Involvement of NFκB signaling in mediating the effects of GRK5 on neural stem cells

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Involvement of NFκB signaling in mediating the effects of GRK5 on neural stem cells Yun Zhang, Guang-li Shen, Li-juan Shangguan, Yang Yu, Mao-lin Hen Department of Neurology, Beijing Shijitan Hospital, Capital Medical University, 10 TieYi Road, Haidian District, Beijing 100038, PR China

art i cle i nfo

ab st rac t

Article history:

Nuclear factor κB (NFκB) signaling plays ubiquitous roles in inflammation, immune response

Accepted 21 February 2015

and neurogenesis. G protein-coupled receptor kinase 5 (GRK5) can protect neurons from degeneration. GRK5 also mediates tumor necrosis factor-α (TNFα)-induced NFκB signaling through the phosphorylation of IκBα. Here, we show that NFκB signaling is involved in neural

Keywords:

stem cell (NSC) differentiation. The IκBα/p65 pathway was activated by phorbol myristate

Neuron stem cell G protein-coupled receptor NFκB

acetate (PMA), a stimulator of protein kinase C (PKC). Once the NFκB was activated, the initial stage of neural differentiation was induced, with an increased level of GRK5 in NSCs. This finding was reversed in response to the NFκB inhibitor N-acetyl cysteine (NAC). To evaluate

Neurogenesis

the effect of GRK5-NFκB signaling crosstalk on NSC neurogenesis and apoptosis, GRK5 was

Apoptosis

knocked down by siRNAs in cell culture. SiRNAs against GRK5 not only impaired neural differentiation and axogenesis, but also induced apoptosis of NSC. GRK5 knockdown affected the transcription of NFκB, phosphorylation of the liver kinase B1 (LKB1) and the activity of caspase 3, thereby modulated neurogenesis and apoptosis. Taken together, our findings reveal a novel function of GRK5 in neurogenesis and provide insight into the molecular mechanisms underlying neurodevelopmental disorders and neurodegenerative diseases. & 2015 Elsevier B.V. All rights reserved.

1.

Encinas et al., 2013; Furutachi et al., 2013). Although many

Introduction

extrinsic factors and intrinsic proteins have been identified in Abnormal neurogenesis contributes to a variety of disorders, including neurodevelopmental defects, stroke and neurodegenerative diseases (Lazarov and Marr, 2013). During adult neurogenesis, NSCs follow a differentiation pattern that is similar to embryonic neurogenesis (Gonzalez-Perez et al., 2012;

Abbreviations: AD,

Alzheimer's disease; GPCR,

IL-1β, interleukin-1β; NAC,

the regulation of neurogenesis, the underlying signaling pathways and molecular mechanisms remain poorly understood (Govindarajan and Kempermann, 2014). G protein-coupled receptor (GPCR) kinases (GRKs) regulate GPCR signaling by inducing receptor desensitization. Among

G protein-coupled receptor; GRK, G-protein receptor kinase; IL-6,

N-acetyl cysteine; NFκB,

nuclear factor κB; NPCs,

PKA, protein kinase A; PMA, phorbol myristate acetate; TNFα, n Corresponding author. E-mail address: [email protected] (M.-l. He).

neural progenitor cells; NSCs,

interleukin-6;

neuron stem cells;

tumor necrosis factor-α

http://dx.doi.org/10.1016/j.brainres.2015.02.041 0006-8993/& 2015 Elsevier B.V. All rights reserved.

Please cite this article as: Zhang, Y., et al., Involvement of NFκB signaling in mediating the effects of GRK5 on neural stem cells. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.02.041

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the GRK family members, GRK5 is ubiquitously expressed in mammalian tissues. GRK5 can regulate GPCR signaling via 7-transmembrane receptors, several of which have been implicated in neurodegenerative diseases (Liu et al., 2010; Suo and Li, 2010). Recent studies have demonstrated that GRK5 expression is decreased in Alzheimer's disease (AD) (Suo et al., 2004; Suo and Li, 2010). Furthermore, GRK5 deficiency alone can enhance axonal defects and promote apoptosis during aging, leading to dementia (Suo et al., 2007). Interestingly, GRK5 has been shown to interact with and affect NFκB signaling. NFκB proteins are a family of ubiquitously expressed transcription factors that regulate cellular proliferation, apoptosis and cell cycle progression (Imielski et al., 2012). GRK5 can mediate the phosphorylation of IκBα in Raw264.7 macrophages (Patial et al., 2009). As IκBα is one of the key factors in NFκB activation (Brasier, 2006; Imielski et al., 2012), TNFα-induced NFκB signaling may be mediated by GRK5 (Patial et al., 2009). In contrast, recent studies have demonstrated that NFκB plays a critical role in the regulation of GRK5 transcription in myocytes (Crampton and O'Keeffe, 2013). It is inferred that a reciprocal link may present between these two systems. Although some aspects of GRK5-dependent signaling have been well established (Suo et al., 2004; Suo and Li, 2010), its function in neurogenesis and apoptosis remains unclear. Based on previous studies, we hypothesized that GRK5 could enhance NFκB transcriptional activity and consequently promote neural differentiation and inhibit apoptosis of NSCs. In this study, we treated primary NSCs obtained from E14.5 rat embryos brain with specific GRK5 siRNA or activators of the NFκB pathway. We investigated whether GRK5 affects IκBα cellular levels and NFκB transcriptional activity. Moreover, we assessed the effects of GRK5 on NFκB-dependent phenotypes in vitro, such as NSCs proliferation and differentiation, axogenesis and apoptosis. Our observations provide new insight into the functional role of GRK5.

2.

2.2. Alterations in NFκB expression affect GRK5 protein levels and neural differentiation of NSCs/NPCs (neural progenitor cells) Moreover, we examined the effect of PMA and an NFκB inhibitor (NAC) on GRK5 protein levels and the differentiation of NSCs. We cultured NSCs in suspension culture in the presence of PMA or NAC alone or in combination for 24 h prior to differentiation. There were four groups in this study: control (without PMA nor NAC treatment), PMA(20 nM) treatment, NAC(5 mM) treatment, and PMAþNAC treatment (where NSCs were maintained in the presence of PMAþNAC) (Islam and Koch, 2012). Western blot was used to determine the effect of PMA7NAC on the protein levels of NFκB and GRK5 in NSCs. PMA increased GRK5, p65 and p50 protein levels (Fig. 2A and B, Po0.01). And NAC partially attenuated PMA-induced increase in GRK5, p65 and p50 levels (Fig. 2A and B, Po0.01). We deduced that inhibition of NFκB with NAC cannot entirely block the stimulation of PMA on this pathway. Decreased expression of p50 and p65 when NFκB was inhibited by NAC

Results

2.1. PMA increases GRK5 mRNA levels and protein levels in NSCs The NFκB proteins are a family of transcription factors that consist of five members: p65 (RelA), RelB, c-Rel, NFκB1 (p50 and its precursor 105) and NFκB2 (p52 and its precursor p100). The p65–p50 heterodimer is transcriptional activator of the canonical NFκB pathway. While the p50–p50 homodimer can repress the expression of their target genes (Yamamoto and Gaynor, 2004). PMA is known to activate several PKC isoforms, which can regulate the activation of NFκB in several tissue types (Churchill et al., 2008). Using RT-PCR, we determined the effects of PMA on mRNA expression of GRK5 in NSCs. As shown in Fig. 1A, 20 nM of PMA induced the expression of p65 and p50 within 1 h, and this effect was maintained for over 24 h. Importantly, GRK5 level increased after NSCs were treated with PMA (Fig. 1A). Moreover, Western blot analysis showed enhanced p65, p50 and GRK5 expression at 24 h postPMA treatment (Fig. 1B and C, Po0.01). These results suggest that PMA can increase the expression of NFκB and GRK5.

Fig. 1 – Effects of PMA on the expression of GRK5 and NFκB in cultured NSCs. NSCs were incubated with 20 nM PMA for different times. (A) GRK5, p65 and p50 mRNA levels were quantified using real-time PCR. The units were arbitrary with the control, and expressed as means7SD (N¼ 6). (B) GRK5, p65 and p50 NFκB proteins were analyzed using Western blot with β-actin as a loading control. (C) Semiquantitative analyses for Western blot in panel (B). nn, Po0.01 versus control; Student's t-test (N¼ 6).

Please cite this article as: Zhang, Y., et al., Involvement of NFκB signaling in mediating the effects of GRK5 on neural stem cells. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.02.041

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Fig. 2 – Alteration in NFκB expression affects GRK5 protein level and NSCs neural differentiation. (A) NSCs were incubated in the absence or presence of PMA, or NAC, or in combination for 24 h. The GRK5, p50 and p65 NFκB protein levels were analyzed using Western blotting, with β-actin as a loading control. (B) Semi-quantitative analyses of the Western blot in panel (A). All protein bands were quantitatively analyzed as a one band per lane. The units are arbitrary with the control, and expressed as means7SD (N¼ 6). (C) Alteration of NFκB function affects the neural differentiation of NSCs. Immunofluorescence analysis highlighted cells expressing nestin at day 3 (a–d) or Tuj1 at day 7 (e–h) in different groups. (i) 50.7% of the cells were nestinþ in the control, fewer (39.2%) nestinþ cells in PMA group, but more (68.3%) nestinþ cells in NAC group. (j) 55.9% of the cells were Tuj1þ in the control group, more (75.9%) in the PMA group, but less (36.3%) in the NAC group. N¼6–8 per group. n, Po0.05; nn, Po0.01 versus control. ##, Po0.01 versus PMA group. &&, Po0.01 versus NAC group. Tuj1: green. nestin: red. The nuclei were stained with DAPI. Bar, 50 μm. Please cite this article as: Zhang, Y., et al., Involvement of NFκB signaling in mediating the effects of GRK5 on neural stem cells. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.02.041

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was consistent with the involvement of NFκB in the induction of GSK5 expression. A neurosphere is a culture system composed of free-floating clusters of NSCs, which provides a method to investigate NPCs in vitro. Neurosphere was transferred into NeuroCult NSC differentiation medium for culture. Immunofluorescence analyses indicated that the differentiated neurons in the four groups were positive for Tuj1. The immunofluorescence analysis revealed cells expressing nestin at day 3 (Fig. 2C). In the NACs group 68.2% of the cells was nestinþ, which was significantly higher than the control (50.7%) (Fig. 2C, Po0.01). In the PMA treatment group, the percentage of nestinþ cells was 39.2%, which was lower than the control (Fig. 2C, Po0.01). Furthermore, many mature Tuj1þ neurons appeared at 7 days. In the PMA group, 75.9% of the cells were Tuj1þ, which was significantly higher than the control (55.9%) (Fig. 2C, Po0.01). In the NAC treatment group, the average percentage of Tuj1þ cells was only 36.3%, which was lower than the control (Fig. 2C, Po0.01). These data indicated that changes in the expression of NFκB signaling could regulate GRK5 expression and neural differentiation.

2.3. Activation of GRK5-NFκB signaling was involved in NSC/NPC neural differentiation To explore the function of GRK5 in neural differentiation of NSCs/NPCs, we treated NSCs with siRNA against GRK5. After transfection, the cells were treated with or without PMA for 24 h prior to differentiation. There were four groups in this study: the control siRNA, control siRNAþPMA, GRK5 siRNA, and GRK5 siRNAþPMA treatment. Western blot analysis was performed to determine the effect of PMA and GRK5 siRNA on the proteins levels of NFκB and GRK5 in NSCs. The cells treated with GRK5 siRNA showed a significant reduction in GRK5 levels compared with the control (Fig. 3). Importantly, the loss of GRK5 corresponded to a reduction of p65 protein in NSCs/NPCs (Fig. 3). The immunoblotting analysis showed the robust phosphorylation of IκBα with PMA treatment (Fig. 3A), whereas silencing of GRK5 partially prevented the PMAinduced phosphorylation of IκBα (Fig. 3, Po0.01). We compared p65 protein between the GRK5 siRNAþPMA and the GRK5 siRNA alone. PMA could still stimulate the expression of p65 protein even when GRK5 was blocked (Fig. 3, Po0.01). It was deduced that PMA stimulated NFκB signaling not only through GRK5, but also other signaling. The experiment showed a correlation between decreased GRK5 expression and expression of p65. Neurospheres were transferred into NeuroCult NSC differentiation medium for further culture. The majority of neurospheres attached to the plate following transfer into differentiation medium for 24 h. Immunofluorescence analyses indicated that the undifferentiated aggregates in the four groups were all positive for Sox2 (sex determining region Y-box 2). Sox2 is a transcription factor that is essential for maintaining self-renewal, or pluripotency, of undifferentiated embryonic stem cells. Sox2 plays a critical role in maintenance of NSCs. On day 1, 89.2% of the cells were Sox2þ in the GRK5 siRNA group, which was significantly higher than the control (78.9%) (Fig. 4A and B, Po0.01). In the PMA treatment group, the Sox2þ cells was 55.4%, which was lower than the control (Fig. 4A and B, Po0.01). After the NSCs/NPCs were subjected to

differentiation for 7 days, very few Sox2þ cells remained. Moreover, many mature Tuj1þ neurons appeared at 7 days. At day 7, 84.1% of the cells were Tuj1þ in the PMA group, which was significantly higher than the control (62.1%) (Fig. 4A and C, Po0.01). There were 53.2% of Tuj1þ cells in the GRK5 siRNA group which was lower than the control (Fig. 4A and C, Po0.05). The number of Tuj1þ cells in the GRK5 siRNAþPMA group was significantly higher than that of GRK5 siRNA group (Fig. 4A and C, Po0.01). The data indicated that GRK5 and PMA were involved in neural differentiation, suggesting that GRK5– NFκB Signaling plays a role in the neural differentiation of NSCs/NPCs.

2.4. Axogenesis was reduced in NSCs/NPCs using GRK5 siRNA To analyze the relationship between PMA and GRK5 with regard to axogenesis in NSCs/NPCs, PMA- or GRK5-induced morphological changes were examined for neuronal polarity. Tau1 was used as an axonal marker. Axon length was reduced in the neurons in the GRK5 siRNA group (89.6712.2 mm) compared to the control (114.4715.6 mm) (Fig. 4Ai, Ak and D, Po0.01). Moreover, the average axon length of neurons in the GRK5 siRNAþPMA group (118.0714.3 mm) was shorter than the control siRNAþPMA group (156.9722.0 mm) (Fig. 4Ak, Al and D, Po0.01). However, the average axon length of neurons in the GRK5 siRNAþPMA group was longer than that in the GRK5 siRNA group (Fig. 4Aj, Al and D, Po0.01). There was no significant difference in the average axon length of neurons between the

Fig. 3 – Knockdown of GRK5 affects NFκB protein levels and phosphorylation of LKB1 ps431 in NSCs/NPCs. (A) Representative Western blots for GRK5, p50, p65, pIκBα, and LKB1 ps431. (B) Semi-quantitative analyses of the Western blots in panel (A). All protein bands were quantitatively analyzed as a one band per lane. The units are arbitrary with control, and expressed as means7SD (N ¼6). *, Po0.05; **, Po0.01 versus control. ##, Po0.01 versus PMA group. &&, Po0.01 versus GRK5 siRNA group.

Please cite this article as: Zhang, Y., et al., Involvement of NFκB signaling in mediating the effects of GRK5 on neural stem cells. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.02.041

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GRK5 siRNAþPMA group and the control (Fig. 4Ai, Al and D, P40.05). LKB1 is a substrate of PKA and an important player in axogenesis (Imielski et al., 2012). Since phosphorylation of LKB1-S431 is a crucial step for initiation of axon outgrowth, we analyzed phosphorylation of LKB1-S431 after PMA treatment or GRK5 knockdown. We found that phosphorylation of LKB1 at Ser 431 was reduced after GRK5 knockdown (Fig. 3, Po0.05), which is consistent with a previous study in NFκB-deficient mice

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(Imielski et al., 2012). NF-κB controls the transcription of FOXO1 and PKA, regulating axogenesis. Taken together, we presumed that GRK5 might control the transcription of PKA and regulate axogenesis via NFκB.

2.5.

GRK5 knockdown increased apoptosis in NSCs/NPCs

Apoptosis on day 7 in NSCs/NPCs of different groups was assessed by fluorescence using fluorescein-conjugated Annexin-V. The

Fig. 4 – Activation of NFκB-GRK5 signaling is required for the neural differentiation of NSCs/NPCs. (A) Immunofluorescence analysis highlighted the cells coexpressing DAPI and Sox2 at day 1 (a–d) or Tuj1 at day 7 (e–h) in different groups. (i–l) Tau1 expression as an axonal marker in different groups at day 7. (B) Quantitative analysis showing a significant increase in Sox2þ NSCs/NPCs in the GRK5 siRNA group and a significant decrease in Sox2þ NSCs/NPCs in the PMA group. (C) Quantitative analysis showing a significant increase in Tuj1þ NSCs/NPCs in the PMA group and a significant decrease of Tuj1þ NSCs/NPCs in the GRK5 siRNA group. (D) Quantitative analysis showing that the axon length was reduced in the GRK5 siRNA group but extended in the PMA group. N¼ 6–8 per group. n, Po0.05; nn, Po0.01 versus control. #, Po0.05; ##, Po0.01 versus PMA group. &&, Po0.01 versus GRK5 siRNA group. Bar, 50 μm. Please cite this article as: Zhang, Y., et al., Involvement of NFκB signaling in mediating the effects of GRK5 on neural stem cells. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.02.041

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cells of the GRK5 siRNA group exhibited a more robust fluorescence than the control siRNA cells (Fig. 5A). Furthermore, there were more Annexin-Vþ neurons in the GRK5 siRNAþPMA group than the control siRNA or PMA group. These results are consistent with a previous study in GRK5 KO mice (Suo et al., 2007).

Caspases are cysteine proteases that cleave a set of cellular proteins to initiate apoptotic cascade. The expression and activity of pro-apoptotic molecules were examined. We found that PMA significantly inhibited caspase-3 activity, whereas knockdown of GRK5 significantly increased the levels of caspase-3 (Fig. 5D). There was no difference between the GRK5 siRNA and GRK5 siRNAþPMA group (Fig. 5D), where GRK5 siRNA fully blocked the effect of PMA on caspase-3. Because the Bcl-2 family is an important regulator in various apoptosis pathways, changes in the expression of either the pro-apoptotic or anti-apoptotic Bcl-2 family members can affect apoptosis. To explore whether GRK5 inhibits apoptosis by modulating Bcl-2 family, the protein level of Bcl-2 was determined using Western blotting. As shown in Fig. 5B and C, PMA increased Bcl-2 expression (1.570.1-fold of the control), whereas GRK5 siRNA downregulated Bcl-2 (0.670.07-fold of the control). In addition, PMA treatment restored the Bcl-2 level in the GRK5 siRNA group (Fig. 5B and C, Po0.01). Those results indicated that both GRK5 and NFκB could inhibit the apoptosis of NSCs/NPCs.

3.

Fig. 5 – Knockdown of GRK5 affects NSCs/NPCs apoptosis. (A) Annexin-V staining showing apoptosis with respect to neurons with propidium iodide and DAPI as controls. Cells coexpressing Annexin-V (green) with propidium iodide (red) were found in the GRK5 siRNA group. (B) Cells were harvested and then analyzed using Western blot with antibodies against Bcl-2. (C) Semi-quantitative analyses of the Western blot in panel (B). (D) Caspase-3 activity in neurons. The units were arbitrary with the control, and expressed as mean7S.D from six independent experiments. nn, Po0.01 versus control. ##, Po0.01 versus PMA group. &&, Po0.01 versus GRK5 siRNA group. Bar, 50 μm.

Discussion

We examined the potential effect of PMA (a PKC activator) on regulating the expression of GRK5 and NFκB in NSCs. PMA induced the expression of p65, p50, and GRK5 in NSCs. N-acetyl cysteine, an inhibitor of NFκB, exerted an opposite effect on their expression. Previous studies have shown that p50 and p65 subunit of NFκB can directly bind to GRK5 DNA in the nucleus and promote mRNA expression using EMSA or ChIP assays in myocytes (Islam and Koch, 2012). We treated NSCs with siRNA against GRK5 to analyze the function of GRK5 in NFκB activation. The loss of GRK5 was accompanied by a decrease in p65 protein in NSCs (Fig. 3). The activation of NFκB signaling was evident via the degradation of IκBα, as well as the phosphorylation and nuclear translocation of p65. Immunoblotting analyses showed strong phosphorylation of IκBα by PMA. Silencing of GRK5 partly attenuated PMA-induced IκBα phosphorylation (Fig. 3A). Although the regulation of NFκB by GRK5 is still controversial (Sorriento et al., 2008), our data indicated that GRK5 might influence p65 expression in NSCs. It was reported that the differentiation of neurospheres from rat embryos induced an up-regulation of GRK5 (Gurevich et al., 2004). Although the effects of GRK5 on cytogenesis have been extensively studied, the function of GRK5 in neurogenesis remains unclear. Here, we propose that GRK5-NFκB signaling pathway is involved in neurogenesis. Immunofluorescence analyses indicated that a higher number of Sox2þ cells presented in response to GRK5 knockdown at day 1. Moreover, the number of Sox2þ cells in the PMA treatment group was less than the control (Fig. 4). There was a higher number of Tuj1þ cells in the PMA group than the control at day 7. However, the number of Tuj1þ cells in the GRK5 siRNA group was less than the control at day 7 (Fig. 4). NAC decreased GRK5 protein levels when NSCs were treated with PMA (Fig. 2). The average percentage of Tuj1þ cells in the NAC treatment group was lower than the control (Fig. 2). Previous studies have demonstrated that NFκB is necessary for axon formation, survival and integration in the neuronal network in immature neurons (Imielski et al., 2012). Moreover,

Please cite this article as: Zhang, Y., et al., Involvement of NFκB signaling in mediating the effects of GRK5 on neural stem cells. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.02.041

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NFκB can stimulate NSCs/NPCs to release pro-inflammatory cytokines, such as TNFα, IL-6 and IL-1β, to regulate neural differentiation (Piotrowska et al., 2006; Das and Basu, 2008; Whitney et al., 2009; Schölzke et al., 2011). TNFα can induce neural differentiation via NFκB activation at a very early stage of neurogenesis (Zhang et al., 2012). Several studies have identified CCAAT/enhancer binding protein (C/EBP) β as an effector of NFκB signaling for the modulation of early neurogenesis. C/EBPβ promotes the neuronal linage differentiation of NPCs by binding to the promoter cis-elements of the neuronal markers Tuj1 and NeuroD (Paquin et al., 2005). In contrast, other reports have shown that the increased expression of C/EBPβ and signal transducer and activator of transcription (STAT) 3C in NSCs prevents neuronal differentiation (Uittenbogaard et al., 2007). NFκB activation may downregulate C/EBPβ expression and initiate NSC differentiation. However, the mechanism by which NFκB signaling suppresses C/EBPβ remains unclear (Zhang et al., 2012). The role of NFκB in neurogenesis is controversial, however, we provided evidence that GRK5 may mediate the differentiation of NSCs at very early stages of neurogenesis by activating NFκB signaling. We further examined the relationship between NFκB and GRK5 in axogenesis. Axon length was significantly reduced after GRK5 knockdown. The forkhead transcription factor Foxo1 and PKA kinase cascade have been shown to potentially integrate both axogenesis and neuronal survival (Imielski et al., 2012). LKB1 is a substrate of PKA and an important player in axogenesis (Barnes et al., 2007; Shelly et al., 2007). The phosphorylation of LKB1, which is a crucial step for the initiation of axon outgrowth, is decreased in GRK5-deficient NSCs/NPCs (Fig. 3). Taken together, GRK5-NFκB signaling may cooperate during two consecutive stages of neurogenesis, the proliferation/differentiation of NSCs/NPCs and the axon specification of young neurons. The function of GRK5 in apoptosis has been widely studied. GRK5 was reported to phosphorylate p53 and regulate p53mediated gene expression and apoptosis in response to DNA damage (Chen et al., 2010). Recently, active caspase-3þ cholinoceptive neurons were found in the hippocampus of GRK5 knockout (KO) mice but not wild-type (WT) mice (Suo et al., 2007). Thus, we investigated whether GRK5 could affect apoptosis of NSCs/NPCs. GRK5 knockdown sensitized apoptosis in cultured NSCs/NPCs (Fig. 5). Importantly, we found that GRK5 knockdown increased the levels of caspase-3 and strongly inhibited the expression of Bcl-2 in NSCs (Fig. 5). NFκB was shown to induce the expression of several different genes that protect neurons against apoptosis, including inhibitor-of-apoptosis proteins (IAPs), manganese superoxide dismutase (Mn-SOD) and Bcl-2 (Mattson and Camandola, 2001). Our results showed that GRK5 siRNA fully blocked effects of PMA on caspase-3 and partly inhibited effects of PMA on anti-apoptotic Bcl-2 expression (Fig. 5).We found that GRK5 siRNA inhibited anti-apoptotic Bcl2 expression and upregulated pro-apoptotic caspase-3 activity. Moreover, the anti-apoptotic effect of GRK5 could also be due to the maintaining activity of protein kinase A (PKA), a vital signaling pathway for cell survival and apoptotic resistance. M2 signaling is known to reduce cAMP levels and to downregulate PKA activity (Pemberton et al., 2000; Malbon et al., 2004; Tasken and Aandahl, 2004; Kiermayer et al., 2005). Accumulating evidence has indicated that M2 is primarily a presynaptic

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autoreceptor that inhibits ACh release. Impaired M2 desensitization by GRK5 silencing may result in prolonged presynaptic M2 signaling and consequently in reduced cAMP levels (Levey, 1996; Zhang et al., 2002). GRK5 dysfunction also promotes a deleterious cycle that further increases β-amyloid accumulation and exaggerates tau hyperphosphorylation which can significantly induce apoptosis. Apoptosis caused by GRK5 dysfunction may offset the anti-apoptotic effect of Bcl-2 induced by PMA. These results suggested that GRK5-NFκB signaling may play a role in regulating neuron apoptosis during neurogenesis. The mechanism by which GRK5 suppresses neuron apoptosis remains further investigation using in vivo models of neurodegenerative diseases. Taken together, our study and previous reports indicate that the GRK5 and NFkB pathways have partially overlapping roles in affecting NSCs differentiation, axogenesis and apoptosis. Importantly, our data may unveil novel aspects of the functions of GRK5 in neurogenesis.

4.

Experimental procedures

4.1.

Ethics statement

Animal use was in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. All experimental procedures were approved by the Animal Experiment Administration Committee of Beijing ShiJiTan Hospital, Capital Medical University.

4.2.

Neuronal cultures

Dissociated hippocampal neuronal cultures were prepared as previously described (Schwamborn and Püschel, 2004). Briefly, the hippocampus was dissected from E14.5 rat embryos and dissociated. The dissociated cells were cultured at a density of 2  105 cells per milliliter in NeuroCult NSC proliferation medium containing 20 ng/ml epidermal growth factor (EGF) and 10 ng/ml basic fibroblast growth factor (bFGF) (PeproTech, Inc.). The medium was changed every other day, and primary neurospheres were formed within 3–7 days. For adherent monolayer culture, primary neurospheres were dissociated with accutase and plated in plates or coverslips coated with poly-ornithine (Sigma) and fibronectin (R&D). Half of the medium was changed every other day.

4.3.

NFκB treatment

Neurospheres were maintained in NeuroCult NSC differentiation medium in the presence or absence of the PKC stimulator PMA (20 nM), or NFκB signaling inhibitor NAC(5 mM) or in combination for 24 h (Islam and Koch, 2012).

4.4.

GRK5 gene silencing

GRK5 siRNA sequences (against mouse GRK5) and control siRNA-scrambled sequences were purchased from Dharmacon (Dharmacon Research Inc., Lafayette, CO). Treated cells were analyzed for knockdown using Western blotting after 48 h of transfection.

Please cite this article as: Zhang, Y., et al., Involvement of NFκB signaling in mediating the effects of GRK5 on neural stem cells. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.02.041

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4.5. RNA isolation and reverse transcriptase real-time quantitative PCR Total RNA was isolated from NSCs using Trizol Reagent (Invitrogen) according to the manufacturer's instructions. The RNA integrity was evaluated following agarose gel electrophoresis, and the RNA concentration was determined using UV spectroscopy. RNA samples were treated with DNase I (Promega) for 30 min at 37 1C to remove DNA contamination. Reverse transcription of equal amounts of total RNA samples was performed using the reverse transcription kit (k1622) (Fermentas). A real-time PCR analysis was performed on the LightCycler480 (Roche) using the SYBR Green I kit (Roche). Each sample was tested in triplicate. Quantitative calculations of the gene of interest versus GAPDH were performed using the ΔΔCt method. The results were analyzed using the Genex Microsoft Excel plugin program (Bio-Rad). Validated primer sets directed against GRK5 (forward, 50 -GAAGGTTAAGCGGGAAGAGG-30 ; reverse, 50 TCCAGGCGCTTAAAGTTCAT-30 ), p65 (forward, 50 -CAA GTG CCT TAA TAG CAG GGC AAA-30 ; reverse, 50 –AGA GCT AGA AAG AGC AAG AGT CCA AT-30 ) and GAPDH (forward, 50 –TGAAGCAGGCATCTGAGGG-30 ; reverse, 50 –CGAAGGTGGAAGAGTGGGA G-30 ) were used for quantitative PCR amplification (Inoue et al., 1998; Islam and Koch, 2012).

4.6.

Immunofluorescence of cultured cells

Standard immunofluorescence was performed. Primary antibodies included an anti-nestin antibody (Sigma, N5413), antiSox2 antibody (Sigma, S9072), anti-Tau1 antibody (Abcam, ab32057) and anti-Tuj1 antibody (Covance Research Products, PRB-435 P). The cells were fixed for 20 min in 4% paraformaldehyde/PBS. After three rinses, the cells were treated with 0.5% Triton X-100 PBS for 20 min and blocked with 10% serum or 2% bovine serum albumin/PBS for 1 h. The cells were incubated with primary antibodies overnight at 4 1C. After rinsing three times, the cells were incubated with secondary antibodies at room temperature for 1 h and with 40 -6-diamidino-2-phenylindole (DAPI, Sigma) for 5 min. After three rinses with PBS, the cells were mounted with a coverslip using anti-fading aqueous mounting media (Biomeda) and analyzed under a fluorescence microscope.

4.7.

Western blotting

The protein concentration was determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA). All protein samples were denatured, electrophoresed on SDS/polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). An anti-GRK5 antibody (Santa Cruz Biotechnology, sc-565), anti-NFκB p65 antibody (Santa Cruz Biotechnology, sc-372), anti-NFκB p50 antibody (Santa Cruz Biotechnology, sc-7178), anti-bcl-2 antibody (Santa Cruz Biotechnology, sc-492), anti-LKB1antibody (Santa Cruz Biotechnology, sc-32245), anti-p-IκBα antibody (Santa Cruz Biotechnology, sc101714) and anti-β-actin antibody (Santa Cruz Biotechnology, sc1616) were detected by first incubating with the corresponding antibodies, followed by incubating with secondary antibodies. The β-actin served as a positive control for total protein. The bands were visualized using the enhanced chemiluminescence

kit (ECL, Amersham Biosciences, UK). The relative intensity of the bands was scanned and quantified using UVIPhoto and UVISoft UVIBand software V97.04 (UVI, UK).

4.8.

Assays for apoptosis and dying neurons

Apoptosis was analyzed using Western blotting with the Bcl-2 antibody and Annexin-V-FLUOS staining kit (Roche). To determine the activity of caspase-3, NSCs were lysed in lysis buffer and analyzed using a colorimetric caspase-3 assay kit (Chemicon International Co.) according to the manufacturer's instructions. Briefly, the cells were collected, washed with PBS and resuspended in a cell lysis buffer. After incubation on ice for 10 min, the lysates were centrifuged for 20 min at 12,000  g; the supernatants were collected, and the protein concentrations were determined. The cell lysates (100 μg) were mixed with reaction buffer containing the DEVD-pNA substrate (200 μM) for caspase-3 activity. The absorbance of each well was measured at 460 nm using an ELISA reader (Yu et al., 2012).

4.9.

Quantification and statistical analysis

Neuronal morphology was analyzed by staining with anti-Tuj1 and using the WASABI software (Hamamatsu), ImageJ (NIH) and Adobe Photoshop. The length of the axons was determined using Spot software (Diagnostic Instruments). The population of nestinexpressing cells, Sox2-expressing cells and Tuj1-expressing cells was counted from the total number of differentiated cells (DAPIlabeled). An automated stage was used and operated using Image-Pro Plus (IPP). Six to eight coverslips were counted in each group. A statistical analysis was performed using the SPSS statistical package. All values are expressed as the mean7SD. The differences were analyzed using Student's t-test or one-way ANOVA (when comparing means among more than 2 groups). A P value ofo0.05 was considered to be significant.

Conflict of interests No competing financial interest exists

Acknowledgments The present study was supported by a grant from the National Natural Science Foundation of China (No. 81200849), and a grant from the Beijing Municipal Natural Science Foundation (No. 7112068).

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