Ischemic Injury-Induced CaMKIIδ and CaMKIIγ Confer Neuroprotection Through the NF-κB Signaling Pathway
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Ca2+/calmodulin-dependent protein kinase II (CaMKII) has long been implicated in neuronal injury caused by acute ischemia/reperfusion (I/R). However, its precise role and regulatory mechanisms remain obscure. Here, we investigated the role of the CaMKII family in neuronal survival during I/R. Our data indicated that CAMK2D/CaMKIIδ and CAMK2G/CaMKIIγ were selectively upregulated in a time-dependent manner at both transcriptional and protein levels after acute ischemia. Overexpression of CaMKIIδ promoted neuronal survival, while their depletion exacerbated ischemic neuronal death. Similar to CaMKIIδ, knockdown of CAMKIIγ resulted in significant neuronal death after I/R. We further identified CaMKIIδ2 as the subtype that is selectively induced by I/R in primary neurons. The induction of CaMKIIδ was controlled in part by a pair of long non-coding RNAs (lncRNAs), C2dat1 and C2dat2. C2dat2, similar to C2dat1, was upregulated by I/R and cooperated with C2dat1 to modulate CaMKIIδ expression. Knockdown of C2dat1/2 blocked OGD/R-induced CaMKIIδ expression and decreased neuronal survival but did not affect the levels of CaMKIIγ, indicating specific targeting of CAMK2D by C2dat1/2. Mechanistically, I/R-induced CaMKIIδ and CaMKIIγ caused the upregulation of IKKα/β and further activation of the NF-κB signaling pathway to protect neurons from ischemic damage. Genetically, downregulating p65 subunit of NF-κB in mice increased I/R-induced neuronal death by blocking the activity of CaMKII/IKK/IκBα/NF-κB signaling axis. In summary, CaMKIIδ and CaMKIIγ are novel I/R-induced genes that promote neuronal survival during ischemic injury. The upregulation of these CaMKII kinases led to activation of the NF-κB signaling pathway, which protects neurons from ischemic damage.
KeywordsStroke In vitro ischemia CaMKII Long non-coding RNA Neuroprotection
polymerase chain reaction
long non-coding RNA
CAMK2D-associated transcript 1
CAMK2D-associated transcript 2
central nervous system
calcium/calmodulin-dependent kinase II
IκB kinase complex
middle cerebral artery occlusion
mouse Neuro 2A
Stroke is a worldwide health problem that leads to high rates of death and neurological disability in adults. Focal cerebral ischemia is a type of stroke that inflicts brain damages by reducing blood flow and halting oxygen supply to the brain. The mechanisms underlying cerebral ischemia injury are complex and still less understood. Despite the therapeutic advances in recent years, there remain limited treatment options for this disease.
The family of calcium/calmodulin-dependent kinase II (CaMKII) comprises of four isoforms (CaMKIIα, β, γ, δ) encoded by different genes that display distinct and overlapping expression [1, 2, 3]. The CaMKII holoenzyme is a homo- or hetero-multimer that is assembled by 8 to 12 isoforms. It is by far the most abundant protein kinase in the brain. CaMKII is multifunctional and plays important roles in synaptic transmission and plasticity that affect behavior, learning, and memory. At molecular level, it modulates a wide range of cellular processes including calcium (Ca2+) homeostasis, gene transcription, receptor function, and cytoskeletal alterations [1, 4, 5, 6, 7, 8, 9]. It has been implicated in both neuronal death and survival, and its precise role in brain ischemia remains to be determined. I/R injury induces many genes and pathways involved in inflammation, apoptosis, and oxidative stress . Among them, the nuclear factor-κB (NF-κB) signaling pathway is a major mediator of these complex biological processes, and dysregulation of NF-κB transcriptional activity has been linked to neurodegenerative diseases and numerous inflammatory conditions . The regulatory mechanisms and functions of the NF-κB pathway remain to be fully elucidated in cerebral ischemia.
The regulation of individual CaMKII isoform expression and activity is complex and not fully understood. We have previously reported a novel CAMK2D-associated long non-coding RNA (lncRNA) – C2dat1 in neurons. C2dat1 is induced by I/R and targets CAMK2D for upregulation in murine neuronal cells and mouse I/R models. Increased CaMKIIδ promotes neuronal survival by activating the NF-κB signaling pathway . Compared to CaMKIIδ, CAMK2G/CaMKIIγ is a less-known member of the CaMKII family and its role in the CNS has just begun to emerge. It has been shown that the upregulation of CaMKIIγ facilitates local anesthetic-induced nerve injury . CaMKIIγ transports sequestered Ca2+/calmodulin from the neuronal surface to the nucleus and triggers a highly cooperative activation of the nuclear CaMK cascade [5, 14]. Taken together, despite intense studies on CaMKII holoenzyme in post-insult neurons, its precise role, signaling mechanisms, and the specific isoforms involved in the process remain to be fully defined.
In this study, we conducted a thorough analysis on the CaMKII family kinases in murine neuronal cells and primary neurons subjected to I/R or in vitro ischemia. Our study revealed selective induction of CaMKIIδ and CaMKIIγ genes and proteins by ischemia. The upregulation of CaMKIIγ and CaMKIIδ acts to protect neurons from ischemic damage. We found that two novel lncRNAs, C2dat1 and C2dat2, mediated ischemia-induced upregulation of CaMKIIδ. Further, CaMKIIδ and CaMKIIγ activated the NF-κB signaling pathway to promote neuronal survival. This study shed lights to neuroprotective mechanisms evoked after ischemic injury in the brain.
Ischemia/Reperfusion (I/R) Induced Time-Dependent Upregulation of CAMK2D/CaMKIIδ and CAMK2G/CaMKIIγ
In contrast to N2a, in rat primary cortical neurons, CAMK2G and CAMK2A mRNAs were detected at higher levels as compared to CAMK2B and CAMK2D at the basal state (Fig. S1D). In response to OGD/R, a time-dependent upregulation of CAMK2D (~ 3-fold) was similarly detected, which peaked at 12 and 24 h and returned to baseline at 48 h, followed by another peak at 72 h. Additionally, distinct from N2a cells, we also observed an approximately 7-fold increase of CAMK2G that peaked at 24 h. No changes of CAMK2A andCAMK2B were observed (Fig. 1c). At the protein level, CaMKIIγ was the predominant CaMKII isoform detected using the pan-CaMKII antibody, while the levels of other isoforms were lower in primary neurons (Figs. 1d and S1D). In accordance with increased CAMK2G, CaMKIIγ was persistently upregulated over time. There was also a significant increase of CaMKIIδ detected by the pan-CaMKII antibody, which was validated using a CaMKIIδ-selective antibody in primary neurons (Fig. 1e). In contrast, no changes of CaMKIIα and CaMKIIβ were observed in neurons (Fig. 1d). Collectively, our data indicated that OGD/R selectively induced the upregulation of CAMK2D/CaMKIIδ and CAMK2G/CaMKIIγ in primary neurons, implying a potential role of these kinases in ischemic-associated biological processes in neuronal cells.
Differential Expression and Induction of CAMK2D Subtypes by OGD/R in N2a Cells and Primary Neurons
CaMKIIδ and CaMKIIγ Promoted Neuronal Survival in Response to In Vitro Ischemia
The effect of CaMKIIγ on neuronal survival was determined by knockdown of endogenous CAMK2G using CAMK2G siRNA (si-CAMK2G) in rat primary neurons. As shown in Fig. 4d, e, knockdown of CAMK2G enhanced ischemic cell death after OGD/R as compared to the control cells transfected with non-targeting siRNA (si-NT). Thus, similar to CaMKIIδ, the induction of CaMKIIγ by I/R also protected neurons from ischemic damage.
Ischemia-Induced C2dat1 and C2dat2 lncRNAs Were Required for Upregulation of CAMK2D, but not CAMK2G, in Neuronal Cells
Next, murine neuronal cell survival was examined. Knockdown of C2dat1 or C2dat2 exacerbated OGD/R-induced neuronal cell death compared with the control (si-NT) in N2a (Fig. S2B) and murine primary neurons (Fig. 5d, e). Moreover, knockdown of both C2dat1 and C2dat2 resulted in greater cell death than knockdown each lncRNA alone, reaching approximately 60% cell death at 24 h post-OGD/R in N2a cells (Fig. S2B). Thus, C2dat1 or C2dat2 cooperated to promote neuronal survival by upregulating CAMK2D expression on post-ischemic neurons.
CaMKIIδ Acted Through the NF-κB Signaling Pathway to Promote Neuronal Survival
Knockdown of C2dat1 and C2dat2 Decreased CaMKIIδ Expression and Inhibited the NF-κB Signaling Activity. The NF-κB Signaling Pathway Also Acted Downstream of CaMKIIγ
The effect of CAMK2G knockdown on the NF-κB signaling pathways was also examined in neurons. Our data showed that knockdown of CAMK2G similarly blocked the activation of the NF-κB signaling activity by downregulating IKKα, IKKβ, and NF-κB expression, blocking IκBα degradation, and reduced Bcl-xL expression in primary neurons. Meanwhile, knockdown of C2dat1 and C2dat2 blocked the upregulation of CaMKIIδ but did not affect the expression of CaMKIIγ (Fig. 7a, top lane), implying that the C2dat lncRNAs selectively targeted CAMK2D. Interestingly, knockdown of CAMK2G appeared not only to have depleted CaMKIIγ but also reduced the expression of CaMKIIδ (Fig. 7a). These results again verified that C2dat1 and C2dat2 specifically targeted CAMK2D, and both CaMKIIδ and CaMKIIγ signaled through the NF-κB signaling pathway in primary neurons.
Loss of p65 NF-κB Facilitated Ischemia-Induced Neuronal Death and Decreased CaMKIIδ and CaMKIIγ Expression
CaMKII as one of the most abundant protein kinase families in the CNS has long been implicated in cerebral ischemia, but its function in neuronal survival remains controversial. Meanwhile, the specific isoforms involved and their regulatory mechanisms are still largely unknown. This study aims at deciphering the function and signaling mechanisms of CaMKII kinases in ischemic damage in the brain. We reported for the first time selective induction of two CaMKII isoforms, namely CaMKIIδ and CaMKIIγ, by I/R. In particular, CaMKIIγ has not been associated with cerebral ischemia in the past. Our study also revealed novel lncRNA-mediated regulation of CaMKIIδ expression and identified C2dat2 as a novel I/R-induced lncRNA, which cooperated with C2dat1 to modulate the expression of CAMK2D in response to I/R. Our data further demonstrated that CaMKIIδ and CaMKIIγ activate the NF-κB signaling pathways, which acted to promote neuronal survival during I/R. The neuroprotective roles of CaMKIIδ and CaMKIIγ may benefit therapeutic intervention of I/R-induced neuronal injury.
It is a classical view that the predominant CaMKII isoforms in brain are CaMKIIα and CaMKIIβ, the former is the dominant form in forebrain, the latter in the cerebellum . It is believed that CaMKIIδ and CaMKIIγ are ubiquitously distributed, but their levels in the brain are much lower than CaMKIIα and CaMKIIβ [3, 17, 18, 19]. Therefore, CaMKIIδ and CaMKIIγ have received less attention in the neuronal system. Here, for the first time, we demonstrated that I/R selectively induced both CaMKIIδ and CaMKIIγ in primary neurons. Using the pan-CaMKII antibody that recognizes all four isoforms of CaMKII, we found that CaMKIIδ, but not CaMKIIα or CaMKIIβ, was the predominant isoform expressed in N2a neuronal cells. In contrast, in murine primary neurons, CaMKIIγ was shown to be the most abundant isoform, followed by CaMKIIα and then CaMKIIδ/β. The low expression of CaMKIIδ/β in isolated neurons challenges the traditional view described above, although it is possible that this was attributed to the low reactivity of the pan-antibody towards CaMKIIδ/β. Nonetheless, the high expression of CaMKIIγ and its persistent upregulation by I/R was indeed a novel finding that implies a potentially important role of this isoform in neurons. Moreover, the fold of upregulation of CAMK2G was much greater than CAMK2D at the peak time (7- vs. 3~4-fold), although the difference was smaller at protein levels. Overall, these findings suggest that CaMKIIγ may be functionally important to neurons in the brain. We also further investigated the specific CaMKIIδ isoforms expressed in the neurons and N2a cells, and their response to OGD/R. Interestingly, we found that all four subtypes of CaMKIIδ were upregulated by OGD/R in N2a cells, while only CaMKIIδ2 was upregulated by OGD/R in neurons. The selective induction of CaMKIIδ2 is intriguing, which could be due to (1) different subtypes of CaMKIIδ was expressed in N2a and primary neurons with CaMKIIδ2 being the predominant form in neurons (others are minimally expressed); (2) the mechanism that mediated the upregulation of CaMKIIδ2 may be different in the neurons as compared to N2a, although knockdown of C2dat1 or 2 did not differentially impact the subtypes induced by OGD/R in neurons vs. N2a cells (data not shown). This (difference in CaMKIIδ subtypes induced by OGD/R in neurons vs. N2a) may also explain the different levels of CaMKIIδ detected by pan-CaMKII antibody in N2a vs. primary neurons, since the antibody may preferentially recognize one subtype of CaMKIIδ but not the others. To test this, we re-examined the levels of CaMKIIδ using a CaMKIIδ-specific antibody (presumably recognizing most subtypes of CaMKIIδ). This antibody was able to detect CaMKIIδ with greater sensitivity and demonstrated high expression of CaMKIIδ in neurons as well as N2a (Fig. S1). In contrast, CAMK2A/CaMKIIα and CAMK2B/CaMKIIβ were not induced by OGD/R, which is in line with the finding that activated CaMKIIα did not play any significant role in regulating ischemic cell death . Taken together, our study demonstrated high expression and selective induction of CaMKIIδ and CaMKIIγ by I/R in neuronal cells. These findings have identified CaMKIIδ and CaMKIIγ as new players in ischemic injury in the brain.
Our previous study reported the discovery of C2dat1, which was induced by I/R in murine MCAO models and neuronal cells . In this study, we described the identification of another novel CAMK2D-associated lncRNA - C2dat2, which had similar function as C2dat1 despite targeting a different region of the CAMK2D gene. Knockdown of C2dat1 and/or C2dat2 increased neuronal cell death and selectively abolished OGD/R-induced CAMK2D/CaMKIIδ upregulation, but did not affect the expression of CAMK2G gene and CaMKIIγ protein, implying this regulation is likely isotype specific as it exclusively targets CAMK2D, and there are no overlapping sequences between CAMK2D and those of CAMK2A, 2B, and 2G.
Ischemic insult to the brain not only causes progressive neuronal cell death but also induces adaptive responses which act to reduce damage and promote neuronal survival. Mechanisms underlying these prosurvival adaptive responses are not fully understood. Our study has identified the NF-κB pathway as one of the major signaling pathways activated downstream of CaMKIIδ and CaMKIIγ. We showed that increased NF-κB signaling activity conferred neuroprotection in N2a and murine primary neurons. NF-κB is a master regulator of inflammation and immune responses . It is potently activated in brain tissues from stroke patients and ischemic insulted animals . The activation of NF-κB induces a variety of genes that could either promote or inhibit cell survival . Although many studies have identified the NF-κB pathway as a pro-inflammatory signaling pathway to promote neuronal cell death in response to injury, neuroprotective role of NF-κB has also been reported in ischemic injury . The pro-survival effect of NF-κB in neurons is mediated through the upregulation of several antiapoptotic proteins, including superoxide dismutase , Bcl-2, and Bcl-xL . Studies have shown that the spatial and temporal control of NF-κB activation may determine whether it promotes neuronal death or survival ; however, the precise mechanisms underlying its effect in ischemic injury remains to be fully defined [24, 27, 28, 29]. Using neurons obtained from p65+/− knockout mice, we showed the knockdown of NF-κB p65 moderately exacerbated OGD/R-induced neuronal cell death. The loss of p65 also resulted in downregulation of CaMKIIδ and CaMKIIγ, implying NF-κB p65 may feedback regulating the expression of CaMKIIδ and CaMKIIγ. This could be a mechanism that maximizes the neuroprotective activity of this signaling pathway. Further analysis is needed to reveal the underlying mechanisms.
In summary, our study has demonstrated I/R-induced selective upregulation of CaMKIIδ and CaMKIIγ and revealed important mechanisms that mediated the upregulation of CaMKIIδ. Our functional analysis demonstrated the neuroprotective role of both CaMKIIδ and CaMKIIγ in neuronal cells through activation of the NF-κB signaling pathway. This study sheds lights upon mechanisms of neuroprotection and recovery following cerebral ischemia, and provided novel molecular targets and signaling pathways that may be exploited for treatment of ischemic injuries in the brain.
Materials and Methods
Animals and Primary Cortical Neuron Preparation
Wild type C57BL/6J mice at 8–10 weeks old (25–30 g body weight, male, female) were used for middle cerebral artery occlusion (MCAO). Animals were housed in a temperature- and humidity-controlled animal facility with a 12-h light-dark cycle. Food and water were available ad libitum. The NF-κB p65 knockout mouse colony was described previously . Genotyping of p65 knockout mice was described in SI Materials and Methods. All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For preparation of rat primary cortical neurons, embryonic day 18 (E18) wild-type Sprague-Dawley rat pups, E15–17 wild-type C57BL/6J mouse pups, or E15–17 NF-κB p65 knockout mouse pups were used for primary cortical neuron preparation and culture as described previously . Details of primary neuron preparation are given in SI Materials and Methods.
Transient focal cerebral ischemia was induced in mice by intraluminal occlusion of the left middle cerebral artery (MCAO) for 50 min as described previously . Details of MCAO surgery and tissue collection and processing are provided in SI Materials and Methods.
Cell Lines, In Vitro Ischemia by Oxygen-Glucose Deprivation/Reoxygenation (OGD/R), and Cell Survival Assays
N2a cells (ATCC) and primary cortical neurons were subjected to in vitro ischemia by oxygen and glucose deprivation for 3 h (N2a) or 1 h (primary neurons), followed by incubation in glucose-containing medium under normoxic conditions for various times . N2a cells survival was assayed using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) . Primary neuronal cell viability was assessed by calcein-AM/propidium iodide (PI) staining assay as previously described [31, 33]. Details of cell culture, OGD/R, and cell survival assays are provided in SI Materials and Methods.
Plasmid and siRNA Transfection, RNA Extraction, Real-Time RT-qPCR, Western Blotting Analysis
Myc-DDK-CaMKIIδ plasmid (OriGene) plasmid and CAMK2D- or CAMK2G-targeted siRNAs were transfected using standard procedures according the manufacturers’ instructions. Total RNAs from tissues or cells were extracted using TRIzol LS Reagent or RNeasy mini kit (Qiagen). Sequences of real-time polymerase chain reaction (PCR) primers are provided in Table S1. Details of transfection procedures, RNA extraction, real-time RT-qPCR, and Western blotting analysis are given in SI Materials and Methods.
All measurements were performed by investigators who were blinded to the experimental conditions. Data was analyzed by using the Student’s t test for comparison between two groups (two-tailed) and one-way or two-way ANOVA for multiple groups followed by Bonferroni’s multiple comparisons test. All statistical analysis was done using GraphPad Prism IV software (GraphPad Software, La Jolla, CA, USA). All values are shown as the mean ± SEM of at least three independent experiments. A p value < 0.05 was considered statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant).
The author is particularly indebted to Drs. Zhenlong Zhao, Shuying Dong, Mohammad Iqbal Hossain Bhuiyan, Jinjun Zhao, Dengming Lai, Linsong Chen, Huan Liu, Junting Cai, Fengyan Zhao, Victoria Veroli, and Zhongling Zhang for their technical support and helpful discussions during the study. This work was supported in part by the National Institutes of Health grant R21NS096946 (Wang, QJ), Department of Defense award PC150190 (Wang, QJ), NIH NS038118 (Sun). National Nature Science Foundation of China grant NSFC81701185 (Xu) and Science and Technology Planning Project of Guangdong Province 2016A020215120 (Ye).
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
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