RET signaling does not modulate MPTP toxicity but is required for regeneration of dopaminergic axon terminals

Abstract

Activation of the RET (rearranged during transfection) receptor by glial cell-line-derived neurotrophic factor (GDNF) has been identified as an important differentiation and survival factor for dopaminergic neurons of the midbrain in preclinical experiments. These encouraging results have led to clinical trials of GDNF in patients with Parkinson's disease, which have resulted in conflicting findings. To investigate the potential benefit of Ret-dependent signaling on the challenged dopaminergic system, we tested the effect of tissue-selective ablation of the Ret gene on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity in mice, the most widely used animal model for Parkinson's disease. Ablation of Ret did not modify the MPTP-induced loss of dopaminergic neurons in the substantia nigra pars compacta and the dopaminergic innervation of the striatum at 14 days. However, Ret ablation abolished the regeneration of dopaminergic fibers and terminals, as well as the partial recovery of striatal dopamine concentrations, that was observed in control mice between days 14 and 90 after MPTP treatment. We therefore conclude that RET signaling has no influence on the survival of dopaminergic neurons in the MPTP model of Parkinson's disease but rather facilitates the regeneration of dopaminergic axon terminals.

Fig. 1.

MPTP-induced loss of TH-positive SNpc neurons. (A–C) In saline-treated control mice, there was no difference in the number of TH-positive SNpc neurons among the genotypes DAT-Cre (A), Ret^lx (B), and DAT-Ret^lx/lx (C). (D–I) In addition, MPTP-induced depletion of TH-positive SNpc neurons did not differ among the genotypes DAT-Cre (D and G), Ret^lx (E and H), and DAT-Ret^lx/lx (F and I) at 14 days (D–F) and 90 days (G–I). Furthermore, there was no difference between 14 and 90 days for each of the three genotypes.

Fig. 2.

MPTP-induced loss of TH-positive striatal fibers. In saline-treated control mice, there was no difference in the number of striatal TH-positive fibers among the genotypes DAT-Cre (A and A′), Ret^lx (B and B′), and DAT-Ret^lx/lx (C and C′). In addition, MPTP-induced depletion of TH-positive striatal fibers did not differ among the genotypes DAT-Cre (D and D′), Ret^lx (E and E′), and DAT-Ret^lx/lx (F and F′) at 14 days. Striatal fibers showed regeneration between 14 and 90 days in DAT-Cre (G and G′) and Ret^lx (H and H′) mice but not in DAT-Ret^lx/lx mice (I and I′). (A–I) The entire frontal sections through the striatum stained for TH by using DAB. (A′–I′) Representative higher-resolution images used for the quantification of striatal fibers. (J and K) TH was stained by immunofluorescence. (Scale bar, 25 μm.) The bar graphs summarize the data for the dorsal (J) and ventral (K) striatum from three to four mice per group. Numbers are means ± SD; ANOVA followed by Tukey's post hoc test. n.s., nonsignificant.

Fig. 3.

DAT fiber density in the striatum. (A–C) At 90 days, there was no difference in the amount of striatal DAT-positive fibers for saline-treated DAT-Cre (A), Ret^lx (B), and DAT-Ret^lx/lx (C) mice. (D–F) At 90 days after MPTP treatment, DAT staining was reduced in DAT-Ret^lx/lx mice (F) but partially recovered in DAT-Cre (D) and Ret^lx mice (E). (Scale bar, 25 μm.) (G and H) The bar graphs summarize the data for the dorsal (G) and ventral (H) striatum from three to four mice per group at 90 days after vehicle or MPTP treatment. Numbers are means ± SD. ***, P < 0.0001 compared with MPTP-treated DAT-Cre or Ret^lx mice; ANOVA followed by Tukey's post hoc test.

Fig. 4.

Striatal catecholamine concentrations. MPTP reduces striatal dopamine (A), DOPAC (B), and HVA (C) concentrations, which partially recover between 14 and 90 days after MPTP treatment in DAT-Cre and Ret^lx but not in DAT-Ret^lx/lx mice. Numbers are means ± SD. For dopamine, DOPAC, and HVA, there were no significant differences among genotypes at 14 days after MPTP treatment. At 90 days, dopamine, DOPAC, and HVA from DAT-Ret^lx/lx mice were different from Ret^lx mice (P < 0.01) but not from DAT-Cre mice (two-way ANOVA followed by Tukey's post hoc test).

Fig. 5.

MPTP-induced gliosis. (A–C) Immunohistochemical staining for GFAP in the striatum in DAT-Cre (A), Ret^lx (B), and DAT-Ret^lx/lx (C) mice. (D–F) There is a robust increase in GFAP-positive reactive astrocytes at 14 days after MPTP treatment, with no difference among genotypes. (G–I) At 90 days, the number of GFAP-positive astrocytes is back to baseline levels. (Scale bar, 100 μm.)