Zebrafish GDNF and its co-receptor GFRα1 activate the human RET receptor and promote the survival of dopaminergic neurons in vitro

Abstract

Glial cell line-derived neurotrophic factor (GDNF) is a ligand that activates, through co-receptor GDNF family receptor alpha-1 (GFRα1) and receptor tyrosine kinase “RET”, several signaling pathways crucial in the development and sustainment of multiple neuronal populations. We decided to study whether non-mammalian orthologs of these three proteins have conserved their function: can they activate the human counterparts? Using the baculovirus expression system, we expressed and purified Danio rerio RET, and its binding partners GFRα1 and GDNF, and Drosophila melanogaster RET and two isoforms of co-receptor GDNF receptor-like. Our results report high-level insect cell expression of post-translationally modified and dimerized zebrafish RET and its binding partners. We also found that zebrafish GFRα1 and GDNF are comparably active as mammalian cell-produced ones. We also report the first measurements of the affinity of the complex to RET in solution: at least for zebrafish, the Kd for GFRα1-GDNF binding RET is 5.9 μM. Surprisingly, we also found that zebrafish GDNF as well as zebrafish GFRα1 robustly activated human RET signaling and promoted the survival of cultured mouse dopaminergic neurons with comparable efficiency to mammalian GDNF, unlike E. coli-produced human proteins. These results contradict previous studies suggesting that mammalian GFRα1 and GDNF cannot bind and activate non-mammalian RET and vice versa.

GDNF receptor agonist supports dopamine neurons in vitro and protects their function in animal model of Parkinson’s

“…1J-K). In this assay GDNF usually increases luciferase activity in GFRα1/RET expressing cells by 50-100 fold (Sidorova et al , 2010, 2017; Saarenpää et al , 2017).…”

“…GDNF (10 ng/ml) increased the number of TH-positive cells in culture by 1.8 fold (P = 0.0207, Dunnett’s post hoc test) (Figure 2A, B). Important to note, that the bell-shaped dose-response curves are common for neurotrophic factors in general and for GDNF in particular (Hou et al , 1996; Saarenpää et al , 2017). Therefore, the lack of increase in dopamine neuron survival in response to the higher doses of BT13 is expected.…”

“…3) in the striatum of naïve mice at the dose of 750 μg. It is important to note, that the concentrations of the positive control proteins in all experiments were selected on the basis of previous results and biological activity in screening assay in order to produce clear effects in studied systems (Trupp et al , 1996; Sidorova et al , 2010, 2017; Runeberg-Roos et al , 2016; Saarenpää et al , 2017). Nevertheless, the potency of BT13 was two-four orders of magnitude lower: GDNF supported the survival of cultured dopamine neurons in approximately 0.3 nM concentration and reduced the number of amphetamine-induced turns at about 8 μM concentration.…”

“…The effect of BT13 on survival of näive dopamine neurons was assessed using the method described previously (Planken et al , 2010; Saarenpää et al , 2017). Dopamine neurons were labelled with an antibody against the key enzyme of dopamine synthesis, tyrosine hydroxylase (TH) (1:500, Millipore Cat# MAB318, RRID:AB_2201528), on 5 th day in vitro (5 th DIV) followed by secondary antibody conjugated with fluorophore.…”

Binding of EphrinA5 to RET receptor tyrosine kinase: An in vitro study

“…Especially for efn-As, they have an overall sequence similarity as high as 80% among their orthologs ( S1 Fig ). Our recent study found that zebrafish GDNF/GFRα1 could activate human RET, revealing the structural and functional conservation of RET from an evolutionary perspective [ 24 ]. Because mammalian RETs are prone to misfold [ 25 , 26 ] and thus difficult to produce, we wanted to determine if the binding of RET to efn-A5 is conserved in lower vertebrates, for example zebrafish, and characterize their binding behavior to elucidate the underlying molecular mechanism of the reverse signaling of efn-A5.…”