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. 2012;7(2):e32019.
doi: 10.1371/journal.pone.0032019. Epub 2012 Feb 20.

Ephrin-A1-mediated Dopaminergic Neurogenesis and Angiogenesis in a Rat Model of Parkinson's Disease

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Free PMC article

Ephrin-A1-mediated Dopaminergic Neurogenesis and Angiogenesis in a Rat Model of Parkinson's Disease

Xuefeng Jing et al. PLoS One. .
Free PMC article

Abstract

Cells of the neural stem cell lineage in the adult subventricular zone (SVZ) respond to brain insult by increasing their numbers and migrating through the rostral migratory stream. However, in most areas of the brain other than the SVZ and the subgranular zone of the dentate gyrus, such a regenerative response is extremely weak. Even these two neurogenic regions do not show extensive regenerative responses to repair tissue damage, suggesting the presence of an intrinsic inhibitory microenvironment (niche) for stem cells. In the present study, we assessed the effects of injection of clustered ephrin-A1-Fc into the lateral ventricle of rats with unilateral nigrostriatal dopamine depletion. Ephrin-A1-Fc clustered by anti-IgG(Fc) antibody was injected stereotaxically into the ipsilateral lateral ventricle of rats with unilateral nigrostriatal lesions induced by 6-hydroxydopamine, and histologic analysis and behavioral tests were performed. Clustered ephrin-A1-Fc transformed the subventricular niche, increasing bromodeoxyuridine-positive cells in the subventricular area, and the cells then migrated to the striatum and differentiated to dopaminergic neurons and astrocytes. In addition, clustered ephrin-A1-Fc enhanced angiogenesis in the striatum on the injected side. Along with histologic improvements, behavioral derangement improved dramatically. These findings indicate that the subventricular niche possesses a mechanism for regulating both stem cell and angiogenic responses via an EphA-mediated signal. We conclude that activation of EphA receptor-mediated signaling by clustered ephrin-A1-Fc from within the lateral ventricle could potentially be utilized in the treatment of neurodegenerative diseases such as Parkinson's disease.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of EphA–related signaling molecules in subventricular zone cells.
(A) RT-PCR for Ephas, Efnas, Fgfrs, Ephexin1, and Frs2α. G: Glyceraldehyde 6-phosphate dehydrogenase. For Efna1 and Efna4, liver RNA was also used as control. (B) Detection of EphA receptors that binds to ephrin-A1-Fc in rat brain lysate. Rat brain subventricular cell lysate was incubated with ephrin-A1-Fc, precipitated by protein A-agarose, and immunoblotted with the antibodies for EphAs. (C) EphA4 phosphorylation in tissue surrounding the lateral ventricle. In rats with lesioned unilaterally in the nigrostriatal dopaminergic pathway, subventricular tissue was collected 18 hours after single injection of clustered IgG(Fc) (3 µg), unclustered ephrin-A1-Fc (Un-A1) (3 µg), clustered ephrin-A1-Fc (C-A1) (3 µg), or FGF2 (100 ng). The tissue lysate (450 µg protein) was immunoprecipitated with anti-EphA4 antibody followed by immunoblotting with anti-phosphotyrosine (pY) antibody and anti-EphA4 antibody.
Figure 2
Figure 2. Effect of clustered ephrin-A1-Fc injection in rats with chemical lesions in the unilateral nigrostriatal dopaminergic pathway.
(A) Effect of intrastriatal injection of clustered ephrin-A1-Fc on the regeneration of dopaminergic cells. Rats with unilateral nigrostriatal lesions were injected with clustered IgG(Fc) (upper panels) or ephrin-A1-Fc (lower panels) into the lesioned side of the striatum near the subventricular zone. Rats were killed 6 weeks after injection. Brains were sectioned coronally, and sections were stained immunohistochemically for tyrosine hydroxylase (TH). Yellow arrows indicate the course of needles used for injection of clustered IgG(Fc) or ephrin-A1-Fc. Scale bar: 100 µm. (B) Entire striatal coronal view of TH–positive cells. Clustered IgG(Fc) or ephrin-A1-Fc was injected into the ventricle ipsilateral to the lesioned side, and coronal sections of the entire striatum were stained for TH. Upper panels, clustered IgG(Fc)-injected rat; lower panels, clustered ephrin-A1-Fc-injected rat. Scale bar: 500 µm.
Figure 3
Figure 3. Behavioral effects of clustered ephrin-A1-Fc injection into the lateral ventricle.
Effect of a single injection (3 µg) (A) or a 1-week infusion with a micro-osmotic pump (B) of clustered ephrin-A1-Fc (2 µg/day) or control (clustered IgG[Fc]). Production of rats with unilateral nigrostriatal lesions and evaluation of behavior after intraperitoneal injection of apomorphine were performed as described in the Materials and Methods. (A) Behavior was analyzed just before and 6 weeks after injection of clustered IgG(Fc) or ephrin-A1-Fc. In each animal, the value of rotation frequency at 6-week point was adjusted to that before injection. Mean of the adjusted values in control animals at 6-week point was set at 100%. Error bars represent SD. *p<0.01 (n = 7). (B) Behavioral analysis was performed in lesioned rats infused with clustered IgG(Fc) (n = 5; white bars) or clustered ephrin-A1-Fc (n = 5; black bars) every 4 weeks up to 12 weeks from the start of infusion. In each group, the mean rotation value before infusion was taken as 100%. Error bars represent SD. *p<0.01 compared to the control.
Figure 4
Figure 4. Distribution of BrdU(+) cells in the striatum after intraventricular injection of clustered ephrin-A1-Fc.
(A) Pulse-chase experiments of BrdU labeling of proliferating cells. Unilaterally lesioned rats were injected with a single dose of clustered ephrin-A1-Fc (3 µg) into the lateral ventricle of the lesioned side, followed by 3 intraperitoneal injections of BrdU at 6-hour intervals. Coronal sections of brains were examined 1, 7, and 14 days after injection of clustered ephrin-A1-Fc. BrdU staining is shown as fine green dots in the striatum. TH staining was used to detect the lesioned side of the brain (not shown). Lesioned sides are shown on the left, and normal sides are shown on the right. The numbers of days after clustered ephrin-A1-Fc injection (Day 0) are shown above the panels. Upper panels represent the anterior part of the striatum, and lower panels represent the middle region of the striatum. Vertical white bars show the intra-striatal distribution front of BrdU(+) cells. (B) Enlarged photomicrographs of the numbered regions in (A). (C) The same as (A) except that clustered IgG(Fc) (3 µg) instead of clustered ephrin-A1-Fc was injected as control. Only the middle region of the striatum is shown. (D) Enlarged photomicrographs of the numbered regions of (C). Scale bar: 100 µm. These are the typical results of 3 separate experiments for each treatment.
Figure 5
Figure 5. Tracking BrdU(+) cells following intraventricular infusion of clustered ephrin-A1-Fc.
Unilaterally lesioned rats were infused with clustered IgG(Fc) or clustered ephrin-A1-Fc into the lateral ventricle of the lesioned side for 1 week with simultaneous intraperitoneal injection of BrdU. (A) Number of BrdU(+) cells in the striatum counted with a stereologic counting system. n = 4 for the clustered IgG(Fc)-infused and ephrin-A1-Fc-infused groups. Error bars represent SD. *p<0.05 (Mann Whitney test, n = 4) compared to clustered IgG(Fc). (B) Tracking the cells labeled with BrdU and CM-DiI in the striatum. Brain slices were stained for BrdU and with Wheat Germ Agglutinin (WGA), and confocal 3D micrographs were taken at 1-µm intervals. Then, ten serial confocal micrographs were compiled for one all-in-focus micrograph, and the number of cells labeled with BrdU or co-labeled with both BrdU and CM-DiI was counted in defined areas as described in the Materials and Methods. **p<0.01 (n = 8) compared to IgG(Fc). (C) Tracking of the cells labeled with BrdU and CM-DiI in the olfactory bulb. Study protocols are the same as in (B). The labeled cells in the granule cell layer of the olfactory bulb were counted. *p<0.05 (n = 8) compared to IgG(Fc). (D) A typical confocal micrograph showing the relative localization of BrdU (blue), WGA (green) and CM-DiI (red) in a cell level in the striatum. Scale bar, 20 µm; arrowheads, cells labeled with both BrdU and CM-DiI.
Figure 6
Figure 6. Effect of intraventricular infusion of clustered ephrin-A1-Fc on the distribution of immunostained cells in the striatum.
Unilaterally lesioned rats were treated as for Fig. 5. BrdU is shown in green, and GFAP in red. (A) Magnification of the rectangular inset in Fig S3. White arrowheads indicate the cells positive for both BrdU and GFAP outside of the SVZ. Scale bar: 50 µm. (B) BrdU(+) cells and BrdU(+)&GFAP(+) cells were counted as described in the Materials and Methods. Total numbers of BrdU(+) cells in 8 animals are shown on the left, and percentages of GFAP(+) cells among BrdU(+) cells are shown on the right. Error bars represent SD. *p<0.01 (n = 8) compared to control (IgG[Fc]). (C) Triple staining of the lesioned side of the subventricular region after infusion of clustered ephrin-A1-Fc or IgG(Fc). CD24, green; GFAP, red; BrdU, blue. Scale bar: 20 µm. (D) Staining for MASH1 (green) and BrdU (red) in and around the SVZ 6 weeks after infusion of clustered ephrin-A1-Fc. White arrowheads indicate the cells positive for both MASH1 and BrdU. (E) Staining for Doublecortin (DCX) (red) and BrdU (green) in and around the SVZ 6 weeks after infusion of clustered ephrin-A1-Fc. White arrowheads indicate the cells positive for both DCX and BrdU. (F) Staining for Nestin (red) and BrdU (green) in and around the SVZ 6 weeks after infusion of clustered ephrin-A1-Fc. In (D), (E) and (F), the lateral ventricle is toward the right side of the panel, and the dotted line indicates the border between the lateral ventricle and the ependymal cell layer. Scale bar: 20 µm.
Figure 7
Figure 7. Differentiation of BrdU(+) cells to neuronal cells in the striatum.
The brain of a unilaterally lesioned rat treated as in Fig. 6 was sectioned coronally and immunostained for NeuN and BrdU. (A) Left panel, number of cells positive for BrdU alone (white bars) and for both BrdU and NeuN (black bars) in the striatum. Cell numbers were counted in 6 animals using the dynamic cell count program of the Keyence fluorescence microscopy system. *p<0.01 (n = 6) compared to values for the side of clustered ephrin-A1-Fc infusion. Right panel, ratio of the number of NeuN(+)&BrdU(+)cells to that of BrdU(+) cells. *p<0.01 (n = 6) compared to values for the side of clustered ephrin-A1-Fc infusion. Error bars indicate SD. (B) BrdU(+) cells differentiating to the TH(+) neuronal lineage as detected by confocal microscopy. BrdU(+) nuclei are shown in green, and cytoplasmic TH in red. Merged regions are shown in yellow. 3D confocal photomicrographs are indicated by vertical (red) and horizontal (green) lines. Blue lines indicate the depth of the confocally sectioned plane relative to the top surface located close to the enface micrograph. Scale bar: 20 µm. (C) Co-localization of dopamine transporter (DAT) and TH in a regenerated neuron fiber. Sections were stained for DAT (green) and TH (red), and analyzed by confocal microscopy. Scale bar: 10 µm.
Figure 8
Figure 8. Effect of clustered ephrin-A1-Fc on vascular formation in the rat striatum.
(A) Distribution of BrdU(+) endothelial cells. Brain of the unilaterally lesioned rats 6 weeks after infusion of clustered ephrin-A1-Fc were sectioned coronally and stained for BrdU and Rat Endothelial Cell Antigen-1 (RECA-1). Numbers of the BrdU(+) cells and BrdU(+)&RECA-1(+) cells were counted as describe in the Materials and Methods. Total numbers of BrdU(+) cells in 8 animals are shown on the top, and percentages of RECA-1(+) cells among BrdU(+) cells are shown on the bottom. Error bars represent SD. *p<0.01 (n = 8) compared to control (IgG[Fc]). (B) Magnified confocal micrographs of insets in Fig S5. Scale bar: 50 µm. (C) Quantification of endothelial cell area. Coronal sections of striatum were stained for RECA-1 as in Fig S5, and the RECA-1 stained area was quantified using an ImageJ computer program (NIH). The areas taken for measurements were as described in the Materials and Methods. The values from 6 animals were analyzed statistically. *p<0.01 (n = 6). Error bars represent SD. (D) Co-localization of BrdU, CM-DiI, and RECA-1 in endothelial cells of the striatum. Sections taken from the rats treated as in Fig. 5B with intraventricular CM-DiI injection were stained for BrdU and RECA-1 and subjected to 3D confocal microscopy. Micrographs are the all-in-focus compilation of 12 confocal micrographs at 0.5 µm intervals.

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