Neuroglobin promotes neurogenesis through Wnt signaling pathway

Abstract

Neuroglobin (Ngb) has been demonstrated by our lab and others to be neuroprotective against neurological disorders including stroke. However, the roles of Ngb in neurogenesis remain elusive. Neurogenesis can occur in adulthood and can be induced by pathological conditions in the brain such as stroke, and significantly contributes to functional recovery, thus enhancing endogenous neurogenesis may be a promising therapeutic strategy for neurodegenerative diseases. In this study we aimed to investigate the roles of Ngb in neurogenesis using Lentivirus overexpressing Ngb (Lv-Ngb). We show that Ngb overexpression promoted the proliferation of neural progenitor cells (NPC) marked by increased neurosphere number and size. Ngb overexpression also enhanced neuronal differentiation of cultured NPC under differentiation conditions. Moreover, subventricular injection of Lv-Ngb in mice after middle cerebral artery occlusion (MCAO) increased PSA-NCAM positive neuroblastoma cells and Tuj1 positive immature neurons, suggesting that Ngb overexpression promotes neurogenesis in mice brain after stroke. We further show that the pro-neurogenesis effect of Ngb overexpression might be mediated through Dvl1 up-regulation, and subsequent activation of Wnt signaling, indicated by increased nuclear localization of beta-catenin. These results suggest that Ngb may play an important role in promoting neurogenesis in neurodegenerative diseases such as stroke, which may eventually benefit the development of stroke therapeutics targeting neurogenesis through Ngb upregulation.

Fig. 1. Cultured neurospheres have phenotypes of neural progenitor cells.

Neurosphere was cultured from isolated adult mouse SVZ region. Neurospheres were formed after 7 days. Neurosphere phenotype was examined by immunostaining for neural progenitor cell marker nestin and doublecortin

Fig. 2. Ngb overexpression promotes NPC proliferation.

Lentivirus containing Ngb (Lv-Ngb) or GFP (Lv-GFP) were transduced to neurosphere at day 2 of culture. NPC proliferation was examined by measuring neurosphere number and size, and BrdU corporation 6 days later. a Lv-GFP transduction into neurosphere and microscopic imaging of GFP; b The efficiency of Lv-Ngb transduction was tested by measuring Ngb protein level in neurosphere using Western blot. (n = 4, * p < 0.05vs Lv-GFP). c Representative images of cultured neurosphere; d Quantification of relative neurosphere number, e Quantification of relative neurosphere size (n = 6, *p < 0.05 vs Lv-GFP); f Representative images of BrdU immunostaining; g Quantification of the percentage of BrdU positive cell (n = 4, *p < 0.05 vs Lv-GFP)

Fig. 3. Ngb overexpression promotes neuronal differentiation of cultured NPC.

NPC was cultured in differentiation medium (DMEM/F12 + 2%FBS) and Lentivirus containing Ngb or GFP gene was transduced on day 2 of culture. Differentiated cells were examined by immunocytochemistry using anti-Tuj1 (immature neuron maker) and anti-GFAP antibodies. NPC differentiation was examined by immunostaining for Tuj1 (immature neuron) and GFAP (astrocytes). a Representative images of Tuj1 and GFAP immunostaining; b Quantification of percentages Tuj1 positive cells, c Quantification of average neurite length (n = 4,* p < 0.05 vs Lv-GFP)

Fig. 4. Wnt signaling is involved in the promotional effect of Ngb overexpression on neurogenesis.

To investigate the mechanisms of the neurogenesis promotional effect by Ngb overexpression, we examined the protein levels of Dvl1 and β-catenin, the two key components of Wnt signaling, after Lv-Ngb transduction in cultured neurosphere. a Representative Western blot images for Dvl1 and β-catenin using cell lysate of cultured NPCs. β-actin was used as loading control. b Quantification of Dvl1 protein level; c Immunostaining for β-catenin in cultured NPC; d Representative Western blot images and e quantification of β-catenin in isolated nuclear proteins. Histone-H3 was used as nucleus loading control (n = 4,* p < 0.05 vs Lv-GFP). We further used a Wnt inhibitor, IWR-endo, to test the involvement of Wnt signaling in the neurogenesis promotional effect by Ngb overexpression; f Relative neurosphere number after Lv-Ngb transduction and IWR-endo treatment; g Relative neurosphere size after Lv-Ngb transduction and IWR-endo treatment; h The percentage of Tuj positive cell numbers in NPC differentiation after Lv-Ngb transduction and IWR-endo treatment (n = 4, *p < 0.05 vs Lv-GFP, ^#p < 0.05 vs Lv-Ngb)

Fig. 5. Ngb overexpression improved neurogenesis in mice after MCAO.

To test the effect of Ngb overexpression on neurogenesis after stroke, we performed transient MCAO in mouse and administered Lv-Ngb or Lv-GFP through ICV injection at 2 days after stroke. a Ngb protein level was measured by Western blot to confirm Ngb overexpression by Lv-Ngb transduction at 5 days post stroke. NPC proliferation was assessed at 7 days post stroke using PSA-NCAM immunostaining. b PSA-NCAM immunostaining in SVZ area; c Quantification of PSA-NCAM positive cells in SVZ regions; d PSA-NCAM immunostaining in peri-infarct cortex region; e Quantification of PSA-NCAM positive cells in SVZ regions. Furthermore, differentiated immature neurons were assessed at 10 days post stroke using Tuj1 immunostaining. f Tuj1 immunostaining in hippocampus region; g Quantification of Tuj1 positive cells in hippocampus region; h Tuj1 immunostaining in peri-infarct cortex region; i Quantification of Tuj1 positive cells in peri-infarct cortex area. (n = 6,* p < 0.05 vs sham, ^#p < 0.05 vs MCAO Lv-GFP)

Fig. 6. Effect of Ngb overexpression on Dvl1 and β-catenin expression after stroke.

To further investigate the involvement of Wnt signaling in Ngb-overexpression-induced neurogenesis, we tested the protein levels of Dvl1 and β-catenin in peri-infarct mice brain at 5 days after stroke. a Representative images of Western blot for Dvl1 and β-catenin from peri-infarct brain lysate; b, c Quantification of Dvl1 and β-catenin; d Representative images of Western blot for β-catenin from isolated nuclear protein; e Quantification of β-catenin in nuclear extract. (n = 4, *p < 0.05 vs sham, ^#p < 0.05 vs MCAO Lv-GFP)