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Here we report the generation and characterization of two distinct monoclonal antibodies, G-90 and B-1531, specific to glial cell line-derived neurotrophic factor (GDNF). ELISA results confirmed that G-90 and B-1531 both recognize GDNF. Western blots showed that G-90 recognized only the GDNF dimer, whereas B-1531 recognized both the monomer and dimer. Peptide competition ELISA (PCE) and BIAcore data suggested that G-90 and B-1531 recognize different epitopes: PCE confirmed that B-1531 binds to NH2-terminal peptides between amino acids 18 and 37, whereas G-90 does not; BIAcore data showed that B-1531 binds to the NH2 terminus of GDNF, whereas G-90 does not. G-90, in a concentration-dependent manner, completely neutralized the GDNF-induced increases of choline acetyltransferase in cultured motoneuron and of dopamine uptake and morphological differentiation in dopaminergic neuron cultures. B-1531 had no neutralizing effects. GDNF-induced Ret autophosphorylation in NGR-38 cells was completely neutralized by G-90, whereas B-1531 had a moderate effect. These data show that G-90 and B-1531 are specific antibodies to GDNF. The data also suggest that the NH2 terminus of GDNF is not critical for activity. Partial inhibition of Ret phosphorylation is insufficient to downregulate GDNF-induced biological activity.

Purpose. The purpose of this paper is to determine the significance of cyclic imide formation of an aspartic acid residue during storage on the pharmaceutical quality of a recombinant human glial cell line-derived neurotrophic factor (rhGDNF) formulation.

Recently, a novel glial cell line-derived neurotrophic factor (GDNF) has been identified, cloned, and shown to have potent survival- and growth-promoting activity on fetal rat midbrain dopaminergic neurons in cell culture. In this study, we document marked and long-lasting effects on adult rat midbrain dopaminergic neurons in vivo after intracranial administration. A single injection of this factor into the substantia nigra elicited a dose-dependent increase in both spontaneous and amphetamine-induced motor activity, and a decrease in food consumption, lasting 7-10 days. Using immunocytochemistry, we found sprouting of tyrosine hydroxylase-positive neurites towards the injection site, and increased tyrosine hydroxylase immunoreactivity of the ipsilateral striatum was produced by GDNF. There was also a marked and dose-dependent increase in dopamine turnover in the substantia nigra and striatum, and in ipsilateral dopamine levels in the substantia nigra. Little or no effects of GDNF were seen on norepinephrine or serotonin levels. The neurochemical changes on dopaminergic afferents persist for at least 3 weeks after a single intracranial injection of 10 micrograms. Taken together, these data suggest that this glial cell line-derived factor has a potent influence on adult rat dopamine neurons and may have a potentially important role as a trophic factor for these neurons.

Recombinant human nerve growth factor (rhNGF) secreted by insect cells was purified by ion-exchange and reversed-phase chromatography to near homogeneity. The N-terminus of the secreted molecule was analogous to that of mouse salivary gland NGF. In its native conformation, the insect cell produced rhNGF molecules were homodimers consisting of 120 amino acid polypeptide chains. Mature rhNGF was found not to be significantly glycosylated (less than 0.08 mol of N-acetylglucosamine/mol of protein). The rhNGF was homogeneous with regard to molecular weight and amino acid sequence. Isoelectric focusing resolved the rhNGF into one major and one minor component. Because rhNGF from insect cells can be obtained in large quantities, purified to near homogeneity, and is similar to natural NGF with regard to physicochemical properties and biological activity, it is suitable for further evaluation in animal models as a therapeutic molecule for neurodegenerative diseases such as Alzheimer's disease.

Vanilloid receptor 1 (TRPV1), a membrane-associated cation channel, is activated by the pungent vanilloid from chili peppers, capsaicin, and the ultra potent vanilloid from Euphorbia resinifera, resiniferatoxin (RTX), as well as by physical stimuli (heat and protons) and proposed endogenous ligands (anandamide, N-arachidonyldopamine, N-oleoyldopamine, and products of lipoxygenase). Only limited information is available in TRPV1 on the residues that contribute to vanilloid activation. Interestingly, rabbits have been suggested to be insensitive to capsaicin and have been shown to lack detectable [(3)H]RTX binding in membranes prepared from their dorsal root ganglia. We have cloned rabbit TRPV1 (oTRPV1) and report that it exhibits high homology to rat and human TRPV1. Like its mammalian orthologs, oTRPV1 is selectively expressed in sensory neurons and is sensitive to protons and heat activation but is 100-fold less sensitive to vanilloid activation than either rat or human. Here we identify key residues (Met(547) and Thr(550)) in transmembrane regions 3 and 4 (TM3/4) of rat and human TRPV1 that confer vanilloid sensitivity, [(3)H]RTX binding and competitive antagonist binding to rabbit TRPV1. We also show that these residues differentially affect ligand recognition as well as the assays of functional response versus ligand binding. Furthermore, these residues account for the reported pharmacological differences of RTX, PPAHV (phorbol 12-phenyl-acetate 13-acetate 20-homovanillate) and capsazepine between human and rat TRPV1. Based on our data we propose a model of the TM3/4 region of TRPV1 bound to capsaicin or RTX that may aid in the development of potent TRPV1 antagonists with utility in the treatment of sensory disorders.

A potent neurotrophic factor that enhances survival of midbrain dopaminergic neurons was purified and cloned. Glial cell line-derived neurotrophic factor (GDNF) is a glycosylated, disulfide-bonded homodimer that is a distantly related member of the transforming growth factor-beta superfamily. In embryonic midbrain cultures, recombinant human GDNF promoted the survival and morphological differentiation of dopaminergic neurons and increased their high-affinity dopamine uptake. These effects were relatively specific; GDNF did not increase total neuron or astrocyte numbers nor did it increase transmitter uptake by gamma-aminobutyric-containing and serotonergic neurons. GDNF may have utility in the treatment of Parkinson's disease, which is marked by progressive degeneration of midbrain dopaminergic neurons.

Vanilloid receptor subtype 1 (VR1) is a ligand-gated channel that can be activated by capsaicin and other vanilloids as well as by protons and heat. In the present study, we have analyzed the oligomeric state of VR1. Co-immunoprecipitation of differently tagged VR1 molecules indicated that VR1 can form oligomers. Using two different heterologous VR1 expression systems as well as endogenous VR1 expressed in dorsal root ganglion cells, we analyzed oligomer formation using perfluoro-octanoic acid polyacrylamide gel electrophoresis. Results were confirmed both with chemical cross-linking agents as well as through endogenous cross-linking mediated by transglutaminase. Our results clearly show that VR1 forms multimers in each of the expression systems with a homotetramer as a predominant form. The oligomeric structure of VR1 may contribute to the complexity of VR1 pharmacology. Finally, differences in glycosylation between the systems were observed, indicating the need for caution in the use of the heterologous expression systems for analysis of VR1 properties.

The proto-oncogene Ret, a membrane-associated receptor protein tyrosine kinase, has recently been shown to be a component of the glial cell line-derived neurotrophic factor (GDNF) receptor complex. GDNF has potent dopaminergic neurotrophic properties and has been suggested as a treatment for Parkinson's disease (PD). In this study, tissue sections of human substantia nigra (SN) from normal and PD cases were examined to determine the pattern of Ret expression in this region, and whether there was continued Ret expression in surviving dopaminergic neurons in PD cases. Using a polyclonal antibody to the amino terminal of Ret, immunoreactivity was localized in the SN to dopaminergic neurons. The antibody predominantly identified punctate deposits within cells. A similar pattern of immunoreactivity was observed in rat and monkey SN neurons. In neurologically normal cases, immunoreactivity was detected in many of the SN neurons. In all the PD cases studied, continued expression of Ret was observed in many of the surviving dopaminergic neurons. In certain cases, it was also detected on cells with the morphology of microglia. Ret expression by microglia was confirmed by immunoblot analysis on the human THP-1 macrophage type cell line. However, these cells did not express the mRNA for GDNFRalpha, the other component of the GDNF receptor complex.

The survival of isolated neurons from chick embryo ciliary, sympathetic, and dorsal root ganglia is greatly enhanced by concentrations of extracellular potassium that significantly depolarize the neurons (ED 50 = 20–25mM). The survival-promoting effect of elevated potassium on each of these 3 types of neurons appears to be the result of the opening of voltage-gated calcium channels. The dihydropyridine, Bay K 8644, which increases calcium influx through L-type voltage-gated calcium channels in neurons, strongly potentiated the survival-promoting action of elevated potassium (ED 50 = 10.8 ± 7.0mM). In contrast, chemically closely related dihydropyridines, PN200-110 (ED 50 = 0.33 ± 0.15nM) and nitrendipine (ED 50 = 1.3 ± 0.3nM), which block calcium influx through the same voltage-gated channels, completely inhibited potassium-mediated neuronal survival. Chemically different agents that also block calcium influx through voltage-gated channels also inhibited potassium-mediated neuronal survival: the phenylalkylamine verapamil (ED 50 = 0.78 ± 0.38 μM), the benzothiazepine diltiazem (ED 50 = 1.7 μM), and the inorganic ion cadmium (ED 50 = 5.8 μM). These calcium-channel blockers are not simply toxic to neurons, since they did not inhibit neuronal survival mediated by the neurotrophic proteins, nerve growth factor, basic fibroblast growth factor, or ciliary neurotrophic factor, also suggesting that voltage-gated calcium channels are not involved in the action of these factors. These results suggest that neuronal survival in elevated potassium in ciliary, sympathetic, and dorsal root ganglion neurons is the result of calcium influx through dihydropyridine-sensitive, L-type voltage-gated calcium channels. These findings are discussed in relation to the neuronal toxicity of excitatory amino acids which is also thought to occur through increased calcium influx.

Ciliary neurotrophic factor (CNTF) is one of a small number of proteins with neurotrophic activities distinct from nerve growth factor (NGF). CNTF has now been purified and cloned and the primary structure of CNTF from rabbit sciatic nerve has been determined. Biologically active CNTF has been transiently expressed from a rabbit complementary DNA clone. CNTF is a neural effector without significant sequence homologies to any previously reported protein.