Glial cell line-derived neurotrophic factor receptor Rearranged during transfection agonist supports dopamine neurons in Vitro and enhances dopamine release In Vivo.

Arun Kumar Mahato,Andrii Domanskyi,Juho‐Matti Renko,Tanel Visnapuu,Jaakko Kopra,Ilari Korhonen,Nita Pulkkinen,Mati Karelson,Raimo K Tuominen,Merja H Voutilainen,Maxim M Bespalov,Mart Saarma,Eric Ronken,Yulia A Sidorova,T Petteri Piepponen

doi:10.1002/mds.27943

Abstract

BackgroundMotor symptoms of Parkinson's disease (PD) are caused by degeneration and progressive loss of nigrostriatal dopamine neurons. Currently, no cure for this disease is available. Existing drugs alleviate PD symptoms but fail to halt neurodegeneration. Glial cell line–derived neurotrophic factor (GDNF) is able to protect and repair dopamine neurons in vitro and in animal models of PD, but the clinical use of GDNF is complicated by its pharmacokinetic properties. The present study aimed to evaluate the neuronal effects of a blood‐brain‐barrier penetrating small molecule GDNF receptor Rearranged in Transfection agonist, BT13, in the dopamine system.MethodsWe characterized the ability of BT13 to activate RET in immortalized cells, to support the survival of cultured dopamine neurons, to protect cultured dopamine neurons against neurotoxin‐induced cell death, to activate intracellular signaling pathways both in vitro and in vivo , and to regulate dopamine release in the mouse striatum as well as BT13's distribution in the brain.ResultsBT13 potently activates RET and downstream signaling cascades such as Extracellular Signal Regulated Kinase and AKT in immortalized cells. It supports the survival of cultured dopamine neurons from wild‐type but not from RET‐knockout mice. BT13 protects cultured dopamine neurons from 6‐Hydroxydopamine (6‐OHDA) and 1‐methyl‐4‐phenylpyridinium (MPP+)–induced cell death only if they express RET. In addition, BT13 is absorbed in the brain, activates intracellular signaling cascades in dopamine neurons both in vitro and in vivo, and also stimulates the release of dopamine in the mouse striatum.ConclusionThe GDNF receptor RET agonist BT13 demonstrates the potential for further development of novel disease‐modifying treatments against PD. © 2019 The Authors. Movement Disorders published by Wiley Periodicals LLC. on behalf of International Parkinson and Movement Disorder Society.

Highlights

BT13 had similar efficacy to GDNF family ligands (GFLs) in REarranged during Transfection (RET), ERK, and AKT phosphorylation assays (Fig. 1A–I); in integral luciferase assay the effect of BT13 was much more modest compared with Glial cell line–derived neurotrophic factor (GDNF) or soluble GDNF/GFRα1 complex (Fig. 1J,K28,30)
This can be partly explained by the quick metabolism of BT13.28 The efficacy of BT13 (100 nM and 1 μM) was comparable with that of GDNF in its ability to support the survival of cultured dopamine neurons (Fig. 2), to protect them from 6-OHDA (Fig. 3A) and MPP+ -induced cell death (Fig. 3B), and to increase phosphorylated ERK (pERK) and Protein S6 (pS6) levels both in vitro (Fig. 4) and in vivo (Fig. 5)
The potency of BT13 was 2 to 3 orders of magnitude lower than that of GDNF, that is, GDNF supported the survival of cultured dopamine neurons at approximately the 0.3 nM concentration

Summary

Results

BT13 Stimulates RET Phosphorylation and Downstream Intracellular Signaling in Immortalized Cells. In the cells expressing GFP/RET, an increased level of pRET was observed in response to 50 μM (1.7-fold increase, P < 0.0001) and 100 μM (2.1-fold increase, P < 0.0001, 1-way ANOVA with Dunnett’s post hoc test for all comparisons) BT13 (Fig. 1A-F). BT13 increased the number of TH-positive cells in wild-type embryonic midbrain culture by 1.4 fold (0.1 μM, P < 0.0001) and 1.5 fold (1 μM, P = 0.0002) and GDNF (10 ng/ml) by 1.8 fold (P = 0.0207, Repeated Measures (RM) ANOVA with Dunnett’s post hoc test for all comparisons; Fig. 2A,B). In the MPP+ toxin model, BT13 (1 μM) increased the number of TH-positive neurons by 1.3 fold (P = 0.0068) and GDNF (10 ng/ml) by 1.2 fold (P = 0.0389, 1-way ANOVA with Dunnett’s post hoc test for all comparisons). An analysis of dopamine and dopamine metabolite content in the midbrain revealed a statistically significant increase in the concentration of HVA and a trend toward an increase in dopamine and DOPAC concentrations in rats receiving intravenous BT13 (Fig. S3)

Materials and Methods

Discussion