOD1 silencing and suggest that critical thresholds of protein knockdown and transduction across various cell types are required to translate local neuroprotective effects into functional improvements.

Effects of transplanted GDNF gene modified marrow stromal cells on focal cerebral ischemia in rats

Objective: To evaluate the therapeutic effect of transplanted glial cell derived neurotrophic factor (GDNF) modified marrow stromal cells (MSCs) on an experimental ischemic brain injury based on the behavioral, morphological, and immunohistochemical observations. Methods: The MSCs from four-week newborn rats were cultured in vitro. The cerebral ischemia and reperfusion model was established in adult Sprague–Dawley (SD) rats by using the suture method. Three days after model establishment, the animals were injected with prepared MSCs via their caudal veins. The animals were then divided into a sham-operation group, ischemia group, MSCs transplantation group, or GDNF+ MSCs transplantation group and were scored for their neurobehavioral manifestations at 3, 14, and 28 days after the transplantation was performed. At this time, the survival condition of intracerebral transplanted cells was measured by laser confocal microscopy while the effect of transplantation on the Generic Digital Beam Former (GDNF) expression in the ischemic brain tissue was evaluated. Results: The MSCs cells transfected with GDNF gene were characterized by green fluorescence. Three days after the transplantation, the animals that underwent the cell transplantation showed significantly better behavioral data than the controls. Fourteen days after transplantation, the animals transplanted with GDNF gene modified MSCs were better than those transplanted with common MSCs. As compared with common MSCs transplantation, GDNF+MSCs transplantation was significantly more effective in reducing apoptotic cell volume and enhancing Bcl-2 expression, which was favorable for the ischemic brain injury. Conclusions: Transplanted GDNF modified MSCs can improve the nervous function and have a protective effect on the ischemic brain injury through reducing apoptotic cell volume and enhancing the expression of anti-apoptotic gene Bcl-2.

Basic fibroblast growth factor promotes glial cell-derived neurotrophic factor gene expression mediated by activation of ERK5 in rat C6 glioma cells

Extracellular signal-regulated kinases (ERKs) play important physiological roles including proliferation, differentiation and gene expression. ERK5 contains kinase domain that shares homology with ERK1/2 and the T-E-Y activation motif at amino-terminal half, whereas the extended carboxy-terminal half is unique. Because the physiological role of ERK5 in glial cells remains unclear, we examined the involvement of ERK5 in expression of neurotrophic factors and cytokines in rat C6 glioma cells, comparing it with ERK1/2. Basic fibroblast growth factor (bFGF) induced both ERK5 and ERK1/2 phosphorylation in a time- and concentration-dependent manner. Among the neurotrophic factors and cytokines, bFGF induced significant gene expression of glial cell-derived neurotrophic factor (GDNF). The GDNF gene expression and protein synthesis induced by bFGF were blocked by BIX02189 and PD98059 that selectively inhibit ERK5 and ERK1/2 signaling, respectively. The effect was also blocked by overexpression of a dominant-negative MEK5 mutant, indicating that GDNF expression induced by bFGF requires both ERK5 and ERK1/2. Because GDNF gene expression is regulated by various transcription factors, we examined the activity of these factors. We demonstrated that phosphorylation of cAMP-response element-binding protein at Ser 133 was induced by bFGF, which was blocked by BIX02189 and PD98059. Expression of c-fos, a major component of activator protein-1, and early growth response-1 was enhanced by bFGF, and expression of these genes was blocked by BIX02189, PD98059 and overexpression of dominant-negative MEK5. Taking these results together, bFGF promotes GDNF expression accompanied by the activation of ERK5, ERK1/2 and their downstream transcription factors in C6 glioma cells.

Regeneration of the MPTP-lesioned dopaminergic system after convection-enhanced delivery of AAV2-GDNF.

Clinical studies to date have failed to establish therapeutic benefit of glial cell-derived neurotrophic factor (GDNF) in Parkinson's disease (PD). In contrast to previous nonclinical neuroprotective reports, this study shows clinically relevant and long-lasting regeneration of the dopaminergic system in rhesus macaques lesioned with 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine 3-6 months before GDNF gene delivery (AAV2-GDNF). The observed progressive amelioration of functional deficits, recovery of dopamine, and regrowth of fibers to the striatal neuropil demonstrate that high GDNF expression in the putamen promotes restoration of the dopaminergic system in a primate model of advanced PD. Extensive distribution of GDNF within the putamen and transport to the severely lesioned substantia nigra, after convection-enhanced delivery of AAV2-GDNF into the putamen, indicates anterograde transport via striatonigral connections and is anticipated to occur in PD patients. Overall, these data demonstrate nonclinical neurorestoration after putaminal infusion of AAV2-GDNF and suggest that clinical investigation in PD patients is warranted.

Identification of GDNF Gene Sequence Variations in Patients with Medullary Sponge Kidney Disease

Medullary sponge kidney (MSK) is a rare nephropathy characterized by cystic anomalies of precalyceal ducts, nephrocalcinosis, renal stones, and tubule dysfunctions. Its association with various malformations and cases of familial aggregation supports the conviction that genetic factors are involved, but no genetic studies have been conducted to date. It is hypothesized that MSK is due to a disruption at the "ureteric bud/metanephric blastema" interface caused by critical developmental genes functioning abnormally. Fifty-five apparently sporadic MSK patients were analyzed by direct DNA sequencing of all exons and exon-intron boundaries of glial cell-derived neurotrophic factor (GDNF) gene and rearranged during transfection (RET) gene, which have a leading role in renal development. Two novel variants were found in heterozygosity in the MSK case population: GDNF{ENST00000344622}:c.-45G>C and c.-27+18G>A in a putative binding domain for paired-box 2 transcription factor. As a whole, eight patients showed these variations: four patients carried the c.[-45G>C; -27+18G>A] complex allele, and the others had the c.-27+18G>A alone. A case-control study revealed that these two alleles were significantly associated with MSK. Five of the eight cases were found to be familial, and the allele variants cosegregated with the disease in a seemingly dominant pattern of inheritance. Patients revealed no mutations in the RET gene. This is the first report identifying GDNF gene sequence variations in patients with MSK and suggesting a role for this gene in the pathogenesis of some cases of the disease.

Culture and characterization of and lentiviral vectors mediated glial cell derived neurotrophic factor expression in mesenchymal stem cells from human umbilical cord blood

To isolate and culture mesenchymal stem cells from umbilical cord blood (UCB-MSCs), study its biological characterization in vitro, transfect UCB-MSCs using lentiviral vectors encoding glial cell derived neurotrophic factor (GDNF) gene, evaluate the biological function change of UCB-MSCs, and detect GDNF expression level in vitro. We isolated monocyte by Ficoll density gradient, separated two kinds of adherent cells through different trypsin digestion time, and detected the cells surface markers by fluorescence activated cell sorting when it was proliferated for P7 passages. At the same time, we sub-cloned GDNF gene into lentiviral vectors and packaged lentiviral supernatant through three plasmids co-transfection method, then transfected the UCB-MSCs using lentiviral vectors encoding GDNF at different multiplicity of infection, and evaluated the change of biological function by observing the ability of proliferation and differentiation, morphology, and the cells surface markers. We detected the GDNF mRNA and protein expression level by using real-time polymerase chain reaction (real-time PCR) and enzyme-link immunosorbent assay (ELISA). The UCB-MSCs were successfully isolated and cultured in vitro, and induced it to differentiate into fat cells. FACS results showed that the UCB-MSCs expressed CD90, CD73, and CD105 positively, and CD14, CD34, CD45, CD19, HLA-DR, Stro-1, and CD106 negatively. Real-time PCR and ELISA showed that the expressions of GDNF protein and mRNA were correlated with the copy number of transfected cells: high copy number of transfected cells were associated with high GDNF expression. The biological characterization of UCB-MSCs did not obviously change after sub-cloning with GDNF. UCB-MSCs was successfully isolated and cultured in vitro. By transfecting UCB-MSCs with GDNF gene-containing lentiviral vectors, the secretion of GDNF protein and mRNA expression level can be controlled by the copy number of transfected cells, and thus make it constantly express GDNF at high level.

Peripheral Gene Transfer of Glial Cell-Derived Neurotrophic Factor Ameliorates Neuropathic Deficits in Diabetic Rats

Deprivation of neurotrophic factors contributes to the pathogenesis of diabetic neuropathy. However, the role of glial cell-derived neurotrophic factor (GDNF) in the pathogenesis of diabetic neuropathy remains unclear. The present study evaluated the pathogenic role of GDNF deficiency and the therapeutic potential of GDNF gene transfer for diabetic neuropathy. After injection of streptozotocin (STZ) for 2 weeks, diabetic rats displayed significant alteration in electrophysiological parameters, which was associated with structural changes and defective myelination in the sciatic nerves. The early diabetic neuropathy was accompanied by attenuation of the GDNF/GFRalpha1/Akt signaling cascade and depletion of sensory neuropeptides in the peripheral nerves. After detection of neuropathy, intramuscular GDNF gene transfer reversed the deficiency of GDNF/Akt signaling in the sciatic nerve and improved the neurological functions of diabetic rats. Moreover, GDNF gene delivery alleviated the axonal demyelination and restored the sensory neuropeptide levels in the sciatic nerve of diabetic rats. In summary, peripheral GDNF gene delivery ameliorates the diabetes-induced downregulation of the GDNF signaling complex in the peripheral nervous system and holds promises for treatment of diabetic neuropathy.

Common variants of the glial cell-derived neurotrophic factor gene do not influence kidney size of the healthy newborn

Glial cell-derived neurotrophic factor (GDNF) plays an important role in renal development, serving as a trophic factor for outgrowth of the ureteric bud and its continued arborisation. Our previous studies have shown that common variants of the human paired-box 2 (PAX2) gene (a transcriptional activator of GDNF) and rearranged during transfection (RET) gene (encoding the cognate receptor for GDNF) are associated with a subtle reduction in the kidney size of newborns. Since heterozygosity for a mutant GDNF allele causes mild renal hypoplasia and modest hypertension in mice, we considered the possibility that common variants of the GDNF gene might also contribute to renal hypoplasia in humans. We studied the relationship between newborn renal size or umbilical cord cystatin C and 19 common GDNF gene variants [minor allele frequency (MAF) >5%], three single nucleotide polymorphisms (SNPs) related to a putative PAX binding site and one rare SNP (rs36119840 A/G) which changes an amino acid (R93W), based on data from the haplotype map of the human genome (HapMap). However, none of these 23 SNPs was associated with reduced newborn kidney size or function. Among the 163 Caucasians in our cohort, none had the R93W allele.