Combined Nurr1 and Foxa2 roles in the therapy of Parkinson's disease.
In addition to their proposed cell-autonomous actions in mDA neurons, forced expression of these factors in neighboring glia synergistically protects degenerating mDA neurons in a paracrine mode.As a consequence of these bimodal actions, adeno-associated virus (AAV)-mediated gene delivery of Nurr1 and Foxa2 in a PD mouse model markedly protected mDA neurons and motor behaviors associated with nigrostriatal DA neurotransmission.The effects of the combined gene delivery were dramatic, highly reproducible, and sustained for at least 1 year, suggesting that expression of these factors is a promising approach in PD therapy.
Affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, Seoul, Korea Hanyang Biomedical Research Institute, Hanyang University, Seoul, Korea Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Korea.
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fig05: Therapeutic effects of AAV-mediated gene delivery of Nurr1 + Foxa2 in the mouse PD modelA Experimental scheme. As shown, PD model mice were generated by IP injection of MPTP, and AAVs expressing Nurr1 and Foxa2 (NF-AAV) or AAVs expressing empty vector (control, C-AAV) were stereotaxically injected 3 days prior to the initial MPTP treatment.B–E Cytoprotective and behavioral effects of NF expression were assessed in PD mice unilaterally injected with NF-AAVs on the right side of midbrains. C-AAVs were injected into the left midbrains of the same mice. TH+ mDA neuron numbers in the midbrain (B) and TH+ fiber intensities in the striatum (C) of the NF-AAV-injected right sides were compared with those of the corresponding left sides after 8 weeks. Insets of (B), high-powered images of the areas indicated by red arrowheads in the C-AAV (left) and NF-AAV-injected (right) sides. TH+ cell numbers, *P = 0.000078, n = 7, paired Student's t-test. The sectioned striatal tissues were immunofluorescently stained for TH, and TH+ fiber intensities were estimated from mean fluorescence intensities (MFI). n = 56 microscopic fields at a magnification of 200× each for the left and right striatum. *P = 0.000003, paired Student's t-test. Behavioral asymmetry in the mice unilaterally injected with NF-AAVs (right side) was assessed using the apomorphine-induced rotation test (D) after 4 and 8 weeks. The cylinder test (E) was carried out after 8 weeks to determine left forelimb movement. As a control, the behaviors were tested in PD mice bilaterally injected with C-AAV. *P = 0.028 (rotation test), 0.043 (cylinder test); one-way ANOVA followed by Bonferroni post hoc test.F–L Behaviors of the PD mice injected with NF-AAVs bilaterally at both sides of the midbrain were assessed using the pole (J), beam (K), and locomotor (L) tests. *P = 0.004 (pole), 0.044 (beam), and 0.045 (locomotor); one-way ANOVA followed by Bonferroni post hoc test. Shown in (F–I) is a representative TH-stained midbrain (F, H) and striatum (G, I) of mice bilaterally injected with C-AAVs (F, G) and NF-AAVs (H, I).M, N Sustained mDA neuroprotective effects. Shown in (M) is a confocal image of TH/HA-stained cells 6 months after NF-AAV injection (Foxa2 gene tagged with HA). Inset, z-stacked image of the boxed area along the y-axis (right) and x-axis (lower). TH+ cells in the right (NF-AAV) and left (C-AAV injected) midbrains were counted 6 and 12 months after AAV injection (N). *P = 0.000019 (6M), 0.000066 (12M), n = 6–7, paired Student's t-test.O, P NF-AAV-mediated mDA neuroprotective effects were further assessed with the experimental schedule (O), in which AAV injections were subjected after three consecutive MPTP injections. Shown in (P) is TH+ cell counts of the left and right sides of the midbrains. *P = 0.000012 (D14), 0.000036 (1M), 0.000027 (2M), n = 6–7, paired Student's t-test.
Based on our in vitro data, we examined whether forced expression of Nurr1 and Foxa2 could forestall degeneration of mDA neurons in PD. To this end, we chose the best-characterized MPTP mouse PD model (Beal, 2001) using a subchronic systemic approach (MPTP injection of 30 mg/kg, i.p., for five consecutive days) and the adeno-associated viral (AAV) system for gene delivery (schematized in Fig5A and O). Because of AAV's low immunogenicity, lack of cytotoxic response, and ability to infect non-dividing cells, clinical trials using it are currently underway in many disorders including PD (for reviews, see McCown, 2011; Weinberg et al, 2013). By injecting green fluorescent protein (GFP)-expressing AAVs into mouse midbrains, we confirmed expression of the transgene in our target cells expressing TH (DA neurons), GFAP (astrocyte), and Iba-1 (micro-glia) (Supplementary Fig S3).