Article Figures & Data
Figures
- Figure 1.
The effect of CDNF and GDNF alone or their coadministration on amphetamine-induced rotation. A, Experimental design. Rats were administered 6-OHDA (20 µg) unilaterally in the striatum. Four weeks later, the rats were given a unilateral intrastriatal injection of CDNF (1 µg), GDNF (1 µg), CDNF (2.5 µg), GDNF (2.5 µg), CDNF (5 µg), or GDNF (5 µg) alone or combination of them. The rotational behavior was measured 3, 6, 8, 10, and 12 weeks after lesion. B, Amphetamine-induced ipsilateral rotations at 12 weeks after lesion in rats treated with vehicle, CDNF (2.5 µg), GDNF (1 µg), or combinations of GDNF (1 µg) with either CDNF (1 µg), CDNF (2.5 µg), or CDNF (5 µg). C, Amphetamine-induced ipsilateral rotations at 12 weeks after lesion in rats treated with vehicle, CDNF (1 µg), alone, or in combination with GDNF (1 µg), GDNF (2.5 µg), or GDNF (5 µg). D, Amphetamine-induced ipsilateral rotations at 12 weeks after lesion in rats treated with vehicle, CDNF (2.5 µg) and GDNF (2.5 µg) alone or in combination; CDNF (5 µg) and GDNF (5 µg) alone or in combination. Means ± SEM are shown; n = 8–10 in each group. p < 0.05 Tukey/Kramer post hoc analysis after one-way ANOVA. *p < 0.05, **p < 0.01.
- Figure 2.
Number of TH-positive and Nissl-positive cells in the SNpc and OD of TH-positive fibers in the STR. A, The effect of CDNF (2.5 μg) alone and GDNF (1 μg) alone, or the combination of CDNF (2.5 μg) + GDNF (1 μg) on TH-positive cell bodies in the SNpc analyzed at 12 weeks after lesion. The effect of the combination of CDNF (2.5 μg) and GDNF (1 μg) differed significantly from the vehicle-treated group (p < 0.05). B, Analyses of Nissl-stained cell bodies in SNpc. Results were in line with TH-positive cell body counts from SNpc. C, The effect of CDNF (2.5 μg) alone, GDNF (1 μg) alone, or the combination of CDNF (2.5 μg) and GDNF (1 μg) on TH-positive fibers in the STR. CDNF and the combination of CDNF and GDNF partly rescue TH-positive fibers in the STR. D, Representative TH-stained striatal sections for each treatment. Mean ± SEM in (A, B) n = 7-12 and in (C) n = 7-12 in each group. Tukey/Kramer post hoc analysis after one-way ANOVA. *p < 0.05, ****p < 0.0001.
- Figure 3.
Different effects of CDNF and GDNF on ERK1/2 and PI3K/AKT pathways in naïve rats. Ability of CDNF and GDNF to activate MAPK or PI3K/AKT/mTOR pathways was studied injecting CDNF (2.5 µg), GDNF (1 µg), or their combination into the left STR (LSTR) of naïve rats (A–D). Sham-operated rats received PBS into LSTR. A, B, In rats dissected 1 h after NTF injection, GDNF activated ERK1/2 and PI3K/AKT pathway whereas CDNF had no effect. D, In rats dissected 4 h after CDNF injection the PI3K/AKT pathway was activated in nonlesioned brains (D), whereas the ERK1/2 pathway was not affected (C). Representative Western blot images of ERK1/2, p-ERK1/2, PI3K/AKT, and p-PI3K/AKT are shown. Mean ± SEM, n = 3-6 in each group. Tukey/Kramer post hoc analysis after one-way ANOVA.
- Figure 4.
Differences in activation of ERK1/2 and PI3K/AKT pathways after intrastriatal injection of CDNF and GDNF or their combination in 6-OHDA-lesioned rats. When the rats were dissected 4 h after the NTF injection, the MAPK or PI3K/AKT pathway was not activated in 6-OHDA-lesioned rats. CDNF (2.5 µg) did not have significant effect on MAPK (A) or PI3K/AKT/mTOR (B) pathways. CDNF (2.5 µg) increased ribosomal protein S6 phosphorylation in the STR in comparison to rats treated with GDNF (1 µg) (C). The number of pErk-positive cells in STR was increased by all treatments (D), while there was no effect on OD of pAkt-positive fibers in STR (E). Tukey/Kramer post hoc analysis after one-way ANOVA. *p < 0.05, **p < 0.01.
- Figure 5.
Effects of CDNF and GDNF and their combination on ER stress-triggered UPR markers in cultured DA neurons and in rat 6-OHDA model in vivo. A–C, E13 dopamine neurons were cultured 5-7 d with GDNF (50 ng/ml). Then, the cultures were treated with thapsigargin (200 nM) to induce ER stress. CDNF (100 ng/ml), GDNF (50 ng/ml), or their combination was added to the cultures at the same time. The expression levels were normalized to the levels of β-actin in the same samples. CDNF and the combination reduced expression of ATF6 mRNA (A). Levels of Xbp1-sp (B) and GPR-78 (C) were not changed. D--F, Effect of CDNF and GDNF on ER stress markers in the rat 6-OHDA model. D, Experimental design for ER stress markers analyses. Rats were administered 6-OHDA (20 µg) unilaterally into the left striatum (L). Four weeks later, the rats received a single injection of either vehicle (PBS), CDNF (2.5 µg), GDNF (1 µg), or their combination into the lesioned striatum. Rats were killed 4 h after NTF injection, and striata were dissected for Western blot analyses. E, Injection of CDNF decreases GRP78 protein levels in striata of 6-OHDA-lesioned rats compared with PBS (p = 0.2) or GDNF treatment (p < 0.05). The level of expression is expressed as % of intact contralateral site of the brain. Representative Western blot images of GRP78 expression. Control shows basal level of GRP78 in a naïve rat brain. β-tubulin is used as loading control. F, CDNF-treated animals showed a decrease in the activation of P-eIF2α compared with the vehicle-treated rats. Representative Western blot images of P-eIF2α and total eIF2α. Control shows basal level of P-eIF2α and total eIF2α in a naïve rat brain. Mean ± SEM. n = 5-6 in each group. Tukey/Kramer post hoc analysis after one-way ANOVA. **p < 0.01.
- Figure 6.
Effect of CDNF, GDNF, and their combination on TH- and DAT protein levels in 6-OHDA-lesioned and sham-lesioned brain. Four weeks after lesioning, sham-lesioned or 6-OHDA-lesioned rats received an intrastriatal injection of vehicle (VEH), CDNF, GDNF, or a combination of CDNF and GDNF. Eight weeks after lesioning, rat SN and striatum (STR) protein expression were analyzed from total protein samples (13 µg for SN samples and 15 µg for STR samples, respectively) using Western blotting. 6-OHDA lesion resulted in a marked decrease in both TH and DAT protein levels in the SN (A, B) and STR (C, D). None of the treatments were able to significantly affect the TH and DAT protein levels after sham or 6-OHDA lesion, although GDNF alone or in combination with CDNF tended to decrease the levels of TH in sham-lesioned STR (C). Results are shown as the group mean ± SEM of protein band density relative to the density of a control sample (pooled from four naive STR or SN) and β-actin. n = 3-5 in each group. Tukey/Kramer post hoc analysis after one-way ANOVA. TH-levels in SN (A) and striatum (C) and DAT-levels in SN (B) and STR (D) did not show statistically significant changes.
Tables
Symbol in text Dataset Data structure Type of test p value a Fig. 1B, ipsilateral rotations at 12 weeks: GDNF (1 µg) vs PBS at 12 weeks Normal distribution One-way ANOVA, Tukey 0.006 b Fig. 1B, ipsilateral rotations at 12 weeks: GDNF (1 µg) + CDNF (1 µg) vs PBS at 12 weeks Normal distribution One-way ANOVA, Tukey 0.0306 c Fig. 1B, ipsilateral rotations at 12 weeks, GDNF (1 µg) + CDNF (2.5 µg) vs PBS at 12 weeks Normal distribution One-way ANOVA, Tukey 0.001 d Fig. 1B, ipsilateral rotations at 10 weeks: CDNF (2.5 µg) vs PBS at 12 weeks Normal distribution One-way ANOVA, Tukey 0.029 e Fig. 1D, ipsilateral rotations at 12 weeks, GDNF (2.5 µg) vs PBS at 12 weeks after lesion Normal distribution One-way ANOVA, Tukey 0.0104 f Fig. 1D, ipsilateral rotations at 12 weeks, GDNF (2.5 µg) + CDNF (2.5 µg) vs PBS at 12 weeks after lesion Normal distribution One-way ANOVA, Tukey 0.0113 g Fig. 2A, TH cell numbers, GDNF (1 µg) + GDNF (2.5 µg) vs VEH Normal distribution One-way ANOVA, Tukey 0.046 h Fig. 2B, Nissl-positive cells, All NTF-treated groups differed from VEH Normal distribution One-way ANOVA, Tukey 0.0001 i Fig. 4C, OD of pS6 staining in STR. CDNF (2.5 µg) vs GDNF (1 µg) Normal distribution One-way ANOVA, Tukey 0.0184 j Fig. 4D, pERK-positive cells in STR. All NTF-treated groups differed from VEH Normal distribution One-way ANOVA, Tukey 0.0077 k Fig. 5A, level of ATF6 expression in DA neuron cultures, CDNF (100 ng/ml) vs control, no factor Normal distribution One-way ANOVA, Tukey 0.04 l Fig. 5A, level of ATF6 expression in DA neuron cultures, combination [CDNF (100 ng/ml) + GDNF (50 ng/ml)] vs control Normal distribution One-way ANOVA, Tukey 0.0102 m Fig. 5E, GRP78 expression, CDNF (2.5 µg) vs GDNF (1 µg) Normal distribution One-way ANOVA, Tukey 0.014
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