IKKβ is expressed in the adult hippocampal NSCs and was knocked down in adult hippocampal NSCs of GFAP-CreERT2/IKKβflox/flox mice
To determine the expression of IKKβ in NSCs from the hippocampal DG or primary cultured adult hippocampal NSCs, IHC and ICC analyses were performed. These analyses revealed IKKβ expression in the hippocampal DG cells positive for the NSC markers, Nestin, GFAP, and SOX2 (Fig. 1A–C) and in Nestin- and SOX2-positive primary cultured adult hippocampal NSCs (Supp Fig. 1D–E). To assess changes in downstream pathways upon IKKβ deletion in hippocampal NSCs of the GFAP-CreERT2/IKKβflox/flox mice, western blot analysis was used to evaluate NF-κB signaling in the hippocampal DG samples from vehicle-treated (control) and IKKβ cKD mice. This analysis revealed that the expression of IKKβ, phospho-IKKβ (pIKKβ), phospho-IκB (pIκB), and phospho-RelA (pRelA) were significantly lower by 48%, 36%, 39.5%, and 42%, respectively, in the IKKβ cKD mice when compared with the vehicle-treated control mice (Fig. 1F–G), indicating effective IKKβ/NF-κB conditional knockdown in the hippocampal DG.
Locomotion, spatial learning, and memory are enhanced in GFAP-CreERT2/IKKβflox/flox mice
Locomotor activity evaluation using the open-field test revealed that locomotion was significantly increased in the IKKβ cKD mice when compared with vehicle-treated control mice (Fig. 2A). We observed that the IKKβ cKD group spent more time at the center than the vehicle-treated control group (Fig. 2B–C). Moreover, evaluation of spontaneous alternation in the Y-maze test, which represents the willingness of the mice to investigate new environments, revealed that the percentage of spontaneous alternation was similar between vehicle-treated and IKKβ cKD mice (Fig. 2D). However, the IKKβ cKD group showed significantly more arm entries throughout the Y-maze test than the vehicle-treated control mice (Fig. 2E), suggesting hyperactivity in the IKKβ cKD mice, which was consistent with the open-field test results. These findings suggest that IKKβ disrupts locomotor activity in mice.
Several memory tasks were used to evaluate learning and memory function in IKKβ cKD mice. First, we tested spatial working memory and cognitive flexibility. The one-hour interval object location test of short-term memory revealed that the preference for the novel object location (NOL) was significantly increased in the IKKβ cKD mice when compared with the vehicle-treated control mice (Fig. 2F), suggesting significantly improved short-term location memory in the IKKβ cKD mice. In the 24-hour interval object location test, the IKKβ cKD mice showed that they could remember the fixed object and spent significantly more time sniffing the relocated object, whereas the vehicle-treated control group spent equal time sniffing both locations (Fig. 2G). Total object exploration time was similar between the vehicle-treated and the IKKβ cKD mice during NOL tests (Supp Fig. 1A–C). These data suggest that IKKβ could inhibit mouse short-term and long-term spatial memory. Secondly, we assessed spatial memory using the Morris water maze test. During training trials, swimming speed was not significantly different between the vehicle-treated mice and IKKβ cKD mice (Supp Fig. 2). In the acquisition phase, the two groups did not differ in how quickly they learned the location of the hidden platform (Fig. 2H). In the probe test after the acquisition phase, both the vehicle-treated mice and IKKβ cKD mice showed significantly increased target quadrant occupancy when compared with any other quadrant occupancy (Fig. 2I). Moreover, when the platform position was shifted, escape latency analysis revealed that IKKβ cKD mice were significantly faster than the vehicle-treated mice throughout the test (Fig. 2J−L). These data suggest that although IKKβ cKD does not alter spatial learning in the training phase of the water maze test, it promotes spatial memory in the probe and reversal phases.
Adult hippocampal NSC proliferation is increased in GFAP-CreERT2/IKKβflox/flox mice
First, we assessed adult hippocampal NSC proliferation by counting the number of BrdU (S-phase marker) positive cells in the SGZ of the hippocampal DG in the wildtype (WT) and vehicle (sunflower oil) treated mice 2.5 hours after a single BrdU injection. Because the number of BrdU-positive cells was not significantly different in WT vs vehicle-treated mice (Supp Fig. 3A–B), we used vehicle-treated mice as the control group in subsequent experiments. To investigate the role of IKKβ in hippocampal NSC proliferation in the adult hippocampal DG, we first examined the proliferation of SOX2-positive cells (a marker for actively proliferating type 2a NSCs) in the vehicle-treated mice and IKKβ cKD mice. This analysis revealed that the number of BrdU/SOX2 double-positive cells was significantly higher 2.5 hours after BrdU administration in the IKKβ cKD mice when compared with vehicle-treated control mice (Fig. 3A–B), suggesting that IKKβ inhibits the proliferation of type 2a NSCs in the hippocampal DG. Next, we performed experiments after one and three days to evaluate the overall BrdU-positive cell proliferation in the hippocampal DG. Notably, the total number of BrdU-positive cells in the hippocampal DG was significantly higher in IKKβ cKD mice at one and three days, when compared with the vehicle-treated control mice, suggesting that IKKβ inhibits NSC proliferation in the adult hippocampal DG (Fig. 3C–D). This study provides valuable insight into the critical role of IKKβ in regulating adult NSCs proliferation in the hippocampus, particularly in exerting inhibitory effects on type 2a NSCs and the overall NSC population in the DG.
To investigate the molecular mechanism by which IKKβ affects the proliferation of adult mouse hippocampal NSCs, we counted the Ki67 (proliferation marker) positive cells and evaluated the expression of cell cycle regulators in primary cultured adult hippocampal NSCs after IKKβ shRNA transfection. First, we subjected primary cultured adult hippocampal NSCs to ICC to determine SOX2, Nestin, and Nanog (NSC marker) expression and flow cytometry to determine Nestin and Nanog expression. To verify the adult hippocampal NSC phenotype, we measured the expression of SOX2, Nestin, and Nanog by immunostaining (Supp Fig. 4A−C), and flow cytometry analysis demonstrated 98% of Nestin-positive cells and 83% of Nanog-positive cells (Supp Fig. 4D). The number of Ki67-positive cells with SOX2 co-expression in the adult hippocampal NSCs was significantly increased in the IKKβ shRNA-transfected group when compared with the control or the control shRNA-transfected group (Fig. 3E–F). The expression of cyclin D1 and CDK4 (G1-phase cell cycle progression markers) and cyclin E1 and CDK2 (S-phase cell cycle progression markers), was significantly upregulated in the IKKβ shRNA-transfected group when compared with the control- or control shRNA-transfected group, while the expression of the cell cycle inhibitors, p15Ink4B (G1 phase) and p27Kip1 (S phase), was significantly downregulated (Fig. 2G–H). Taken together, these results suggest that IKKβ decreases hippocampal NSC proliferation by inhibiting the cell cycle.
The survival of adult hippocampal NSCs is decreased by regulating cleaved caspase-3 and the Bax family in GFAP-CreERT2/IKKβflox/flox mice
To determine the role of IKKβ in the survival of proliferating NSCs in the hippocampal DG, the number of BrdU-positive cells in the hippocampal DG was counted on days 5, 14, and 28 after five consecutive BrdU injections into vehicle-treated control and IKKβ cKD mice. This analysis revealed that the number of BrdU-positive cells was significantly increased on days 5, 14, and 28 in the IKKβ cKD mice when compared with vehicle-treated control mice (Fig. 4A–B). Next, we evaluated the survival rate of proliferating NSCs by counting the number of BrdU-positive cells three hours after the five consecutive days of BrdU injection as well as 28 days after. The survival rate of proliferating NSCs from between 5 and 28 days BrdU-positive cells was significantly enhanced in the IKKβ cKD mice when compared with the vehicle-treated control mice (Fig. 4C). Moreover, to assess the role of IKKβ in the apoptosis of adult NSCs in the hippocampal DG, we performed immunostaining for cleaved caspase-3 (a pro-apoptotic protein) and western blot analysis of Bax and cytochrome c (a pro-apoptotic protein) or Bcl-2 (an anti-apoptotic protein). This analysis revealed that the number of cleaved caspase-3-positive cells was significantly decreased in the IKKβ cKD mice when compared with vehicle-treated control mice (Fig. 4D–E). In addition, Bax, cytochrome c, and cleaved caspase-3 were significantly downregulated in the hippocampal DG of IKKβ cKD mice when compared with the vehicle-treated control group, whereas Bcl-2 was significantly upregulated (Fig. 4F). These results indicate that IKKβ reduces the survival of NSCs in the hippocampal DG by promoting apoptosis and underscore its pivotal role in modulating key factors associated with NSC survival and apoptosis across various stages of neurogenesis.
Adult hippocampal NSC neuronal differentiation is increased in GFAP-CreERT2/IKKβflox/flox mice
To investigate the role of IKKβ in the neural differentiation of adult NSCs in the hippocampal DG, double IHC analysis was used to detect cells that were positive for doublecortin (DCX), an immature neuronal marker, and BrdU or neuronal nuclei (NeuN), a mature neuronal marker, and BrdU. The number of DCX/BrdU double-positive cells was significantly increased in the IKKβ cKD mice when compared with vehicle-treated control mice (Fig. 5A–B). Furthermore, the ratio of DCX/BrdU double-positive cells to BrdU-positive cells was significantly increased in the IKKβ cKD mice when compared with vehicle-treated control mice (Fig. 5C). Similarly, the number of NeuN/BrdU double-positive cells was significantly increased in the IKKβ cKD mice when compared with vehicle-treated control mice (Fig. 5D–E) and the ratio of NeuN/BrdU double-positive cells to BrdU-positive cells was significantly higher in the IKKβ cKD (Fig. 4F). However, the ratio of GFAP/BrdU double-positive cells (gliogenesis) to BrdU-positive cells was not significantly different between the IKKβ cKD mice vs the vehicle-treated mice (Supp Fig. 3C–D). Collectively, these data reveal that IKKβ interferes with immature and mature neural differentiation of adult NSCs in the hippocampal DG.
Neuronal differentiation of hippocampal NSCs is suppressed by downregulating β-catenin and NeuroD1 expression in GFAP-CreERT2/IKKβflox/flox mice
To examine whether IKKβ regulates the neuronal differentiation of NSCs through the β-catenin and NeuroD1 pathways in the hippocampal DG, we used IHC and western blot analysis to assess β-catenin and NeuroD1 levels and found that their expression was significantly upregulated in the hippocampal DG of IKKβ cKD mice when compared with the vehicle-treated control group (Fig. 6A–B). To investigate the potential direct interaction between NF-κB and β-catenin in the hippocampal DG, we performed Co-IP. This revealed that when cell lysates were immunoprecipitated with an antibody against NF-κB, β-catenin was also found in the pellet, indicating their physical association, whereas no protein bands were observed in cell lysates that were not immunoprecipitated (Fig. 6C). These findings support the hypothesis that NF-κB directly interacts with β-catenin in the hippocampal DG and have significant implications for the intricate signaling networks that control NSC-derived neural differentiation in the adult hippocampus. IHC analysis showed that the ratio of DCX (a marker of immature neurons)/NeuroD1 double-positive cells to DCX-positive cells in the hippocampal DG was significantly increased in the IKKβ cKD group when compared with the vehicle-treated control group (Fig. 6D–E). These results indicate that IKKβ may be involved in NeuroD1-induced neural differentiation derived from NSCs associated with β-catenin in the hippocampal DG.
In addition, we assessed the expression of β-catenin and NeuroD1 in isolated mouse hippocampal NSCs transfected with IKKβ or β-catenin shRNA using western blot and ICC analyses. Western blot revealed that when compared with control- or control shRNA-transfected adult hippocampal NSCs in the neural differentiation condition, the expression of IKKβ, pIKKβ, and pRelA was significantly downregulated in the IKKβ shRNA-transfected adult hippocampal NSCs, whereas β-catenin, NeuroD1, and βIII-tubulin were significantly upregulated (Fig. 7A–B). ICC revealed that the ratio of βIII-tubulin/NeuroD1 double-positive cells to βIII-tubulin-positive cells was significantly increased in the IKKβ shRNA-transfected adult hippocampal NSCs when compared with the control- or control shRNA-transfected adult hippocampal NSCs in the neural differentiation condition for seven days (Fig. 7C–D). However, the expression of β-catenin, NeuroD1, and βIII-tubulin was significantly downregulated in the β-catenin shRNA-transfected adult hippocampal NSCs when compared with the control- or control shRNA-transfected adult hippocampal NSCs (Fig. 7E–F). ICC analysis showed that the number of βIII-tubulin-positive cells was significantly decreased in the β-catenin shRNA-transfected adult hippocampal NSCs when compared with the control- or control shRNA-transfected adult hippocampal NSCs in the neural differentiation condition (Fig. 7G). Overall, these results suggest that IKKβ negatively regulates neural differentiation from adult hippocampal NSCs by inhibiting the β-catenin and NeuroD1 pathways.