Dopamine-induced proliferation of adult neural precursor cells in the mammalian subventricular zone is mediated through EGF

Abstract

A reduction in dopaminergic innervation of the subventricular zone (SVZ) is responsible for the impaired proliferation of its resident precursor cells in this region in Parkinson's disease (PD). Here, we show that this effect involves EGF, but not FGF2. In particular, we demonstrate that dopamine increases the proliferation of SVZ-derived cells by releasing EGF in a PKC-dependent manner in vitro and that activation of the EGF receptor (EGFR) is required for this effect. We also show that dopamine selectively expands the GFAP(+) multipotent stem cell population in vitro by promoting their self-renewal. Furthermore, in vivo dopamine depletion leads to a decrease in precursor cell proliferation in the SVZ concomitant with a reduction in local EGF production, which is reversed through the administration of the dopamine precursor levodopa (L-DOPA). Finally, we show that EGFR(+) cells are depleted in the SVZ of human PD patients compared with age-matched controls. We have therefore demonstrated a unique role for EGF as a mediator of dopamine-induced precursor cell proliferation in the SVZ, which has potential implications for future therapies in PD.

Fig. 1.

Adult SVZ-derived cells are responsive to dopamine receptor stimulation. Immunolabeling of expanded adult SVZ-derived cultures. (A) D2L receptor (green) and EGFR (red) demonstrates colocalization; Hoescht nuclei are blue. (Scale bar: 60 μm.) (B and C) Cell viability of cultures exposed to dopamine at differing concentrations assessed by MTT assay (B) and LDH assay (C). *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus control cultures. Values in the LDH assay are expressed as the mean ± SEM % of control levels.

Fig. 2.

Dopamine increases proliferation of adult SVZ-NSCs in vitro. (A) The number of BrdU⁺ cells in dopamine-treated cultures was significantly increased compared with control cultures. (B) Representative photomicrograph of BrdU⁺ (green) and hoescht⁺ cells (blue) after dopamine stimulation. (Scale bar: 60 μm). (C) Significant increase in the number of newborn cells adopting a GFAP⁺ phenotype after dopamine exposure. (D) Representative photomicrograph of GFAP⁺ (red), BrdU⁺ (green), and hoescht⁺ cells (blue). *, P < 0.05; **, P < 0.01. (Scale bar: 60 μm.) (E) Clonal analysis of dopamine-responsive NSCs using limiting dilution analysis, with starting cell numbers ranging from 1 to 500 per well. The slope of the line reflects the proportion of cells plated that form neurospheres, i.e., display NSC characteristics. Linear regression values show that dopamine-treated cultures contain significantly more NSCs compared with untreated control cultures (***, P < 0.001). (F) Triple immunolabeling of a single neurosphere derived from dopamine-responsive NSCs demonstrate multipotentiality with the genesis of neurons (red), astrocytes (blue), and oligodendrocytes (green). (Scale bar: 160 μm.)

Fig. 3.

Dopamine stimulates the release of EGF and EGFR activation from adult SVZ NPCs. (A) EGF release after 24-h treatment with dopamine ± PKC inhibitor, compared with control using an ELISA. ***, P < 0.001; *, P < 0.05. (B) Cell cycle analysis of NPCs using propidium iodide shows that the percentage of cells in S phase treated with dopamine alone is increased and lost when used in combination with an EGFR inhibitor (AG1478). ***, P < 0.001.

Fig. 4.

Elimination of dopaminergic fibers decreases proliferation in the SVZ caused by reduced local EGF production, and restoration of dopamine levels in the SVZ through l-DOPA treatment restores EGF levels to baseline. (A) Experimental paradigm. The dopaminergic nigrostriatal tract was lesioned by 6-OHDA. Two weeks later, animals were challenged with amphetamine, then1 week later animals received BrdU daily for 6 days, ± l-DOPA for the same 6 days and were killed 1 day or 21 days later. 2/52 and 3/52 represents 2 and 3 weeks later, respectively. 6/7 represents 6 days later. (B) Numbers of BrdU⁺ cells in the SVZ 1 day after the last BrdU injection. *, P < 0.05 versus control group; #, P < 0.001 versus untreated lesion group. A significant interaction effect was observed between treatment and lesion (***, P < 0.001). (C) Representative Western blots showing a significant decrease in EGF levels in the SVZ of lesioned animals, which could be restored to control levels with l-DOPA. The blotted membrane was reprobed with α-TH to show that the dopaminergic projections in the SVZ were depleted and the α-actin antibody as loading control. (D) Optical densitometry of band intensities for EGF normalized to actin (n = 3 per group) showing that EGF levels in lesioned animals were significantly decreased relative to the other 3 treatment groups. **, P < 0.05. (E) Representative confocal image shows colocalization of BrdU and NeuN in the OB of an l-DOPA-treated lesioned rodent. (Scale bar: 5 μm.) (F) 6-OHDA-lesioned animals show a significant reduction in the percentage of BrdU⁺ cells expressing NeuN in the OB 21 days after the last BrdU injection (***, P < 0.001). This was restored by l-DOPA (#, P < 0.001 versus untreated lesion group.)

Fig. 5.

The number of EGFR⁺ cells in the SVZ of PD patients is significantly decreased compared with age-matched controls. (A) EGFR⁺ cells are present in the human SVZ, as demonstrated by a representative control human SVZ section at 10× (Left) and 63× (Right) magnification. (Scale bar: 60 μm.) (B) A representative SVZ section from a patient that died with PD at 10× (Left) and 63× (Right) magnification. The arrow points to an EGFR⁺ cell. STR, striatum; E, ependymal layer; LV, lateral ventricle. (C) The number of EGFR⁺ cells is significantly decreased in the SVZ of PD patients compared with age-and sex-matched controls (n = 6 per group). ***, P < 0.001.