Ret is essential to mediate GDNF's neuroprotective and neuroregenerative effect in a Parkinson disease mouse model

Abstract

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF's neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF's effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD.

Figure 1

Tools and paradigm to address Ret function in mediating GDNF neuroprotective and regenerative effect in the MPTP mouse model of PD. (a–f) Coronal striatum sections are shown for the three mouse genotypes (DAT-Cre, Ret^lx/lx and DAT-Ret^lx/lx) which were transduced with an adeno-associated virus of the serotype 5 encoding EGFP under the GFAP promoter to target astrocytes (AAV5-EGFP). (a–c) EGFP expression in the striatum of the left brain hemisphere at low resolution (scale bar, 500 μm). (d–f) EGFP expression in green co-localizes in astrocytes immunohistochemically stained in red with antibodies against GFAP (scale bar, 20 μm). (g) Schema shows absolute values of GDNF measured by ELISA in pg/mg tissue (red numbers) and fold of GDNF overexpression (blue numbers) of AAV5-GDNF injected mice in comparison to AAV5-EGFP injected wild-type animals with 40 pg GDNF/mg tissue in the striatum and 18 pg GDNF/mg tissue in the nigra. Only 1.3% of the GDNF expressed in the infected striatum is retrograde transported into the ipsilateral substantia nigra (green number). (h) Schema illustrating the experimental paradigm used

Figure 2

Stereological quantification of nigral dopaminergic neurons. Numbers of TH and Nissl-positive nigral neurons are shown for the three genotypes (Dat-Cre, Ret^lx/lx and DAT-Ret^lx/lx) 2 weeks post MPTP treatment (a) and 3 months after MPTP treatment (b) and control mice which instead received saline (NaCl) (a and b). For each mouse the numbers on the side with mock injection (inj), AAV5-EGFP injection (EGFP) or AAV5-GDNF (GDNF) and on the non-injected contralateral side (con) are shown (n=6 animals per group)

Figure 3

Quantification of striatal dopaminergic fiber density. TH immunoreactivity of striatal tissue sections is shown in a for DAT-Cre mice (upper panel), Ret^lx/lx mice (middle panel) and DAT-Ret^lx/lx mice (lower panel) at 2 weeks after MPTP treatment (first two columns) and 3 months after MPTP treatment (last two columns), for the injected hemispheres (inj., columns 1 and 3) and the contralateral hemispheres (con, columns 2 and 4). The quantification of striatal TH fiber density is presented in (b) for 2 weeks, and in c for 3 months after MPTP application. Scale bar, 40 μm in (a) (n=6 animals per group)

Figure 4

Quantification of striatal dopamine levels. Striatal dopamine levels assessed by HPLC are shown for control animals (NaCl), for animal receiving MPTP and AAV5-EGFP (MPTP+EGFP), and for animals receiving MPTP and AAV5-GDNF (MPTP+GDNF) for the vector injected (inj) and the contralateral hemisphere (con). Data are shown from the three genotypes (Dat-Cre, Ret^lx/lx mice and DAT-Ret^lx/lx) at 2 weeks (a) and at 3 months post MPTP treatment (b) (n=6 animals per group)