Failure of Diphtheria Toxin Model to Induce Parkinson-Like Behavior in Mice

Abstract

Rodent models of Parkinson's disease are based on transgenic expression of mutant synuclein, deletion of PD genes, injections of MPTP or rotenone, or seeding of synuclein fibrils. The models show histopathologic features of PD such as Lewi bodies but mostly only subtle in vivo manifestations or systemic toxicity. The models only partly mimic a predominant loss of dopaminergic neurons in the substantia nigra. We therefore generated mice that express the transgenic diphtheria toxin receptor (DTR) specifically in DA neurons by crossing DAT-Cre mice with Rosa26 loxP-STOP-loxP DTR mice. After defining a well-tolerated DTx dose, DAT-DTR and DTR-flfl controls were subjected to non-toxic DTx treatment (5 × 100 pg/g) and subsequent histology and behavioral tests. DAT protein levels were reduced in the midbrain, and tyrosine hydroxylase-positive neurons were reduced in the substantia nigra, whereas the pan-neuronal marker NeuN was not affected. Despite the promising histologic results, there was no difference in motor function tests or open field behavior. These are tests in which double mutant Pink1^-/-SNCA^A53T Parkinson mice show behavioral abnormalities. Higher doses of DTx were toxic in both groups. The data suggest that DTx treatment in mice with Cre/loxP-driven DAT-DTR expression leads to partial ablation of DA-neurons but without PD-reminiscent behavioral correlates.

Keywords: Cre-recombinase; SLC6a3; diphtheria toxin; dopamine transporter; motor functions; open field behavior; tyrosine hydroxylase.

Figure 1

Efficacy of DTx treatment of DAT-DTR mice versus control mice. (A) Exemplary agarose gel showing a typical genotyping result. (B) Survival curve of mice injected with 100 ng/mouse per dose (≈4 ng/g) DTx. Details of mice and sample sized per dose in Supplementary Table S1. (C) Body weight at baseline and 30 d after treatment with low non-toxic dose of 100 pg/g/d DTx for five consecutive days (cumulative dose 0.5 ng/g). All mice survived. Each scatter is a mouse (n = 12, 8 and 10). (D) Quantitative RT-PCR of DAT/Slc6a3 RNA in the midbrain after 5 × 0.1 ng/g DTx treatment (triplicate samples of n = 3–4 mice). (E,F) Western blot analysis and quantification of DAT/Slc6a3 protein in the midbrain after 5 × 0.1 ng/g DTx (example shows n = 2 per group). For C–F, mice were 11–13 weeks at the onset of DTX, and tissue was obtained at 30 d.

Figure 2

Immunofluorescence analysis of tyrosine hydroxylase (TH) as a marker for DA neurons in the substantia nigra (SN). The pan-neuronal marker NeuN was used to label all neurons. DAPI was used as counterstain of nuclei. Mice were treated with 5 × 0.1 ng/g DTx. Mice were 9–13 weeks old at the onset of DTx and were sacrificed 6–7 weeks after the last DTx dose. The image shows examples of n = 4–6 mice per group (one side or both sides). Scale bar 200 µm.

Figure 3

Quantification and statistical comparison of TH and NeuN immunofluorescence. (A,B) Box/scatter plot and Paired data analysis of the TH and NeuN immunopositive areas determined with the Particle Counter in FIJI ImageJ. The scatter represents images of the left and right SN of 4–6 mice per group. Data of TH and NeuN were compared by ANOVA and subsequent unpaired, two-tailed t-test for TH and NeuN separately. The asterisk shows a significant result with p < 0.05. (C,D) Estimation plots for TH and NeuN showing the group difference obtained via t-tests.

Figure 4

Behavior in DTx-treated mice. (A–C) Travel paths and the times spent in the center compartment and the border compartment in a classical open field test (OFT). The behavior was tested at baseline (0d) and 30d after DTx treatment with 0.1 ng/g/d for 5 consecutive days. Exploration of the center dropped in all mice in the 30 d retest without difference between groups. Each scatter is a mouse, sample size n = 4–6. (D–F) Motor coordination analysis in the pole test and accelerating Rotarod test and body weights at baseline and 30 d after 5 × 0.1 ng/g DTx. Mice were 13–16 weeks old at the onset of DTx. Sample size n = 4–6 per group.

Figure 5

Paired analysis of test-retest behavior in the open field test. (A–C) Paired analysis of test-retest OFT behavior of individual mice was used to assess learning. Data were compared with 2-way ANOVA and revealed a significant effect of time (i.e., test versus retest) but not of the between-subject factor group. Sample sizes n = 4–6 per group.

Figure 6

Behavior in genetic Parkinson Pink1^−/−SNCA^A53T double mutant mice. (A) Running time on an accelerating Rotarod. (B–D) Travel paths and times spent in the center and border compartment in the open field test (OFT). Pink1^−/−SNCA^A53T were hyperactively running along the walls. Mice were 12–15 months of age. Each scatter is a mouse; n = 18 per group. Data were compared with t-tests; asterisks indicate significant differences between genotypes.