Adeno-associated Virus-mediated Ezh2 Knockdown Reduced the Increment of Newborn Neurons Induced by Forebrain Ischemia in Gerbil Dentate Gyrus

Abstract

It is established that neurogenesis of dentate gyrus is increased after ischemic insult, although the regulatory mechanisms have not yet been elucidated. In this study, we focused on Ezh2 which suppresses gene expression through catalyzing trimethylation of lysine 27 of histone 3. Male gerbils were injected with adeno-associated virus (AAV) carrying shRNA targeting to Ezh2 into right dentate gyrus 2 weeks prior to forebrain ischemia. One week after ischemia, animals were injected with thymidine analogue to label proliferating cells. Three weeks after ischemia, animals were killed for histological analysis. AAV-mediated knockdown of Ezh2 significantly decreased the ischemia-induced increment of proliferating cells, and the proliferated cells after ischemia showed significantly longer migration from subgranular zone (SGZ), compared to the control group. Furthermore, the number of neural stem cells in SGZ significantly decreased after ischemia with Ezh2 knockdown group. Of note, Ezh2 knockdown did not affect the number of proliferating cells or the migration from SGZ in the non-ischemic condition. Our data showed that, specifically after ischemia, Ezh2 knockdown shifted the balance between self-renewal and differentiation toward differentiation in adult dentate gyrus.

Keywords: Adeno-associated virus vector; Gerbil; Neurogenesis; Polycomb group complex; Transient forebrain ischemia.

Fig. 1

AAV vector carrying Ezh2 shRNA was expressed whole DG of hippocampus. a Construct of RNA-mediated gene silencing system delivered by AAV vector. b Four different shRNA constructs were transduced in gerbil fibroma cells for 48 h. Ezh2 shRNA-4 decreased Ezh2 expression by 60–70%. c Experimental design of this study. d Representative images of DG treated with Ezh2 shRNA-4 3 weeks after transient forebrain ischemia. The mature neuron marker, NeuN, shows the granule cell layer. The GFP signals were prominent in cell bodies and dendrites of neurons. AAV, adeno-associated virus; CMV, cytomegalovirus promoter; EdU, 5-ethynyl-2′-deoxyuridine; EmGFP, emerald green fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindole; GCL, granule cell layer; SGZ, subgranular zone. Scale bar, 200 μm in low magnification images and 20 μm in high magnification images (in the yellow rectangulars). Data represent mean ± SD

Fig. 2

Ezh2 knockdown abolished increment of neurogenesis and promoted neural differentiation after transient forebrain ischemia. a Representative images of dentate gyrus treated with Ezh2 shRNA-4 3 weeks after transient forebrain ischemia. EdU-positive cells were prominent in subgranular zone and granule cell layer. b Quantification of EdU-positive cells in the dentate gyrus. Transient forebrain ischemia significantly increased EdU-positive cells of the negative shRNA group. On the other hand, Ezh2 knockdown abolished the post-ischemic increase of the EdU-positive cells. c Quantification of distances between subgranular zone and EdU-positive cells. Transient forebrain ischemia increased the distance between subgranular zone and EdU-positive cells both in negative shRNA (not significant) and Ezh2 RNA groups (p < 0.001). d Representative images of measurement method of distances between subgranular zone and EdU-positive cells (two-way arrows). DAPI, 4′,6-diamidino-2-phenylindole; EdU, 5-ethynyl-2′-deoxyuridine; GCL, granule cell layer; GFP, green fluorescent protein; SGZ, subgranular zone. Scale bar = 200 μm in low magnification images (a), 20 μm in high magnification images (in the yellow rectangulars, a), and 20 μm in d. ANOVA followed by Tukey. Data represent mean ± SD. * p < 0.05. *** p < 0.001. N.S., not significant

Fig. 3

Ezh2 knockdown decreased neural stem cells in SGZ after forebrain ischemia. a Representative images of dentate gyrus treated with Ezh2 shRNA-4 3 weeks after transient forebrain ischemia. Sox2-positive cells were prominent in subgranular zone. b Quantification of Sox2-positive cells in the dentate gyrus. Transient forebrain ischemia significantly decreased Sox2-positive cells of negative shRNA group (p < 0.05) and Ezh2 shRNA group (p < 0.01). GCL, granule cell layer; GFP, green fluorescent protein; SGZ, subgranular zone. Scale bar = 200 μm in low magnification images and 20 μm in high magnification images (in the yellow rectangular). ANOVA followed by Tukey. Data represent mean ± SD. * p < 0.05. ** p < 0.01. N.S., not significant

Fig. 4

The concept of this study. After birth, neural progenitor cells in SGZ are capable of proliferation and multi-potential differentiation but are unable to self-renew. One week after birth, these progenitors are committed to neuronal lineage. Two weeks after birth, they differentiate and migrate into GCL. It takes about 2 months from birth of neural progenitor cells to mature granule cells fully incorporated into the hippocampal circuits. Our data suggested that, specifically after ischemia, Ezh2 knockdown shifted the balance between self-renewal and differentiation toward differentiation in adult DG. EdU, 5-ethynyl-2′-deoxyuridine; GCL, granule cell layer; SGZ, subgranular zone