CDNF protects the nigrostriatal dopamine system and promotes recovery after MPTP treatment in mice

Abstract

Cerebral dopamine neurotrophic factor (CDNF) is a recently discovered protein, which belongs to the evolutionarily conserved CDNF/MANF family of neurotrophic factors. The degeneration of dopamine neurons following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment is well characterized, and efficacy in this model is considered a standard criterion for development of parkinsonian therapies. MPTP is a neurotoxin, which produces parkinsonian symptoms in humans and in C57/Bl6 mice. To date, there are no reports about the effects of CDNF on dopamine neuron survival or function in the MPTP rodent model, a critical gap. Therefore, we studied whether CDNF has neuroprotective and neurorestorative properties for the nigrostriatal dopamine system after MPTP injections in C57/Bl6 mice. We found that bilateral striatal CDNF injections, given 20 h before MPTP, improved horizontal and vertical motor behavior. CDNF pretreatment increased tyrosine hydroxylase (TH) immunoreactivity in the striatum and in the substantia nigra pars reticulata (SNpr), as well as the number of TH-positive cells in substantia nigra pars compacta (SNpc). Posttreatment with CDNF, given 1 week after MPTP injections, increased horizontal and vertical motor behavior of mice, as well as dopamine fiber densities in the striatum and the number of TH-positive cells in SNpc. CDNF did not alter any of the analyzed dopaminergic biomarkers or locomotor behavior in MPTP-untreated animals. We conclude that striatal CDNF administration is both neuroprotective and neurorestorative for the TH-positive cells in the nigrostriatal dopamine system in the MPTP model, which supports the development of CDNF-based treatment for Parkinson's disease.

Figure 1. Striatal cerebral dopamine neurotrophic factor (CDNF) injections protect midbrain dopamine circuitry against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Mice were balanced into two groups according to their 24-h locomotor behavior (blue columns in A and B left panel). Bilateral CDNF 10 μg or PBS injections were given into the striatum 20 h prior to four MPTP injections. White columns and square symbols are PBS + MPTP; black columns and square symbols are CDNF + MPTP. Locomotor behavior and tyrosine hydroxylase (TH) immunoreactivity were measured 2 weeks later. (A) Distance traveled (cm). (B) Vertical activity after MPTP treatment. (C–E) MPTP and PBS white columns and MPTP and CDNF black columns. (C) TH optical density in the striatum. (D) TH optical density in the substantia nigra pars reticulata (SNpr). (E) TH-positive cells in the substantia nigra pars compacta (SNpc). Representative photomicrographs from striatum from normal control (naive), PBS-, and CDNF-treated mice. (A–C) n = 6–9; (D, E) n = 5–8. *Significant difference ( p < 0.05) between PBS- and CDNF-treated mice 2 weeks after MPTP treatment, in (A) and (B) with Bonferroni post hoc test and in (C–E) with Student’s t-test.

Figure 2. NeuN fluorescence intensity after MPTP treatment in CDNF and PBS injected mice

Bilateral CDNF 10 μg or PBS injections were given into the striatum 20 h prior to four MPTP injections. Mice were perfused and the brains were fixed with 4% paraformaldehyde (PFA) 2 weeks later and NeuN (neuronal nuclei) and tyrosine hydroxylase (TH) immunoreactivity were carried. Analysis of NeuN fluorescence intensity in SNpc showed no differences between PBS + MPTP-treated mice and CDNF + MPTP-treated mice. The data are shown in relation to nonlesioned naive mice. Scale bar: 0.5 mm. In the bar graph n = 4 per group.

Figure 3. Effects of striatal CDNF injections in MPTP-naive animals

Mice were balanced according to their behavior and placed into two groups (A–E). MPTP-naive mice were injected bilaterally into striatum with either PBS or CDNF 10 μg. Two weeks later striata were collected and TH and dopamine transporter (DAT) levels were analyzed by Western blotting. (A, D) TH levels in the striatum in normal naive, PBS-, and CDNF-treated mice. (D) Lane 1: Cerebral cortex negative control, lane 2: naive, lane 3: PBS, lane 4: CDNF. TH (58.5 kDa) shown in red and actin (42 kDa) in green. (B) TH levels in substantia nigra (SN). (C, E) DAT levels in striatum. (E) Lane 1: Cerebral cortex negative control, lane 2: naive, lane 3: PBS, lane 4: CDNF. DAT (68.5 kDa) shown in red and actin in green. Rats were given unilateral intrastriatal injections of vehicle, CDNF (10 μg), or glial cell line-derived neurotrophic factor (GDNF; 10 μg). (F) Two weeks later animals were given d-amphetamine (2.5 mg/kg, SC) and locomotor activity was measured for 2 h. Activity is shown as related to the control group. (G) Number of TH-positive cells in substantia nigra pars compacta shown in relation to the contralateral untreated side. (H) TH optical density in substantia nigra pars reticulata shown as normalized to the control group.

Figure 4. Striatal CDNF injections restore midbrain dopamine circuitry in the MPTP toxicity model

In the neurorestoration experiment, mice were balanced into two groups (PBS and CDNF 10 μg) according to their 24-h behavior 1 week after MPTP injections. Bilateral PBS and CDNF injections were given into striatum and locomotor behavior and TH immunoreactivity were measured 1 week later. (A) Distance traveled. (B) Vertical activity. (C) TH optical density in the striatum. (D) TH optical density in the SNpr. (E) TH-positive cells in the SNpc. (A, B, E) n = 13–16, (C, D) n = 4. White bars and symbols represent PBS and black bars and symbols CDNF. The upper photomicrograph in (C) is from the striatum of a CDNF-treated mouse and lower photomicrograph from a PBS-treated mouse.