Paraquat and MPTP induce neurodegeneration and alteration in the expression profile of microRNAs: the role of transcription factor Nrf2

Abstract

Both transcription factors (TFs) and microRNAs (miRNAs) can exert a widespread impact on gene expression. In the present study, we investigated the role of Nrf2 in paraquat-induced intracorporeal neurodegeneration and miRNA expression by exposing Nrf2 wild-type and knockout mice to paraquat (PQ) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Exposure to 10 mg/kg PQ or 30 mg/kg MPTP caused damage to nerve cells in the substantia nigra (SN) in both Nrf2 (+/+) and Nrf2 (-/-) ICR mice, which included cell morphological changes, detectable apoptosis and a significant reduction in the number of dopaminergic (DA) neurons. When mice were exposed to the same PQ dose of 10 mg/kg, significant fewer tyrosine hydroxylase (TH)-positive DA neurons were observed in the Nrf2 (-/-) mice than that in the Nrf2 (+/+) mice. Both Nrf2 deficiency and PQ or MPTP exposure could alter miRNA expression profile in the SN, suggesting the potential involvement of Nrf2 in the PQ-induced or MPTP-induced miRNA expression alteration. The expression of miR-380-3p was altered by the Nrf2-MPTP interaction effect. miR-380-3p/Sp3-mRNA pathway is likely part of the mechanism of MPTP-induced neurodegeneration. Collectively, our results corroborated the protective role of Nrf2 and also demonstrated the essential interaction of Nrf2 with miRNAs in intracorporal neurodegeneration induced by neurotoxicants.

Fig. 1

HE staining in the SN of Nrf2 (+/+) and Nrf2 (−/−) mice after treatment with PQ, MPTP and saline for neuropathological changes. Each microgram (100×) contains a view of SN from each group (n = 3). Representative damaged cells were shown in bottom left corner of the panel (400×)

Fig. 2

Neuropathological changes in the SN of Nrf2 (+/+) or Nrf2 (−/−) mice after treatment with PQ, MPTP or saline (n = 3). a Each microgram (100×) shows a representative perspective view of SN immunostained with TH from each group. b The loss of TH-positive DA neurons induced by PQ or MPTP in each group, reflected by numbers of residual neurons. ▲ Nrf2 (+/+) PQ10 vs. Nrf2 (+/+) saline (p < 0.01), ★ Nrf2 (+/+) MPTP30 vs. Nrf2 (+/+) saline (p < 0.01), ● Nrf2 (−/−) PQ10 vs. Nrf2 (−/−) saline (p < 0.01), ■ Nrf2 (−/−) MPTP30 vs. Nrf2 (−/−) saline (p < 0.01), ▽ Nrf2 (−/−) PQ10 vs. Nrf2 (+/+) PQ10 (p < 0.05), ◎ Nrf2 (−/−) MPTP30 vs. Nrf2 (+/+) MPTP30 (p < 0.05)

Fig. 3

Cell apoptosis in the SN of Nrf2 (+/+) and Nrf2 (−/−) mice after treatment with PQ, MPTP or saline (n = 3). a Each microgram (400×) shows a representative view of apoptosis in the SN of each group. b TUNEL-positive rate in each group. We counted the number of TUNEL staining positive cells and the number of total cells, and calculated the TUNEL staining positive rate (the number of TUNEL positive cells/total number of cells x 100%). ▲ Nrf2 (+/+) PQ10 vs. Nrf2 (+/+) saline (p < 0.01), ■Nrf2 (+/+) MPTP30 vs. Nrf2 (+/+) saline (p < 0.01), ◆Nrf2 (−/−) PQ10 vs. Nrf2 (−/−) saline (p < 0.01), ●Nrf2 (−/−) MPTP30 vs. Nrf2 (−/−) saline (p < 0.01)

Fig. 4

Expression of Nrf2 protein in the SN of Nrf2 (+/+) or Nrf2 (−/−) mice after treatment with PQ, MPTP or saline (n = 3). Each microgram shows a representative view of an Nrf2-immunostained slice from the SN (400×). Nrf2 protein staining has confirmed results of both Nrf2 knockout and response of Nrf2 to PQ/MPTP exposure in mice

Fig. 5

The expression level of miR-380-3p in the SN of Nrf2 (+/+) and Nrf2 (−/−)after treatment with PQ, MPTP or saline. The arrows indicated the signal of LNA™ probes for miR-380-3p. a The expression intensity of miR-380-3p analyzed by LNA–ISH hybridization in the SN of Nrf2 (+/+) ICR mice after saline, PQ or MPTP treatment (n = 3); each microgram (400×) shows a representative view of a slice of the SN from each group. b The expression intensity of miR-380-3p analyzed by LNA–ISH hybridization in Nrf2 (−/−) ICR mice SN after saline, PQ or MPTP treatment (n = 3); each microgram (400×) shows a representative view of a slice of the SN from each group. c The expression levels of miR-380-3p analyzed by real-time PCR in Nrf2 (+/+) and Nrf2 (−/−) SN after treatment with PQ, MPTP or saline (n = 4). ◆ Nrf2 (+/+) PQ5 vs. Ap (p < 0.01), ▲ Nrf2 (+/+) PQ10 vs. Nrf2 (+/+) saline (p < 0.01), ★ Nrf2 (+/+) MPTP30 vs. Nrf2 (+/+) saline (p < 0.01), □Nrf2 (−/−) PQ5 vs. Nrf2 (−/−) saline (p < 0.01), ●Nrf2 (−/−) PQ10 vs. Nrf2 (−/−) saline (p < 0.01), ■ Nrf2 (−/−) MPTP30 vs. Nrf2 (−/−) saline (p < 0.01), ◎ Nrf2 (−/−) MPTP30 vs. Nrf2 (+/+) MPTP30 (p < 0.01)

Fig. 6

Analysis of common target genes of miR-380-3p. a Vennplot for the predictions of miR-380-3p target genes by Microcosm, Targetscan and Miranda. b The seven predicted common target genes