The Role and Mechanism of Transglutaminase 2 in Regulating Hippocampal Neurogenesis after Traumatic Brain Injury

Abstract

Traumatic brain injury usually results in neuronal loss and cognitive deficits. Promoting endogenous neurogenesis has been considered as a viable treatment option to improve functional recovery after TBI. However, neural stem/progenitor cells (NSPCs) in neurogenic regions are often unable to migrate and differentiate into mature neurons at the injury site. Transglutaminase 2 (TGM2) has been identified as a crucial component of neurogenic niche, and significantly dysregulated after TBI. Therefore, we speculate that TGM2 may play an important role in neurogenesis after TBI, and strategies targeting TGM2 to promote endogenous neural regeneration may be applied in TBI therapy. Using a tamoxifen-induced Tgm2 conditional knockout mouse line and a mouse model of stab wound injury, we investigated the role and mechanism of TGM2 in regulating hippocampal neurogenesis after TBI. We found that Tgm2 was highly expressed in adult NSPCs and up-regulated after TBI. Conditional deletion of Tgm2 resulted in the impaired proliferation and differentiation of NSPCs, while Tgm2 overexpression enhanced the abilities of self-renewal, proliferation, differentiation, and migration of NSPCs after TBI. Importantly, injection of lentivirus overexpressing TGM2 significantly promoted hippocampal neurogenesis after TBI. Therefore, TGM2 is a key regulator of hippocampal neurogenesis and a pivotal therapeutic target for intervention following TBI.

Keywords: hippocampus; neurogenesis; neuronal stem/progenitor cell; transglutaminase 2; traumatic brain injury.

Figure 1

TGM2 is expressed in hippocampal NSPCs and upregulated after TBI. (a) Representative images of TGM2 immunostaining in the subgranular zone (SGZ) of Nestin-creER^T2;tdTamato mice. The regions within the dotted white boxes are shown in a higher magnification 3D view (right panel). Scale bars: 50 µm (left and middle panels), 5 µm (right panel). (b) Schematic (upper panel) and representative image (lower panel) of Nestin-creER^T2;tdTamato mice at 7 days post-injury (dpi). Scale bar: 100 µm. (c,d) Representative images (c) and quantification (d) of TGM2 immunostaining in SGZ at 7dpi. Scale bar: 20 μm. (e) Quantification of Tgm2 mRNA levels in SGZ at 7 dpi by real-time qPCR. n = 4 mice per group. Data are represented as means ± SEM; two-tailed t-test, * p < 0.05, ** p < 0.01.

Figure 2

Deletion of TGM2 in NSPCs inhibits hippocampal neurogenesis after TBI in vivo. (a) Schematic illustration for analyzing neurogenesis in vivo. (b–d) Representative images (b) and quantification (c,d) of BrdU (red) and DCX (green) immunostainings of dentate gurus of Tgm2 iKO and WT littermates at 7 dpi. Arrowheads indicate BrdU⁺DCX⁺ cells. Scale bar: 50 µm. (e–g) Representative images (e) and quantification (f,g) of BrdU⁺ cells (f) and BrdU⁺NeuN⁺ cells (g) in dentate gurus of Tgm2 iKO and WT littermates at 21 dpi. n = 4 mice per group. Data are represented as means ± SEM; two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

TGM2 promotes neurogenesis of NSPCs in in vitro. (a) Schematic illustration for analyzing proliferation of NSPCs in vitro. Cultured E13.5 NSPCs were transduced by lenti-NC, lenti-Tgm2-OE, or lenti-shTgm2 for 48 h, then treated with control medium or microglial conditioned medium (MCM) for 48 h, followed by BrdU labeling. The ratio of BrdU⁺GFP⁺/GFP⁺ cells was quantified. (b,c) Representative images (b) and quantification (c) of BrdU immunostaining of lenti-NC, lenti-Tgm2-OE or lenti-shTgm2-transduced NSPCs. Scale bars: 20 µm. (d) Schematic illustration for analyzing differentiation of NSPCs in vitro. Cultured E13.5 NSPCs were transduced by lenti-NC, lenti-Tgm2-OE, or lenti-shTgm2 for 48 h, then switched to differentiation medium treated with control medium or MCM for 48 h. The ratio of Tuj1⁺GFP⁺/GFP⁺ and GFAP⁺ GFP⁺/GFP⁺ cells were quantified. (e,f) Representative images (e) and quantification (f) of Tuj1 and GFAP immunostaining of the differentiation of NSPCs which were transduced with lenti-NC, lenti-Tgm2-OE or lenti-shTgm2 virus. Scale bars: 20 µm. n = 5 cultures. Data are represented as means ± SEM; two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Overexpression of TGM2 promotes neurogenesis after TBI. (a) Schematic illustration for analyzing neurogenesis in TBI hippocampi which were injected with lenti-NC, lenti-Tgm2-OE, or lenti-shTgm2 virus. (b–g) Representative images (b,c) and quantification (d–g) of BrdU (blue), DCX (pink) or NeuN (pink) immunostaining of hippocampal sections from Nestin-CreER^T2;tdTomato mice which were injected with lentivirus, tamoxifen, and BrdU at given time windows. Percentages of BrdU⁺ GFP⁺ tdTamato⁺ cells among GFP⁺ tdTamato⁺ cells were quantified to examine proliferative potential of lentivirus-transduced NSPCs at 7 dpi (c) and 21 dpi (f), respectively. Percentages of BrdU⁺ GFP⁺ tdTamato⁺ DCX⁺ cells (newborn immature neurons) or BrdU⁺ GFP⁺ tdTamato⁺ NeuN⁺ cells (newborn mature neurons) among BrdU⁺ GFP⁺ tdTamato⁺ cells were quantified to determine the ability of neuronal differentiation of lentivirus-transduced NSPCs at 7 dpi (d) and 21 dpi (g), respectively. Scale bars: 50 µm. n = 4 mice per group. Data are represented as means ± SEM; two-tailed t-test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Tgm2-deletion alters the expression of genes related to cell proliferation and neural differentiation. (a) Workflow diagram for transcriptome sequencing of Tgm2 iKO and WT NSPCs under proliferating or differentiating conditions. (b) Volcano plot illustrating the differentially expressed genes (DEGs) between Tgm2 iKO and WT NSPCs under differentiating conditions. Red, upregulated genes; Blue, downregulated genes. (c) Bar plot depicting the significantly enriched GO terms (biological processes, BP) and KEGG pathways of downregulated genes during neural differentiation of TGM2-null NSPCs. (d) Volcano plot illustrating the DEGs between Tgm2 iKO and WT NSPCs under proliferating conditions. Red, upregulated genes; blue, downregulated genes. (e) Bar plot depicting the significantly enriched GO terms (biological processes, BP) and KEGG pathways of downregulated genes during proliferation of TGM2-null NSPCs. (f) Venn plot illustrating the co-downregulated genes in processes of proliferation and differentiation. (g) Netplot depicting the linkages of TOP 30 co-downregulated genes as a network in processes of cell proliferation and neural differentiation.

Figure 6

Deletion of Tgm2 dysregulates the expression of genes associated with the proliferation and differentiation of NSPCs. (a) Heat map diagrams of differentially expressed Notch and MAPK signaling-pathway genes between WT and Tgm2 iKO NSPCs in the differentiation process, as well as co-downregulated genes in processes of proliferation and differentiation of Tgm2 iKO NSPCs. (b–d) Quantitative PCR analysis validated the downregulation of genes associated with Notch signaling (b), MAPK and PI3-Akt signaling pathways (c) and co-downregulated genes in both proliferation and differentiation processes (d) using a new culture of Tgm2 iKO NSPCs under differentiating conditions. n = 4 cultures. Data are represented as means ± SEM; two-tailed t-test, * p < 0.05, ** p < 0.01, *** p < 0.001.