Visual detection of glial cell line-derived neurotrophic factor based on a molecular translator and isothermal strand-displacement polymerization reaction.

BACKGROUND: Glial cell line-derived neurotrophic factor (GDNF) is a small protein that potently promotes the survival of many types of neurons. Detection of GDNF is vital to monitoring the survival of sympathetic and sensory neurons. However, the specific method for GDNF detection is also un-discovered. The purpose of this study is to explore the method for protein detection of GDNF. METHODS: A novel visual detection method based on a molecular translator and isothermal strand-displacement polymerization reaction (ISDPR) has been proposed for the detection of GDNF. In this study, a molecular translator was employed to convert the input protein to output deoxyribonucleic acid signal, which was further amplified by ISDPR. The product of ISDPR was detected by a lateral flow biosensor within 30 minutes. RESULTS: This novel visual detection method based on a molecular translator and ISDPR has very high sensitivity and selectivity, with a dynamic response ranging from 1 pg/mL to 10 ng/mL, and the detection limit was 1 pg/mL of GDNF. CONCLUSION: This novel visual detection method exhibits high sensitivity and selectivity, which is very simple and universal for GDNF detection to help disease therapy in clinical practice.

A novel immunoprecipitation strategy identifies a unique functional mimic of the glial cell line-derived neurotrophic factor family ligands in the pathogen Trypanosoma cruzi.

The journey of the Chagas' disease parasite Trypanosoma cruzi in the human body usually starts in the skin after an insect bite, when trypomastigotes get through the extracellular matrix to bind specific surface receptors in the epidermis and dermis to enter cells, where they differentiate and replicate. As the infection spreads to the heart, nervous system, and other parts of the body via the circulatory system, the parasite must also cope with additional receptors in the immune system and vascular endothelium. The molecular underpinnings that govern host cell receptor recognition by T. cruzi counterreceptors remain largely unknown. Here, we describe an immunoprecipitation strategy designed to concurrently identify host receptors and complementing parasite counterreceptors. Extracellular domains of growth factor receptors fused to human immunoglobulin G (IgG) Fc were incubated with parasite lysates, immunoprecipitated on protein G-Sepharose, and eluted with Laemmli sample buffer. Possible T. cruzi counterreceptors pulled down by the receptor-Fc bait were visualized on immunoblots probed with multispecific high-affinity IgG from chronic chagasic sera and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels stained with silver or Coomassie blue. In screening receptors important for nervous system repair, this parasite counterreceptor immunoprecipitation (PcIP) assay identified 7 to 11 polypeptides (molecular masses, 14 kDa to 55 kDa) that bound to the coreceptors of glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) GFRalpha-1, -2, and -3. Binding was specific because the T. cruzi mimic of host GFLs, named TGFL, did not react with GFL coreceptor tyrosine kinase RET and with other neurotrophic receptors. The polypeptides were located on the parasite outer membrane and bound noncovalently to each other. TGFL eluted from the GFL receptor/protein G affinity column with 0.5 M NaCl, pH 7.5, and potently promoted neurite outgrowth and cell survival in a GFL-sensitive mouse pheochromocytoma cell line. Given that GFLs are neuron survival factors crucial for development and maintenance of central and peripheral nervous systems, it may be that T. cruzi mimicry of host GFLs helps in mutually beneficial host repair of infected and damaged nervous tissue. As there are >30 growth factor receptor-Fc chimeras commercially available, this PcIP assay can be readily adapted to identify receptors/counterreceptors in other T. cruzi invasion sites and in other infections such as Lyme disease, amebiasis, and schistosomiasis.

Immunolocalization of glial cell-derived neurotrophic factor (GDNF) and its receptor GFR-alpha1 in varicocele-induced rat testis.

The aim of the study was to determine the immunolocalisation of glial cell-derived neurotrophic factor (GDNF) and its receptor (GFRalpha1) in testicular dysfunction induced by experimental left varicocele. Male Wistar rats were divided randomly into two groups: a varicocele-induced group and a sham-operated group for 9, 11 and 13 weeks (each group n=6). After orchiectomy, part of the left testis from each animal was fixed, processed and embedded in paraffin wax for immunohistochemistry and the other part was fixed for ultrastructural investigations. GDNF immunoreactivity was localized in the interstitial space in Leydig cell cytoplasm and there was no significant difference (P=0.5) between the varicocele-induced groups at the various time points. GFRalpha1 localization was perinuclear in spermatids and cytoplasmic in Leydig cells. The decrease of GFRalpha1 immunoreactivity was significant (P=0.001) in varicocele-induced testis at 13 weeks when compared with the age-matched sham group. This is the first study to describe the immunolocalization patterns of GDNF and GFRalpha1 in a rat model of varicocele. Although there was no change in GDNF labelling at the different time points after varicocele, GFRalpha1 was significantly decreased in the 13-week group. Distribution of GDNF and its receptor GFRalpha1 in normal and varicocele-induced rat testes suggests both autocrine and paracrine regulation of spermatogenesis.

Localization of nerve growth factor, neurotrophin-3, and glial cell line-derived neurotrophic factor in nestin-expressing reactive astrocytes in the caudate-putamen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated C57/Bl mice.

To address the hypothesis that reactive astrocytes in the basal ganglia of an animal model of Parkinson's disease serve neurotrophic roles, we studied the expression pattern of neurotrophic factors in the basal ganglia of C57/Bl mice that had been treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce the degeneration of nigral dopamine neurons and parkinsonism. MPTP induced significant neuronal degeneration in the substantia nigra pars compacta as detected with Fluoro-Jade B staining, and this was accompanied by an increase in nestin-expressing astrocytes within the caudate-putamen. The number of nestin-positive reactive astrocytes in the caudate-putamen peaked within 3-5 days following MPTP treatment and then declined progressively toward the basal level by 21 days after treatment. Immunofluorescence and confocal microscopy confirmed coexpression of nestin or Ki-67 (cell proliferation marker) in glial fibrillary acid protein-positive astrocytes in the caudate-putamen. Double immunolabeling further revealed immunoreactivities for nerve growth factor (NGF), neurotrophin-3 (NT3), and glial cell line-derived neurotrophic factor (GDNF) in nestin-positive reactive astrocytes. Semiquantification of data obtained from mice 5 days after MPTP injection indicated that the majority of nestin-expressing cells expressed NGF (92%), NT3 (90%), or GDNF (86%). Our results present novel evidence of neurotrophic features among reactive astrocytes in the dopamine-depleted striatum. These nestin-expressing reactive astrocytes may therefore play neurotrophic roles in neural remodeling of the basal ganglia in Parkinson's disease.

GDNF family ligand immunoreactivity in the gut of teleostean fish.

Glial-derived neurotrophic factor (GDNF), neurturin (NRTN), persephin (PSPN), and artemin (ARTN) are a group of proteins belonging to the GDNF family ligands (GFLs). GDNF, NRTN, and ARTN support the survival of central, peripheral, and autonomic neuron populations, while PSPN supports the survival of only several central neuron populations. A common receptor, RET, modulates the action of this family and a co-receptor, GFRalpha, determines RET ligand specificity. GDNF and NRTN appear to be essential for enteric nervous system (ENS) development in mammals, zebrafish, and other teleostean species. GFLs are also essential for the maintenance and plasticity of adult mammalian ENS. In this study, the distribution pattern of GFLs in the intestine of five adult fish (bass, gilt-head, scorpionfish, trout, and zebrafish) was evaluated by immunochemical and immunocytochemical analysis. The results demonstrated the presence of GDNF, NRTN, and ARTN in the gut of all species studied. They appeared to be spread in the ENS and/or endocrine cells of the intestine. These findings suggest that the presence of GFLs in fish gut is not only limited to developmental period, but could be also involved in the enteric physiology of adult species.