Glial Cell Line-derived Neurotropic Factor Research Articles

Effects of 1,25-Vitamin D3 and 24,25-Vitamin D3 on Corneal Nerve Regeneration in Diabetic Mice.

Corneal nerve homeostasis is essential for the functional integrity of the ocular surface. Vitamin D deficiency (VDD) and vitamin D receptor knockout (VDR KO) have been found to reduce corneal nerve density in diabetic mice. This is the first study to comprehensively examine the influence of vitamin D on nerve regeneration following corneal epithelial injury in diabetic mice. Corneal nerve regeneration was significantly retarded by diabetes, VDR KO, and VDD, and it was accelerated following topical 1,25 Vit D and 24,25 Vit D administration. Furthermore, topical 1,25 Vit D and 24,25 Vit D increased nerve growth factor, glial cell line-derived neurotropic factor, and neurotropin-3 protein expression, and it increased secretion of GDNF protein from human corneal epithelial cells. CD45+ cells and macrophage numbers were significantly decreased, and vitamin D increased CD45+ cell and macrophage recruitment in these wounded diabetic mouse corneas. The accelerated nerve regeneration observed in these corneas following topical 1,25 Vit D and 24,25 Vit D administration may be related to the vitamin D-stimulated expression, secretion of neurotrophic factors, and recruitment of immune cells.

Influence of functional-assembled-FGL polypeptide timbering material on expression of glial cell line-derived neurotropic growth factor mRNA in damaged rat’s spinal cord

Spinal cord injury is one of most serious complications when there is spine injury in patients. Spinal cord injury clinically involves anesthesia and mobility loss in leg in most of patients. Rats were herein molded and divided into groups, and Basso-Beattie-Bresnahan (BBB) method was adopted to assess the motor function of rats. The quantities of cassyl aspartic acid-specified protease (Caspase-3), neurofilament protein-200 (NF-200) and Glial Fibrillary Acidic Protein (GFAP)-positive cell were detected by immunohistochemical staining method. The expression of Glial cell line-derived neurotropic growth factor (GDNF) mRNA in spinal cord tissue was detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) method. The quantity of Caspase-3 positive cell and area of glial scar in the fiber grating laser-National Security (FGL-NS) group were significantly reduced compared with those from the other two groups. The quantities of NF-200 positive cell, graded result of Basso-Beattie-Bresnahan method and content of GDNF mRNA in the FGL-NS group were increased compared to those from the other two groups. The FGL-NS improved the expression of GDNF mRNA so as to repair the damaged neuronal cells.

Biological Activity In Vitro, Absorption, BBB Penetration, and Tolerability of Nanoformulation of BT44:RET Agonist with Disease-Modifying Potential for the Treatment of Neurodegeneration.

BT44 is a novel, second-generation glial cell line-derived neurotropic factor mimetic with improved biological activity and is a lead compound for the treatment of neurodegenerative disorders. Like many other small molecules, it suffers from intrinsic poor aqueous solubility, posing significant hurdles at various levels for its preclinical development and clinical translation. Herein, we report a poly(2-oxazoline)s (POx)-based BT44 micellar nanoformulation with an ultrahigh drug-loading capacity of 47 wt %. The BT44 nanoformulation was comprehensively characterized by 1H NMR spectroscopy, differential scanning calorimetry (DSC), powder X-ray diffraction (XRD), dynamic light scattering (DLS), and cryo-transmission/scanning electron microscopy (cryo-TEM/SEM). The DSC, XRD, and redispersion studies collectively confirmed that the BT44 formulation can be stored as a lyophilized powder and can be redispersed upon need. The DLS suggested that the redispersed formulation is suitable for parenteral administration (Dh ≈ 70 nm). The cryo-TEM measurements showed the presence of wormlike structures in both the plain polymer and the BT44 formulation. The BT44 formulation retained biological activity in immortalized cells and in cultured dopamine neurons. The micellar nanoformulation of BT44 exhibited improved absorption (after subcutaneous injection) and blood-brain barrier (BBB) penetration, and no acute toxic effects in mice were observed. In conclusion, herein, we have developed an ultrahigh BT44-loaded aqueous injectable nanoformulation, which can be used to pave the way for its preclinical and clinical development for the management of neurodegenerative disorders.

Association of Glial Cell-Line Derived Neurotrophic Factor and Nerve Growth Factor with Duration of Untreated Psychosis and Clinical Symptoms in Drug-Naive Schizophrenia.

The neurodevelopmental hypothesis is one of the most-emphasized hypotheses in the etiology of schizophrenia. Nerve growth factor (NGF) and glial cell-line derived neurotropic factor (GDNF) are neurotrophic factors that provide growth, differentiation, and survival in nerve cells in the development process. In this study, we aimed to compare the GDNF and NGF levels of schizophrenia patients with healthy controls and to analyze the relationship between the Positive and Negative Syndrome Scale (PANSS) scores, serum GDNF and NGF levels and the duration of untreated psychosis (DUP) of the patients. The study involved 45 patients with a diagnosis of schizophrenia, who had never used any antipsychotic drug, and 45 age- and sex-matched healthy participants. The participants filled a sociodemographic data form. The PANSS was applied to evaluate the clinical conditions. Before the initiation of the treatment, serum samples were collected from the patients. The difference between the GDNF and NGF levels of the patient group and control group was statistically significant. The serum GDNF and NGF levels in schizophrenia patients were lower than healthy controls. No correlation was found between the DUP and serum GDNF and NGF levels. There was a positive correlation between general psychopathology and negative scores of PANSS and the DUP of patients. GDNF and NGF levels seem to be indicators of schizophrenia and its progress; nevertheless, we still do not have sufficient information about these neurotrophic factors. The results of our study indicate that the neurodevelopmental changes occurring at the early stages of the illness prominently affect the progress of disease, highlighting the importance of treatment in the early stages of disease.

MRI-Guided Focused Ultrasound-Induced Blood Brain Barrier Disruption to Deliver Glial Cell Line Derived Neurotropic Factor Proteins into Brain to Treat Rat Depression.

Glial cell line derived neurotropic factor (GDNF) plays a crucial role in the development and maintenance of glial cells, serotonergic and dopaminergic neurons. A positively therapeutic effect has been demonstrated on some animal neurodegenerative diseases. However, the inability to deliver the protein across blood brain barrier (BBB) into damaged brain region limits its clinical application. Here, we developed GDNF-loaded microbubbles (MBs) and achieved a local and precise delivery of GDNF into the brain through MRI-guided focused ultrasound-induced BBB disruption. To demonstrate the therapeutic effect, rat depression model was developed by chronic mild stress treatment. Typical depression behaviors were confirmed. MRI-guided focused ultrasound was used to irradiate the GDNF-loaded MBs. Obvious BBB opening was observed in the treated rat brains and a significant higher GDNF concentration was detected in the ultrasound-treated brain tissues. Behavioral tests demonstrated the increased GDNF could reverse the depressive-like behaviors induced by chronic mild stress, improve the expression of 5-HT 1B receptor and the protein p11, and increase the number of 5-HT or TPH2 immunoreactive neurons. In conclusion, our study provided an effective approach to deliver GDNF proteins into brain to treat rat depression through MRI-guided focused ultrasound-induced destruction of blood-brain barrier.

Dopaminergic induction of human dental pulp stem cells by photobiomodulation: comparison of 660nm laser light and polychromatic light in the nir

Human dental pulp stem cells (hDPSCs) are able to differentiate into dopaminergic neurons and help the maintenance of partially degenerated neurons, which makes them as an alternative cell source for treatment of Parkinsons' disease (PD) patients. Here, the effect of photobiomodulation with polychromatic light source in the near infrared (NIR) range (600–1200 nm) or low level 660 nm diode laser light on hDPSCs during dopaminergic induction was investigated. Real time RT-qPCR analysis indicated that expressions of brain derived neurotrophic factor (BDNF), glial cell line derived neurotropic factor (GNDF), matrix associated protein 2 (MAP2), nuclear receptor related 1 protein (NURR1) and dopamine transporter (DAT) were increased, especially in the first 7 days of dopaminergic induction when 660 nm laser light was applied with a total energy density of 1.6 J/cm2. The activity of polychromatic light on hDPSCs depended on the differentiation media and protein type. BDNF, GDNF, NURR-1 and MAP2 expressions were increased in the presence of pre-induction factors, and decreased when the post-induction factors were added into the culture medium. In contrast with all these promising results, the dopaminergically induced hDPSCs did not show any functional characteristics of dopaminergic neurons and died after they were transferred to a new laminin coated culture plates. In conclusion, the expression of dopaminergic neuron protective protein mRNAs in hDPSCs was increased by photobiomodulation in defined conditions. However, the cells were not able to differentiate into functional dopaminergic neurons either in control or in photobiomodulated groups that are prone to cell death and exhibit immature dopaminergic neuron characteristics.

Scaffold protein Lin7 family in membrane skeletal protein complex in mouse seminiferous tubules.

The membrane skeletal complex, protein 4.1G-membrane palmitoylated protein 6 (MPP6), is localized in spermatogonia and early spermatocytes of mouse seminiferous tubules. In this study, we investigated the Lin7 family of scaffolding proteins, which interact with MPP6. By immunohistochemistry, Lin7a and Lin7c were localized in germ cells, and Lin7c had especially strong staining in spermatogonia and early spermatocytes, characterized by staging of seminiferous tubules. By immunoelectron microscopy, Lin7 localization appeared under cell membranes in germ cells. The Lin7 staining pattern in seminiferous tubules was partially similar to that of 4.1G, cell adhesion molecule 1 (CADM1), and melanoma cell adhesion molecule (MCAM). Lin7-positive cells included type A spermatogonia, as revealed by double staining for Lin28a. Lin7 staining became weaker in MPP6-deficient mice by immunohistochemistry and western blotting, indicating that MPP6 transports and maintains Lin7 in germ cells. The histology of seminiferous tubules was unchanged in MPP6-deficient mice compared to that of wild-type mice. In cultured spermatogonial stem cells maintained with glial cell line-derived neurotropic factor (GDNF), Lin7 was clearly expressed and immunolocalized along cell membranes, especially at cell-cell junctions. Thus, Lin7 protein is expressed in germ cells, and Lin7, particularly Lin7c, is a useful marker for early spermatogenesis.

Long-term culture of undifferentiated spermatogonia isolated from immature and adult bovine testes.

Undifferentiated spermatogonia eventually differentiate in the testis to produce haploid sperm. Within this cell population, there is a small number of spermatogonial stem cells (SSCs). SSCs are rare cells in the testis, and their cellular characteristics are poorly understood. Establishment of undifferentiated cell line would provide an indispensable tool for studying their biological nature and spermiogenesis/spermatogenesis in vitro. However, there have been few reports on the long-term culture of undifferentiated spermatogonia in species other than rodents. Here, we report the derivation and long-term in vitro culture of undifferentiated spermatogonia cell lines from immature and adult bovine testes. Cell lines from immature testes were maintained in serum-free culture conditions in the presence of glial-cell-line-derived neurotropic factor (GDNF) and bovine leukemia inhibitory factor (bLIF). These cell lines have embryonic stem (ES)-like cell morphology, express pluripotent-stem-cell-specific and germ-cell-specific markers at the protein and mRNA levels, and contributed to the inner cell mass (ICM) of embryos in the blastocyst stage. Meanwhile, cell lines established from adult testes were maintained in low-serum media in the presence of 6-bromoindirubin-3'-oxime (BIO). These cell lines have characteristics resembling those of previously reported male mouse germ cell lines as confirmed by their botryoidally aggregated morphology, as well as the expression of germ-cell-specific markers and pluripotent stem cell markers. These findings could be useful for the development of long-term culture of undifferentiated spermatogonia, which could aid in conservation of species and improvement of livestock production through genome editing technology.

192 Culture Conditions Supporting Long-Term Expansion of Bovine Spermatogonial Stem Cells Isolated from Adult and Immature Testes

Spermatogonial stem cells (SSC) self-renew and differentiate into spermatocytes to produce haploid sperm. Because SSC are a small population of adult stem cells in the testis, numerous studies have been reported to derive cell lines from cultured SSC. It has been reported that neonatal and adult mouse SSC can be cultured in vitro over the long term. Male germline stem (GS) cells, embryonic stem (ES)-like cells, and multipotent male germline stem (MGS) cells were derivated from mouse SSC. However, in domestic species including cattle, information about in vitro culture of SSC is mainly available in the neonatal and immature animal. To our knowledge, there are no reports about long-term culture of SSC isolated from adult bovine testis. In this report, we established culture conditions to maintain SSC isolated from adult and immature testes. The SSC were isolated by 3-step enzymatic digestion and enriched by Percoll gradient centrifugation. For adult testicular cell suspensions, SSC were further enriched by differential plating on precoated gelatin dish. After Percoll gradient centrifugation, we found differential expression of SSC markers (GFRα-1 and UCHL-1) in the isolated cells from immature and adult testis. The RT-PCR results also confirmed the expression of differentiated spermatogonia markers (SYCP3 and STRA-8) in adult testicular cell suspensions. It suggests that isolated testicular germ cell population from adult testis are more heterogeneous than those of immature testis. The SSC isolated from adult testes were cultured in low-serum media containing 6-bromoindirubin-3′-oxime (BIO), an inhibitor of glycogen synthase kinase-3α (GSK3), and subsequently the cultures were maintained in the medium containing glial cell line-derived neurotropic factor (GDNF). The cell lines have characteristics resembling mouse GS cell lines as confirmed by their grape-like shape morphology, the expression of SSC markers (UCHL-1, DBA, and GFRa-1), and pluripotent stem cell markers (POU5F1, SOX2, KLF4). The SSC from immature testes were proliferated for more than 3 months in serum-free culture conditions in the presence of GDNF and bovine leukemia inhibitory factor (LIF). The cell lines had ES-like cell morphology, expressed pluripotent stem cell markers and SSC-specific markers. They differentiated in vitro into 3 germ layers confirmed by the expression of ectoderm (NESTIN), mesoderm (BMP4), and endoderm (GATA-6) markers by RT-PCR and neuron like-cells confirmed by the expression of glial fibrillary acidic protein (GFAP) by immunofluorescence analysis. In conclusion, these findings indicate an efficient method to enrich SSC without cell sorting method and different long-term culture systems subsequently established to maintain SSC from adult and immature testes. Furthermore, our data would be useful for further studies that aim to preserve endangered species and improve livestock production through genome editing technology.

Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2.

The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 μ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions.

GDNF-transfected macrophages produce potent neuroprotective effects in Parkinson's disease mouse model.

The pathobiology of Parkinson's disease (PD) is associated with the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) projecting to the striatum. Currently, there are no treatments that can halt or reverse the course of PD; only palliative therapies, such as replacement strategies for missing neurotransmitters, exist. Thus, the successful brain delivery of neurotrophic factors that promote neuronal survival and reverse the disease progression is crucial. We demonstrated earlier systemically administered autologous macrophages can deliver nanoformulated antioxidant, catalase, to the SNpc providing potent anti-inflammatory effects in PD mouse models. Here we evaluated genetically-modified macrophages for active targeted brain delivery of glial cell-line derived neurotropic factor (GDNF). To capitalize on the beneficial properties afforded by alternatively activated macrophages, transfected with GDNF-encoded pDNA cells were further differentiated toward regenerative M2 phenotype. A systemic administration of GDNF-expressing macrophages significantly ameliorated neurodegeneration and neuroinflammation in PD mice. Behavioral studies confirmed neuroprotective effects of the macrophage-based drug delivery system. One of the suggested mechanisms of therapeutic effects is the release of exosomes containing the expressed neurotropic factor followed by the efficient GDNF transfer to target neurons. Such formulations can serve as a new technology based on cell-mediated active delivery of therapeutic proteins that attenuate and reverse progression of PD, and ultimately provide hope for those patients who are already significantly disabled by the disease.

The reparative response to cross-linked collagen-based scaffolds in a rat spinal cord gap model

Prior work demonstrated the improvement of peripheral nerve regeneration in gaps implanted with collagen scaffold-filled collagen tubes, compared with nerve autografts, and the promise of such implants for treating gaps in spinal cord injury (SCI) in rats. The objective of this study was to investigate collagen implants alone and incorporating select therapeutic agents in a 5-mm full-resection gap model in the rat spinal cord. Two studies were performed, one with a 6-week time point and one with a 2-week time point. For the 6-week study the groups included: (1) untreated control, (2) dehydrothermally (DHT)-cross-linked collagen scaffold, (3) DHT-cross-linked collagen scaffold seeded with adult rat neural stem cells (NSCs), and (4) DHT-cross-linked collagen scaffold incorporating plasmid encoding glial cell line-derived neurotropic factor (pGDNF). The 2-week study groups were: (1) nontreated control, (2) DHT-cross-linked collagen scaffold; (3) DHT-cross-linked collagen scaffold containing laminin; and (4) carbodiimide-cross-linked collagen scaffold containing laminin. The tissue filling the defect of all groups at 6 weeks was largely composed of fibrous scar; however, the tissue was generally more favorably aligned with the long axis of the spinal cord in all of the treatment groups, but not in the control group. Quantification of the percentage of animals per group containing cystic cavities in the defect showed a trend toward fewer rats with cysts in the groups in which the scaffolds were implanted compared to control. All of the collagen implants were clearly visible and mostly intact after 2 weeks. A band of fibrous tissue filling the control gaps was not seen in the collagen implant groups. In all of the groups there was a narrowing of the spinal canal within the gap as a result of surrounding soft tissue collapse into the defect. The narrowing of the spinal canal occurred to a greater extent in the control and DHT scaffold alone groups compared to the DHT scaffold/laminin and EDAC scaffold/laminin groups. Collagen biomaterials can be useful in the treatment of SCI to: favorably align the reparative tissue with the long axis of the spinal cord; potentially reduce the formation of fluid-filled cysts; serve as a delivery vehicle for NSCs and the gene for GDNF; and impede the collapse of musculature and connective tissue into the defect.

Gene Expression Profiling of Rat Testis Development During the Early Post‐Natal Stages

Spermatogenesis is a complex biological process that requires precise regulation of gene expression in the germ cells and their surrounding somatic cells. Some testis-specific genes are involved in different stages of spermatogenesis; however, the precise mechanisms of stage-specific spermatogenesis are still not elucidated. In this study, we first examined the expression patterns of SYCP3, Tnp2, CDH1, glial cell-line-derived neurotropic factor (GDNF) and GFRA1 mRNAs on post-natal days (PNDs) 2, 4, 6, 8, 10, 12, 15, 20, 25 and 30 in rat testis. SYCP3 mRNA was firstly detected from PND 15, while Tnp2 transcript was only found on PND 30. CDH1 mRNA was highly expressed before PND 6, but decreased dramatically on PND 8, then gradually increased until it started to decrease after 12 dpp. Low GDNF and GFRA1 mRNAs were found before PND 6, but gradually increased to the peak on PND 12, then gradually decreased to low level. According to the expression patterns of CDH1, GDNF and GFRA1, we hypothesized that PNDs 6-10 are critical period in the early spermatogenesis. We, therefore, explored gene expression pattern on PNDs 6, 8 and 10 using cDNA microarray. 700 (PND 8 vs PND 6), 4519 (PND 10 vs PND 8), and 4298 (PND 10 vs PND 6) differentially expressed genes (≥ 2-fold) were identified from the comparisons, which cover thousands of gene ontology categories (GO terms) and hundreds of signalling pathways. High consistency between microarray data and quantative real-time PCR (qRT-PCR) was verified from five genes (LOC686076, Trib3, Cxcl6, LOC682508 and C2cd4d). These data provide more information to understand the precisely regulatory mechanism at the early stage of spermatogenesis.

Intervertebral Disc Degeneration-induced Expression of Pain-related Molecules

Discogenic low back pain has been shown to develop into chronic intractable pain due to an unknown pathogenesis. To study the mechanism of discogenic pain, we analyzed the serial expression of pain-related molecules in the dorsal root ganglia (DRG) and thalamus using a newly developed rat model of disc degeneration. Ten microliters of complete Freund's adjuvant was injected into the L5-6 disc of male Sprague-Dawley rats for 10 minutes using a 26-gauge needle. Using a behavioral test, rats with significant pain were selected and subsequently serial gene expression of pain-related molecules in the DRG and the thalamus was analyzed by reverse transcriptase polymerase chain reaction. The expression of tumor necrosis factor-α and interleukin-1β significantly increased at 4 and 8 weeks in the DRG of rats with pain. Furthermore, interleukin-6 was significantly increased at 4 weeks in the DRG; however, these cytokines did not show a significant change in the thalamus. Calcitonin gene-related peptide and substance P were significantly increased in DRG at 4 and 8 weeks and in the thalamus at 2 and 4 weeks. The level of nerve growth factor-β did not significantly increase in the DRG or thalamus, whereas glial cell line-derived neurotropic factor (GDNF) was significantly increased at 2 weeks and was sustained through 8 weeks in both the DRG and thalamus. The disc degeneration rat model described herein led to significant pain of a chronic nature. The gradual and persistent increase of GDNF in both the thalamus and DRG suggests that GDNF might be a key factor in the development of intractable, chronic discogenic pain.

Prolonged administration of proguanil induces reproductive toxicity in male rats

The toxicity of proguanil was studied on reproductive activities in male rats using in vivo and in vitro methods. Groups of ten to twelve-week-old rats were administered proguanil (2.9 mg/kg body weight) daily for 5 days and 6 weeks respectively. Thereafter, body and reproductive organ weights were taken, sperm parameters were analyzed, while the histology of the testis and epididymis were carried out. Also, serum levels of testosterone, luteinizing hormone and follicle stimulating hormone were determined. Sertoli cells obtained from sixteen to eighteen day-old-rats were cultured and treated with 0.3 µM to 10 µM of proguanil for 5 days after which Sertoli cell viability and nuclei integrity were determined. Also, the genetic expressions of transferrin and Glial cell line-derived neurotrophic factor (G.D.N.F.) were assessed. The data obtained were analyzed using the analysis of variance (ANOVA) with Students-Newman-Keuls multiple comparison test. The results showed duration dependent significant decrease in body and organ weights and also in sperm parameters. Serum testosterone level was significantly decreased while luteinizing hormone and follicle stimulating hormone showed no significant change. Duration dependent degeneration was also observed in the testis and epididymis of treated rats. These changes were restored in recovery experiment. Viability and DNA integrity of treated Sertoli cells reduced in a dose and duration dependent manner. However, the genetic expressions of transferrin and Glial cell line-derived neurotropic factor were normal. These results show that proguanil could induce infertility in rats which may act by distorting the blood-testis barrier formed by the Sertoli cells.

Progress of tumor markers of children's germ cell tumor

Tumor marker is a hot subject of research in oncology field,which would promote the diagnosis and treatment in clinic.Tumor markers of children's germ cell tumor include α-fetoprotein,glypican 3,SALL4,glial cell line-derived neurotropic factor receptorα-1(GFRα-1),Ki-67,octamer-binding transcription factor4,CD117,D2-40 and CD30.SALL4 would be used as a new tumor marker for germ cell tumor,specially for yolk sac tumor.GFRα-1 can be helpful for the diagnosis of immature teratoma. Key words: Tumor marker; Germ cell tumor; Children

Effect of Intranigral Injection of GDNF and EGF on the Survival and Possible Differentiation Fate of Progenitors and Immature Neurons in 6-OHDA-Lesioned Rats

We investigated the survival and the possible differentiation fate of the progenitors and immature neurons in the pars compacta of the substantia nigra (SNc) by intranigral injection of a glial cell line-derived neurotropic factor (GDNF) or glial cell line-derived neurotropic factor plus epidermal growth factor (EGF + GDNF) in 6-hydroxydopamine (6-OHDA)-lesioned rats. First, we performed behavioral tests by postural asymmetry and forelimb akinesia on the rats injected with 6-OHDA in striatumat day 7, and selected the qualified model according to the results. Then, intranigral GDNF or EGF + GDNF treatment was administered in the qualified PD model rats. On day 21, behavioral tests were performed with these rats; and then the rats were sacrificed for analyses of β-tubulin isotype-III (Tuj1), nestin, glial fibrillary acidic protein (GFAP), and tyrosine hydroxylase (TH) by immunohistochemistry and Western blotting. The results indicated that GDNF could promote the survival of the progenitor cells and immature neurons in rat SNc following 6-OHDA lesion. Moreover, EGF is capable of enhancing the survival effect of GDNF on the progenitor cells and immature neurons in SNc. On day 21, rapid functional recovery from the lesion-induced behavioral asymmetries was observed in the GDNF or EGF + GDNF treated rats, and the numbers of TH-positive neurons increased in SNc, suggesting that the rats might generate new dopaminergic neurons. Thus, our study provides the new insight that the progenitors and immature neurons in SNc of 6-OHDA-lesioned rats might be able to differentiate toward the dopaminergic neurons fate subsequent to treatment with GDNF or EGF + GDNF.

Ape1/Ref-1 Stimulates GDNF/GFRα1-mediated Downstream Signaling and Neuroblastoma Proliferation

We previously reported that glial cell line-derived neurotropic factor (GDNF) receptor alpha1 (GFRalpha1) is a direct target of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1). In the present study, we further analyzed the physiological roles of Ape1/Ref-1-induced GFRalpha1 expression in Neuro2a mouse neuroblastoma cells. Ape1/Ref-1 expression caused the clustering of GFRalpha1 immunoreactivity in lipid rafts in response to GDNF. We also found that Ret, a downstream target of GFRalpha1, was functionally activated by GDNF in Ape1/Ref-1-expressing cells. Moreover, GDNF promoted the proliferation of Ape1/Ref-1-expressing Neuro2a cells. Furthermore, GFRalpha1-specific RNA experiments demonstrated that the downregulation of GFRalpha1 by siRNA in Ape1/Ref-1-expressing cells impaired the ability of GDNF to phosphorylate Akt and PLCgamma-1 and to stimulate cellular proliferation. These results show an association between Ape1/Ref-1 and GDNF/GFRalpha signaling, and suggest a potential molecular mechanism for the involvement of Ape1/Ref-1 in neuronal proliferation.

Expression of Glial Cell Line–Derived Neurotropic Factor Receptor α-1 in Immature Teratomas

The immaturity of teratomas is usually manifested as immature neuroepithelium. The amount of immature neuroepithelium has been correlated with the survival of adult patients with ovarian immature teratoma. To date, no immunohistochemical marker has been found to facilitate the identification of immature teratoma. In this study, we evaluated the expression of glial cell line-derived neurotropic factor receptor alpha-1 (GFRalpha-1) for this purpose. We retrieved 38 cases of germ cell tumors: 26 cases contained immature teratoma, of which 24 had immature neuroepithelium and showed strong membrane staining for GFRalpha-1. No significant staining was seen in other components including embryonal carcinoma, seminoma, yolk sac tumor, choriocarcinoma, immature mesenchyme, and intratubular germ cell neoplasia. Immunohistochemical staining for GFRalpha-1 in immature neuroepithelium may facilitate its identification.

Role of glial cell-line derived neurotropic factor family receptor α2 in the actions of the glucagon-like peptides on the murine intestine

The intestinal glucagon-like peptides GLP-1 and GLP-2 inhibit intestinal motility, whereas GLP-2 also stimulates growth of the intestinal mucosa. However, the mechanisms of action of these peptides in the intestine remain poorly characterized. To determine the role of the enteric nervous system in the actions of GLP-1 and GLP-2 on the intestine, the glial cell line-derived neurotropic factor family receptor alpha(2) (GFRalpha2) knockout (KO) mouse was employed. The mice exhibited decreased cholinergic staining, as well as reduced mRNA transcripts for substance P-ergic excitatory motoneurons in the enteric nervous system (ENS) (P < 0.05). Examination of parameters of intestinal growth (including small and large intestinal weight and small intestinal villus height, crypt depth, and crypt cell proliferation) demonstrated no differences between wild-type and KO mice in either basal or GLP-2-stimulated mucosal growth. Nonetheless, KO mice exhibited reduced numbers of synaptophysin-positive enteroendocrine cells (P < 0.05), as well as a markedly impaired basal gastrointestinal (GI) transit rate (P < 0.05). Furthermore, acute administration of GLP-1 and GLP-2 significantly inhibited transit rates in wild-type mice (P < 0.05-0.01) but had no effect in GFRalpha2 KO mice. Despite these changes, expression of mRNA transcripts for the GLP receptors was not reduced in the ENS of KO animals, suggesting that GLP-1 and -2 modulate intestinal transit through enhancement of inhibitory input to cholinergic/substance P-ergic excitatory motoneurons. Together, these findings demonstrate a role for GFRalpha2-expressing enteric neurons in the downstream signaling of the glucagon-like peptides to inhibit GI motility, but not in intestinal growth.