Cerebral Dopamine Neurotrophic Factor Diffuses Around the Brainstem and Does Not Undergo Anterograde Transport After Injection to the Substantia Nigra

Abstract

Cerebral dopamine neurotrophic factor (CDNF) has shown therapeutic potential in rodent and non-human primate models of Parkinson's disease by protecting the dopamine neurons from degeneration and even restoring their phenotype and function. Previously, neurorestorative efficacy of CDNF in the 6-hydroxydopamine (6-OHDA) model of Parkinson's disease as well as diffusion of the protein in the striatum (STR) has been demonstrated and studied. Here, experiments were performed to characterize the diffusion and transport of supra-nigral CDNF in non-lesioned rats. We injected recombinant human CDNF to the substantia nigra (SN) of naïve male Wistar rats and analyzed the brains 2, 6, and 24 h after injections. We performed immunohistochemical stainings using an antibody specific to human CDNF and radioactivity measurements after injecting iodinated CDNF. Unlike the previously reported striatonigral retrograde transport seen after striatal injection, active anterograde transport of CDNF to the STR could not be detected after nigral injection. There was, however, clear diffusion of CDNF to the brain areas surrounding the SN, and CDNF colocalized with tyrosine hydroxylase (TH)-positive neurons. Overall, our results provide insight on how CDNF injected to the SN may act in this region of the brain.

Keywords: Parkinson’s disease; cerebral dopamine neurotrophic factor; diffusion; striatum; substantia nigra; transport.

FIGURE 1

Diffusion of ¹²⁵I CDNF when injected to the STR or SN of naive rats. (A,B) Percentage of the total radioactivity (calculated based on radioactivity of all brain areas measured) after injecting radiolabeled CDNF (¹²⁵I-CDNF) into the STR of naive rats and measuring 24 h later, ipsilateral regions (A) and contralateral (B). (C,D) Percentage of the total radioactivity (calculated based on radioactivity of all brain areas measured) after injecting radiolabeled CDNF into the SN of naive rats and measuring 24 h later, ipsilateral regions (C) and contralateral (D). (E,F) 2000× or 10 000× molar excess of cold (unlabeled) CDNF was given together with radiolabeled CDNF to SN of naive rats and measured 24h later. Ipsilateral regions (E) and contralateral (F). (n = 5–6/brain region) Bars represent mean ± SEM. ^∗∗∗∗p = < 0.0001. FCTX, frontal cortex; CTX, cortex; STR, striatum; HC, hippocampus; SN, substantia nigra; GP, globus pallidus; STN, subthalamic nucleus.

FIGURE 2

Diffusion of human CDNF after injection to the SN in naive rat brain at different time points and volumes. CDNF (3 μg in 4 μl) was injected to the SN of rats and perfused (A) 2, (B) 6, or (C) 24 h later. Human CDNF (hCDNF) immunostaining of a representative posterior, nigral, thalamic, and striatal section for each time point. N = 2/time point. Diffusion of human CDNF (3 μg) when injected into the SN in the volume of 1 or 4 μl. Rats were perfused 2 h after the injection. (D) Increasing magnification of CDNF injected side versus the uninjected side of the injected area. (E) 40× maginification of injected area. Black arrowheads indicate dark brown puncta staining of CDNF.

FIGURE 3

Representative confocal images of CDNF-injected rat substantia nigra with tyrosine hydroxylase (TH) neurons or parvalbumin (PV) neurons to demonstrate colocalization. (A) Representative confocal images from a rat brain injected with human CDNF into the substantia nigra (SN) and perfused 2 h later with costaining for CDNF and TH. Total of six rats were injected and stained for the colocalization study. White arrows in the injected side indicate CDNF immunoreactive puncta (cyan dots). Uninjected side representative image taken with identical settings to account for possible background. Top panel 10× objective, lower panel 63× objective. (B) Representative confocal images from rats injected with human CDNF into the SN and perfused 2 h later with costaining for CDNF and PV. Uninjected side representive image taken with identical settings to account for possible background. Top panels 10× objective, lower panels 63× objective. N = 6.