Article Figures & Data
Figures
- Figure 1.
Stability of exogenous CDNF protein in rat brain. A, Sections from rhCDNF-infused (20 µg) hemispheres were double stained with anti-CDNF and anti-NeuN antibodies, and each time point is represented by sections from two different animals. The time between rhCDNF infusion and perfusion of the animal is indicated below the images. A high level of anti-CDNF IR was detectable at positions up to 2 mm caudally from the infusion site, but there was very little spreading of rhCDNF to the contralateral uninfused hemisphere (data not shown). Scale bar, 2 mm. B, Intactness of rhCDNF protein at 6 h after its infusion into rat brain. Lysates of rat brain at 15 min and 6 h after the infusion of rhCDNF were analyzed by immunoblotting with anti-CDNF antibody. Two animals were analyzed for each time point. Double staining with anti-α-tubulin antibody was used to ensure equal loading. Fifty nanograms of purified rhCDNF protein served as a positive control. CL, Contralateral uninfused hemisphere.
- Figure 2.
Subcellular localization patterns of infused rhCDNF in rat brain. Representative images of anti-CDNF IR at the 2 h time point, as observed by confocal fluorescence microscopy (n = 4). A–E, Sections from the rhCDNF-infused (20 µg) or contralateral uninfused hemisphere were double stained with anti-CDNF antibodies (A, B, D) and anti-NeuN antibodies (C, E). F, G, Sections from rhCDNF-infused hemisphere were double stained with anti-CDNF antibodies (F) and anti-parvalbumin antibodies (G). White arrows mark NeuN-positive cells that exhibit only the punctate CDNF-IR pattern, arrowheads mark cells that also exhibit diffuse anti-CDNF IR in the cytoplasm, the asterisk marks a neuron with strong cytoplasmic anti-CDNF IR, and a non-neuronal cell is marked with letter “x.” DAPI was used to stain nuclei (shown in blue). Scale bar, 10 µm.
- Figure 3.
Subcellular patterns of anti-GFP immunoreactivity in rat brain 2 h after the infusion of recombinant GFP, as observed by confocal fluorescence microscopy. A–C, Sections from the GFP-infused or contralateral uninfused hemisphere were double stained with anti-GFP antibodies (A, C) and anti-NeuN antibodies (B). Arrows mark NeuN-positive cells with punctate anti-GFP IR pattern, and the arrowhead marks a cell that exhibits strong peripheral anti-GFP IR and diffuse anti-GFP IR in the cytoplasm. DAPI was used to stain nuclei (shown in blue). Scale bar, 10 µm.
- Figure 4.
Immunoelectron microscopy of rhCDNF immunoreactivity in rat striatum 2 h after its intracerebral infusion. Shown are the representative images. A, B, Immunolabeling of infused rhCDNF can be seen in an early endosome (A) and in a multivesicular body (B). No labeling was observed in the ER, Golgi apparatus, or mitochondria. The inset in A shows a closeup of the anti-CDNF IR structure. C, Anti-CDNF IR in the extracellular space. mt, Mitochondrion; er, ER; g, Golgi apparatus. Scale bar, 250 nm.
- Figure 5.
Localization of retrogradely transported rhCDNF in dopaminergic neurons of SNc. A–D, Representative confocal microscopy images of anti-CDNF IR in SNc 6 h after infusion of 10 µg rhCDNF into the striatum (n = 5, 6). Sections from uninfused (A), rhCDNF-infused (B, C), or 6-OHDA-treated rhCDNF-infused hemispheres (D) at the position of midbrain were double stained with anti-CDNF (green) and anti-TH (magenta) antibodies. E–H, Chromogenic detection of anti-CDNF IR was used to analyze the SNc on sections from uninfused (E), rhCDNF-infused (F), or 6-OHDA-treated rhCDNF-infused hemispheres (G; n = 5–6). Sections from CDNF-deficient mice were used to reveal unspecific staining by the anti-CDNF antibody (H). Arrowheads mark the nuclei of non-neuronal cells nonspecifically binding the anti-CDNF antibody. Arrows mark some of the cells that contain punctate anti-CDNF IR. DAPI was used to stain nuclei in A–D (shown in blue). Scale bar, 20 µm.
- Figure 6.
Loss of TH-positive nigrostriatal projections of DA neurons upon treatment with 6-OHDA. Images of anti-TH- or anti-CDNF-stained sections from brains collected 6 h after intrastriatal rhCDNF infusion (10 µg). On the right-hand side is the brain from a rat that had undergone intrastriatal infusion of 6-OHDA (20 µg) 3 d before. Scale bar, 1 mm.
- Figure 7.
Spreading and clearance of rhCDNF after intrastriatal infusion. Representative images of anti-CDNF IR at different time points after infusion into striatum. Coronal sections from the level of A/P −0.3 are shown. LD, Low dose (3.7 µg) of rhCDNF; HD, high dose (16 µg) of rhCDNF; STR, striatum. Scale bar, 1 mm.
- Figure 8.
Quantitative analysis of rhCDNF clearance after intrastriatal infusion. A, Quantitative analysis of CDNF immunofluorescence in the striatum. Data are shown separately for the left hemisphere, which received the high dose of 16 μg of rhCDNF, and the right hemisphere, which received the low dose of 3.7 μg of rhCDNF. Mean ± SEM values are shown; n = 3 hemispheres per time point. B, Levels of rhCDNF in the striatum. C, Levels of rhCDNF in the substantia nigra, as measured by ELISA. Mean ± SEM values are shown; n = 5-6 hemispheres per time point.
Tables
- Table 1:
Quantification of the observed anti-CDNF IR patterns at 2 h postinfusion from sections costained with antibodies to CDNF and NeuN
Punctate Weak diffuse (cytoplasmic) Strong diffuse
(cytoplasmic)Strong (peripheral) Cortex (70–130 cells scored per animal) 96.1 ± 3.4% 22.9 ± 13.3% NQ 3.2 ± 2.0% Caudate/putamen (69–107 cells scored per animal) 95.7 ± 3.7% 19.3 ± 8.2% NQ 3.3 ± 1.7% Randomly selected NeuN-positive cells in the caudate/putamen and cortex of the rhCDNF-infused hemisphere from four rats were scored on the basis of their anti-CDNF IR pattern. The number of cells matching the classification criteria is presented as a percentage of the total number of NeuN-positive cells scored (mean ± SD is shown, n = 4). NQ, Not quantified.
High dose Low dose Ratio Cmax (ng/mg) 140.6 20.6 6.8 AUC0-t (µg/mg x min) 1.143 0.208 5.5 Half-life (h) 5.51 5.55
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