Generation of dopamine neurons with improved cell survival and phenotype maintenance using a degradation-resistant nurr1 mutant

Abstract

Nurr1 is a transcription factor specific for the development and maintenance of the midbrain dopamine (DA) neurons. Exogenous Nurr1 in neural precursor (NP) cells induces the differentiation of DA neurons in vitro that are capable of reversing motor dysfunctions in a rodent model for Parkinson disease. The promise of this therapeutic approach, however, is unclear due to poor cell survival and phenotype loss of DA cells after transplantation. We herein demonstrate that Nurr1 proteins undergo ubiquitin-proteasome-system-mediated degradation in differentiating NP cells. The degradation process is activated by a direct Akt-mediated phosphorylation of Nurr1 proteins and can be prevented by abolishing the Akt-target sequence in Nurr1 (Nurr1(Akt)). Overexpression of Nurr1(Akt) in NP cells yielded DA neurons in which Nurr1 protein levels were maintained for prolonged periods. The sustained Nurr1 expression endowed the Nurr1(Akt)-induced DA neurons with resistance to toxic stimuli, enhanced survival, and sustained DA phenotypes in vitro and in vivo after transplantation.

Figure 1

UPS-mediated protein degradation is responsible for the decrease in Nurr1+/TH+ cells during the differentiation of neural precursor cells in vitro. Neural precursor cells were cultured from nondopaminergic rat embryonic cortices at embryonic day 13, and transduced with Nurr1 or LacZ (control). On the day after transduction, differentiation of Nurr1-transduced precursors into DA neurons was induced for 12 days by withdrawing the mitogen bFGF. (A–L): Representative microscopic images for Nurr1+ (A–D) and Nurr1+/TH+ (E–H) cells from Nurr1-transduced cultures, and β-gal-stained cells (I–L) from LacZ-transduced cells over the in vitro differentiation period. Scale bar = 20 μm. Insets of (E–H) show enlarged views of the regions indicated by arrows. (M): Percent changes of Nurr1+, TH+, and β-gal+ cells from three independent cultures. Significant differences were found from the value of %Nurr1+ cells at differentiation day 0 (Diff0)* and from the %TH+ cells of Diff 4# (p < .01). Nurr1 protein (N) and mRNA (O) levels at Diff0, Diff3, and Diff8 were further determined.(P–R): UPS-mediated degradation of Nurr1 proteins. In the presence of the protein synthesis inhibitor cycloheximide (40 μg/ml), Nurr1+ cells (P) and Nurr1 protein levels (Q) were determined in the cortical precursor cells treated with the proteasome inhibitors MG132 or lactacystin (0, 1, and 10 μM) at differentiation day 6. (R): Immunoprecipitation assay for Ub and Nurr1 protein binding. *Significantly different from the untreated cultures (p < .01, n = 3 independent experiments).

Figure 2

Basic fibroblast growth factor (bFGF) is specific to maintenance of Nurr1 protein stability. To maintain similar levels of cell-to-cell contact, which may influence Nurr1 protein stability, cortical precursors transduced with Nurr1 were cultured for 2 days in the form of floating cell aggregates (neurospheres) in the absence (A, D) or presences of the mitogens bFGF (B, E) or epidermal growth factor (C, F), and then Nurr1 protein levels were determined (G). Scale bars = 40 μm. In addition, neurospheres treated with various cytokines were plated on FN-coated surfaces and were stained against Nurr1. (H): Percent decreases of Nurr1+ cells for 1 day of in vitro culture. Nurr1-transduced precursor cells were left untreated (no tx) or treated with the factors and inhibitors indicated and the percent decreases were calculated by percent changes of Nurr1+ cell numbers before and 1 day after the treatments. *Significantly different from the untreated control (p < .01, n = 3, Student t test).

Figure 3

Raf-MEK and PI3K-Akt intracellular pathways downstream of basic fibroblast growth factor (bFGF) have opposing actions in the regulation of Nurr1 protein decay. (A–C): Opposing roles of extracellular regulated kinase (ERK) and Akt in the regulation of Nurr1 protein stability. (A, B): Time-course changes of ERK and Akt signal activations for 84 hours after bFGF withdrawal. The activated ERK and Akt levels were estimated by the ratios of pERK/ERK and pAkt/Akt, respectively. Each dot and bar in (B) represents the mean and SEM of the activated protein level (relative to that of time point 0) scanned from five Western blot analyses performed with three sets of independent cultures. (C, D): Nurr1 protein levels in the cultures treated with FGFR1 blocker (SU5402), Raf-ERK inhibitor (PD98059), dn-raf, or PI3K-Akt inhibitors (LY294002, wortmannin) were compared with the untreated control. (E): Effects of Raf-ERK and PI3K-Akt signals in Nurr1 protein stability. HEK-293 cells transfected with Nurr1 were treated with cycloheximide in the absence or presence of PD98059 or LY294002 and then harvested for a Western blot assay at the times indicated.

Figure 4

Enhanced maintenance of TH+ DA cells generated by Nurr1^Akt transduction. (A): Direct protein binding of Akt to Nurr1. Cortical neural precursor (NP) cells transduced with Flag-Nurr1 were immunoprecipitated by incubation with Flag antibody and immunoblotted with Akt antibody. (B): Sequence alignment of Nurr1 proteins around the consensus Akt phosphorylation site. (C): Abolishment of Akt phosphorylation in Nurr1^Akt mutant proteins in which serine 347 is transformed into alanine. Rat cortical NP cells transduced with Flag-Nurr1^WT or Flag-Nurr1^Akt were harvested, immunoprecipitated with Flag antibody, and then immunoblotted by phospho-(Ser/Thr) Akt substrate antibody. (D): Abolishment of the Akt phosphorylation site in Nurr1 enhances protein stability. Nurr1 protein levels were compared in the cultures transfected by Nurr1^WT and Nurr1^Akt during 6 hours of cycloheximide (100 μg/ml) treatment. (E): Decreased ubiquitinylation in the Nurr1 Akt phosphorylation mutant. Immunoprecipitation assays for Ub and Nurr1 protein binding were performed as described in Figure 1. (F–S): Maintenance of Nurr1+/TH+ DA neurons in cell cultures transduced with Nurr1^Akt. (F–Q): Representative microscopic images for Nurr1+ (F–K) and Nurr1+/TH+ (L–Q) cells from the cultures transduced with Nurr1^WT and Nurr1^Akt over the differentiation period in vitro. Scale bar = 20 μm. Percent Nurr1+ and TH+ cells are depicted in (R) and (S), respectively. *Significantly different from the respective Nurr1^WT values for the same differentiation days (p < .01).

Figure 5

Nurr1^Akt-transduced NP cells yield dopamine (DA) neurons that are resistant to toxic stimuli and show enhanced survival in vitro and in vivo after transplantation. (A–L): Cellular toxicity induced by H₂O₂ (50–500 μM) or 6-OHDA (50–200 uM) treatments. After 6 days of in vitro differentiation, cells were treated with the toxins for 8 hours, and cell viability was determined by the MTT assay (I, K), PI staining (E–H), and directly counting TH+ cells (J, L). Scale bar = 20 μm. *Significantly different from the respective Nurr1^WT values of the same toxin concentrations (p < .01, n = 3 independent experiments). (A–D) and (E–H) are the representative images of TH+ cells and PI-stained cells, respectively, which demonstrate the difference in H₂O₂-induced cell toxicity in Nurr1^WT- and Nurr1^Akt-transduced cultures. (M-V): In vivo survival of TH+/Nurr1+ cells after transplantation. Representative images for TH+ (N, Q), Nurr1+ (M, P), and TH+/Nurr1+ (O, R) cells in the grafts generated by Nurr1^WT- (M–O) and Nurr1^Akt- (P–R) transduced precursors. Quantification of graft volumes (S), total donor cells (T), Nurr1+ (U), and TH+ (V) cell numbers in the grafts are shown. Scale bar = 20 μm. *Significantly different from the respective Nurr1^WT values (p < .01, n = 5 for each value, Student's t test).