Ischemic Injury-Induced CaMKIIδ and CaMKIIγ Confer Neuroprotection Through the NF-κB Signaling Pathway

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. Electronic supplementary material The online version of this article (10.1007/s12035-018-1198-2) contains supplementary material, which is available to authorized users.

. Knockdown of C2dat2 or C2dat1/2 inhibited the activation of the NF-κB signaling pathway in N2a cells.
(A) Knockdown of C2dat2 (si-C2dat2) or both C2dat1 and C2dat2 (si-C2dat1+2) blocked the upregulation of CaMKIIδ and the activation of the NF-κB signaling pathway induced by OGD/R in N2a cells. N2a cells were transfected with si-C2dat2, si-C2dat1 +si-C2dat2, or si-NT. Two days after transfection, the cells were subjected to OGD/R. Representative images from one of at least three independent experiments were shown. (B) Quantification of the levels of IKKα, IKKβ, NF-κB and Bcl-xL by densitometry analysis. The mean ± S.E.M. from three independent experiments were calculated and plotted. ns, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 vs. control by One-Way ANOVA. con, control. Table S1. List of real time PCR primers used in the study.

Genotyping of p65 Knockout Mice
Mouse genotyping was conducted by polymerase chain reaction (PCR) using REDExtract-N-Amp™ Tissue PCR Kit according to the manufacturers' instructions (Sigma, St Louis, Missouri) (15). Mouse livers were cut into pieces (2-3 mm) and the mixtures of extraction solution and tissue preparation solution were added. The samples were incubated at room temperature for 30 min and then boiled for 3 min. The neutralization solution buffer was then added. The PCR reaction was set up by mixing 10 µl REDExtract-N-Amp Tissue PCR Red Mix, 6 µl of the primers, and 4 µl samples. The PCR primers for p65 were: CCTATAGAGGAGCAGCGCGGG (sense), AATCGGATGTGAGAGGACAGG (antisense for WT), AAATGTGTCAGTTTCATAGCCTGAAGAACG (antisense for p651/2). The thermal cycling conditions were as follows: an initial denaturation step at 94 °C for 5 min; 35 cycles including denaturation at 94 °C for 45 s; annealing at 54 °C for 45 s; extension at 72 °C for 90 s; and then a final extension at 72 °C for 5 min. The amplified PCR product was analyzed by standard agarose gel electrophoresis.

MCAO surgery
Transient focal cerebral ischemia was induced in mice by intraluminal occlusion of the left middle cerebral artery (MCAO) for 50 min as described previously (31). Achievement of ischemia was confirmed by monitoring regional cerebral blood flow with laser speckle contrast imager (Pericam). For reperfusion, the suture was withdrawn after the MCAO. Sham-operated mice underwent the similar operations to expose the carotid arteries without the suture insertion. After recovery, animals were returned to their cages with free access to food and water. Mice were sacrificed at 24h, 48h, 72h and 96h after reperfusion. Mouse brains were removed quickly for biochemical analysis. Part of the brain tissue was snap frozen in isopentane for cryostat sectioning. Serial coronal sections (1 mm apart) were stained with 2, 3, 5-triphenyltetrazolium chloride monohydrate (TTC; Sigma-Aldrich, St. Louis, MO) for assessing size of infarction. Tissues from the ischemic core and surrounding penumbra were isolated and snap frozen in liquid N2 for western blotting analysis.

Primary cortical neuron preparation and culture
The primary neuron culture was prepared as described previously (31). The pregnant rats and mice were anesthetized with 5% isoflurane and euthanized. Fetuses were removed and the cortices dissected from individual pups in ice-cold Hank's Balanced Salt Solution. The tissues were treated with 200 Units papain solution at 37°C for 4 min followed by 3 washes with trypsin inhibitor at 37°C for 4 min each. Tissues were mechanically dissociated through pipetting up and down several times until no obvious particles were seen. The cells were then plated at a density of 60 × 10 3 cells/cm 2 in Poly-D-Lysine-coated culture dishes with neurobasal medium containing B-27 supplements, L-Glutamine and penicillin/streptomycin (100 U/mL / 0.1 mg/mL). Neuronal Cultures were incubated at 37°C in an incubator with 5% CO2 and neurons grown for 8-10 days were used in the study.

Cell lines and cell culture
Mouse Neuro 2A (N2a) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultured in Minimum Essential Medium Eagle (MEM) with Eagle's salt and L-glutamine supplemented with 10% FBS (Invitrogen, Carlsbad, CA) at 37 °C with 5% CO2.

In vitro ischemia by oxygen-glucose deprivation/reoxygenation (OGD/R)
To mimic ischemic-like conditions in vitro, the growth medium was replaced with deoxygenated glucose-free Hanks Balanced Salt Solution. Cells were placed into a temperature-controlled (37±1°C) anaerobic chamber (Forma Scientific, Marietta, OH) that contains a gas mixture composed of 1% O2, 5% CO2 and 94% N2 for 3 h (N2a) or 1 h (primary neuronal cells). After OGD exposure, the medium was replaced with the original glucose-containing medium and cultured under normoxic condition for different times at 37 °C with 5% CO2. Control cell cultures were not deprived of oxygen and glucose and always placed in normoxic glucosecontaining medium, or harvest immediately after OGD treatment (t=0 min). OGD conditions were optimized to ensure cell viability before (con) and immediately after OGD (t=0 min) does not differ more than 5-10%.

Cell Survival Assays
N2a cells survival was assayed by Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan), according to the manufacturers' instructions. Briefly, N2a cells were plated at a density of 1 × 10 5 cells per well in 24-well plates. After siRNA transfection and/or drug treatment, CCK-8 solution containing a highly water-soluble tetrazolium salt WST-8 [2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] was added to cells in each well, followed by incubation for 2 h. Cell proliferation/viability was determined by measuring the OD at 450 nm. Percent over control was calculated as a measure of cell viability. Primary neuronal cell viability was assessed by propidium iodide (PI) uptake and retention of calcein-AM based on the methods as previously described (31, 33). Cultured neurons were washed with 1xPBS and incubated with 20 nM calcein-AM viability dye (Invitrogen, Carlsbad, CA) and 1 µg/ml PI in PBS at 37°C for 30 min. Images were captured under an Olympus 1X71 inverted system microscope with 10X objective lens, and the Calcein-AM or PI stained cells were analyzed by Image J software (NIH, Bethesda, MD). Cells were counted in at least 10 random fields in each image. Cell viability was expressed as the ratio of calcein-AM-positive cells to the sum of calcein-AM-positive and PI-positive cells.

Plasmid and siRNA transfection
Myc-DDK-CaMKIIδ plasmid was obtained from OriGene Technologies (Rockville, MD) was transfect with Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA). For primary neuron cells, the P3 Primary Cell 4D-Nucleofector reagent was used for siRNAs or plasmid transfection according to the manufacturers' instructions (Lonza, Walkersville, MD). The DsiRNAs of CAMK2D and CAMK2G, C2dat1-and C2dat2-targeted siRNAs, and a control non-targeting siRNA (negative control) were from Integrated DNA Technologies (IDT, Coralville, IA). The siRNAs were transfected into N2a cells using DharmaFECT transfection reagent (Lafayette, CO) or Cell Line Nucleofector V reagent (Lonza Walkersville, MD) according to the manufacturers' instructions. 24h or 48 h after transfection, N2a cells and primary neurons were subjected to OGD treatment, followed by reoxygenation under normoxic culture conditions for various times (0, 12 and 24 h).

Preparation of samples and RNA extraction
Total RNAs from N2a or primary neurons were extracted using TRIzol LS Reagent or RNeasy mini kit (QIAGEN, Germantown, MD). OD260 and OD280 of total RNA were measured by Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT). RNA purity was evaluated based on OD260/280 ratio and RNA integrity was assessed using standard denaturing agarose gel electrophoresis. The total amount of RNA obtained did not differ significantly between the samples.

Primers
A list of real-time PCR primers used in the study and their sequences are provided in Table S1.

Real-time RT-qPCR
cDNA synthesis from 1 μg total RNAs from N2a cells or 0.3 μg of total RNAs from primary neurons were carried out using the iScript cDNA synthesis kit (Bio-Rad, Richmond, CA). The levels of C2dat1, C2dat2, CAMK2A, CAMK2B, CAMK2G and CAMK2D transcripts was analyzed by real-time PCR using the SsoFast EvaGreen Supermix on CFX96 Real-Time PCR Detection System (Bio-Rad, Richmond, CA). Data were normalized using GAPDH as an internal control.

Western blotting analysis
Cells were harvested and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 5 mM EGTA, 20 μM leupeptin, 1 mM AEBSF, 1 mM NaVO3, 10 mM NaF and 1 × protein inhibitor cocktail). The brain tissue was homogenized with Dounce hand homogenizers and lysed in homogenization buffer (40 mM Tris, pH 7.4, 2mM EDTA, 640mM Sucrose, 40mM Na pyrophosphate, 20mM NaF, 40mM b-glycerophosphate, 0.4 mM NaVO3, and 1x protein inhibitor cocktail). Western blot analysis was carried out according to standard procedures. Briefly, 15 μg of proteins were separated on SDS-PAGE and transferred onto nitrocellulose membranes. After pre-blocking, the membranes were incubated with the appropriate primary antibody at 1:1000 dilutions at 4 °C overnight. After washing, the membranes were incubated with secondary antibody at room temperature for 1 h. Proteins were detected with the enhanced chemiluminescence (ECL) kit (Thermo Fisher). The images were captured on X-ray films and band intensity was quantified by Image J software (NIH, Bethesda, MD).