IKKβ inhibits cognitive memory and adult hippocampal neurogenesis via the β-catenin pathway

IKKβ inhibits cognitive memory and adult hippocampal neurogenesis via the β-catenin pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article IKKβ inhibits cognitive memory and adult hippocampal neurogenesis via the β-catenin pathway Kyung-Joo Seong, Bo-Ram Mun, Shintae Kim, Won-Seok Choi, Sung Joong Lee, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4591233/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The role of IκB kinase β (IKKβ) and its underlying mechanism in regulating cognitive memory and hippocampal neurogenesis were investigated using GFAP-CreERT2/IKKβ flox/flox transgenic mice, where the IKKβ gene is specifically knocked down in hippocampal neural stem cells (NSCs) to generate IKKβ conditional knockdown (cKD) mice. Our findings indicate that IKKβ cKD led to increased exploratory activity in the open-field test, promoted hyperactivity in the Y-maze test, and enhanced spatial learning and memory function in the object location and the Morris water maze tests. Furthermore, IKKβ cKD increased the proliferation of adult hippocampal NSCs by upregulating positive cell cycle regulators through the inhibition of negative cell cycle regulators. Neuronal differentiation of adult hippocampal NSCs was also increased by IKKβ cKD, affecting β-catenin signaling and Neurogenic differentiation 1. Additionally, IKKβ cKD enhanced NSC survival, as indicated by decreased cleaved caspase-3 levels and diminished Bax and Cytochrome c expression in the hippocampal dentate gyrus. These findings indicate that in hippocampal NSCs, IKKβ inhibits locomotion, cognitive function, and adult hippocampal neurogenesis by suppressing the β-catenin signaling pathway. Our findings highlight a key role for IKKβ in the inhibition of cognitive function and decrease in hippocampal neurogenesis through NF-κB signaling in adult NSCs. neural stem cell neurogenesis hippocampus memory IKKβ β-catenin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The IκB kinase (IKK) family is made up of the members, IKKα, IKKβ, and IKKγ (also known as nuclear factor kappa B [NF-κB] essential modulator), which act upstream of NF-κB, and are crucial regulators of the NF-κB signaling pathway [ 1 – 3 ]. The NF-κB complex, which is composed of p50 and RelA, is a key regulator of inflammation, cell survival, apoptosis, and neural plasticity in the canonical pathway [ 4 , 5 ]. Canonical NF-κB signaling is the most extensively studied NF-κB pathway [ 6 ] and it can be mediated by the activation of Toll-like receptor (TLR) 4 [ 7 ] and inflammatory factors like lipopolysaccharide [ 8 ] and tumor necrosis factor-alpha (TNF-α) [ 9 ]. IKKβ, an inhibitor of κB kinase, is required for NF-κB activation in the canonical pathway [ 10 ], whereas IKKα is essential for the activation of the noncanonical NF-κB signaling pathway [ 11 ]. Previous studies have underscored the diverse roles of IKKβ in various cell types, indicating its involvement in essential cellular functions. The absence of neuronal IKKβ is associated with the amelioration of Alzheimer’s disease pathology in amyloid precursor protein and tau transgenic mice [ 12 ]. Tamoxifen-induced IKKβ knockout and NF-κB inhibition in striatal neurons increase striatal neurodegeneration in mice with Huntington’s disease. [ 13 ]. IKK/NF-κB signaling inhibition promotes mesenchymal lineage specification and enriches functional mesenchymal stromal cells from human embryonic stem cells [ 14 ]. Hence, IKKβ/NF-κB activation may have negative and positive effects on neurogenesis, depending on physiological or pathophysiological conditions. Adult neurogenesis, the process of producing new neurons from neural stem cells (NSCs), occurs throughout life in the dentate gyrus (DG) of the hippocampus [ 15 ]. The hippocampus, which is crucial for regulating locomotor activity and spatial recognition, benefits from the continuous generation of new neurons through neurogenesis [ 16 , 17 ]. Radial glial cells are a prominent subtype of NSCs localized within the subgranular zone (SGZ) of the DG [ 18 ]. Remarkably, radial glial cells have a distinctive molecular profile that includes glial fibrillary acidic protein (GFAP) and stem cell markers, notably SRY-Box transcription Factor 2 (SOX2) and the neuroepithelial stem cell protein, Nestin [ 19 , 20 ]. This unique expression pattern confers radial glial cells with the remarkable ability to differentiate into neurons and astrocytes, thus substantially contributing to the hippocampal microenvironment and cognitive function [ 21 ]. Adult hippocampal neurogenesis is finely regulated by a multitude of endogenous and exogenous factors. Notably, physical activity and environmental enrichment promote neurogenesis and cognitive improvements [ 22 ], whereas systemic diseases and inflammation can have negative effects on neurogenic processes [ 23 ], thereby impacting hippocampal function and plasticity. As key factors in the intricate process of adult neurogenesis, radial glial cells have significant implications for brain plasticity and the maintenance of brain health [ 24 ]. However, despite considerable progress in understanding adult neurogenesis, the effects of radial glial cells in genetic mutation conditions are not fully established. NF-κB signaling is well known to regulate adult NSC proliferation and neuronal differentiation [ 25 , 26 ]. However, the impact of NF-κB signaling on hippocampal neurogenesis, specifically in hippocampal memory formation, is controversial. While previous studies have reported that NF-κB/RelA inhibition increases memory function and hippocampal or hypothalamic NSC proliferation and survival [ 27 , 28 ], the lack of NF-κB c-Rel impairs hippocampus-dependent fear memory [ 29 ]. This suggests a complex role for NF-κB signaling in the context of hippocampal memory formation and neurogenesis. Although molecular and functional studies indicate that NF-κB plays an important role in regulating adult neurogenesis in the hippocampus or hypothalamus, the role and underlining mechanism of IKKβ, the upstream regulator of NF-κB, in regulating memory function and adult hippocampal neurogenesis is unknown. The β-catenin pathway is involved in various stages of development, including the proliferation and differentiation of various stem cells [ 30 – 32 ]. β-catenin, a key downstream effector of the Wnt signaling pathway, increases the neuronal differentiation of NSCs in the hippocampal DG [ 33 , 34 ] by upregulating the expression of neuronal differentiation 1 (NeuroD1), a downstream target of β-catenin [ 35 , 36 ]. NeuroD1 overexpression is shown to promote neuronal differentiation in adult hippocampal NSCs, whereas its downregulation decreases the maturation of newborn neurons [ 37 ]. Taken together, it can be speculated that β-catenin regulates adult neurogenesis from NSCs in the hippocampal DG through the NeuroD1 transcription factor, which is responsible for CNS development. Some studies have reported that in human cancer cell, NF-κB regulates Wnt5a transcription by controlling the Wnt5a promoter B [ 38 ]. IKKβ physically interacts with β-catenin and decreases β-catenin-dependent transcriptional activation in SW480 cells [ 39 ]. β-catenin also interacts with NF-κB and NF-κB suppresses β-catenin expression in human breast cancer cells [ 40 ]. Wnt/β-catenin is a well-established key regulator of neurogenesis and it intricately interacts with various signaling pathways, including Notch, Shh, and NF-κB pathways [ 41 , 42 ]. However, despite extensive studies of the interplay between Wnt/β-catenin and the Notch, Shh, and NF-κB pathways, there is a significant gap in our understanding, particularly in the context of hippocampal neurogenesis. Specifically, the intricate regulatory dynamics of β-catenin signaling, under the control of IKKβ–NF-κB, in hippocampal neurogenesis, are not fully elucidated. IKKβ knockout causes embryonic lethality on embryonic day 14.5 because of liver degeneration [ 43 ]. This study sought to address this knowledge gap by providing valuable insight into the complex regulatory mechanisms governing neurodevelopment in the hippocampus. Here, we investigate the role and underlying mechanism of IKKβ in regulating cognitive function and hippocampal neurogenesis using GFAP-CreERT2/IKKβ flox/flox transgenic mice, in which IKKβ knockdown in adult hippocampal NSCs was induced by tamoxifen-inducible Cre recombinase or in primary cultured adult hippocampal NSCs transfected with IKKβ shRNA. Materials and methods Animals Seven-week-old GFAP-CreERT2/IKKβ flox/flox male mice, hereafter called IKKβ-conditional knockdown (IKKβ cKD) were provided by Dr. Lee Sung Joong (Seoul National University, Korea). The mice were maintained in standard conditions (22°C, 55% humidity, and a 12-hour light/dark cycle) with free access to food and water. The mice were divided into the IKKβ cKD group, which received intraperitoneal (I.P.) tamoxifen injection to knock down IKKβ in hippocampal NSCs, and the vehicle-treated control group, which was intraperitoneally injected with sunflower oil (vehicle). In this study, approximately 150 animals were sacrificed across the experiment groups, consisting of 70 mice in the control group and 80 mice in the IKKβ cKD group. All animal experiments were approved by the Animal Care and Use Committee of Chonnam National University. Treatment with Tamoxifen Tamoxifen (Sigma–Aldrich, T5648) was prepared in physiological sunflower oil (Sigma–Aldrich, S5007). The mice were I.P.-injected with tamoxifen (2 mg in 200 µl) once daily for five consecutive days. An equal concentration of ethanol in sunflower seed oil was I.P.-injected as negative control. All experiments began 7 days after the last tamoxifen injection [ 44 ]. 5-bromo-2’-deoxyuridine (BrdU) injection BrdU (Thermo Fisher, 000103) was dissolved in physiological saline and I.P.-injected into the mice at 50 mg/kg. To assess NSC proliferation in the DG, mice were sacrificed at three hours after a single BrdU injection. To assess the immature neural differentiation from BrdU-positive cells, mice were sacrificed 2.5 hours, one day, and three days after a single BrdU injection. To assess the survival rate of the newborn cells generated from NSCs or the mature neural differentiation rate of the NSCs, mice were sacrificed 28 days after BrdU injection once daily for five consecutive days. Brain tissue preparation for immunohistochemistry (IHC) Animals were transcardially perfused with 0.1 M PBS, followed by fresh cold 4% (v/v) paraformaldehyde in 0.1 M PBS. Brains were then collected and fixed overnight in 4% paraformaldehyde in 0.1 M PBS, at 4°C, and washed for six hours in PBS at 4°C. Sucrose-saturated brains were then embedded in O.C.T. compound (Leica Biosystems, 3801480), freeze-chilled in isopentane (-25°C), and stored at -80°C until sectioning. Brains were cryosectioned coronally at a thickness of 40 µm using a cryostat (Leica Microsystems, Model CM3050) and stored in a cryoprotectant solution (25% v/v ethylene glycol, 25% v/v glycerol, and 0.05 M sodium phosphate buffer) at -20°C until IHC processing. Brain sections were meticulously arranged in nine-well plates. For staining, two wells were chosen, and within each well, 10 sections were selected and fixed onto microscope slides for subsequent staining, as we previously described [ 8 ], before quantification. Adult hippocampus NSC culture and transfection with IKKβ or β-catenin shRNA plasmid Hippocampal NSCs were obtained from seven-week-old C57BL/6 mice. Briefly, under an Olympus SZ51 stereomicroscope (Olympus Life Science, USA), the skin, skull, and brain membrane were cleaned and the hippocampal part of the brain was dissected in cell dissociation media made of 10% (v/v) 10X HBSS (Thermo Fisher, 14065), 1.95 g of HEPES (Sigma–Aldrich, H3375), and 0.01 g phenol red (Sigma–Aldrich, P3532) in 500 ml of distilled water. The hippocampi were then enzymatically dissociated in the dissociation media for 30 minutes, followed by mechanical trituration for thorough cell dissociation. The dissociated cells were then washed with 1X PBS to eliminate residual dissociation agents and plated on 10-cm dishes pre-coated with 5 µg/ml of poly-L-ornithine (Sigma–Aldrich, P3655) and 5 µg/ml of fibronectin (Sigma–Aldrich, F1141) in serum-free N2 medium (12 g DMEM/F12 [Life Technologies, 12500-039], 1.69 g of sodium bicarbonate [Sigma–Aldrich, S5761], 0.073 g of L-glutamine [Sigma–Aldrich, G8540], 25 mg of insulin [Sigma–Aldrich, 11882], 1.55 g of D(+) glucose [Thermo Fisher, G8270], 0.1 mM of progesterone [Thermo Fisher, P8783], 1 mM of putrescine [Thermo Fisher, P6024], 0.03 µM of selenite [Thermo Fisher, S5261), 100 mg of apo-transferrin [Thermo Fisher, pro-325], and 1% (v/v) of P/S (WELGENE, LS 202-02) in 1 L of distilled water). The medium was replaced daily with fresh bFGF (Sigma-Aldrich, F0291) containing N2 medium. The NSCs were sub-cultured in N2 medium in coated dishes. After passaging the NSCs, they were induced for neural differentiation using the following protocol: 1) cells were detached with HBSS medium, resuspended in an equal volume of N2 medium, and centrifuged, 2) they were then seeded at a density of 1x10 5 cells in a 60-mm plate in 3 ml N2 medium containing bFGF, 3) after incubation at 37°C with 5% CO 2 for one day, the medium was switched to neural differentiation medium (Stem Cell Technology, 05835), 4) half of the neural differentiation medium was replaced every other day or when the neural differentiation medium’s color changed, 5) after culture in the neural differentiation medium five days, neural differentiation was assessed using flow cytometry. IKKβ and β-catenin shRNA plasmids (Santa Cruz Biotechnology, sc-35645-SH, sc-29210-SH) were transfected using Lipofectamine 3000 (Invitrogen, L3000001) according to the manufacturer’s instructions. To this end, plasmid DNA mixed with both Lipofectamine (10 µl) and Opti-MEM (250 µl) containing shRNA was added to NSCs cultured in 60-mm dishes in antibiotic-free media. The cells were then cultured at 37°C with 5% CO 2 for 24 hours, followed by medium replacement with shRNA-free medium and further culture for one week in neuronal differentiation medium. Immunophenotypic characterization of adult hippocampal NSCs Primary cultured hippocampal NSCs were washed with PBS containing 0.4% (v/v) Triton X-100 (PBST) and incubated with Alexa Fluor® 488-conjugated anti-Nestin (Abcam, ab307131) or PE-conjugated anti-Nanog (CST, #14955) antibodies (both at 1:500) for one hour at room temperature. Data acquisition was done on a FACS Caliber flow cytometer (Becton Dickinson, USA) and analyzed using the FACS Caliber software. IHC and ICC staining For IHC, brain sections were washed in sodium phosphate buffer and mounted on charged glass slides. BrdU-immunostained sections were pretreated with 2 N HCl for 30 minutes at 37°C and then neutralized with PBS, followed by incubation with primary antibodies. Sections were incubated for 60 minutes with 5% (v/v) normal horse serum in PBST. The sections and coated dishes (for ICC) were incubated overnight at 4°C with the primary antibodies in the same buffer solution. The following primary antibodies were used: rat anti-BrdU (Abcam, ab6326) at 1:200, goat anti-DCX (Abcam, ab235153) at 1:1000, rabbit anti-βⅢ tubulin (Abcam, ab52623) at 1:500, mouse anti-NeuN (Sigma–Aldrich, MAB377) at 1:100, rabbit anti-GFAP (Abcam, ab7260) at1:5000, mouse anti-Nestin (Abcam, ab18102) at 1:50, rabbit anti-SOX2 (Abcam, ab92494) at 1:1000, rabbit anti-Nanog (Abcam, ab214549) at 1:500, mouse anti-Ki67 (Abcam, ab279653) at 1:1000, and goat anti-NeuroD1 (Abcam, ab235145) at 1:500. The sections were then incubated for two hours with secondary antibodies conjugated with Alexa Fluor 405 (Thermo Fisher, A31553, A31556), 488 (Thermo Fisher, A11001, A11059), or 546 (Thermo Fisher, A21089), all at 1:1000. The sections were then washed thrice with PBST and counterstained with DAPI at 10 mg/ml (Vector Labs, H1200) for 30 minutes before being mounted. Quantification and image analysis IHC images were acquired on a confocal microscope (Zeiss, Thornwood, NY, USA) equipped with an argon/krypton laser (488 nm), two helium/neon lasers (543 and 633 nm), and a Coherent laser (Coherent, Santa Clara, CA, USA), using the 20X objective lens. ImageJ version 1.4 (NIH, USA) was used for image analysis. Image quantification was done automatically by setting a standardized threshold (which was kept the same for all images to maintain consistency) to distinguish positive staining from the background. Integrated density and other relevant metrics were measured. Brain sections were meticulously arranged in nine-well plates. The quantification of antibody staining involved counting BrdU-positive cells across the rostro-caudal extent of the granule cell layer. The values obtained were then multiplied by 10 (slice series) to obtain the total cell count per GCL. For BrdU double-immunostaining with other markers such as DCX, NeuN, GFAP, or NeuroD1, every section (spaced 480 µm apart) from both the proliferation and differentiation groups was utilized. Double-labeling was confirmed through a three-dimensional reconstruction of the z-series. Composite figures were assembled using Adobe Photoshop version CS6 (Adobe Systems, San Jose, CA, USA). Western blot analysis Anesthetized seven-week-old mice were perfused with PBS, followed by brain collection. Next, the DG was dissected from the hippocampi under an Olympus SZ51 stereo microscope. DG samples or primary cultured hippocampal NSC samples were then homogenized in RIPA lysis buffer (Sigma–Aldrich, R0278) using a sample homogenizer (T10 basic ULTRA-TURRAX®, IKA, Korea). Protein concentration was then quantified using a BCA protein assay kit (Thermo Fisher Scientific) and equal amounts (30 ug/ml) of each protein sample were separated using SDS-PAGE. Proteins were then transferred onto nitrocellulose membranes using a semi-dry transfer system and then blocked to prevent non-specific binding. Membranes were then incubated with primary antibodies against IKKβ (CST, #8943), pIKKβ (CST, #2078), IκB (CST, #4814), anti-pIκB (CST, #2859), cyclin D1(CST, #55506), cyclin E1 (CST, #4129), CDK2 (CST, #18048), CDK4 (CST, #12790), p15Ink4B (CST, #36303), p27Kip1 (CST, #3688), actin (CST, #3700), RelA (Abcam, ab288751), pRelA (Abcam, ab76302), β-catenin (Abcam, ab184919), βIII tubulin (Abcam, ab18207), and anti-NeuroD1 (Abcam, ab213725), all at 1:1000. After washing off unbound primary antibody, membranes were incubated with species-specific horseradish peroxidase-conjugated secondary antibodies. Partitioning the membrane based on the molecular weights of the target proteins of interest for Western blot analysis, we efficiently reused the membrane for subsequent quantification analyses by employing a stripping method. The protein signal was then developed using an enhanced chemiluminescence reagent and detected on a Fusion FX imaging system (Vilber Lourmat, Eberhardzell, Germany). Protein levels were determined using densitometric analysis of the protein bands, and actin was used as the loading control for normalization. Western blot analysis was performed in triplicate with multiple samples to ensure reproducibility and reliability of the results. Co-immunoprecipitation (Co-IP) Co-IP was used to investigate the physical association between β-catenin and NF-κB in the DG and primary cultured NSCs derived from the hippocampus. For Co-IP, we used an NF-κB-specific antibody (anti-RelA; Abcam, ab288751) at 1 µg/ml to selectively bind the NF-κB protein to specific antibodies immobilized on protein A/G magnetic beads (20 µg/ml, Santa Cruz, Protein A/G PLUS-Agarose, sc-2003). To initiate the process, protein lysate (1000 µg) was incubated with the antibody-conjugated beads at 4°C overnight, with gentle agitation to enhance protein-protein interactions specificity and facilitate the formation of immunocomplexes between NF-κB and its interacting partners, including β-catenin. Next, the beads were washed thrice, 10 minutes per wash, with cold immunoprecipitation lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, and 1 mM EDTA, pH 7.4) to remove nonspecifically bound proteins and debris. The immunocomplexes were then eluted from the beads using SDS-PAGE sample buffer, heated at 95°C for five minutes, and then briefly centrifuged. The eluted samples were then subjected to western blot analysis. The membranes were then blocked with 5% non-fat milk in TBST (tris-buffered saline containing 0.1% Tween 20) for one hour at room temperature, followed by overnight incubation at 4°C with primary antibodies against β-catenin (Abcam, ab184919), RelA (Abcam, ab288751), and β-actin (CST, #3700), all at 1:1000. After several washes with TBST, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (1:10,000, Invitrogen, #31430, #31402, #G-21234) for one hour at room temperature. Finally, the protein signals were developed using an enhanced chemiluminescence reagent (Cytiva, RPN2209), and imaged and analyzed using a Fusion FX system (Vilber Lourmat, Eberhardzell, Germany). Quantitative real-time reverse transcription PCR Total RNA was extracted using TRIzol reagent (Thermo Fisher, 15596026), and reverse transcription was done using a system containing the Moloney murine leukemia virus reverse transcriptase (Promega, M1701) following the manufacturer’s instructions. PCR was performed on a Palm-Cycler thermal cycler (Corbett Life Science, Sydney, Australia). Real-time amplification of the cDNA was done on a Rotor-Gene 3000 System (Corbett Research, Mortlake, Australia) using a SYBR Green PCR Master Mix (Sigma–Aldrich, KK4602) and the following conditions: incubation for five minutes at 95°C, followed by 30 cycles of denaturation for 15 seconds at 95°C, annealing for 15 seconds at 62°C, and extension for 15 seconds at 72°C. The following primers were used: mouse NeuroD1 (5’-ATGACCAAATCATACAGCGAGAG-3’ and 5’-CTGCCTCGTGTTCCTCGT-3’), mouse β-catenin (5’-CTGCGGGGATGGTTGGAAG-3’ and 5’-CTCTCTCGGAGCCAATGCAA-3’), and mouse β-actin (5’-ACCCGAGCTTAGCGACCAT-3’ and 5’-CACTCTGCGATACGCTGCT-3’). Relative mRNA levels were calculated using a standard curve generated from the cDNA dilutions. Mean cycle threshold (C t ) values from quadruplicate measurements were used to calculate gene expression and normalized to β-actin (internal control). Relative gene expression levels were calculated using the Corbett Robotics Rotor-Gene software (Rotor-Gene 6 version 6.1, Build 90, Australia). Behavioral tests Open-field test Mice were placed in an open-field arena (40 × 40 × 36 cm) for 20 minutes. Between tests, the arena was cleaned with 70% ethanol for disinfection and 1% acetic acid to eliminate scent. Locomotor activity and anxiety levels were analyzed using the ANY-maze software system (Stoelting, Wood Dale, USA). Object location test . Mice were placed in the open-field arena (40 × 40 × 36 cm) with two identical objects and allowed to freely explore the apparatus for eight minutes (training phase). Testing sessions were conducted two or 24 hours after the training phase. In the test phase, mice were again exposed to the two objects (A), except one of the objects was moved to a novel location (B or C). The object was moved to a third location after 24 hours, while the other object remained in its training phase location. Mice were allowed to explore the environment for eight minutes and the time spent exploring and sniffing both objects was recorded. To analyze memory performance, a preference index (%) was calculated as follows: [moved object (B) time/both objects (A + B) time] × 100. Exploration was analyzed during the training and testing phases. Morris water maze test. To evaluate spatial memory, visual cues were attached to find a submerged platform (10.5 × 8 × 6.5 cm) in a circular swimming pool (diameter: 110 cm) filled with water (22 ± 1°C). Analyses included a three-day acquisition phase, in which each mouse started at three randomly determined positions with four swimming trials per day, with 30-minute intervals between trials. If mice failed to arrive at the platform within 40 seconds, a researcher would guide them to the platform, where they remained for 10 seconds. Probe trials (80 seconds) on day four measured reference memory to determine whether the animals had a preference for the platform area in the absence of the platform. The three-day acquisition phase, during which mice were trained to establish the platform’s location, was followed by a two-day reversal phase, during which the platform was moved to the opposite side, to assess relearning and cognitive flexibility. Analyses included escape latency (i.e., time spent before arriving at the platform) and time spent in each quadrant in the probe phase. All tests were recorded and analyzed using the ANY-maze software. Y-maze test. Each mouse was allowed to freely explore the Y-maze for seven minutes. The alternation ratio was calculated as follows: [(number of possible alternations) / (total number of arms entered − 2)]. Statistical analyses All data are presented as mean ± SEM. P < 0.05 indicates statistically significant differences. Statistical analyses were performed using one-way analysis of variance (ANOVA), followed by a Student−Newman−Keuls test for multiple comparisons or a Student’s t - test on GraphPad Prism version 6 (GraphPad Software, USA). Results IKKβ is expressed in the adult hippocampal NSCs and was knocked down in adult hippocampal NSCs of GFAP-CreERT2/IKKβ flox/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. 1 A–C) and in Nestin- and SOX2-positive primary cultured adult hippocampal NSCs (Supp Fig. 1 D–E). To assess changes in downstream pathways upon IKKβ deletion in hippocampal NSCs of the GFAP-CreERT2/IKKβ flox/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. 1 F–G), indicating effective IKKβ/NF-κB conditional knockdown in the hippocampal DG. Locomotion, spatial learning, and memory are enhanced in GFAP-CreERT2/IKKβ flox/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. 2 A). We observed that the IKKβ cKD group spent more time at the center than the vehicle-treated control group (Fig. 2 B–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. 2 D). However, the IKKβ cKD group showed significantly more arm entries throughout the Y-maze test than the vehicle-treated control mice (Fig. 2 E), 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. 2 F), 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. 2 G). Total object exploration time was similar between the vehicle-treated and the IKKβ cKD mice during NOL tests (Supp Fig. 1 A–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. 2 H). 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. 2 I). 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. 2 J−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/IKKβ flox/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. 3 A–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. 3 A–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. 3 C–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. 4 A−C), and flow cytometry analysis demonstrated 98% of Nestin-positive cells and 83% of Nanog-positive cells (Supp Fig. 4 D). 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. 3 E–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, p15 Ink4B (G1 phase) and p27 Kip1 (S phase), was significantly downregulated (Fig. 2 G–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/IKKβ flox/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. 4 A–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. 4 C). 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. 4 D–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. 4 F). 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/IKKβ flox/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. 5 A–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. 5 C). 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. 5 D–E) and the ratio of NeuN/BrdU double-positive cells to BrdU-positive cells was significantly higher in the IKKβ cKD (Fig. 4 F). 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. 3 C–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/IKKβ flox/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. 6 A–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. 6 C). 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. 6 D–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. 7 A–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. 7 C–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. 7 E–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. 7 G). Overall, these results suggest that IKKβ negatively regulates neural differentiation from adult hippocampal NSCs by inhibiting the β-catenin and NeuroD1 pathways. Discussion In this study, we show that IKKβ is expressed in adult hippocampal NSCs and that the expression of IKKβ (IκB kinase), pIKKβ, pIκB (NF-κB inhibitor), and pRelA (an NF-κB family member) were significantly downregulated in the hippocampal DG of IKKβ cKD mice when compared with vehicle-treated control mice. Our data demonstrate that IKKβ signaling is suppressed in the adult hippocampal DG in GFAP-CreERT2/IKKβ flox/flox mice and that IKKβ knockdown suppressed NF-κB signaling. Moreover, in vitro analyses showed that IKKβ/NF-κB expression was downregulated in IKKβ shRNA-transfected adult hippocampal NSCs. These data indicate that GFAP-CreERT2/IKKβ flox/flox mice and primary cultured hippocampal NSCs transfected with IKKβ shRNA can be used to investigate the role of IKKβ in hippocampal neurogenesis derived from brain NSCs. Our investigation focused on the role of IKKβ of NSCs in hippocampal neurogenesis, with emphasis on its pivotal role as a canonical regulator of the intricate NF-κB signaling pathway, which governs diverse cellular responses. The specific involvement of IKKβ in phosphorylating IκBα highlights its significance in the canonical pathway [2], distinct from the role played by IKKα, which is reported to be predominantly involved in the non-canonical pathway[ 45 , 46 ]. In contrast to the canonical pathway, where IKKβ plays the central role, the non-canonical pathway, which is dominated by IKKα, is characterized by a different set of regulatory mechanisms. IKKβ activation initiates the phosphorylation and subsequent degradation of inhibitory IκB proteins, facilitating the nuclear translocation of NF-κB and the expression of genes associated with immunity, inflammation, and cell survival[ 47 , 48 ]. This strategic focus aligns with extensive literature underscoring IKKβ’s central role in canonical NF-κB signaling and is further supported by the availability of selective inhibitors like BAY11-7082 for precise experimental modulation [49]. Hence, our study sought to elucidate the implication of IKKβ activation in the regulation of hippocampal neurogenesis and to provide insight into its canonical functions in the NF-κB pathway. This investigation utilized GFAP-CreERT2 transgenic mice, an effective model for studying the neurogenesis associated with radial stem cells in the brain [ 50 ]. The successful implementation of GFAP-CreERT2 mice allowed the comprehensive examination of the intricate mechanisms underlying neural stem cell differentiation and its relevance in brain function. The characterization of GFAP-CreERT2 mice demonstrates that CreERT2 is robustly and specifically expressed in radial glial cells. Inducible labeling of radial stem cells is achieved through tamoxifen administration, which allows their lineage tracing during neurogenesis [ 51 ]. The selective expression of CreERT2 in radial glial cells enabled specific targeting and labeling of this cell population within the hippocampus. Furthermore, a quantitative analysis indicated that radial stem cells significantly contribute to adult hippocampal neurogenesis, with labeled neurons integrating into the existing neural circuitry [ 52 ]. Notably, correlation analyses revealed a positive relationship between neurogenesis from labeled radial stem cells and behavioral outcomes, suggesting a potential role in influencing behavior [ 53 ]. NF-κB is involved in the regulation of learning and memory, and neurogenesis in the adult brain [ 25 , 54 ]. However, the role of IKKβ, an essential upstream NF-κB regulator, and its underlying mechanism in the regulation of hippocampal learning and memory or hippocampal neurogenesis from NSCs, have not been previously studied. Thus, we investigated the role of IKKβ in locomotion, learning, and memory function in the hippocampus using various memory tests on GFAP-CreERT2/IKKβ flox/flox mice. Using the open-field test, we show that when compared with vehicle-treated mice, IKKβ cKD mice exhibited significantly higher locomotion and spent more time on outer zone to center zone entries. In addition, the total number of arm entries in the Y-maze test was significantly increased in the IKKβ cKD mice when compared with the vehicle-treated mice. These results suggest that IKKβ inhibits locomotion and hyperactivity, which is consistent with the reported reduction in locomotor activity in NF-κB p50-deficient mice [ 55 ]. However, increased NF-kB signaling in mice with constitutively active IKK2 expression [ 56 ] and HDAC7 overexpression [ 57 ] reduced mouse locomotion and activity, which is consistent with our results. However, further studies in other brain regions, including the prefrontal cortex or hypothalamus, are needed to determine whether hyperactivity and locomotion changes in IKKβ cKD mice are caused by changes in these parts of the brain. In this study, in both the one and 24-hour interval object location tests, the time spent sniffing the relocated object was significantly longer in the IKKβ cKD mice when compared with vehicle-treated mice. Furthermore, escape latency in the probe test was significantly less in the IKKβ cKD mice than in the vehicle-treated mice. These results indicate that IKKβ suppresses learning and memory performance in the hippocampus, and mirror previous findings on NF-κB-mediated aging-related decline in memory function [ 58 ]. Furthermore, there is conflicting evidence regarding the role of NF-κB signaling in NSC proliferation. The inhibitory effect of NF-κB signaling on NSC proliferation has been shown in cortical NSCs derived from RelA/p50 double-knockout mice [ 59 ]. In addition, NF-κB pathway blockade via IκBα overexpression decreases the proliferation of NSCs derived from the subventricular zone [ 60 ]. This inconsistency underscores the complexity of NF-κB signaling and its diverse effects on neural stem cell regulation. Therefore, we investigated the role of IKKβ, upstream of NF-κB, in adult NSC proliferation in the hippocampal DG. Conditional IKKβ knockdown in GFAP-CreERT2/IKKβ flox/flox mice significantly increased the number of BrdU-positive cells in the hippocampal SGZ when compared with the vehicle-treated control group. Moreover, our in vitro results demonstrate that IKKβ knockdown in adult hippocampal NSCs using shRNA significantly increased the number of Ki67-positive cells when compared with the control- or control shRNA-transfected adult hippocampal NSCs. Furthermore, we found that when compared with the control- or control shRNA-transfected groups, the expression of positive cell cycle regulators in the G1 (cyclinD1 and CDK4) and S (cyclinE1 and CDK2) phases was upregulated in the IKKβ shRNA-transfected adult hippocampal NSCs, whereas the expression of negative cell cycle regulators in the G1 (p15 Ink4B ) and S (p27 Kip1 ) phases was downregulated. Taken together, these results indicate that IKKβ inhibits the proliferation of adult hippocampal NSCs, which is consistent with reports that NF-κB is involved in increasing the proliferation of cortical or SVZ NSCs [ 59 , 60 ]. NF-κB signaling is known to control cell survival and stem cell apoptosis. Previous studies indicate that NF-κB activation impairs cell survival of hypothalamic NSCs in obese mice [ 28 ] and increases embryonic stem cell apoptosis [ 61 ]. In this study, conditional IKKβ knockdown in GFAP-CreERT2/IKKβ flox/flox mice significantly increased the survival of NSCs in the hippocampal DG when compared with the vehicle-treated control group. In addition, conditional IKKβ knockdown in GFAP-CreERT2/IKKβ flox/flox mice significantly decreased the number of cleaved caspase 3-positive cells and the expression of Bax, cytochrome c, and cleaved caspase 3 in the hippocampal DG when compared with the vehicle-treated control group. These results suggest that IKKβ inhibits NSC survival by inducing apoptosis in the hippocampal DG, which is consistent with the reported involvement of IKKβ and NF-κB activation in aging-related hypothalamic decline in NSC survival [ 58 ]. NF-κB regulates neural differentiation from NSCs and has been shown to inhibit neuronal differentiation of hypothalamus NSCs through Notch signaling [ 28 ], whereas TLR2 or TLR5 increases neuronal differentiation through NF-κB activation in neural progenitor cells [ 62 , 63 ], and p50 increases neural NSC differentiation in the hippocampal DG [ 64 ]. Because the role of IKKβ in regulating neural differentiation of adult hippocampal NSCs is not fully understood, we investigated whether it regulates neural differentiation from hippocampal NSCs in vivo and in vitro . Our study shows that conditional IKKβ knockdown in GFAP-CreERT2/IKKβ flox/flox mice significantly increased the number of DCX/BrdU and NeuN/BrdU double-positive cells when compared with the vehicle-treated control group. Moreover, in vitro analysis revealed that in cultured adult hippocampal NSCs in the neural differentiation condition, shRNA-mediated IKKβ knockdown significantly increased the number of βIII-tubulin-positive cells when compared with the control- or control shRNA-transfected groups. These results suggest that IKKβ inhibits the neural differentiation of adult NSCs in the hippocampal DG. β-catenin, a key component of the Wnt signaling pathway, plays a pivotal role in the control of stem cell differentiation, especially during hippocampal neurogenesis. Through its dynamic interactions with other transcriptional regulators, including Neurogenin-1 and NeuroD, β-catenin effectively promotes neural lineage commitment and neuronal maturation [ 65 , 66 ]. Studies have shown that β-catenin overexpression increases neural differentiation in cortical NSCs [ 67 ], thus highlighting its role as a crucial factor in directing stem cells toward a neuronal fate [ 68 ]. Moreover, investigations in adult hippocampal NSCs have shown that the overexpression of NeuroD1, a downstream β-catenin target, enhances neuronal differentiation and maturation, further implicating β-catenin in neurogenesis [ 66 ]. Moreover, the study of the interaction between NF-κB and β-catenin in the context of various cellular processes indicates that in cancer cells, NF-κB directly interacts with the Wnt5a promoter, leading to the activation of the TNF-α and TLR pathways [ 41 ] and that NF-κB activation induces β-catenin ubiquitination and inhibits osteogenic differentiation of mesenchymal stem cells [ 69 ]. However, the potential mechanism underlying the interaction between IKKβ and β-catenin in hippocampal neurogenesis from NSCs has not been elucidated. Moreover, NF-κB activation has been identified as a crucial factor during neural differentiation derived from hippocampal NSCs. Upon activation, NF-κB forms a physical complex with β-catenin, significantly reducing β-catenin transactivation activity and target gene expression in breast cancer cells [ 40 ]. Importantly, NF-κB activation has been closely associated with the downregulation of β-catenin expression, underlining the regulatory significance of this mechanism in neural differentiation [ 70 ]. Therefore, we investigated whether IKKβ regulates Wnt3a/β-catenin signaling and NeuroD1 expression during neural differentiation from hippocampal NSCs. Our results show that the conditional knockdown of IKKβ in GFAP-CreERT2/IKKβ flox/flox mice significantly increased β-catenin and NeuroD1 expression in the hippocampal DG when compared with vehicle-treated control mice. Moreover, immunoprecipitation analysis of the potential regulatory role of NF-κB in β-catenin regulation in the hippocampal DG revealed a physical interaction between NF-κB and β-catenin, which holds significant implications for the intricate signaling networks that direct neural differentiation from NSCs in the adult hippocampus. Additionally, we found that shRNA-mediated IKKβ downregulation in cultured adult hippocampal NSCs significantly upregulated the expression of β-catenin and NeuroD1 when compared with the control- or control shRNA-transfected adult hippocampal NSCs in the differentiation condition. Furthermore, this study shows that shRNA-mediated IKKβ downregulation in adult hippocampal NSCs in vitro significantly increased the number of NeuroD1 and βIII-tubulin double-positive cells when compared with the control- or control shRNA-transfected adult hippocampal NSCs in the differentiation condition. Moreover, shRNA-mediated β-catenin downregulation significantly decreased the expression of NeuroD1 and βIII-tubulin, as well as the number of βIII-tubulin-positive cells in adult hippocampal NSCs in vitro . Taken together, these results suggest that IKKβ suppresses hippocampal NSC neuronal differentiation by downregulating β-catenin and NeuroD1 signaling. In summary, our data indicate that IKKβ reduces learning and memory performance, inhibits adult hippocampal NSC proliferation by suppressing the cell cycle, and decreases cell survival by increasing apoptosis. Moreover, IKKβ inhibits hippocampal NSC neuronal differentiation by decreasing NeuroD1 expression through β-catenin inhibition. Our findings are the first evidence that IKKβ inhibits hippocampal neurogenesis by decreasing proliferation and neural differentiation and increasing apoptosis in hippocampal NSCs. Our study has revealed that IKKβ regulation plays a crucial role in adult hippocampal NSCs, highlighting its potential as a promising target for neurogenesis and enhanced cognitive function. These exciting findings not only provide new insights into the intricate molecular processes governing neural development but also highlight targeting IKKβ as a potential therapeutic strategy against a range of neurological disorders. Abbreviations Bax bcl-2-associated X Bcl-2 b-cell lymphoma 2 BrdU bromodeoxyuridine cKD conditional knockdown CreERT2 cre recombinase – estrogen receptor T2 DG dentate gyrus DCX doublecortin GFAP glial fibrillary acidic protein IKKβ IκB kinaseβ Co-IP co-immunoprecipitation NeuroD1 neurogenic differentiation 1 NeuN neuronal nuclear protein NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells NSC neural stem cell SGZ subgranular zone Declarations Data and materials availability All the data underlying this study are available from the corresponding author upon reasonable request. Declarations Conflict of interests The authors declare no competing interests. Ethics approval The laboratory animal welfare and ethics committee of Chonnam University approved all animal experiments (approval number: CNU IACUC-YB-32). All animal experiments adhered to the Institutional Animal Care & Use Committee. Consent for publication All authors have approved the submitted version. Author contributions K.J.S. designed and performed all experiments and analyzed data. S.J.L provided experimental transgenic mice. B.R.M. and W.S.C. performed behavior test analysis. K.J.S., S.T.K., J.J.Y., and W.J.K. wrote the manuscript. All authors read and approved the final manuscript. Acknowledgments We are grateful to Prof. Sung Joong Lee (Seoul National University, Korea) for providing the GFAP-CreERT2/IKKβ flox/flox mice. References Liu T, Zhang L, Joo D, Sun S-C (2017) NF-κB signaling in inflammation. Signal Transduct Target Therapy 2(1):17023 Yu H, Lin L, Zhang Z, Zhang H, Hu H (2020) Targeting NF-κB pathway for the therapy of diseases: mechanism and clinical study. Signal Transduct Target Ther 5(1):209 Kawai T, Akira S (2007) Signaling to NF-kappaB by Toll-like receptors. Trends Mol Med 13(11):460–469 Perkins ND (2007) Integrating cell-signalling pathways with NF-kappaB and IKK function. 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As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4591233","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":315344529,"identity":"7cf94318-b375-42b9-860c-32f4f2d0823b","order_by":0,"name":"Kyung-Joo Seong","email":"","orcid":"","institution":"Chonnam National University School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Kyung-Joo","middleName":"","lastName":"Seong","suffix":""},{"id":315344530,"identity":"240df55a-a6b4-49d3-9b49-70c91b5b7094","order_by":1,"name":"Bo-Ram Mun","email":"","orcid":"","institution":"Chonnam National University School of Biological Sciences and Technology","correspondingAuthor":false,"prefix":"","firstName":"Bo-Ram","middleName":"","lastName":"Mun","suffix":""},{"id":315344531,"identity":"95716f60-95c8-4b52-a7a7-14a4b3b9d658","order_by":2,"name":"Shintae Kim","email":"","orcid":"","institution":"Chonnam National University School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Shintae","middleName":"","lastName":"Kim","suffix":""},{"id":315344532,"identity":"e80f4fd6-5aac-486c-8723-2de6c6ba6c42","order_by":3,"name":"Won-Seok Choi","email":"","orcid":"","institution":"Chonnam National University School of Biological Sciences and Technology","correspondingAuthor":false,"prefix":"","firstName":"Won-Seok","middleName":"","lastName":"Choi","suffix":""},{"id":315344533,"identity":"42e1978b-e599-489a-b3fd-45f143f1b232","order_by":4,"name":"Sung Joong Lee","email":"","orcid":"","institution":"Seoul National University","correspondingAuthor":false,"prefix":"","firstName":"Sung","middleName":"Joong","lastName":"Lee","suffix":""},{"id":315344534,"identity":"302f1c8a-c4a5-4452-ac41-356442f989e7","order_by":5,"name":"Ji-Yeon Jung","email":"","orcid":"","institution":"Chonnam National University School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Ji-Yeon","middleName":"","lastName":"Jung","suffix":""},{"id":315344535,"identity":"03041923-6e22-4b4a-a0df-33b0c01b9b1a","order_by":6,"name":"Won-Jae Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIie3OMQrCMBTG8U8KcSm61lsUCl29SrNXly4OHTrp5qwgnsEuzk8Lr0sPUBfBGwQEZxPQ2ecmmP/wQuD9SACf7ydjEBbqdSEx6b4jdnuwfF8kJD6xPl/2o3lMwc2guwoIMTWzoypiUkmEvvhMUmorR/SBkAImk5KdI8OHlLiPVY6E9pVeQKbEWZOz0tsmLKKsE5DJhpN7XrJet6vaGBYQROQmA4E9JAAYV26Wol2fz+f7057jkkQ7uB254wAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-6549-744X","institution":"Chonnam National University School of Dentistry","correspondingAuthor":true,"prefix":"","firstName":"Won-Jae","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2024-06-17 01:56:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4591233/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4591233/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59610326,"identity":"44f229b1-080a-43d4-b59f-de522e41f291","added_by":"auto","created_at":"2024-07-03 20:04:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":450039,"visible":true,"origin":"","legend":"\u003cp\u003eIKKβ expression in hippocampal NSCs \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. \u003cstrong\u003eA\u003c/strong\u003e The expression of Nestin (a stem cell marker, green) and IKKβ (red) in the hippocampal DG was assessed using immunohistochemistry. \u003cstrong\u003eB\u003c/strong\u003e The expression of GFAP (a stem cell marker, green) and IKKβ (red) in the hippocampal DG was assessed using immunohistochemistry. \u003cstrong\u003eC\u003c/strong\u003eThe expression of Sox2 (a stem cell marker, green) and IKKβ (red) in the hippocampal DG was assessed using immunohistochemistry. \u003cstrong\u003eD–E\u003c/strong\u003e The expression of IKKβ (green) and Nestin (red) or SOX2 (red) in primary cultured adult hippocampal NSCs was assessed using immunocytochemistry. Nuclei were counterstained with DAPI (blue). \u003cstrong\u003eF\u003c/strong\u003e The expression of IKKβ, pIKKβ, IκB, pIκB, RelA, and pRelA was determined using western blot analysis. \u003cstrong\u003eG\u003c/strong\u003e Quantification was normalized to β-actin (loading control). Data are expressed as mean ± SEM. N = 3 per group. * and ** indicate \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u0026lt; 0.01, respectively. Scale bars: 10 or 50 µm in A–C and 70 µm in E. IKKβ cKD: IKKβ conditional knockdown, ns: not significant.\u003c/p\u003e","description":"","filename":"F1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4591233/v1/25819ca4b1eefc30e50df81a.jpg"},{"id":59610327,"identity":"959ebca4-93e5-435d-99ad-9a43f639327e","added_by":"auto","created_at":"2024-07-03 20:04:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":505856,"visible":true,"origin":"","legend":"\u003cp\u003eIKKβ cKD improves mouse spatial learning and memory. \u003cstrong\u003eA–B\u003c/strong\u003e Locomotor activity and anxiety-like behavior in the open-field test were assessed based on total distance traveled (A) and time at the center (B). \u003cstrong\u003eC \u003c/strong\u003eTotal representative exploratory tracks of the vehicle-treated mice or IKKβ cKD mice in the open-field test. The Y-maze test was used to assess spontaneous exploratory behavior. \u003cstrong\u003eD–E\u003c/strong\u003eEvaluation of the alteration ratio (D)and total arm entry (E). \u003cstrong\u003eF–G\u003c/strong\u003e Vehicle-treated mice and IKKβ cKD mice were tested using the object location test. \u003cstrong\u003eH\u003c/strong\u003e Escape latency during the acquisition and reversal phases was assessed using the Morris water maze test. \u003cstrong\u003eI\u003c/strong\u003eThe proportion of the total time spent in each quadrant. \u003cstrong\u003eJ–K\u003c/strong\u003e Probe test assessment of the time spent by mice in the target quadrant at one (J) and two days (K). \u003cstrong\u003eL\u003c/strong\u003e Representative swim paths in the reversal trials. Data are expressed as mean ± SEM. N = 8 per group. * and *** indicate \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 and \u0026lt; 0.001, respectively. IKKβ cKD: IKKβ conditional knockdown, sec: second, ns: not significant, NOL: novel object location.\u003c/p\u003e","description":"","filename":"F2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4591233/v1/d4e6e637b3b7dafba89df302.jpg"},{"id":59610669,"identity":"7187bd06-e093-4d5f-be14-4677797a7c2d","added_by":"auto","created_at":"2024-07-03 20:12:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":446360,"visible":true,"origin":"","legend":"\u003cp\u003eIKKβ cKD increases the proliferation of adult NSCs in the mouse hippocampal DG. \u003cstrong\u003eA\u003c/strong\u003e Representative image of BrdU-positive cells in the hippocampal DG 2.5 hours after a single BrdU injection. Newly proliferated cells in the SGZ of the DG were labeled with BrdU (green) and SOX2 (a stem cell marker, red). Nuclei were counterstained with DAPI (blue). \u003cstrong\u003eB\u003c/strong\u003e Quantitative analysis of the number of BrdU- and SOX2-positive cells in the SGZ of the hippocampal DG 2.5 hours after a single BrdU injection. \u003cstrong\u003eC\u003c/strong\u003e Representative image of BrdU-positive cells in the hippocampal DG one and three days after a single BrdU injection. Newly proliferated cells in the SGZ of the DG were labeled with BrdU (green). Nuclei were counterstained with DAPI (blue). \u003cstrong\u003eD\u003c/strong\u003e Quantitative analysis of the number of BrdU-positive cells in the SGZ of the hippocampal DG, one and three days after a single BrdU injection, N = 7 per group. \u003cstrong\u003eE\u003c/strong\u003e Representative image of SOX2 (red) and Ki67 (a proliferation marker, green) positive cells in primary cultured adult hippocampal NSCs. Nuclei were counterstained with DAPI (blue). \u003cstrong\u003eF\u003c/strong\u003e The number of Ki67-positive cells was quantified in the control and Con shRNA- and IKKβ shRNA-transfected adult hippocampal NSCs. \u003cstrong\u003eG\u003c/strong\u003e Expression of cell cycle-regulatory markers, including p15\u003csup\u003eInk4B\u003c/sup\u003e, cyclin D1, CDK4, p27\u003csup\u003eKip1\u003c/sup\u003e, cyclin E1, and CDK2 in adult hippocampal NSCs was assessed using western blot analysis. Quantification was normalized to β-actin (loading control), N = 3 per group. Data are expressed as mean ± SEM. ** indicates \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. Scale bars: 100 and 200 μm in A, 50 μm in C, and 40 μm in D. IKKβ cKD: IKKβ conditional knockdown, Con: control. ns: not significant.\u003c/p\u003e","description":"","filename":"F3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4591233/v1/a7150b5b19221918ca2c40e9.jpg"},{"id":59610332,"identity":"9e97d898-259c-4cfe-90a2-6f146198cc8e","added_by":"auto","created_at":"2024-07-03 20:04:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":352375,"visible":true,"origin":"","legend":"\u003cp\u003eIKKβ cKD enhances adult NSC survival in the mouse hippocampal DG. \u003cstrong\u003eA\u003c/strong\u003e Representative image of BrdU immunostaining (green) in the hippocampal DG 5, 14, and 28 days after consecutive BrdU injections for five days. Nuclei were counterstained with DAPI (blue). \u003cstrong\u003eB\u003c/strong\u003e Quantification of BrdU-positive cells in the hippocampal DG. \u003cstrong\u003eC\u003c/strong\u003eQuantification of the survival rate is presented as the ratio of BrdU-positive cells 28 days after consecutive BrdU injections for five days (N = 5 per group). \u003cstrong\u003eD\u003c/strong\u003e Apoptotic cells in the hippocampal DG were labeled with cleaved caspase-3 (red). Nuclei were counterstained with DAPI (blue). \u003cstrong\u003eE\u003c/strong\u003e Quantification of cleaved caspase-3-positive cells in the hippocampal DG (N = 6 per group). \u003cstrong\u003eF\u003c/strong\u003e Western blot analysis of the expression of Bax, Bcl-2, Cyt \u003cem\u003ec\u003c/em\u003e, and cleaved caspase-3. Quantification was normalized to β-actin (loading control), N = 3 per group. Data are expressed as mean ± SEM. * and ** indicate \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 and \u0026lt; 0.01, respectively. Scale bars: 200 μm in A and 100 μm in D. IKKβ cKD: IKKβ conditional knockdown, cCas-3: cleaved caspase-3, Cyt \u003cem\u003ec\u003c/em\u003e: cytochrome \u003cem\u003ec.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"F4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4591233/v1/f73cbb40c40e7630a70a2fdb.jpg"},{"id":59610668,"identity":"69eac3d7-83c1-47c9-91d6-d96e0861d336","added_by":"auto","created_at":"2024-07-03 20:12:33","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":356817,"visible":true,"origin":"","legend":"\u003cp\u003eIKKβ cKD promotes adult NSC neural differentiation in the mouse hippocampal DG. \u003cstrong\u003eA\u003c/strong\u003e Representative image of BrdU (green) and DCX (red) positive cells in the hippocampal DG five days after BrdU injection. BrdU/DCX double-positive cells (yellow) represent newly differentiated immature neurons. \u003cstrong\u003eB\u003c/strong\u003eQuantification of the number of BrdU/DCX double-positive cells in the hippocampal DG. \u003cstrong\u003eC\u003c/strong\u003e The ratio of BrdU/DCX double-positive to BrdU-positive cells in the hippocampal DG. \u003cstrong\u003eD\u003c/strong\u003e Representative images of BrdU (green) and NeuN (red) positive cells in the DG at 28 days after BrdU injections five times. BrdU/NeuN double-positive cells (yellow) represent newly differentiated, mature neurons. \u003cstrong\u003eE\u003c/strong\u003e Quantification of the number of BrdU/NeuN double-positive cells in the hippocampal DG. \u003cstrong\u003eF\u003c/strong\u003e The ratio of BrdU/NeuN double-positive to BrdU-positive cells in the hippocampal DG. Each experiment was independently repeated five times. Data are shown as mean ± SEM, N = 5 per group. * and ** indicate \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u0026lt; 0.01, respectively. Scale bars: 100 μm in A (left), 50 μm in A (right), 100 μm in D (left), and 25 μm in D (right). IKKβ cKD: IKKβ conditional knockdown.\u003c/p\u003e","description":"","filename":"F5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4591233/v1/3dcae8a0c50f24ae85c7c3bd.jpg"},{"id":59610328,"identity":"e5582bca-a1dc-4f35-b010-d69c3a711200","added_by":"auto","created_at":"2024-07-03 20:04:33","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":315288,"visible":true,"origin":"","legend":"\u003cp\u003eIKKβ cKD promotes the neural differentiation of adult NSCs through β-catenin signaling in the mouse hippocampal DG. \u003cstrong\u003eA\u003c/strong\u003eWestern blot analysis of the expression of β-catenin and NeuroD1. \u003cstrong\u003eB\u003c/strong\u003e Quantification was normalized to β-actin (loading control). N = 3 per group. \u003cstrong\u003eC\u003c/strong\u003e RelA (NF-κB) binds to β-catenin. Co-immunoprecipitation analysis of hippocampal DG samples using an anti-RelA antibody. Each binding partner was detected via immunoblotting with the indicated antibodies. \u003cstrong\u003eD\u003c/strong\u003e Representative images showing DCX (an immature neuronal marker, green) and NeuroD1 (a neural differentiation marker, red) immunoreactive cells in the hippocampal DG. \u003cstrong\u003eE\u003c/strong\u003e The ratio of NeuroD1/DCX double-positive to DCX-positive cells in the adult hippocampal DG. N = 5 per group. Data are shown as mean ± SEM. * and ** indicate \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u0026lt; 0.01, respectively. Scale bar: 100 μm in D. IB: immunoblot, IP: immunoprecipitation, IgG: immunoglobulin G, IKKβ cKD: IKKβ conditional knockdown.\u003c/p\u003e","description":"","filename":"F6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4591233/v1/d930ec0fa40d4d58e0a46e8e.jpg"},{"id":59610330,"identity":"73a52935-23d3-4bae-92c7-1a5149700f3c","added_by":"auto","created_at":"2024-07-03 20:04:33","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":287902,"visible":true,"origin":"","legend":"\u003cp\u003eIKKβ inhibits the neural differentiation of adult NSCs derived from the hippocampus through β-catenin signaling \u003cem\u003ein vitro\u003c/em\u003e. \u003cstrong\u003eA\u003c/strong\u003e Neural differentiation in primary cultured adult hippocampal NSCs, with or without IKKβ shRNA transfection, was assessed using western blot analysis to determine the expression of pIKKβ, IKKβ, pRelA, RelA, β-catenin, NeuroD1, and βIII-tubulin. \u003cstrong\u003eB\u003c/strong\u003e Quantification was normalized to β-actin (loading control). N = 3 per group. \u003cstrong\u003eC\u003c/strong\u003e Immunostaining analysis of βIII-tubulin (green) and NeuroD1 (a neural differentiation marker, red) in primary cultured adult hippocampal NSCs with or without IKKβ shRNA transfection. \u003cstrong\u003eD\u003c/strong\u003e The ratio of NeuroD1/βIII-tubulin double-positive to βIII-tubulin-positive cells in adult hippocampal NSCs with or without IKKβ shRNA transfection. N = 6 per group. \u003cstrong\u003eE\u003c/strong\u003e Western blot analysis of the expression of β-catenin, NeuroD1, and βIII-tubulin in adult hippocampal NSCs with or without β-catenin shRNA transfection. \u003cstrong\u003eF\u003c/strong\u003e Quantification was normalized to β-actin (loading control). N = 3 per group. \u003cstrong\u003eG\u003c/strong\u003e Immunostaining analysis of βIII-tubulin in primary cultured adult hippocampal NSCs. Quantification of the number of βIII-tubulin-positive cells in hippocampal NSCs with or without β-catenin shRNA transfection. N = 5 per group. Data are shown as mean ± SEM. * and ** indicate \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and \u0026lt; 0.01, respectively. Scale bars: 150 μm in C and 100 μm in G. Con: control.\u003c/p\u003e","description":"","filename":"F7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4591233/v1/3ccab3c786f30d02d150225d.jpg"},{"id":60031416,"identity":"18ce7dc7-1abc-403b-aa5d-aa35906bb01a","added_by":"auto","created_at":"2024-07-10 20:29:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3597321,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4591233/v1/88973d9f-ebef-4d82-9248-4bbb575e7128.pdf"},{"id":59610334,"identity":"34229bf6-4302-4212-9b1d-9a6091e8a27a","added_by":"auto","created_at":"2024-07-03 20:04:33","extension":"docx","order_by":19,"title":"","display":"","copyAsset":false,"role":"supplement","size":1336311,"visible":true,"origin":"","legend":"","description":"","filename":"renamedc514b.docx","url":"https://assets-eu.researchsquare.com/files/rs-4591233/v1/68229c7eba900245c8d5b364.docx"}],"financialInterests":"","formattedTitle":"IKKβ inhibits cognitive memory and adult hippocampal neurogenesis via the β-catenin pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe IκB kinase (IKK) family is made up of the members, IKKα, IKKβ, and IKKγ (also known as nuclear factor kappa B [NF-κB] essential modulator), which act upstream of NF-κB, and are crucial regulators of the NF-κB signaling pathway [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The NF-κB complex, which is composed of p50 and RelA, is a key regulator of inflammation, cell survival, apoptosis, and neural plasticity in the canonical pathway [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Canonical NF-κB signaling is the most extensively studied NF-κB pathway [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and it can be mediated by the activation of Toll-like receptor (TLR) 4 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and inflammatory factors like lipopolysaccharide [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and tumor necrosis factor-alpha (TNF-α) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. IKKβ, an inhibitor of κB kinase, is required for NF-κB activation in the canonical pathway [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], whereas IKKα is essential for the activation of the noncanonical NF-κB signaling pathway [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Previous studies have underscored the diverse roles of IKKβ in various cell types, indicating its involvement in essential cellular functions. The absence of neuronal IKKβ is associated with the amelioration of Alzheimer\u0026rsquo;s disease pathology in amyloid precursor protein and tau transgenic mice [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Tamoxifen-induced IKKβ knockout and NF-κB inhibition in striatal neurons increase striatal neurodegeneration in mice with Huntington\u0026rsquo;s disease. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. IKK/NF-κB signaling inhibition promotes mesenchymal lineage specification and enriches functional mesenchymal stromal cells from human embryonic stem cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Hence, IKKβ/NF-κB activation may have negative and positive effects on neurogenesis, depending on physiological or pathophysiological conditions.\u003c/p\u003e \u003cp\u003eAdult neurogenesis, the process of producing new neurons from neural stem cells (NSCs), occurs throughout life in the dentate gyrus (DG) of the hippocampus [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The hippocampus, which is crucial for regulating locomotor activity and spatial recognition, benefits from the continuous generation of new neurons through neurogenesis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Radial glial cells are a prominent subtype of NSCs localized within the subgranular zone (SGZ) of the DG [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Remarkably, radial glial cells have a distinctive molecular profile that includes glial fibrillary acidic protein (GFAP) and stem cell markers, notably SRY-Box transcription Factor 2 (SOX2) and the neuroepithelial stem cell protein, Nestin [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This unique expression pattern confers radial glial cells with the remarkable ability to differentiate into neurons and astrocytes, thus substantially contributing to the hippocampal microenvironment and cognitive function [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Adult hippocampal neurogenesis is finely regulated by a multitude of endogenous and exogenous factors. Notably, physical activity and environmental enrichment promote neurogenesis and cognitive improvements [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], whereas systemic diseases and inflammation can have negative effects on neurogenic processes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], thereby impacting hippocampal function and plasticity. As key factors in the intricate process of adult neurogenesis, radial glial cells have significant implications for brain plasticity and the maintenance of brain health [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, despite considerable progress in understanding adult neurogenesis, the effects of radial glial cells in genetic mutation conditions are not fully established.\u003c/p\u003e \u003cp\u003eNF-κB signaling is well known to regulate adult NSC proliferation and neuronal differentiation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, the impact of NF-κB signaling on hippocampal neurogenesis, specifically in hippocampal memory formation, is controversial. While previous studies have reported that NF-κB/RelA inhibition increases memory function and hippocampal or hypothalamic NSC proliferation and survival [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], the lack of NF-κB c-Rel impairs hippocampus-dependent fear memory [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This suggests a complex role for NF-κB signaling in the context of hippocampal memory formation and neurogenesis. Although molecular and functional studies indicate that NF-κB plays an important role in regulating adult neurogenesis in the hippocampus or hypothalamus, the role and underlining mechanism of IKKβ, the upstream regulator of NF-κB, in regulating memory function and adult hippocampal neurogenesis is unknown.\u003c/p\u003e \u003cp\u003eThe β-catenin pathway is involved in various stages of development, including the proliferation and differentiation of various stem cells [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. β-catenin, a key downstream effector of the Wnt signaling pathway, increases the neuronal differentiation of NSCs in the hippocampal DG [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] by upregulating the expression of neuronal differentiation 1 (NeuroD1), a downstream target of β-catenin [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. NeuroD1 overexpression is shown to promote neuronal differentiation in adult hippocampal NSCs, whereas its downregulation decreases the maturation of newborn neurons [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Taken together, it can be speculated that β-catenin regulates adult neurogenesis from NSCs in the hippocampal DG through the NeuroD1 transcription factor, which is responsible for CNS development. Some studies have reported that in human cancer cell, NF-κB regulates Wnt5a transcription by controlling the Wnt5a promoter B [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. IKKβ physically interacts with β-catenin and decreases β-catenin-dependent transcriptional activation in SW480 cells [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. β-catenin also interacts with NF-κB and NF-κB suppresses β-catenin expression in human breast cancer cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWnt/β-catenin is a well-established key regulator of neurogenesis and it intricately interacts with various signaling pathways, including Notch, Shh, and NF-κB pathways [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, despite extensive studies of the interplay between Wnt/β-catenin and the Notch, Shh, and NF-κB pathways, there is a significant gap in our understanding, particularly in the context of hippocampal neurogenesis. Specifically, the intricate regulatory dynamics of β-catenin signaling, under the control of IKKβ\u0026ndash;NF-κB, in hippocampal neurogenesis, are not fully elucidated. IKKβ knockout causes embryonic lethality on embryonic day 14.5 because of liver degeneration [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. This study sought to address this knowledge gap by providing valuable insight into the complex regulatory mechanisms governing neurodevelopment in the hippocampus. Here, we investigate the role and underlying mechanism of IKKβ in regulating cognitive function and hippocampal neurogenesis using \u003cem\u003eGFAP-CreERT2/IKKβ\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e transgenic mice, in which IKKβ knockdown in adult hippocampal NSCs was induced by tamoxifen-inducible Cre recombinase or in primary cultured adult hippocampal NSCs transfected with IKKβ shRNA.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eSeven-week-old \u003cem\u003eGFAP-CreERT2/IKKβ\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e male mice, hereafter called IKKβ-conditional knockdown (IKKβ cKD) were provided by Dr. Lee Sung Joong (Seoul National University, Korea). The mice were maintained in standard conditions (22\u0026deg;C, 55% humidity, and a 12-hour light/dark cycle) with free access to food and water. The mice were divided into the IKKβ cKD group, which received intraperitoneal (I.P.) tamoxifen injection to knock down IKKβ in hippocampal NSCs, and the vehicle-treated control group, which was intraperitoneally injected with sunflower oil (vehicle). In this study, approximately 150 animals were sacrificed across the experiment groups, consisting of 70 mice in the control group and 80 mice in the IKKβ cKD group. All animal experiments were approved by the Animal Care and Use Committee of Chonnam National University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTreatment with Tamoxifen\u003c/h2\u003e \u003cp\u003eTamoxifen (Sigma\u0026ndash;Aldrich, T5648) was prepared in physiological sunflower oil (Sigma\u0026ndash;Aldrich, S5007). The mice were I.P.-injected with tamoxifen (2 mg in 200 \u0026micro;l) once daily for five consecutive days. An equal concentration of ethanol in sunflower seed oil was I.P.-injected as negative control. All experiments began 7 days after the last tamoxifen injection [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e5-bromo-2\u0026rsquo;-deoxyuridine (BrdU) injection\u003c/h2\u003e \u003cp\u003eBrdU (Thermo Fisher, 000103) was dissolved in physiological saline and I.P.-injected into the mice at 50 mg/kg. To assess NSC proliferation in the DG, mice were sacrificed at three hours after a single BrdU injection. To assess the immature neural differentiation from BrdU-positive cells, mice were sacrificed 2.5 hours, one day, and three days after a single BrdU injection. To assess the survival rate of the newborn cells generated from NSCs or the mature neural differentiation rate of the NSCs, mice were sacrificed 28 days after BrdU injection once daily for five consecutive days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBrain tissue preparation for immunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003e Animals were transcardially perfused with 0.1 M PBS, followed by fresh cold 4% (v/v) paraformaldehyde in 0.1 M PBS. Brains were then collected and fixed overnight in 4% paraformaldehyde in 0.1 M PBS, at 4\u0026deg;C, and washed for six hours in PBS at 4\u0026deg;C. Sucrose-saturated brains were then embedded in O.C.T. compound (Leica Biosystems, 3801480), freeze-chilled in isopentane (-25\u0026deg;C), and stored at -80\u0026deg;C until sectioning. Brains were cryosectioned coronally at a thickness of 40 \u0026micro;m using a cryostat (Leica Microsystems, Model CM3050) and stored in a cryoprotectant solution (25% v/v ethylene glycol, 25% v/v glycerol, and 0.05 M sodium phosphate buffer) at -20\u0026deg;C until IHC processing. Brain sections were meticulously arranged in nine-well plates. For staining, two wells were chosen, and within each well, 10 sections were selected and fixed onto microscope slides for subsequent staining, as we previously described [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], before quantification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAdult hippocampus NSC culture and transfection with IKKβ or β-catenin shRNA plasmid\u003c/h2\u003e \u003cp\u003eHippocampal NSCs were obtained from seven-week-old C57BL/6 mice. Briefly, under an Olympus SZ51 stereomicroscope (Olympus Life Science, USA), the skin, skull, and brain membrane were cleaned and the hippocampal part of the brain was dissected in cell dissociation media made of 10% (v/v) 10X HBSS (Thermo Fisher, 14065), 1.95 g of HEPES (Sigma\u0026ndash;Aldrich, H3375), and 0.01 g phenol red (Sigma\u0026ndash;Aldrich, P3532) in 500 ml of distilled water. The hippocampi were then enzymatically dissociated in the dissociation media for 30 minutes, followed by mechanical trituration for thorough cell dissociation. The dissociated cells were then washed with 1X PBS to eliminate residual dissociation agents and plated on 10-cm dishes pre-coated with 5 \u0026micro;g/ml of poly-L-ornithine (Sigma\u0026ndash;Aldrich, P3655) and 5 \u0026micro;g/ml of fibronectin (Sigma\u0026ndash;Aldrich, F1141) in serum-free N2 medium (12 g DMEM/F12 [Life Technologies, 12500-039], 1.69 g of sodium bicarbonate [Sigma\u0026ndash;Aldrich, S5761], 0.073 g of L-glutamine [Sigma\u0026ndash;Aldrich, G8540], 25 mg of insulin [Sigma\u0026ndash;Aldrich, 11882], 1.55 g of D(+) glucose [Thermo Fisher, G8270], 0.1 mM of progesterone [Thermo Fisher, P8783], 1 mM of putrescine [Thermo Fisher, P6024], 0.03 \u0026micro;M of selenite [Thermo Fisher, S5261), 100 mg of apo-transferrin [Thermo Fisher, pro-325], and 1% (v/v) of P/S (WELGENE, LS 202-02) in 1 L of distilled water). The medium was replaced daily with fresh bFGF (Sigma-Aldrich, F0291) containing N2 medium. The NSCs were sub-cultured in N2 medium in coated dishes. After passaging the NSCs, they were induced for neural differentiation using the following protocol: 1) cells were detached with HBSS medium, resuspended in an equal volume of N2 medium, and centrifuged, 2) they were then seeded at a density of 1x10\u003csup\u003e5\u003c/sup\u003e cells in a 60-mm plate in 3 ml N2 medium containing bFGF, 3) after incubation at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for one day, the medium was switched to neural differentiation medium (Stem Cell Technology, 05835), 4) half of the neural differentiation medium was replaced every other day or when the neural differentiation medium\u0026rsquo;s color changed, 5) after culture in the neural differentiation medium five days, neural differentiation was assessed using flow cytometry.\u003c/p\u003e \u003cp\u003e IKKβ and β-catenin shRNA plasmids (Santa Cruz Biotechnology, sc-35645-SH, sc-29210-SH) were transfected using Lipofectamine 3000 (Invitrogen, L3000001) according to the manufacturer\u0026rsquo;s instructions. To this end, plasmid DNA mixed with both Lipofectamine (10 \u0026micro;l) and Opti-MEM (250 \u0026micro;l) containing shRNA was added to NSCs cultured in 60-mm dishes in antibiotic-free media. The cells were then cultured at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hours, followed by medium replacement with shRNA-free medium and further culture for one week in neuronal differentiation medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunophenotypic characterization of adult hippocampal NSCs\u003c/h2\u003e \u003cp\u003ePrimary cultured hippocampal NSCs were washed with PBS containing 0.4% (v/v) Triton X-100 (PBST) and incubated with Alexa Fluor\u0026reg; 488-conjugated anti-Nestin (Abcam, ab307131) or PE-conjugated anti-Nanog (CST, #14955) antibodies (both at 1:500) for one hour at room temperature. Data acquisition was done on a FACS Caliber flow cytometer (Becton Dickinson, USA) and analyzed using the FACS Caliber software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eIHC and ICC staining\u003c/h2\u003e \u003cp\u003eFor IHC, brain sections were washed in sodium phosphate buffer and mounted on charged glass slides. BrdU-immunostained sections were pretreated with 2 N HCl for 30 minutes at 37\u0026deg;C and then neutralized with PBS, followed by incubation with primary antibodies. Sections were incubated for 60 minutes with 5% (v/v) normal horse serum in PBST. The sections and coated dishes (for ICC) were incubated overnight at 4\u0026deg;C with the primary antibodies in the same buffer solution. The following primary antibodies were used: rat anti-BrdU (Abcam, ab6326) at 1:200, goat anti-DCX (Abcam, ab235153) at 1:1000, rabbit anti-βⅢ tubulin (Abcam, ab52623) at 1:500, mouse anti-NeuN (Sigma\u0026ndash;Aldrich, MAB377) at 1:100, rabbit anti-GFAP (Abcam, ab7260) at1:5000, mouse anti-Nestin (Abcam, ab18102) at 1:50, rabbit anti-SOX2 (Abcam, ab92494) at 1:1000, rabbit anti-Nanog (Abcam, ab214549) at 1:500, mouse anti-Ki67 (Abcam, ab279653) at 1:1000, and goat anti-NeuroD1 (Abcam, ab235145) at 1:500. The sections were then incubated for two hours with secondary antibodies conjugated with Alexa Fluor 405 (Thermo Fisher, A31553, A31556), 488 (Thermo Fisher, A11001, A11059), or 546 (Thermo Fisher, A21089), all at 1:1000. The sections were then washed thrice with PBST and counterstained with DAPI at 10 mg/ml (Vector Labs, H1200) for 30 minutes before being mounted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eQuantification and image analysis\u003c/h2\u003e \u003cp\u003eIHC images were acquired on a confocal microscope (Zeiss, Thornwood, NY, USA) equipped with an argon/krypton laser (488 nm), two helium/neon lasers (543 and 633 nm), and a Coherent laser (Coherent, Santa Clara, CA, USA), using the 20X objective lens. ImageJ version 1.4 (NIH, USA) was used for image analysis. Image quantification was done automatically by setting a standardized threshold (which was kept the same for all images to maintain consistency) to distinguish positive staining from the background. Integrated density and other relevant metrics were measured. Brain sections were meticulously arranged in nine-well plates. The quantification of antibody staining involved counting BrdU-positive cells across the rostro-caudal extent of the granule cell layer. The values obtained were then multiplied by 10 (slice series) to obtain the total cell count per GCL. For BrdU double-immunostaining with other markers such as DCX, NeuN, GFAP, or NeuroD1, every section (spaced 480 \u0026micro;m apart) from both the proliferation and differentiation groups was utilized. Double-labeling was confirmed through a three-dimensional reconstruction of the z-series. Composite figures were assembled using Adobe Photoshop version CS6 (Adobe Systems, San Jose, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eAnesthetized seven-week-old mice were perfused with PBS, followed by brain collection. Next, the DG was dissected from the hippocampi under an Olympus SZ51 stereo microscope. DG samples or primary cultured hippocampal NSC samples were then homogenized in RIPA lysis buffer (Sigma\u0026ndash;Aldrich, R0278) using a sample homogenizer (T10 basic ULTRA-TURRAX\u0026reg;, IKA, Korea). Protein concentration was then quantified using a BCA protein assay kit (Thermo Fisher Scientific) and equal amounts (30 ug/ml) of each protein sample were separated using SDS-PAGE. Proteins were then transferred onto nitrocellulose membranes using a semi-dry transfer system and then blocked to prevent non-specific binding. Membranes were then incubated with primary antibodies against IKKβ (CST, #8943), pIKKβ (CST, #2078), IκB (CST, #4814), anti-pIκB (CST, #2859), cyclin D1(CST, #55506), cyclin E1 (CST, #4129), CDK2 (CST, #18048), CDK4 (CST, #12790), p15Ink4B (CST, #36303), p27Kip1 (CST, #3688), actin (CST, #3700), RelA (Abcam, ab288751), pRelA (Abcam, ab76302), β-catenin (Abcam, ab184919), βIII tubulin (Abcam, ab18207), and anti-NeuroD1 (Abcam, ab213725), all at 1:1000. After washing off unbound primary antibody, membranes were incubated with species-specific horseradish peroxidase-conjugated secondary antibodies. Partitioning the membrane based on the molecular weights of the target proteins of interest for Western blot analysis, we efficiently reused the membrane for subsequent quantification analyses by employing a stripping method. The protein signal was then developed using an enhanced chemiluminescence reagent and detected on a Fusion FX imaging system (Vilber Lourmat, Eberhardzell, Germany). Protein levels were determined using densitometric analysis of the protein bands, and actin was used as the loading control for normalization. Western blot analysis was performed in triplicate with multiple samples to ensure reproducibility and reliability of the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eCo-IP was used to investigate the physical association between β-catenin and NF-κB in the DG and primary cultured NSCs derived from the hippocampus. For Co-IP, we used an NF-κB-specific antibody (anti-RelA; Abcam, ab288751) at 1 \u0026micro;g/ml to selectively bind the NF-κB protein to specific antibodies immobilized on protein A/G magnetic beads (20 \u0026micro;g/ml, Santa Cruz, Protein A/G PLUS-Agarose, sc-2003). To initiate the process, protein lysate (1000 \u0026micro;g) was incubated with the antibody-conjugated beads at 4\u0026deg;C overnight, with gentle agitation to enhance protein-protein interactions specificity and facilitate the formation of immunocomplexes between NF-κB and its interacting partners, including β-catenin. Next, the beads were washed thrice, 10 minutes per wash, with cold immunoprecipitation lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, and 1 mM EDTA, pH 7.4) to remove nonspecifically bound proteins and debris. The immunocomplexes were then eluted from the beads using SDS-PAGE sample buffer, heated at 95\u0026deg;C for five minutes, and then briefly centrifuged. The eluted samples were then subjected to western blot analysis. The membranes were then blocked with 5% non-fat milk in TBST (tris-buffered saline containing 0.1% Tween 20) for one hour at room temperature, followed by overnight incubation at 4\u0026deg;C with primary antibodies against β-catenin (Abcam, ab184919), RelA (Abcam, ab288751), and β-actin (CST, #3700), all at 1:1000. After several washes with TBST, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (1:10,000, Invitrogen, #31430, #31402, #G-21234) for one hour at room temperature. Finally, the protein signals were developed using an enhanced chemiluminescence reagent (Cytiva, RPN2209), and imaged and analyzed using a Fusion FX system (Vilber Lourmat, Eberhardzell, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time reverse transcription PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using TRIzol reagent (Thermo Fisher, 15596026), and reverse transcription was done using a system containing the Moloney murine leukemia virus reverse transcriptase (Promega, M1701) following the manufacturer\u0026rsquo;s instructions. PCR was performed on a Palm-Cycler thermal cycler (Corbett Life Science, Sydney, Australia). Real-time amplification of the cDNA was done on a Rotor-Gene 3000 System (Corbett Research, Mortlake, Australia) using a SYBR Green PCR Master Mix (Sigma\u0026ndash;Aldrich, KK4602) and the following conditions: incubation for five minutes at 95\u0026deg;C, followed by 30 cycles of denaturation for 15 seconds at 95\u0026deg;C, annealing for 15 seconds at 62\u0026deg;C, and extension for 15 seconds at 72\u0026deg;C. The following primers were used: mouse NeuroD1 (5\u0026rsquo;-ATGACCAAATCATACAGCGAGAG-3\u0026rsquo; and 5\u0026rsquo;-CTGCCTCGTGTTCCTCGT-3\u0026rsquo;), mouse β-catenin (5\u0026rsquo;-CTGCGGGGATGGTTGGAAG-3\u0026rsquo; and 5\u0026rsquo;-CTCTCTCGGAGCCAATGCAA-3\u0026rsquo;), and mouse β-actin (5\u0026rsquo;-ACCCGAGCTTAGCGACCAT-3\u0026rsquo; and 5\u0026rsquo;-CACTCTGCGATACGCTGCT-3\u0026rsquo;). Relative mRNA levels were calculated using a standard curve generated from the cDNA dilutions. Mean cycle threshold (C\u003csub\u003et\u003c/sub\u003e) values from quadruplicate measurements were used to calculate gene expression and normalized to β-actin (internal control). Relative gene expression levels were calculated using the Corbett Robotics Rotor-Gene software (Rotor-Gene 6 version 6.1, Build 90, Australia).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral tests\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eOpen-field test\u003c/strong\u003e \u003cp\u003eMice were placed in an open-field arena (40 \u0026times; 40 \u0026times; 36 cm) for 20 minutes. Between tests, the arena was cleaned with 70% ethanol for disinfection and 1% acetic acid to eliminate scent. Locomotor activity and anxiety levels were analyzed using the ANY-maze software system (Stoelting, Wood Dale, USA).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eObject location test\u003c/b\u003e. Mice were placed in the open-field arena (40 \u0026times; 40 \u0026times; 36 cm) with two identical objects and allowed to freely explore the apparatus for eight minutes (training phase). Testing sessions were conducted two or 24 hours after the training phase. In the test phase, mice were again exposed to the two objects (A), except one of the objects was moved to a novel location (B or C). The object was moved to a third location after 24 hours, while the other object remained in its training phase location. Mice were allowed to explore the environment for eight minutes and the time spent exploring and sniffing both objects was recorded. To analyze memory performance, a preference index (%) was calculated as follows: [moved object (B) time/both objects (A\u0026thinsp;+\u0026thinsp;B) time] \u0026times; 100. Exploration was analyzed during the training and testing phases.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMorris water maze test.\u003c/b\u003e To evaluate spatial memory, visual cues were attached to find a submerged platform (10.5 \u0026times; 8 \u0026times; 6.5 cm) in a circular swimming pool (diameter: 110 cm) filled with water (22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C). Analyses included a three-day acquisition phase, in which each mouse started at three randomly determined positions with four swimming trials per day, with 30-minute intervals between trials. If mice failed to arrive at the platform within 40 seconds, a researcher would guide them to the platform, where they remained for 10 seconds. Probe trials (80 seconds) on day four measured reference memory to determine whether the animals had a preference for the platform area in the absence of the platform. The three-day acquisition phase, during which mice were trained to establish the platform\u0026rsquo;s location, was followed by a two-day reversal phase, during which the platform was moved to the opposite side, to assess relearning and cognitive flexibility. Analyses included escape latency (i.e., time spent before arriving at the platform) and time spent in each quadrant in the probe phase. All tests were recorded and analyzed using the ANY-maze software.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eY-maze test.\u003c/b\u003e Each mouse was allowed to freely explore the Y-maze for seven minutes. The alternation ratio was calculated as follows: [(number of possible alternations) / (total number of arms entered \u0026minus;\u0026thinsp;2)].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicates statistically significant differences. Statistical analyses were performed using one-way analysis of variance (ANOVA), followed by a Student\u0026minus;Newman\u0026minus;Keuls test for multiple comparisons or a Student\u0026rsquo;s t\u003cem\u003e-\u003c/em\u003etest on GraphPad Prism version 6 (GraphPad Software, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIKKβ is expressed in the adult hippocampal NSCs and was knocked down in adult hippocampal NSCs of\u003c/b\u003e \u003cb\u003eGFAP-CreERT2/IKKβ\u003c/b\u003e\u003csup\u003e\u003cb\u003eflox/flox\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;C) and in Nestin- and SOX2-positive primary cultured adult hippocampal NSCs (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u0026ndash;E). To assess changes in downstream pathways upon IKKβ deletion in hippocampal NSCs of the \u003cem\u003eGFAP-CreERT2/IKKβ\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u0026ndash;G), indicating effective IKKβ/NF-κB conditional knockdown in the hippocampal DG.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLocomotion, spatial learning, and memory are enhanced in\u003c/b\u003e \u003cb\u003eGFAP-CreERT2/IKKβ\u003c/b\u003e\u003csup\u003e\u003cb\u003eflox/flox\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eLocomotor 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We observed that the IKKβ cKD group spent more time at the center than the vehicle-treated control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). However, the IKKβ cKD group showed significantly more arm entries throughout the Y-maze test than the vehicle-treated control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSeveral 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Total object exploration time was similar between the vehicle-treated and the IKKβ cKD mice during NOL tests (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the acquisition phase, the two groups did not differ in how quickly they learned the location of the hidden platform (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ\u0026minus;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.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAdult hippocampal NSC proliferation is increased in\u003c/b\u003e \u003cb\u003eGFAP-CreERT2/IKKβ\u003c/b\u003e\u003csup\u003e\u003cb\u003eflox/flox\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFirst, 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026ndash;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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026minus;C), and flow cytometry analysis demonstrated 98% of Nestin-positive cells and 83% of Nanog-positive cells (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u0026ndash;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, p15\u003csup\u003eInk4B\u003c/sup\u003e (G1 phase) and p27\u003csup\u003eKip1\u003c/sup\u003e (S phase), was significantly downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG\u0026ndash;H). Taken together, these results suggest that IKKβ decreases hippocampal NSC proliferation by inhibiting the cell cycle.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe survival of adult hippocampal NSCs is decreased by regulating cleaved caspase-3 and the Bax family in\u003c/b\u003e \u003cb\u003eGFAP-CreERT2/IKKβ\u003c/b\u003e\u003csup\u003e\u003cb\u003eflox/flox\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026ndash;E). In addition, Bax, cytochrome \u003cem\u003ec\u003c/em\u003e, 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). 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.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAdult hippocampal NSC neuronal differentiation is increased in\u003c/b\u003e \u003cb\u003eGFAP-CreERT2/IKKβ\u003c/b\u003e\u003csup\u003e\u003cb\u003eflox/flox\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026ndash;E) and the ratio of NeuN/BrdU double-positive cells to BrdU-positive cells was significantly higher in the IKKβ cKD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026ndash;D). Collectively, these data reveal that IKKβ interferes with immature and mature neural differentiation of adult NSCs in the hippocampal DG.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNeuronal differentiation of hippocampal NSCs is suppressed by downregulating β-catenin and NeuroD1 expression in\u003c/b\u003e \u003cb\u003eGFAP-CreERT2/IKKβ\u003c/b\u003e\u003csup\u003e\u003cb\u003eflox/flox\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u0026ndash;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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC\u0026ndash;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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE\u0026ndash;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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Overall, these results suggest that IKKβ negatively regulates neural differentiation from adult hippocampal NSCs by inhibiting the β-catenin and NeuroD1 pathways.\u003c/p\u003e "},{"header":"Discussion","content":"\u003cp\u003e In this study, we show that IKKβ is expressed in adult hippocampal NSCs and that the expression of IKKβ (IκB kinase), pIKKβ, pIκB (NF-κB inhibitor), and pRelA (an NF-κB family member) were significantly downregulated in the hippocampal DG of IKKβ cKD mice when compared with vehicle-treated control mice. Our data demonstrate that IKKβ signaling is suppressed in the adult hippocampal DG in \u003cem\u003eGFAP-CreERT2/IKKβ\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice and that IKKβ knockdown suppressed NF-κB signaling. Moreover, \u003cem\u003ein vitro\u003c/em\u003e analyses showed that IKKβ/NF-κB expression was downregulated in IKKβ shRNA-transfected adult hippocampal NSCs. These data indicate that \u003cem\u003eGFAP-CreERT2/IKKβ\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice and primary cultured hippocampal NSCs transfected with IKKβ shRNA can be used to investigate the role of IKKβ in hippocampal neurogenesis derived from brain NSCs. Our investigation focused on the role of IKKβ of NSCs in hippocampal neurogenesis, with emphasis on its pivotal role as a canonical regulator of the intricate NF-κB signaling pathway, which governs diverse cellular responses. The specific involvement of IKKβ in phosphorylating IκBα highlights its significance in the canonical pathway [2], distinct from the role played by IKKα, which is reported to be predominantly involved in the non-canonical pathway[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In contrast to the canonical pathway, where IKKβ plays the central role, the non-canonical pathway, which is dominated by IKKα, is characterized by a different set of regulatory mechanisms. IKKβ activation initiates the phosphorylation and subsequent degradation of inhibitory IκB proteins, facilitating the nuclear translocation of NF-κB and the expression of genes associated with immunity, inflammation, and cell survival[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. This strategic focus aligns with extensive literature underscoring IKKβ\u0026rsquo;s central role in canonical NF-κB signaling and is further supported by the availability of selective inhibitors like BAY11-7082 for precise experimental modulation [49]. Hence, our study sought to elucidate the implication of IKKβ activation in the regulation of hippocampal neurogenesis and to provide insight into its canonical functions in the NF-κB pathway.\u003c/p\u003e \u003cp\u003eThis investigation utilized GFAP-CreERT2 transgenic mice, an effective model for studying the neurogenesis associated with radial stem cells in the brain [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The successful implementation of GFAP-CreERT2 mice allowed the comprehensive examination of the intricate mechanisms underlying neural stem cell differentiation and its relevance in brain function. The characterization of GFAP-CreERT2 mice demonstrates that CreERT2 is robustly and specifically expressed in radial glial cells. Inducible labeling of radial stem cells is achieved through tamoxifen administration, which allows their lineage tracing during neurogenesis [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The selective expression of CreERT2 in radial glial cells enabled specific targeting and labeling of this cell population within the hippocampus. Furthermore, a quantitative analysis indicated that radial stem cells significantly contribute to adult hippocampal neurogenesis, with labeled neurons integrating into the existing neural circuitry [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Notably, correlation analyses revealed a positive relationship between neurogenesis from labeled radial stem cells and behavioral outcomes, suggesting a potential role in influencing behavior [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNF-κB is involved in the regulation of learning and memory, and neurogenesis in the adult brain [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. However, the role of IKKβ, an essential upstream NF-κB regulator, and its underlying mechanism in the regulation of hippocampal learning and memory or hippocampal neurogenesis from NSCs, have not been previously studied. Thus, we investigated the role of IKKβ in locomotion, learning, and memory function in the hippocampus using various memory tests on \u003cem\u003eGFAP-CreERT2/IKKβ\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice. Using the open-field test, we show that when compared with vehicle-treated mice, IKKβ cKD mice exhibited significantly higher locomotion and spent more time on outer zone to center zone entries. In addition, the total number of arm entries in the Y-maze test was significantly increased in the IKKβ cKD mice when compared with the vehicle-treated mice. These results suggest that IKKβ inhibits locomotion and hyperactivity, which is consistent with the reported reduction in locomotor activity in NF-κB p50-deficient mice [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. However, increased NF-kB signaling in mice with constitutively active IKK2 expression [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] and HDAC7 overexpression [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] reduced mouse locomotion and activity, which is consistent with our results. However, further studies in other brain regions, including the prefrontal cortex or hypothalamus, are needed to determine whether hyperactivity and locomotion changes in IKKβ cKD mice are caused by changes in these parts of the brain. In this study, in both the one and 24-hour interval object location tests, the time spent sniffing the relocated object was significantly longer in the IKKβ cKD mice when compared with vehicle-treated mice. Furthermore, escape latency in the probe test was significantly less in the IKKβ cKD mice than in the vehicle-treated mice. These results indicate that IKKβ suppresses learning and memory performance in the hippocampus, and mirror previous findings on NF-κB-mediated aging-related decline in memory function [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, there is conflicting evidence regarding the role of NF-κB signaling in NSC proliferation. The inhibitory effect of NF-κB signaling on NSC proliferation has been shown in cortical NSCs derived from RelA/p50 double-knockout mice [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In addition, NF-κB pathway blockade via IκBα overexpression decreases the proliferation of NSCs derived from the subventricular zone [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. This inconsistency underscores the complexity of NF-κB signaling and its diverse effects on neural stem cell regulation. Therefore, we investigated the role of IKKβ, upstream of NF-κB, in adult NSC proliferation in the hippocampal DG. Conditional IKKβ knockdown in \u003cem\u003eGFAP-CreERT2/IKKβ\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice significantly increased the number of BrdU-positive cells in the hippocampal SGZ when compared with the vehicle-treated control group. Moreover, our \u003cem\u003ein vitro\u003c/em\u003e results demonstrate that IKKβ knockdown in adult hippocampal NSCs using shRNA significantly increased the number of Ki67-positive cells when compared with the control- or control shRNA-transfected adult hippocampal NSCs. Furthermore, we found that when compared with the control- or control shRNA-transfected groups, the expression of positive cell cycle regulators in the G1 (cyclinD1 and CDK4) and S (cyclinE1 and CDK2) phases was upregulated in the IKKβ shRNA-transfected adult hippocampal NSCs, whereas the expression of negative cell cycle regulators in the G1 (p15\u003csup\u003eInk4B\u003c/sup\u003e) and S (p27\u003csup\u003eKip1\u003c/sup\u003e) phases was downregulated. Taken together, these results indicate that IKKβ inhibits the proliferation of adult hippocampal NSCs, which is consistent with reports that NF-κB is involved in increasing the proliferation of cortical or SVZ NSCs [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNF-κB signaling is known to control cell survival and stem cell apoptosis. Previous studies indicate that NF-κB activation impairs cell survival of hypothalamic NSCs in obese mice [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and increases embryonic stem cell apoptosis [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In this study, conditional IKKβ knockdown in \u003cem\u003eGFAP-CreERT2/IKKβ\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice significantly increased the survival of NSCs in the hippocampal DG when compared with the vehicle-treated control group. In addition, conditional IKKβ knockdown in \u003cem\u003eGFAP-CreERT2/IKKβ\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice significantly decreased the number of cleaved caspase 3-positive cells and the expression of Bax, cytochrome c, and cleaved caspase 3 in the hippocampal DG when compared with the vehicle-treated control group. These results suggest that IKKβ inhibits NSC survival by inducing apoptosis in the hippocampal DG, which is consistent with the reported involvement of IKKβ and NF-κB activation in aging-related hypothalamic decline in NSC survival [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNF-κB regulates neural differentiation from NSCs and has been shown to inhibit neuronal differentiation of hypothalamus NSCs through Notch signaling [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], whereas TLR2 or TLR5 increases neuronal differentiation through NF-κB activation in neural progenitor cells [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], and p50 increases neural NSC differentiation in the hippocampal DG [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Because the role of IKKβ in regulating neural differentiation of adult hippocampal NSCs is not fully understood, we investigated whether it regulates neural differentiation from hippocampal NSCs \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. Our study shows that conditional IKKβ knockdown in \u003cem\u003eGFAP-CreERT2/IKKβ\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice significantly increased the number of DCX/BrdU and NeuN/BrdU double-positive cells when compared with the vehicle-treated control group. Moreover, \u003cem\u003ein vitro\u003c/em\u003e analysis revealed that in cultured adult hippocampal NSCs in the neural differentiation condition, shRNA-mediated IKKβ knockdown significantly increased the number of βIII-tubulin-positive cells when compared with the control- or control shRNA-transfected groups. These results suggest that IKKβ inhibits the neural differentiation of adult NSCs in the hippocampal DG.\u003c/p\u003e \u003cp\u003eβ-catenin, a key component of the Wnt signaling pathway, plays a pivotal role in the control of stem cell differentiation, especially during hippocampal neurogenesis. Through its dynamic interactions with other transcriptional regulators, including Neurogenin-1 and NeuroD, β-catenin effectively promotes neural lineage commitment and neuronal maturation [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Studies have shown that β-catenin overexpression increases neural differentiation in cortical NSCs [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], thus highlighting its role as a crucial factor in directing stem cells toward a neuronal fate [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Moreover, investigations in adult hippocampal NSCs have shown that the overexpression of NeuroD1, a downstream β-catenin target, enhances neuronal differentiation and maturation, further implicating β-catenin in neurogenesis [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Moreover, the study of the interaction between NF-κB and β-catenin in the context of various cellular processes indicates that in cancer cells, NF-κB directly interacts with the Wnt5a promoter, leading to the activation of the TNF-α and TLR pathways [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and that NF-κB activation induces β-catenin ubiquitination and inhibits osteogenic differentiation of mesenchymal stem cells [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. However, the potential mechanism underlying the interaction between IKKβ and β-catenin in hippocampal neurogenesis from NSCs has not been elucidated.\u003c/p\u003e \u003cp\u003eMoreover, NF-κB activation has been identified as a crucial factor during neural differentiation derived from hippocampal NSCs. Upon activation, NF-κB forms a physical complex with β-catenin, significantly reducing β-catenin transactivation activity and target gene expression in breast cancer cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Importantly, NF-κB activation has been closely associated with the downregulation of β-catenin expression, underlining the regulatory significance of this mechanism in neural differentiation [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Therefore, we investigated whether IKKβ regulates Wnt3a/β-catenin signaling and NeuroD1 expression during neural differentiation from hippocampal NSCs. Our results show that the conditional knockdown of IKKβ in \u003cem\u003eGFAP-CreERT2/IKKβ\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice significantly increased β-catenin and NeuroD1 expression in the hippocampal DG when compared with vehicle-treated control mice. Moreover, immunoprecipitation analysis of the potential regulatory role of NF-κB in β-catenin regulation in the hippocampal DG revealed a physical interaction between NF-κB and β-catenin, which holds significant implications for the intricate signaling networks that direct neural differentiation from NSCs in the adult hippocampus.\u003c/p\u003e \u003cp\u003eAdditionally, we found that shRNA-mediated IKKβ downregulation in cultured adult hippocampal NSCs significantly upregulated the expression of β-catenin and NeuroD1 when compared with the control- or control shRNA-transfected adult hippocampal NSCs in the differentiation condition. Furthermore, this study shows that shRNA-mediated IKKβ downregulation in adult hippocampal NSCs \u003cem\u003ein vitro\u003c/em\u003e significantly increased the number of NeuroD1 and βIII-tubulin double-positive cells when compared with the control- or control shRNA-transfected adult hippocampal NSCs in the differentiation condition. Moreover, shRNA-mediated β-catenin downregulation significantly decreased the expression of NeuroD1 and βIII-tubulin, as well as the number of βIII-tubulin-positive cells in adult hippocampal NSCs \u003cem\u003ein vitro\u003c/em\u003e. Taken together, these results suggest that IKKβ suppresses hippocampal NSC neuronal differentiation by downregulating β-catenin and NeuroD1 signaling.\u003c/p\u003e \u003cp\u003eIn summary, our data indicate that IKKβ reduces learning and memory performance, inhibits adult hippocampal NSC proliferation by suppressing the cell cycle, and decreases cell survival by increasing apoptosis. Moreover, IKKβ inhibits hippocampal NSC neuronal differentiation by decreasing NeuroD1 expression through β-catenin inhibition. Our findings are the first evidence that IKKβ inhibits hippocampal neurogenesis by decreasing proliferation and neural differentiation and increasing apoptosis in hippocampal NSCs. Our study has revealed that IKKβ regulation plays a crucial role in adult hippocampal NSCs, highlighting its potential as a promising target for neurogenesis and enhanced cognitive function. These exciting findings not only provide new insights into the intricate molecular processes governing neural development but also highlight targeting IKKβ as a potential therapeutic strategy against a range of neurological disorders.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBax\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebcl-2-associated X\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBcl-2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eb-cell lymphoma 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBrdU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebromodeoxyuridine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecKD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003econditional knockdown\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCreERT2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecre recombinase \u0026ndash; estrogen receptor T2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edentate gyrus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDCX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edoublecortin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGFAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglial fibrillary acidic protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIKKβ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIκB kinaseβ\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCo-IP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eco-immunoprecipitation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNeuroD1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eneurogenic differentiation 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNeuN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eneuronal nuclear protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNF-κB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enuclear factor kappa-light-chain-enhancer of activated B cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eneural stem cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSGZ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esubgranular zone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eData and materials availability\u003c/h2\u003e \u003cp\u003eAll the data underlying this study are available from the corresponding author upon reasonable request.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eDeclarations\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eConflict of interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics approval\u003c/strong\u003e \u003cp\u003e The laboratory animal welfare and ethics committee of Chonnam University approved all animal experiments (approval number: CNU IACUC-YB-32). All animal experiments adhered to the Institutional Animal Care \u0026amp; Use Committee.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003e All authors have approved the submitted version.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eK.J.S. designed and performed all experiments and analyzed data. S.J.L provided experimental transgenic mice. B.R.M. and W.S.C. performed behavior test analysis. K.J.S., S.T.K., J.J.Y., and W.J.K. wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe are grateful to Prof. Sung Joong Lee (Seoul National University, Korea) for providing the \u003cem\u003eGFAP-CreERT2/IKKβ\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLiu T, Zhang L, Joo D, Sun S-C (2017) NF-κB signaling in inflammation. Signal Transduct Target Therapy 2(1):17023\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu H, Lin L, Zhang Z, Zhang H, Hu H (2020) Targeting NF-κB pathway for the therapy of diseases: mechanism and clinical study. Signal Transduct Target Ther 5(1):209\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKawai T, Akira S (2007) Signaling to NF-kappaB by Toll-like receptors. 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Cell 152(1\u0026ndash;2):25\u0026ndash;38\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"neural stem cell, neurogenesis, hippocampus, memory, IKKβ, β-catenin","lastPublishedDoi":"10.21203/rs.3.rs-4591233/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4591233/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe role of IκB kinase β (IKKβ) and its underlying mechanism in regulating cognitive memory and hippocampal neurogenesis were investigated using \u003cem\u003eGFAP-CreERT2/IKKβ\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e transgenic mice, where the IKKβ gene is specifically knocked down in hippocampal neural stem cells (NSCs) to generate IKKβ conditional knockdown (cKD) mice. Our findings indicate that IKKβ cKD led to increased exploratory activity in the open-field test, promoted hyperactivity in the Y-maze test, and enhanced spatial learning and memory function in the object location and the Morris water maze tests. Furthermore, IKKβ cKD increased the proliferation of adult hippocampal NSCs by upregulating positive cell cycle regulators through the inhibition of negative cell cycle regulators. Neuronal differentiation of adult hippocampal NSCs was also increased by IKKβ cKD, affecting β-catenin signaling and Neurogenic differentiation 1. Additionally, IKKβ cKD enhanced NSC survival, as indicated by decreased cleaved caspase-3 levels and diminished Bax and Cytochrome c expression in the hippocampal dentate gyrus. These findings indicate that in hippocampal NSCs, IKKβ inhibits locomotion, cognitive function, and adult hippocampal neurogenesis by suppressing the β-catenin signaling pathway. Our findings highlight a key role for IKKβ in the inhibition of cognitive function and decrease in hippocampal neurogenesis through NF-κB signaling in adult NSCs.\u003c/p\u003e","manuscriptTitle":"IKKβ inhibits cognitive memory and adult hippocampal neurogenesis via the β-catenin pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 20:04:28","doi":"10.21203/rs.3.rs-4591233/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7ebe8d63-df4d-475a-b745-899f85717d70","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-10T20:21:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-03 20:04:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4591233","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4591233","identity":"rs-4591233","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}