Nurr1 promotes neurogenesis of dopaminergic neuron and represses inflammatory factors in the transwell coculture system of neural stem cells and microglia

Abstract

Introduction: Neural stem cells (NSCs) are the most promising cells for cell replacement therapy for Parkinson's disease (PD). However, a majority of the transplanted NSCs differentiated into glial cells, thereby limiting the clinical application. Previous studies indicated that chronic neuroinflammation plays a vital role in the degeneration of midbrain DA (mDA) neurons, which suggested the developing potential of therapies for PD by targeting the inflammatory processes. Thus, Nurr1 (nuclear receptor-related factor 1), a transcription factor, has been referred to play a pivotal role in both the differentiation of dopaminergic neurons in embryonic stages and the maintenance of the dopaminergic phenotype throughout life.

Aim: This study investigated the effect of Nurr1 on neuroinflammation and differentiation of NSCs cocultured with primary microglia in the transwell coculture system.

Results: The results showed that Nurr1 exerted anti-inflammatory effects and promoted the differentiation of NSCs into dopaminergic neurons.

Conclusions: The results suggested that Nurr1 protects dopaminergic neurons from neuroinflammation insults by limiting the production of neurotoxic mediators by microglia and maintain the survival of transplanted NSCs. These phenomena provided a new theoretical and experimental foundation for the transplantation of Nurr1-overexpressed NSCs as a potential treatment of PD.

Keywords: Nurr1; coculture; inflammatory; microglia; neural stem cells.

Figure 1

Identification of Nurr1 overexpression in mNSCs and MG. A, The neurosphere from E14.5 rat cerebrum was immunoreactive to the neuroepithelial marker nestin. B, Neurospheres were infected with pLenO‐DCE‐Nurr1 (MOI 200), and GFP‐expressing cells were observed at 72 h. C, Purified microglia were immunoreactive to CD11B. D, Microglia were infected with pLenO‐DCE‐Nurr1 (MOI 2), and GFP‐expressing cells were seen at 72 h. E, F, RT‐PCR and Western blot were performed for detection of Nurr1 expression in mNSCs. G, H, RT‐PCR and Western blot were performed for the detection of Nurr1 expression in microglia. Nuclei of these cells were stained with DAPI. Scale bar in A, C, and D, 20 μm; Scale bar in B, 100 μm. *P < .05, **P < .01 compared to the control group, unpaired Student's t test. The experiment was repeated 3 times in triplicate using independently prepared cells

Figure 2

Differentiation of mNSCs after transduced by pLenO‐DCE‐Nurr1. Seven days after differentiation culture, the reporter protein, GFP, transfected into pLenO‐DCE‐Nurr1 (A) was still detectable in the cells derived from neurospheres. Most of the cells were immunoreactive to Tuj1 (C, D); small arrows in C and D showed the cell bodies of neurons. Scale bar in A–D, 20 μm

Figure 3

Neuroprotective roles by Nurr1‐expressing primary microglia. A‐B, ELISA showed that, as compared to with the LPS group, Nurr1 overexpression microglia suppressed the inflammatory factors, including IL‐1 and TNF‐α. C‐D, Moreover, the neurotrophic factors were increased after infection by pLenO‐DCE‐Nurr1. **P < .01, ****P < .0001, compared to the LPS group, 1‐way ANOVA followed by Bonferroni post hoc test. The experiment was repeated 3 times in triplicate with independent preparation of the cells

Figure 4

Effect of Nurr1 overexpression and MG microenvironment on dopaminergic differentiation NSCs. A, Real‐time PCR showed that, as compared to the other groups, the mRNA expression levels of DAT, TH, Otx2, and Pitx3 were significantly higher in Nurr1‐overexpressed NSCs cocultured with Nurr1‐overexpressed microglia (NmNSC+NMG group). B–C, Western blot showed that, as compared to the other groups, the protein levels of DAT, TH, Otx2, and Pitx3 were high in the NmNSC+NMG group. D–G, Immunohistochemistry showed that, as compared to the other groups, abundant DAT, TH, Otx2, and Pitx3‐positive neurons were observed in the NmNSC+NMG group. *P < .05, **P < .01, ***P < .001, ****P < .0001, compared to the NmNSC+NMG group, 1‐way ANOVA followed by Bonferroni post hoc test. Scale bar in D‐G, 50 μm. The experiment repeated 3 times in triplicate using independently prepared cells