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The present study investigated whether glial cell line-derived neurotrophic factor prevents the progressive striatal degeneration induced by chronic systemic administration of the mitochondrial toxin, 3-nitropropionic acid. In addition, the effects of delayed treatment with glial cell line-derived neurotrophic factor on toxin-induced behavioural and neurochemical deficits were determined. Locomotor activity in rats infused with 3-nitropropionic acid (15 mg/kg/day, for four weeks) via subcutaneous osmotic minipumps was considerably reduced compared to control rats. However, in rats given a single intracerebroventricular injection of 100 micrograms of glial cell line-derived neurotrophic factor, locomotor activity was significantly higher than in rats injected with the vehicle, an effect that was most pronounced at the onset of toxin infusion. Consistent with a protective or restorative effect in this model of striatal neurodegeneration, toxin-induced deficits in markers of neurotransmitter function were attenuated by glial cell line-derived neurotrophic factor. Thus, [3H]GABA uptake and [3H]tiagabine/GABA uptake sites in striatal target tissues (globus pallidus and substantia nigra), as well as [3H]choline uptake, choline acetyltransferase activity and dopamine receptor binding in the striatum were decreased by the toxin and restored to varying degrees by glial cell line-derived neurotrophic factor administration. As with locomotor abnormalities, effects on neurochemical deficits were most prominent when glial cell line-derived neurotrophic factor was given at the start of toxin infusion, but remained significantly higher than in the vehicle-injected rats when given up to two weeks after. Substance P, dynorphin A and [Met]enkephalin levels in the striatal target tissues also were reduced by 3-nitropropionic acid. The results show that glial cell line-derived neurotrophic factor protects striatal neurons from slow excitotoxic cell death resulting from energy deprivation, secondary to mitochondrial dysfunction. Moreover, they suggest that glial cell line-derived neurotrophic factor may be a viable therapeutic agent for slowly progressive central nervous system disorders, like Huntington's disease, that may be caused by secondary excitotoxicity resulting from abnormal energy utilization.

Basic fibroblast growth factor and interleukin-1 beta are known to regulate the expression of other trophic factors and to stimulate reactive gliosis in vivo. S100 beta is a glial-specific putative neurotrophic factor and has been considered a marker of the reactive status of astrocytes. Therefore, we tested the hypothesis that basic fibroblast growth factor-2 and interleukin-1 beta achieve their effects by altering S100 beta gene expression in cultured rat astrocytes using an RNase protection assay. Short-term treatment with basic fibroblast growth factor-2 produced a transient decrease in S100 beta messenger RNA which was followed by an increase after longer term treatment. In contrast, both short- and long-term treatment with interleukin-1 beta suppressed S100 beta messenger RNA. We measured levels of S100 beta nuclear primary transcript to assess whether alterations in transcriptional rate explain the changes in messenger RNA. Our results indicate that changes in transcription account for changes in steady state levels of messenger RNA since basic fibroblast growth factor-2-induced changes in S100 beta primary transcript temporally preceded changes in messenger RNA. We further measured intracellular S100 beta protein levels by enzyme-linked immunosorbent assay to determine whether changes in gene expression were translated into parallel changes in protein. Our results clearly demonstrate that basic fibroblast growth factor-2 and interleukin-1 beta influence the expression of the S100 beta gene, that this regulation appears to occur at the level of transcription, and that alterations in messenger RNA are sometimes, but not always, reflected in changes at the level of protein. These observations suggest that basic fibroblast growth factor-2 may amplify its trophic effects, in part, by influencing the expression of another trophic factor.

The present study determined the effects of intraventricularly administered glial cell line-derived neurotrophic factor on the behavioral and neurochemical sequelae of unilateral excitotoxic lesions of the striatum. Distinct asymmetrical rotational behavior in response to peripheral administration of amphetamine (5 mg/kg) was noted one and two weeks following injections of quinolinic acid (200 nmol) into two sites in the left striatum. In rats given a single intraventricular injection of glial cell line-derived neurotrophic factor (10-1000 micrograms) 30 min before the toxin, amphetamine-induced rotational behavior was significantly attenuated. Analysis of Nissl-stained coronal sections showed marked neuronal loss in the striatum ipsilateral to the quinolinic acid injections, which was at least partially prevented by glial cell line-derived neurotrophic factor D1 and D2 dopamine binding sites in the striatum, the majority of which are localized to subpopulations of GABAergic neurons, were decreased to a similar extent by quinolinic acid. Moreover, the reduction was attenuated by glial cell line-derived neurotrophic factor treatment to a similar degree, suggesting that the two subpopulations of GABAergic striatal output neurons are equally vulnerable to excitotoxic damage. Concomitant changes in neurotransmitter function as a result of the lesion were also observed: [3H]GABA uptake into striatal target tissues (globus pallidus and substantia nigra) was considerably reduced in the lesioned compared to the contralateral unlesioned tissues, as were [3H]choline and [3H]dopamine uptake into striatal synaptosomes. Similarly, striatal choline acetyltransferase activity was decreased by the lesion. Decrements in neuropeptide levels of similar magnitude were evident ipsilateral to the lesion; substance P, met-enkephalin and dynorphin A contents in the globus pallidus and substantia nigra were significantly reduced. Striatal somatostatin and neuropeptide Y levels were not altered. All of the neurochemical deficits induced by striatal quinolinic acid lesions were attenuated by intraventricular delivery of glial cell line-derived neurotrophic factor. Continuous intraventricular infusion of this trophic factor (10 micrograms/day) over a two-week period did not afford notable improvement compared to the single injection of 10 micrograms. In contrast, continuous infusion of brain-derived neurotrophic factor (10 micrograms/day) directly into the striatum did not affect any of the neurochemical parameters studied. However, neurotrophin-3 (10 micrograms/day) delivery into the striatum significantly increased [3H]GABA uptake, but only modestly affected [3H]choline uptake. The results indicate that glial cell line-derived neurotrophic factor counteracts neuronal damage induced by a striatal excitotoxic insult and support its potential use as a treatment for central nervous system disorders that may be a consequence of excitotoxic processes, such as Huntington's disease.

Glial cell line-derived neurotrophic factor was initially identified as a survival factor for developing midbrain dopamine neurons (for reviews, see Refs 17 and 19). Subsequent studies have demonstrated a more wide-spread role for glial cell line-derived neurotrophic factor in the developing and adult CNS. In the adult rat brain, for instance, prior administration of glial cell line-derived neurotrophic factor protects nigrostriatal dopamine neurons from 6-hydroxydopamine-induced damage. When given several weeks after 6-hydroxydopamine injection, glial cell line-derived neurotrophic factor also restores the function of these neurons. Glial cell line-derived neurotrophic factor attenuates excitotoxin-induced cell death in the striatum and hippocampal formation and protective effects of glial cell line-derived neurotrophic factor following axotomy have been reported for spinal motor neurons and basal forebrain cholinergic neurons. These findings suggest that glial cell line-derived neurotrophic factor may be a protective/restorative agent for a diverse population of neurons and imply that it may be a useful therapeutic tool for a variety of neurodegenerative diseases including Parkinson's, Huntington's and Alzheimer's diseases. The potential receptor mediating the pleiotropic effects of glial cell line-derived neurotrophic factor has been characterized only recently as a novel glycosyl-phosphatidylinositol-linked protein, GDNFR-alpha. Because GDNFR-alpha is a cell surface receptor, an additional protein(s) was thought to be involved in the glial cell line-derived neurotrophic factor signalling cascade. The identity of the likely candidate, ret, was inferred initially from indirect evidence. Not only were there remarkable similarities in the distribution of glial cell line-derived neurotrophic factor and the proto-oncogene ret in the developing rat and mouse brain, but also in the phenotype of glial cell line-derived neurotrophic factor knockout mice and mice with ret mutations. Mice with either mutation exhibited pronounced renal and enteric abnormalities, implicating the receptor tyrosine kinase protein product of the ret proto-oncogene as the glial cell line-derived neurotrophic factor signalling protein. More conclusive evidence showing that activation of GDNFR-alpha by glial cell line-derived neurotrophic factor induces phosphorylation of ret has confirmed ret as a signalling protein for glial cell line-derived neurotrophic factor. Preliminary results showing that 6-hydroxydopamine lesions of the substantia nigra markedly reduced ret messenger RNA expression, established its localization to presumably glial cell line-derived neurotrophic factor-responsive dopamine neurons in the nigrostriatal pathway. In contrast, it is not clear whether other glial cell line-derived neurotrophic factor-responsive neurons in the CNS, such as the basal forebrain cholinergic neurons and striatal neurons, also express ret, nor is it evident whether levels of the protein are regulated by disruption of the respective pathways. The present study shows that dense networks of ret immunoreactivity are distributed throughout the nigrostriatal pathway, with lower densities of staining in other brain regions, including the septohippocampal pathway. Following extensive unilateral 6-hydroxydopamine lesions of the medial forebrain bundle, ret immunoreactivity in the substantia nigra and striatum was reduced significantly, to a similar extent as tyrosine hydroxylase immunoreactivity. In contrast, excitotoxic lesions of the striatum, achieved by intrastriatal quinolinic acid injections, resulted in increased ret staining in this brain region. In addition, marked decrements in septal ret immunoreactivity were consequent to complete transections of the fimbria-fornix.