Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system

Abstract

The dopamine (DA) transporter (DAT) is a plasma membrane glycoprotein expressed in dopaminergic (DA-) cells that takes back DA into presynaptic neurons after its release. DAT dysfunction has been involved in different neuro-psychiatric disorders including Parkinson's disease (PD). On the other hand, numerous studies support that the glial cell line-derived neurotrophic factor (GDNF) has a protective effect on DA-cells. However, studies in rodents show that prolonged GDNF over-expression may cause a tyrosine hydroxylase (TH, the limiting enzyme in DA synthesis) decline. The evidence of TH down-regulation suggests that another player in DA handling, DAT, may also be regulated by prolonged GDNF over-expression, and the possibility that this effect is induced at GDNF expression levels lower than those inducing TH down-regulation. This issue was investigated here using intrastriatal injections of a tetracycline-inducible adeno-associated viral vector expressing human GDNF cDNA (AAV-tetON-GDNF) in rats, and doxycycline (DOX; 0.01, 0.03, 0.5 and 3 mg/ml) in the drinking water during 5 weeks. We found that 3 mg/ml DOX promotes an increase in striatal GDNF expression of 12× basal GDNF levels and both DA uptake decrease and TH down-regulation in its native and Ser40 phosphorylated forms. However, 0.5 mg/ml DOX promotes a GDNF expression increase of 3× basal GDNF levels with DA uptake decrease but not TH down-regulation. The use of western-blot under non-reducing conditions, co-immunoprecipitation and in situ proximity ligation assay revealed that the DA uptake decrease is associated with the formation of DAT dimers and an increase in DAT–α-synuclein interactions, without changes in total DAT levels or its compartmental distribution. In conclusion, at appropriate GDNF transduction levels, DA uptake is regulated through DAT protein–protein interactions without interfering with DA synthesis.