Methylene blue promotes quiescence of rat neural progenitor cells

Abstract

Neural stem cell-based treatment holds a new therapeutic opportunity for neurodegenerative disorders. Here, we investigated the effect of methylene blue on proliferation and differentiation of rat neural progenitor cells (NPCs) both in vitro and in vivo. We found that methylene blue inhibited proliferation and promoted quiescence of NPCs in vitro without affecting committed neuronal differentiation. Consistently, intracerebroventricular infusion of methylene blue significantly inhibited NPC proliferation at the subventricular zone (SVZ). Methylene blue inhibited mTOR signaling along with down-regulation of cyclins in NPCs in vitro and in vivo. In summary, our study indicates that methylene blue may delay NPC senescence through enhancing NPCs quiescence.

Keywords: methylene blue; neural progenitor cell; neurogenesis; proliferation; quiescence.

FIGURE 1

MB inhibits NPC proliferation in vitro. (A) NPC apoptosis after 6 day MB treatment. Left: representative dot plots. Right: statistical analysis. (B) Detection of NPC proliferation by Ki67 staining. Left: representative histograms. Right: statistical analysis. (C) Detection of NPC cell cycle by PI staining at 1 day and 3 days after MB treatment. Left: representative histograms. Right: statistical analysis. N = 6 in each group. Ctrl, control; MB, MB-treated cells. *p < 0.05; **p < 0.01; ***p < 0.001 compared with control group.

FIGURE 2

MB down-regulates cyclin expression in NPCs. (A) Detection of mRNA levels of cyclin (D,E,A,B) in NPCs at 24 h after MB treatment. (B) Western blot assay for cyclin expression in NPCs. This is a representative of three independent experiments. (C) Statistical analysis for cyclin protein levels. Expression level was normalized to each control group at 12 h after culture. N = 6 in each group. Ctrl, control; MB, MB-treated cells. *p < 0.05; **p < 0.01; ***p < 0.001 compared with control group.

FIGURE 3

MB maintains neurosphere formation capacity of NPCs. (A–E) Detection of mRNA levels of stem cell, neural and glial markers in passage 1 (P1) NPCs. N = 3 in each group. (F) Serial passage analysis for neurosphere formation. N = 6 in each group. (G) Total cell number counting at the end of each passage. Note that at the beginning of each passage, only 1 × 10⁴ NPCs were seeded. (H) Western blot assay for Nestin, GFAP and DCX expression in P1 NPCs treated with MB for 6 days. Left, representative Western blot image. Right, statistical analysis for expression level of each protein. *p < 0.05; **p < 0.01; ***p < 0.001 compared with control group.

FIGURE 4

MB does not impede committed neuronal differentiation. (A) Q-RTPCR analysis of the expression of differentiation markers in differentiated NPCs at 1 and 7 days after growth factors withdraw. N = 3 per group. ***p < 0.001 compared with day-1 differentiated NPCs. (B) Q-RTPCR for expression of differentiation markers in differentiated NPCs in the presence or absence of MB. N = 3 per group. (C) Map2 staining for differentiated NPCs in the presence or absence of MB. Upper, 50×; lower, 200×. (D) Total cell number counting and Map2⁺ cell number in five microscopy fields (50×). (E) Western blot assay for expression of differentiation markers in differentiated NPCs in the presence or absence of MB. This is a representative of two independent experiments.

FIGURE 5

MB down-regulates mTOR expression via decreasing mTOR transcription. (A) Western blot assay of mTOR phosphorylation in NPCs. This is a representative of two independent experiments. (B) Western blot assay for the expression of mTOR signaling components in P1 NPCs. (C) Statistical analysis for protein levels of mTOR, p70S6K, and 4EBP1 after MB treatment. (D) Western blot assay for mTOR, p70S6K, and 4EBP1 in P1 NPCs treated with vehicle, MB, or MB plus proteasome inhibitor MG132. This is a representative of two independent experiments. (E) Q-RTPCR for mRNA levels of mTOR, p70S6K and 4EBP1 in P1 NPCs treated with MB. N = 3 in each group. *p < 0.05; **p < 0.01, ***p < 0.001 compared with control group.

FIGURE 6

MB restrains NPC proliferation in vivo. (A) Detection of NPC proliferation with Nestin and BrdU staining. Left: representative confocal microscopy images (100×) of Nestin (green) and BrdU (red) at anterior and lateral part ventricular wall of SVZ in MB or vehicle treated rats. Arrows indicate Nestin⁺/BrdU⁺ cells. Right: quantification of Nestin⁺ cells and Nestin⁺/BrdU⁺ cells in the SVZ. (B) Representative confocal microscopy images (400×) of Nestin (green) and BrdU (red) at anterior and lateral part ventricular wall of SVZ. (C) Western blot assay for mTOR, p70S6K and cyclin D1 expression at SVZ in MB or vehicle-treated rats. (D) Statistical analysis for mTOR, p70S6K and cyclin D1 proteins at SVZ in MB or vehicle-treated rats. *p < 0.05; **p < 0.01 compared with control group.