GDNF family ligand RET receptor in the brain of adult zebrafish

RET is a tyrosine kinase receptor, and transduces signaling by family of glial cell line-derived neurotrophic factor ligands (GFLs). RET is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons. RET exists in two main isoforms originated by differential splicing, RET9 and RET51; phylogenetic studies have shown that the RET gene is conserved across vertebrates. The aim of this study was to investigate the RET expression within the brain of zebrafish, using immunohistochemistry, western blotting and RT-PCR. In homogenate brains both RET protein and mRNA were observed. RET immunoreactivity was widespread in neurons and neural processes of all the major regions of the brain. These results demonstrate the occurrence of RET and suggest an involvement of GDNF family ligands in the brain of adult zebrafish.     

Glial cell line-derived neurotrophic factor in Purkinje cells of adult zebrafish: An autocrine mode of action?

Glial cell line-derived neurotrophic factor (GDNF) has a neuroprotective role in Purkinje cells of cerebellum, promoting the survival and the differentiation of these cells. Its signalling is mediated by a receptorial complex GFRalpha1/RET. In the brain of adult zebrafish (Danio rerio) we previously investigated GDNF expression and localization, but no data exist regarding GFRalpha1 and RET presence. Thus, the present study was designed to clarify the morphological relation between GDNF and its receptorial complex GFRalpha1/RET immunoreactivity in the cerebellum of adult zebrafish. The expression of gdnf, GFRalpha1 and ret genes was demonstrated in adult zebrafish cerebellum by a standard RT-PCR. The distribution of GDNF and its receptorial complex GFRalpha1/RET was examined by single and double immunocytochemical stainings. In the valvula and corpus cerebelli GDNF, GFRalpha1 and RET immunoreactivity was seen co-localized in Purkinje cells, identified morphologically and by using an antiserum against a specific marker for these cells, aldolase C enzyme. In the vestibulolateralis lobe, Purkinje neurons were lacking in both the eminentiae granulares and medial caudal lobe. These results demonstrated the expression of the GDNF receptorial complex in adult zebrafish cerebellum and suggest an autocrine mode of action of GDNF in Purkinje cells.     

Potential role of glial cell line-derived neurotrophic factor receptors in Müller glial cells during light-induced retinal degeneration

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN) and their receptors (GFRalpha1, GFRalpha2 and Ret) play an important role in the survival of neurons in the central and peripheral nervous system. For example, GDNF as well as other trophic factors promotes photoreceptor survival during retinal degeneration. Recent studies have proposed that part of neurotophic rescue of photoreceptors may be indirect, mediated by interaction of the neurotrophic factors with other cell types, that in turn release secondary factors that act directly on photoreceptors. In the present study, we examined the GDNF receptor expression in control and light-damaged retina, and found that GFRalpha2 protein is upregulated in retina-specific Müller glial cells during photoreceptor degeneration. We also examined the effect of GDNF or NTN on cultured Müller cells. Exogenous GDNF increased brain-derived neurotrophic factor, basic fibroblast growth factor and GDNF, but not NTN mRNA production. On the other hand, NTN increased NTN, but not GDNF mRNA production in cultured Müller cells. These observations suggest that GDNF, NTN and their receptors are involved in the regulation of trophic factor production in retinal glial cells, and that functional glia-neuron network may utilize GDNF family for the protection of neural cells during retinal degeneration.     

Identification of a novel GDNF mRNA induced by LPS in immune cell lines

Glial cell line-derived neurotrophic factor (GDNF) is a survival factor for many neuronal cell types and has wide ranging effects within the central nervous system. To investigate the expression of the GDNF gene in immune cell lines under inflammatory conditions, we pharmacologically estimated the induction of GDNF mRNA in RAW264.7 cells. RT-PCR analysis revealed that LPS-induced GDNF mRNA in RAW264.7 cells does not include exon 3, which encodes the translational start site of this gene. A novel type of GDNF mRNA cloned by 5'-RACE consisted of the previous exon 4 and its flanking 5' upstream region, akin to a single exon gene. A similar type of human GDNF mRNA was also detected in a human neuroblastoma cell line, SH-SY5Y, without any stimulation. This novel (Ex4) GDNF mRNA was also upregulated by LPS in primary cultured rat macrophages, microglia and astrocytes and was found to exist in mouse brain. Ex4 GDNF protein produced by transfected HEK293 cells was mainly detected in cell lysates, but in conditioned medium only after PMA stimulation. Ex4 GDNF protein was found to exist as an unglycosylated form in both the transfected cells and the conditioned medium while full-type GDNF protein is glycosylated. PMA-stimulated augmentation of unglycosylated Ex4 GDNF protein was demonstrably regulated at the post-translational level.     

Expression and cell localization of brain-derived neurotrophic factor and TrkB during zebrafish retinal development

Brain‐derived neurotrophic factor (BDNF) signaling through TrkB regulates different aspects of neuronal development, including survival, axonal and dendritic growth, and synapse formation. Despite recent advances in our understanding of the functional significance of BDNF and TrkB in the retina, the cell types in the retina that express BDNF and TrkB, and the variations in their levels of expression during development, remain poorly defined. The goal of the present study is to determine the age‐dependent changes in the levels of expression and localization of BDNF and TrkB in the zebrafish retina. Zebrafish retinas from 10 days post‐fertilization (dpf) to 180 dpf were used to perform PCR, Western blot and immunohistochemistry. Both BDNF and TrkB mRNAs, and BDNF and full‐length TrkB proteins were detected at all ages sampled. The localization of these proteins in the retina was very similar at all time points studied. BDNF immunoreactivity was found in the outer nuclear layer, the outer plexiform layer and the inner plexiform layer, whereas TrkB immunoreactivity was observed in the inner plexiform layer and, to a lesser extent, in the ganglion cell layer. These results demonstrate that the pattern of expression of BDNF and TrkB in the retina of zebrafish remains unchanged during postembryonic development and adult life. Because TrkB expression in retina did not change with age, cells expressing TrkB may potentially be able to respond during the entire lifespan of zebrafish to BDNF either exogenously administered or endogenously produced, acting through paracrine mechanisms.     

Artemin-like immunoreactivity in the zebrafish,<Emphasis Type="Italic"> Danio rerio</Emphasis>

Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family. It is a neurotrophic factor that supports neurite migration and outgrowth and survival of the sympathetic and sensory nervous system. Artemin has been studied in human and murine tissues, but no study has been devoted to nonmammalian species. Zebrafish is a teleost fish belonging to the family Ciprinidae, which is becoming an important model species for genetic and developmental studies. Thus, the aim of the present investigation was to evaluate, by immunochemical and immunocytochemical analyses, the tissue distribution pattern of artemin in zebrafish. Different isoforms of artemin with corresponding different molecular weights were detected in the brain, muscle, testis, ovary, kidney, gut, and gills of zebrafish by Western blot analysis. Immunocytochemical analysis showed artemin-like immunoreactivity in different cell types: in glial cells and rare neurons of the central nervous system, taste buds, retina, neuromasts of the lateral line, dorsal root ganglia, sympathetic ganglia, gill epithelium, tubular kidney epithelium, gut epithelium and ganglia, pancreas, thyroid, hypothalamus, testis, and ovary. These results indicate a wide distribution of artemin-like immunoreactivity in adult zebrafish, related to the presence of different forms of artemin. These findings might suggest a complex maturation pattern of artemin, whose forms could also exert different roles in zebrafish tissues.     

Synapsin I expression in the rat retina during postnatal development

Summary The expression of the synapsin I gene was studied during postnatal development of the rat retina at the mRNA and protein levels. In situ hybridization histochemistry showed that synapsin I mRNA was expressed already in nerve cells in the ganglion cell layer of the neonatal retina, while it appeared in neurons of the inner nuclear layer from postnatal day 4 onward. Maximal expression of synapsin I mRNA was observed at P12 in ganglion cells and in neurons of the inner nuclear layer followed by moderate expression in the adult. At the protein level a shift of synapsin I appearance was observed from cytoplasmic to terminal localization during retinal development by immunohistochemistry. In early stages (P4 and P8), synapsin I was seen in neurons of the ganglion cell layer and in neurons of the developing inner nuclear layer as well as in the developing inner plexiform layer. In the developing outer plexiform layer synapsin I was localized only in horizontal cells and in their processes. Its early appearance at P4 indicated the early maturation of this cell type. A shift and strong increase of labelling to the plexiform layers at P12 indicated the localization of synapsin I in synaptic terminals. The inner plexiform layer exhibited a characteristic stratified pattern. Photoreceptor cells never exhibited synapsin I mRNA or synapsin I protein throughout development.Abbreviations GCL ganglion cell layer - INB inner neuroblast layer - INL inner nuclear layer - IPL inner plexiform layer - ONB outer neuroblast layer - ONL outer nuclear layer - OPL outer plexiform layer     

Artemin-like immunoreactivity in the zebrafish, Danio rerio

Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family. It is a neurotrophic factor that supports neurite migration and outgrowth and survival of the sympathetic and sensory nervous system. Artemin has been studied in human and murine tissues, but no study has been devoted to nonmammalian species. Zebrafish is a teleost fish belonging to the family Ciprinidae, which is becoming an important model species for genetic and developmental studies. Thus, the aim of the present investigation was to evaluate, by immunochemical and immunocytochemical analyses, the tissue distribution pattern of artemin in zebrafish. Different isoforms of artemin with corresponding different molecular weights were detected in the brain, muscle, testis, ovary, kidney, gut, and gills of zebrafish by Western blot analysis. Immunocytochemical analysis showed artemin-like immunoreactivity in different cell types: in glial cells and rare neurons of the central nervous system, taste buds, retina, neuromasts of the lateral line, dorsal root ganglia, sympathetic ganglia, gill epithelium, tubular kidney epithelium, gut epithelium and ganglia, pancreas, thyroid, hypothalamus, testis, and ovary. These results indicate a wide distribution of artemin-like immunoreactivity in adult zebrafish, related to the presence of different forms of artemin. These findings might suggest a complex maturation pattern of artemin, whose forms could also exert different roles in zebrafish tissues.     

Regulation of FGF soluble receptor type 1 (SR1) expression and distribution in developing, degenerating, and FGF2-treated retina.

The spatial and temporal patterns of expression and content of the fibroblast growth factor (FGF) soluble receptor SR1, a specific inhibitor of FGF, were investigated during embryonic and postnatal development of the retina in Fisher rats. As early as at embryonic day 18 (E18), SR1 mRNA and protein were detected in the retina. SR1 protein was strongly associated with the differentiating ganglion cells and its distribution paralleled the radial pattern of retinal development, from center to periphery. From E18 to postnatal day 5, the levels of both SR1 mRNA and SR1 protein remained constant. Thereafter, they decreased rapidly, by a factor of 5 in the adult retina. SR1 was labeled in the inner nuclear layer, but never in the photoreceptor nuclei. In the neural retina of RCS dystrophic rats, the levels SR1 mRNA and SR1 protein were 2 to 3 times higher than those in the normal congenic controls, before and during photoreceptor degeneration. These results provide the first evidence that a natural FGF inhibitor is regulated during retina development and degeneration and suggest that changes in SR1 content may be involved in the regulation of FGF activities in retina. This was confirmed in vivo in RCS rats, in which delayed photoreceptor apoptosis by intravitreal injection of FGF2 was accompanied by a downregulation of SR1 expression. Dev Dyn 2000;217:24-36.     

Glial cell line-derived neurotrophic factor (GDNF) enhances sympathetic neurite growth in rat hearts at early developmental stages

Molecular signaling of sympathetic innervation of myocardium is an unresolved issue. The purpose of this study was to investigate the effect of neurotrophic factors on sympathetic neurite growth towards cardiomyocytes. Cardiomyocytes (CMs) and sympathetic neurons (SNs) were isolated from neonatal rat hearts and superior cervical ganglia, and were co-cultured, either in a random or localized way. Neurite growth from SNs toward CMs was assessed by immunohistochemistry for neurofilament M and α-actinin in response to neurotrophic factors-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and a chemical repellent, semaphorin 3A. As a result, GDNF as well as NGF and BDNF stimulated neurite growth. GDNF enhanced neurite outgrowth even under the NGF-depleted culture condition, excluding an indirect effect of GDNF via NGF. Quantification of mRNA and protein by real-time PCR and immunohistochemistry at different developmental stages revealed that GDNF is abundantly expressed in the hearts of embryos and neonates, but not in adult hearts. GDNF plays an important role in inducing cardiac sympathetic innervation at the early developmental stages. A possible role in (re)innervation of injured or transplanted or cultured and transplanted myocardium may deserve investigation.     

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, GFRα, 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.     

Diffusion tensor imaging in acquired blind humans

Prion diseases, also called transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders characterized by neuronal loss, astrogliosis, and spongiform changes in the brain. It is postulated that appearance of astrogliosis may provide the neurotrophic factors to prevent or reduce neuronal cell loss in the pathogenesis of prion diseases. To investigate the role of the glial cell line-derived neurotrophic factor (GDNF), we studied the expression levels of GDNF mRNA and protein in an animal model of prion diseases. The expression levels of GDNF mRNA and protein were significantly increased in the brains of scrapie-infected mice at 100 and 160 days after inoculation with scrapie strain compared with those of control mice. In addition, we found more intensive immunoreactivity of GDNF in the brains of scrapie-infected mice, specifically in the hippocampal astrocytes, than was seen in control mice. These results suggest that GDNF participates in protection against neuronal cell loss and atrophy in neurodegenerative disorders, which may play one of the important roles in the pathogenic mechanisms of prion diseases.     

Nerve growth factor is expressed and stored in central neurons of adult zebrafish

Nerve growth factor (NGF), a member of the neurotrophin family, was initially described as neuronal survival and growth factor, but successively has emerged as an active mediator in many essential functions in the central nervous system of mammals. NGF is synthesized as a precursor pro‐NGF and is cleaved intracellularly into mature NGF. However, recent evidence demonstrates that pro‐NGF is not a simple inactive precursor, but is also secreted outside the cells and can exert multiple roles. Despite the vast literature present in mammals, studies devoted to NGF in the brain of other vertebrate models are scarce. Zebrafish is a teleost fish widely known for developmental genetic studies and is well established as model for translational neuroscience research. Genomic organization of zebrafish and mouse NGF is highly similar, and zebrafish NGF protein has been reported in mature and two‐precursors forms. To add further knowledge on neurotrophic factors in vertebrate brain models, we decided to determine the NGF mRNA and protein distribution in the adult zebrafish brain and to characterize the phenotype of NGF‐positive cells. NGF mRNA was visualized by in situ hybridization on whole‐mount brains. NGF protein distribution was assessed on microtomic sections by using an antiserum against NGF, able to recognize pro‐NGF in adult zebrafish brain as demonstrated also in previous studies. To characterize NGF‐positive cells, anti‐NGF was employed on microtomic slides of aromatase B transgenic zebrafish (where radial glial cells appeared fluorescent) and by means of double‐immunolabeling against NGF/proliferative cell nuclear antigen (PCNA; proliferation marker) and NGF/microtube‐associated protein2 (MAP2; neuronal marker). NGF mRNA and protein were widely distributed in the brain of adult zebrafish, and their pattern of distribution of positive perikaryal was overlapping, both in males and females, with few slight differences. Specifically, the immunoreactivity to the protein was observed in fibers over the entire encephalon. MAP2 immunoreactivity was present in the majority of NGF‐positive cells, throughout the zebrafish brain. PCNA and aromatase B cells were not positive to NGF, but they were closely intermingled with NGF cells. In conclusion, our study demonstrated that mature neurons in the zebrafish brain express NGF mRNA and store pro‐NGF.     

Localization of acidic fibroblast growth factor (aFGF) mRNA in mouse and bovine retina by in situ hybridization.

Acidic fibroblast growth factor (aFGF) mRNA has been detected in adult mouse or bovine retina by in situ hybridization with bovine aFGF cDNA clones. It is localized on ganglion cell layer, inner nuclear layer, photoreceptors and slightly on pigmented epithelium. This synthesis of aFGF in highly specialized retinal cell types is discussed in the framework on current views about the role of FGF in retinal cell biology.     

Cellular and developmental patterns of expression of Ret and glial cell line-derived neurotrophic factor receptor alpha mRNAs

 Glial cell line-derived neurotrophic factor (GDNF) has recently been shown to signal by binding to GDNF receptor-alpha (GDNFR-α), after which the GDNF-GDNFR-α associates with and activates the tyrosine kinase receptor Ret. We have localized Ret messenger RNA (mRNA) in the developing and adult rodent and compared with to the expression of GDNF and GDNFR-α mRNA. Ret mRNA is strongly expressed in dopamine neurons and α-motorneurons as well as in thalamus, ruber and occlumotor nuclei, the habenular complex, septum, cerebellum, and brain stem nuclei. Ret mRNA was also found in several sensory systems, in ganglia, and in nonneuronal tissues such as teeth and vibrissae. Very strong Ret mRNA signals are present in kidney and the gastrointestinal tract, where Ret and GDNF mRNA expression patterns are precisely complementary. The presence of Ret protein was confirmed in adult dopamine neurons using immunohistochemistry. GDNFR-α mRNA was strongly expressed in the developing and adult dopamine neurons. It was also found in neurons in deep layers of cortex cerebri, in hippocampus, septum, the dentate gyrus, tectum, and the developing spinal cord. In the kidney and the gastrointestinal tract, GDNFR-α mRNA and Ret mRNA distribution overlapped. Dorsal root ganglia, cranial ganglia, and developing peripheral nerves were also positive. GDNFR-α was additionally found in sensory areas and in developing teeth. Sensory areas included inner ear, eye, olfactory epithelium, and the vomeronasal organ, as well as developing tongue papillae. The temporospatial pattern of expression of GDNFR-α mRNA did not always match that of Ret mRNA. For instance, GDNFR-α mRNA was also found in the developing ventral striatum, including the olfactory tubercle, and in hippocampus. These areas seemed devoid of Ret mRNA, suggesting that GDNFR-α might also have functions unrelated to Ret. Received: 2 January 1997 / Accepted: 26 February 1997     

GDNF mRNA expression in normal postnatal development, aging, and in weaver mutant mice

Glial cell line-derived neurotrophic factor (GDNF) has been demonstrated to enhance the survival and process outgrowth of mesencephalic dopamine neurons. A nuclease protection assay was utilized to determine whether GDNF mRNA is expressed in the ventral mesencephalon and/or striatum during normal mouse postnatal development. While no GDNF mRNA was detected in the ventral mesencephalon expression was detected in the striatum throughout postnatal development and maturity with the peak of expression being in the second postnatal week. In the process of normal aging no change in the levels of GDNF mRNA was observed in the striatum while a 10-fold increase in glial fibrillary acid protein (GFAP) mRNA was detected in 24-month-old relative to either 4.5- or 11-month-old mice. Further analysis addressed whether there are changes in GDNF gene expression associated with the neurodegeneration of dopamine neurons that occurs in the weaver mutant mouse. A transient 65% increase in the expression of GDNF mRNA was observed in weaver mutant striatum on postnatal day 22. The results of this study suggest that GDNF could provide target derived dopaminergic neurotrophic support and stimulate fiber outgrowth during development and that decreased levels of GDNF expression are not responsible for either aging-associated decreases in dopaminergic neuronal plasticity or neurodegeneration in the weaver mutant mouse     

Immunohistochemical localization of protein kinase C-alpha in the retina of pigs during postnatal development

The cellular localization and protein expression level of protein kinase C (PKC)-alpha was examined in pig retina at different ages. Western blot analysis detected PKC-alpha in the retinas of 3-day-old piglets and indicated significantly increased expression in 6-month-old young adult and 2-year-old adult pigs. Immunohistochemistry of 3-day-old retinas revealed intense PKC-alpha reactivity in the inner plexiform and inner nuclear cell layers, weak reactivity in the ganglion cell layer, and few positive cells in the outer nuclear cell layer. The cellular localization of PKC-alpha in the adult retina was similar, with staining more intense than that in neonates. PKC-alpha was co-localized in some glial fibrillary acidic protein-positive cells and glutamine synthetase-positive cells in the retina. This study demonstrates that the protein level of retinal PKC-alpha is increased with maturation and suggests that PKC-alpha plays a role in signal transduction pathways for postnatal development in porcine retina.     

Absolute requirement of GDNF for adult catecholaminergic neuron survival

GDNF is a potent neurotrophic factor that protects catecholaminergic neurons from toxic damage and induces fiber outgrowth. However, the actual role of endogenous GDNF in the normal adult brain is unknown, even though GDNF-based therapies are considered promising for neurodegenerative disorders. We have generated a conditional GDNF-null mouse to suppress GDNF expression in adulthood, hence avoiding the developmental compensatory modifications masking its true physiologic action. After Gdnf ablation, mice showed a progressive hypokinesia and a selective decrease of brain tyrosine hydroxylase (Th) mRNA, accompanied by pronounced catecholaminergic cell death, affecting most notably the locus coeruleus, which practically disappears; the substantia nigra; and the ventral tegmental area. These data unequivocally demonstrate that GDNF is indispensable for adult catecholaminergic neuron survival and also show that, under physiologic conditions, downregulation of a single trophic factor can produce massive neuronal death.