Effect of 6-hydroxydopamine on striatal GDNF and nigral GFRα1 and RET mRNAs in the adult rat

Abstract

Exogenous GDNF as well as vectors containing the gene for this trophic factor has been shown to be neuroprotective in animal models of Parkinson’s disease. We therefore investigated whether changes in striatal GDNF protein and nigral mRNA levels of its co-receptors GFRα1 and RET occur in response to lesions of dopamine (DA) neurons and examined the temporal profile of these changes as they relate to the loss of dopaminergic markers. Rats were lesioned with 6-hydroxydopamine and sacrificed 3 h to 60 days post-infusion. DA tissue levels in the striatum and tyrosine hydroxylase immunoreactivity in the substantia nigra (SN) and ventral tegmental area (VTA) were used to determine the size of the lesions. GDNF protein was measured in the striatum using radioimmunocytochemistry. In situ hybridization was used to determine alterations in the mRNAs of RET and GFRα1 in the SN and VTA. We observed no persistent changes in GDNF protein in the striatum in response to 6-hydroxydopamine over the 60-day observation period, suggesting that compensatory changes in this trophic factor do not occur in response to injury. Dramatic decreases in RET and GFRα1 were observed in both SN and VTA that were generally correlated with the loss of TH protein and striatal DA content, strongly suggesting that these receptors are located on DA neurons and that the protective effect of GDNF reflects a direct action of the trophic factor on these neurons.

Introduction

Glial cell line-derived neurotrophic factor (GDNF), a member of the TGFβ family of neurotrophic factors, has been shown to reduce both spontaneous dopamine (DA) neuron death and DA neuron loss due to 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in embryonic and neonatal mesencephalic cultures [1], [12], [19], [24], [37], [42]. Although the basal expression of GDNF is very low in adult rodent brains [16], administration of exogenous GDNF or the delivery of GDNF via gene therapy can increase the apparent capacity of DA neurons to release their transmitter [55] and protect and even rescue adult DA neurons from injury caused by 6-OHDA and MPTP [17], [22], [32], [36]. Thus, GDNF or related molecules ultimately may provide a therapy for Parkinson’s disease (PD), a progressive neurodegenerative disorder associated with the loss of DA neurons that project from substantia nigra (SN) to striatum and to a lesser extent from ventral tegmental area (VTA) to nucleus accumbens and prefrontal cortex [8], [23].

In addition to suggesting a therapeutic approach to PD, further understanding of the actions of GDNF may provide insights into the etiology of the disease. First, studies of postmortem brains from patients with PD suggest that loss of GDNF and other neurotrophic factors may be involved [14], [53]. Second, since GDNF and/or its receptors can be increased in response to certain toxic insults [5], [21], [25], [46], [64], PD could represent a failure of GDNF to respond to incipient injury. Thus, a more detailed study of the effects of 6-OHDA-induced lesions of DA neurons on GDNF and the elements of its receptor complex, GFRα1 and RET, should provide us with information regarding the origin of this protein, its response to DA neuronal damage and its sites of action.

In the studies to be presented in this report, we observed that GDNF protein was present in measurable amounts in the adult rat striatum but did not show a sustained increase in response to 6-OHDA. We also found that the expression of both GFRα1 and RET mRNAs in SN and VTA was reduced by 6-OHDA in proportion to the loss in those structures of the rate-limiting enzyme for DA biosynthesis, tyrosine hydroxylase (TH). Thus, our studies suggest that whereas the brain is unable to respond to 6-OHDA by an upregulation of GDNF signaling, DA neurons do have the capacity to be directly affected by exogenous GDNF or the delivery of GDNF through the administration of a gene encoding for this trophic factor.