Robust Neuroprotection by NEMO (IKKγ)-binding Domain Peptide via Anti-inflammatory Effects in the Post-ischemic Brain

Abstract

Microglia exhibit a complex and context-dependent role in the post-ischemic brain, performing both neuroprotective and neurotoxic functions. Among the many factors contributing to pro-inflammatory microglia activation, NF-κB signaling plays a pivotal role. The NEMO (IKKγ)-binding domain (NBD) peptide, an 11-amino-acids cell-permeable peptide spanning the NBD of IKKα and IKKβ, acts as a highly specific inhibitor by preventing NEMO-IKKα/IKKβ complex formation. We investigated the neuroprotective effects of the NBD peptide in a post-ischemic brain using a transient middle cerebral artery occlusion (MCAO) animal model. In in vitro experiments, pre-treatment of BV2 cells (a microglia cell line) with NBD peptide significantly suppressed LPS-induced NEMO-IKKα/IKKβ complex formation, nuclear translocation of p65, and upregulation of numerous pro-inflammatory cytokines expressions. The anti-inflammatory effect was further confirmed in reporter gene assay following reporter plasmid transfection, demonstrating a NBD peptide dose-dependent response. In the post-ischemic brain, intranasal delivery of NBD peptide significantly suppressed NEMO-IKKα/IKKβ complex formation, IκB-α phosphorylation, microglial activation, and cytokine induction. Notably, intranasal administration of NBD peptide 3 h post-MCAO significantly reduced infarct volumes in a dose-dependent manner. A significant reduction in infarct volume was observed by 6 h post-administration, suggesting an extended therapeutic window for the NBD peptide. These neuroprotective effects were accompanied by the attenuation of neurological deficits and motor function impairment, as assessed by rota-rod, beam balance, and corner turn tests. Collectively, these results highlight a robust neuroprotective effect along with long-term outcomes of NBD peptide in the post-ischemic brain, with NBD peptide-mediated blocking of NEMO-IKKα/IKKβ complex formation serving as a key underlying molecular mechanism.

INTRODUCTION

Microglia, a resident innate immune cells of the central nervous system (CNS), play a complex and context-dependent role, exhibiting both neuroprotective and neurotoxic functions in various pathophysiological conditions. Microglia are among the first cells activated following ischemic insult and play a crucial role in the inflammatory response [1, 2]. This microglial activation occurs not only in the ischemic core but also in penumbra. Notably, upon reperfusion, microglia in the penumbra become activated, leading to excessive production of reactive oxygen species (ROS), which amplifies oxidative stress, triggers further inflammation, and ultimately exacerbates neuronal damage [1, 2]. During the acute phase of cerebral ischemia, damage-associated molecular patterns (DAMPs), such as High mobility group box 1 (HMGB1), are released by damaged neurons and activated microglia. These danger signals can further perpetuate the inflammation-ROS cycles in the post-ischemic brain and also enter the systemic circulation, potentially triggering systemic immune responses [3-5].

Along with DAMPs, numerous factors regulate microglia activation, with the nuclear factor κB (NF-κB) signaling pathway playing a crucial role. NF-κB is a key transcription factor to drive neuroinflammation by promoting the expression of numerous proinflammatory cytokines and chemokines under pathological conditions. In the cytoplasm, NF-κB dimers are maintained in an inactive state through their association with inhibitory κB (IκB) proteins, IκB-α [6, 7]. The NF-kB essential modulator (NEMO), also known as IKKγ, serves as a critical regulatory subunit of the IκB Kinase (IKK) complex, a trimeric assembly composed of IKKα, IKKβ, and IKKγ subunits. It promotes NEMO (IKKγ)-IKKα/IKKβ complex formation, which is essential for IκB-α phosphorylation and subsequent NF-κB activation [6-8]. Notably, NEMO exhibits widespread expression across various human tissues, including the brain, while exhibiting relatively low expression in the stomach, small intestine, colon, trachea, bladder, and uterus [9].

NEMO-binding domain (NBD) peptide is an 11-amino-acid peptide encompassing NBD of IKKα/β [10]. This peptide is a highly specific NF-κB inhibitor that prevents the interaction between the regulatory subunit NEMO and the catalytic subunits IKKα/IKKβ, thereby blocking IκBα phosphorylation [11, 12]. NBD peptides have been shown to exhibit anti-inflammatory effect in a variety of inflammatory pathological conditions, including synovial inflammation [13], chronic colitis [14], lipopolysaccharide (LPS)-induced acute pulmonary inflammation [15], Alzheimer’s disease [16], and experimental intracerebral hemorrhage [17]. Although, neuroprotective potential of NBD peptide has been examined in an animal models of transient middle cerebral artery occlusion (MCAO) [18], comprehensive investigations into its neuroprotective efficacy following brain ischemia and the underlying molecular mechanisms in microglia remain limited.

Following cerebral ischemia, NF-κB-mediated inflammatory response plays a pivotal role during the acute to subacute phases. In the present study, we aimed to investigate the neuroprotective role of NBD peptide following cerebral ischemia using a transient MCAO animal model. Specifically, we examined the anti-inflammatory effects of NBD peptide in the post-ischemic brain and in LPS-stimulated microglia, focusing on the underlying molecular mechanism and long-term outcomes.

MATERIALS AND METHODS

Surgical procedure of MCAO induction

Male Sprague–Dawley rats (7~8 weeks old) were obtained from Orient Bio Inc. (Gyeonggi, South Korea) and housed under a 12 h light/dark cycle with ad libitum access to standard food and tap water. All animal experiments were performed in strict accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals (NIH, USA, 2013) and the ARRIVE guidelines (http://www.nc3rs.org/ARRIVE, accessed on August 31, 2021). The experimental protocol was reviewed and approved by the INHA University Institutional Animal Care and Use Committee (INHA-IACUC) prior to the commencement of the study (Approval No. INHA210915-782). MCAO was performed as previously described [3, 5]. Briefly, 8-week-old male Sprague–Dawley rats (250~300 g) were initially anesthetized with 5% isoflurane in a gas mixture of 30% oxygen and 70% nitrous oxide delivered via a close-fitting facemask. Anesthesia was maintained with 0.5% isoflurane in the same gas mixture throughout the surgical procedure. A 4−0 nylon monofilament suture (AILEE, Busan, Korea) with a heat-induced tip (approximately 0.3 mm in diameter) was inserted into the internal carotid artery through the external carotid artery bifurcation and advanced 20~22 mm to occlude the right middle cerebral artery for 1 h. Subsequently, the filament was withdrawn to allow reperfusion for up to 7 days. Sham-operated rats underwent identical surgical procedures, with the exception that the MCA was not occluded.

Intranasal administration

Drugs were administered intranasally, following the protocol described by Kim et al. [5]. Briefly, rats were anesthetized via intramuscular injection of a mixture of ketamine (3.75 mg/100 g body weight) and xylazine hydrochloride (0.5 mg/100 g body weight). Throughout the procedure, the animals were maintained in a supine position at a 90° angle. Drug delivery was performed using a pre-sterilized pipette tip (T-200-Y; Axygen, Union, CA, USA). A total volume of 20 µl of PBS-dissolved compounds, including NBD peptide (TP1615L; TargetMol Chemicals Inc., Boston, USA) or mutant NBD peptide (480030, Merck, Burlington, MA, USA), was carefully administered dropwise into each nostril of anesthetized rats. The administration was conducted in a stepwise manner, with 2-min intervals between successive applications until the full dose was delivered.

Infarct volume assessment

Following euthanasia, whole brains were isolated and coronally sectioned into 2-mm thick slices using a metallic brain matrix (RBM-40000, ASI, Springville, UT, USA). The brain slices were stained using immediately incubated in 2% 2, 3, 5-triphenyl tetrazolium chloride (TTC) solution, following the protocol as previously described [3, 5]. Infarcted areas were quantified using the Scion Image software (Scion Corporation, Frederick, MD, USA). To correct for edema and tissue shrinkage, infarct volumes were calculated using the following formula: contralateral hemisphere volume − (ipsilateral hemisphere volume − measured injury volume). The total infarct volume (mm³) was determined by summing infarct areas across consecutive brain sections.

Modified neurological severity scores (mNSS)

Neurological deficits were evaluated at 24 h post-MCAO using the modified neurological severity score (mNSS). The mNSS assessed motor, sensory, balance, and reflex functions, with total scores ranging from 0 (normal function) to 12 (maximal deficit) [19]. Motor function was assessed using two tests: (1) tail suspension, where forelimb flexion, hindlimb flexion, and head movement (>10° relative to the vertical axis) were each scored on a binary scale (0=normal, 1=deficit), yielding a maximum possible score of 3 within 30 seconds observation period; and (2) assessment of walking ability on a flat surface, scored on a scale of 0~3 (0=normal walking, 1=inability to walk straight, 2=circling toward the paretic side, 3=falling on the paretic side). Sensory function was evaluated using the placing test and proprioceptive test, each scored 0 or 1. Balance ability was assessed using the beam balance test, with scores ranging from 0 to 6: 0=maintaining a steady posture, 1=grasping the side of the beam, 2=hugging the beam with one limb off, 3=hugging the beam with two limbs off or rotating around the beam for more than 60 seconds, 4=falling off the beam within 20~40 seconds, 5=falling off within 20 seconds, and 6=making no attempt to balance or cling to the beam. Reflex function was evaluated based on four criteria, each scored 0 or 1 (maximum score of 4): pinna reflex, corneal reflex, startle reflex, and the presence of seizures, myoclonus, or myodystonia.

Rota-rod test and corner turning test

At 24 h before MCAO, rats were conditioned on a rota-rod unit at a constant 5 rpm until they were able to remain on the rotating spindle for 180 s. Each rat was subjected to a test trial on the rota-rod at 15 rpm, and residence times on the rota-rod were measured at 1, 3, and 7 d post-MCAO. The reported values are the mean of three consecutive trials. For corner turn test, after placing the open-edge of apparatus, permit the animals to advance into the corner. The number of turns to the right (ipsilateral side) or left (contralateral side) from the corner in ten times trials were assessed.

Cell cultures and inhibitor treatment

The BV2 murine microglial cell line was cultured in DMEM (Sigma) supplemented with 5% heat-inactivated FBS (Hyclone), 20 U/ml penicillin, and 20 mg/ml streptomycin in a 5% CO₂ incubator. Cells were stimulated with lipopolysaccharide (LPS; derived from Escherichia coli O111:B4; Sigma-Aldrich, St. Louis, MO, USA). For the inhibitor treatment, the cells were pretreated with NBD peptide or mutant NBD peptide starting 1 h prior to LPS (100 ng/ml) stimulation.

Nitrite measurements

BV2 cells cultures were seeded in 24-well plates at a density of 1×10⁵ cells/ml and stimulated with LPS (100 ng/ml) for 24 h. Nitric oxide (NO) production was assessed by measuring nitrite (NO₂^⁻) levels in the conditioned medium using the Griess reagent assay as previously described [3].

Nuclear and cytoplasm extract preparation

Nuclear extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL, USA) by following the manufacturer’s instructions. BV2 cells and BV2 cells cultures (1×10⁵ cells/ml) were processed for nuclear extraction, and crude nuclear proteins in the supernatant were collected and stored at -70°C until further use.

Plasmid transfection and luciferase assay

BV2 cells were seeded in 24-well plates at a density of 1×10⁵ cells/ml, and all transfection experiments were conducted using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) as previously described [20]. Luciferase activity was normalized to total protein levels derived from the co-transfected plasmid.

Immunoblotting and co-immunoprecipitation assay

Immunoblotting and co-immunoprecipitation studies were carried out as previously described [5, 20]. Immunoblotting was performed with the following primary antibodies: anti-NEMO (1:1,000, ab178872, Abcam, Cambridge, UK), anti-LaminB1 (1:1,000, ab133741, Abcam), anti-GAPDH (1:10,000, 14C10, Cell Signaling Technology, Danvers, MA, USA), anti-p65 (1:1,000, D14E12, Cell Signaling Technology), anti-IκB-α (1:1,000, L35A5, Cell Signaling Technology), anti-phospho-IκB-α (1:1,000, ab24783-50, Abcam), anti-IKKα (1:1,000, 61294T, Cell Signaling Technology), anti-IKKβ (1:1,000, 8943T, Cell Signaling Technology), and anti-iNOS (1:1,000, 15323, Abcam). Co-immunoprecipitation was performed using the Catch and Release v2.0 Reversible Immunoprecipitation System (Merck Millipore) by following the manufacturer’s protocol. Cell or tissue lysates (500 µg of total protein) were immunoprecipitated with 1 μg anti-NEMO (Abcam). The immunoprecipitates or input protein lysates were separated by 10% SDS-PAGE, and then analyzed by immunoblotting.

Immunohistochemistry

Animals were sacrificed 24 h post-MCAO after euthanization, and their brains were fixed by transcardiac perfusion with 4% paraformaldehyde, followed by post-fixation in the same solution overnight at 4°C. Subsequently, brains were sectioned coronally at a thickness of 30 μm using a vibratome. Immunostaining was carried out as previously described [21]. Primary antibodies were incubated with the following dilutions: anti-Iba1(Wako Pure Chemicals, Osaka, Japan) at 1:500. After washing with PBS containing 0.1% Triton X-100, sections were incubated with anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) for anti-Iba1 in PBS for 1 h at room temperature. Immunoreactivity was then visualized using the HRP/3,3′-diaminobenzidine substrate kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions.

Reverse transcription quantitative polymerase chain reaction (RT qPCR)

RNA preparation and real-time PCR were performed as described previously [5, 21]. Quantitative real-time polymerase chain reaction (RT-qPCR) was carried out following the cycling conditions: initial 15 min at 95°C, followed by 55 cycles of 10 sec 95°C, 15 sec at 55°C, and 30 sec at 72°C. The following primer sets were used: 5′-CACCACGCTCTTCTGTCTACTG-3′ (forward) and 5′-GTACTTGGGCAGATTGACCTC-3′ (reverse) for TNF-α; 5′-GGAGAAGCTGTGGCAGCTA-3′ (forward) and 5′-GCTGATGTACCAGTTGGGGA-3′ (reverse) for IL-1β; 5′-GCATCCCAAGTACGAGTGGT-3′ (forward) and 5′-CCATGATGGTCACATTCTGC-3′ (reverse) for iNOS, and 5′-TCATTGACCTCAACTACATGGT-3′ (forward) and 5′-CTAAGCAGTTGGTGGTGCAG-3′ (reverse) for GAPDH. All PCR reactions were performed in quadruplicate, and the average threshold cycle (Ct) values were calculated for each sample. Gene expression levels were normalized to the housekeeping gene GAPDH, and relative gene expression levels were quantified using the Livak (2 − ΔΔCt) method.

Statistical analysis

Statistical analysis was performed, San Diego, CA, USA using PRISM software 10.4 (GraphPad Software). Data were initially assessed for normality and homogeneity of variance (if applicable). For comparisons involving multiple groups, one-way or two-way analysis of variance (ANOVA) was conducted, followed by the Newman–Keuls test was used for post-hoc analysis. Data are presented as mean±SEM and p-values <0.05 was considered statistically significant.

RESULTS

NBD peptide exerts anti-inflammatory effects in activated microglia

To investigate the potential involvement of NBD peptide in the NF-κB signaling pathway in microglia, BV2 cells (a microglia cell line) were stimulated with LPS (100 ng/ml) in the presence or absence of NBD peptide. Firstly, treatment with NBD peptide (0.1, 1, 10, or 50 μM) for 24 h did not induce cytotoxicity in BV2 cells (Fig. 1A). Subsequently, pre-treatment of BV2 cells with NBD peptide, commenced 1 h prior to LPS stimulation, effectively suppressed nitrite production in a dose-dependent manner. In contrast, mutant NBD peptide exhibited no suppressive effects (Fig. 1B). Similarly, the LPS-induced enhancement of iNOS protein levels at 24 h post-stimulation was also significantly reduced by pre-treatment with NBD peptide (5, 10 μM), but not by pre-treatment with mutant NBD peptide (Fig. 1C). Furthermore, a significant and dose-dependent attenuation of the upregulation of pro-inflammatory markers (iNOS, IL-1β, and TNFα) was also observed in NBD peptide-treated cells, but not in mutant NBD peptide-treated cells (Fig. 1D~F). Taken together, these results demonstrate an anti-inflammatory effect of NBD peptide in LPS-stimulated microglia.

Fig. 1.

NBD peptide exerts anti-inflammatory effects in LPS-stimulated microglia. (A, B) BV2 cells were stimulated with LPS (100 ng/ml) in the presence or absence of NBD peptide (NBDp, 0.1, 1, 10, 50 μM) and cell viability (A) and nitrite levels (B) was determined at 24 h after LPS stimulation. (C~F) BV2 cells were stimulated with LPS (100 ng/ml) in the presence or absence of NBDp (5, 10 μM) or mutant NBD peptide (mNBDp, 5 μM), with peptides added 1 h prior to LPS stimulation. iNOS protein levels (C) and the expression of pro-inflammatory markers (TNFα, IL-1β, and iNOS) (D~F) were assessed at 24 h after LPS stimulation using immunoblotting or RT-qPCR, respectively. Data are presented as means±SEMs (n=4). NBD, NEMO (IKKγ)-binding domain; LPS, lipopolysaccharide. *p<0.05, **p<0.01, ***p<0.001 compared to controls and ^##p<0.01, ^###p<0.001, ^$p<0.05, ^$$p<0.01, ^$$$p<0.001 between indicated groups.

NBD peptide inhibits NF-κB signaling pathway activation in LPS-stimulated microglia

We investigated whether the NBD peptide-mediated anti-inflammatory effect observed in the previous section was attributable to NBD peptide’s suppression of the NF-κB signaling pathway in microglia. Co-immunoprecipitation assays revealed that the LPS-induced enhancement of interaction between NEMO and IKKα/β, observed at 3 h post-LPS stimulation, was abolished by NBD peptide but remained unaffected by treatment with the mutant NBD peptide (Fig. 2A, B). Furthermore, NBD peptide treatment significantly reduced the elevated levels of nuclear p65 protein and increased cytosolic levels of IκB-α (Fig. 2C~F), indicating a critical role of NBD peptide in modulating the NF-κB signaling pathway in LPS-stimulated microglia. The inhibition of NF-κB signaling by NBD peptide was further validated using a reporter gene assay with Luc2p/NF-κB-RE, which contains five copies of NF-κB binding site (Fig. 2G). LPS stimulation significantly increased luciferase activity in microglia (6.45-fold), which was attenuated by NBD peptide in a dose-dependent manner (Fig. 2G). Collectively, these results demonstrate that NBD peptide inhibits NF-κB signaling in LPS-stimulated microglia, leading to anti-inflammatory effect.

Fig. 2.

NBD peptide inhibits NEMO-IKKα/IKKβ interaction and subsequent NF-κB activation in LPS-stimulated microglia. BV2 cells were stimulated with LPS (100 ng/ml) in the presence or absence of NBD peptide (NBDp, 5, 10 μM) or mutant NBD peptide (mNBDp, 5 μM), with peptides added 1 h prior to LPS stimulation. (A, B) Co-immunoprecipitation experiments were performed at 3 h post-LPS stimulation with anti-NEMO (for immunoprecipitation) and anti-IKKα- or -IKKβ (for immunoblotting) antibodies. Input controls are shown in the right panel. (C~F) Nuclear p65 (C, E) and cytosolic IκB-α (D, F) levels were determined by immunoblotting at 3 h post-LPS stimulation. (G) NFκB-Luc reporter gene assay was performed in BV2 cells at 24 h after LPS (100 ng/ml) stimulation in the presence of NBDp (5, 10 μM). Data are presented as means±SEMs (n=4). NBD, NEMO (IKKγ)-binding domain; LPS, lipopolysaccharide. **p<0.01, ***p<0.001 compared to controls and ^#p<0.05, ^##p<0.01, ^###p<0.001, $$p<0.01 between indicated groups.

NBD peptide attenuates post-ischemic brain inflammation by inhibiting NF-κB signaling

To confirm the NBD peptide-mediated anti-inflammatory effects in the post-ischemic brain, NBD peptide or mutant NBD peptide was administered intranasally (100 μg/kg) at 3 h post-MCAO. The enhanced interaction between NEMO and IKKα/IKKβ, observed at 24 h post-MCAO, was significantly suppressed by NBD peptide (Fig. 3A, B). Additionally, IκB-α phosphorylation, which was increased at 6 h post-MCAO and further increased at 24 h, was significantly reduced by NBD peptide at 24 h post-MCAO (Fig. 3C). In contrast, the mutant peptide showed no inhibitory effects on NEMO-IKKα/IKKβ complex formation and p65 nuclear translocation (Fig. 3A~C). At 24 h post-MCAO, Iba-1-positive cells displayed morphological characteristics of activated microglia, including enlarged cell bodies and retracted processes in the penumbra region (Fig. 3E, H). However, Iba-1-positive cells largely maintained their ramified morphology in the NBD peptide-treated MCAO group, but not in the mutant peptide-treated group (Fig. 3D~H). Importantly, the upregulation of pro-inflammatory cytokine at 24 h post-MCAO was significantly suppressed in the NBD peptide-administered group but not in the mutant peptide-administered group (Fig. 3I~K). Collectively, these data demonstrate that NBD peptide-mediated inhibition of NF-κB activation plays a critical role in suppressing the pro-inflammatory response after ischemic stroke.

Fig. 3.

NBD peptide suppresses NEMO-IKKα/IKKβ complex formation and exerts anti-inflammatory effects in the post-ischemic brain. NBD peptide (NBDp, 100 µg/kg) or mutant NBD peptide (mNBDp, 100 µg/kg) was administered intranasally 3 h post-MCAO. (A, B) Co-immunoprecipitation assays were performed at 24 h post-MCAO with anti-NEMO antibody (for immunoprecipitation) and anti-IKKα or -IKKβ antibody (for immunoblotting). Input controls are shown in the right panel. (C) Levels of phosphorylated IκBα were assessed by immunoblotting at 6 or 24 h post-MCAO. (D~H) Coronal brain sections obtained at 24 h post-MCAO were immunostained with anti-Iba1 antibody. Representative images are shown (D~G) and the areas of Iba1-positive cells within 0.5 mm² region were quantified (n=12, 12 brain slices from three animals) (H). Scale bars represent 100 µm, and those in the insets represent 20 µm (D~G). (I~K) Pro-inflammatory marker expression levels at 24 h post-MCAO were determined by qPCR. Data are presented as the means±SEM (n=4). Sham, sham-operated rats; MCAO, saline-treated MCAO rats; MCAO+NBDp, NBD peptide-treated MCAO rats; MCAO+mNBDp, mutant NBD peptide-administered MCAO animals. NBD, NEMO (IKKγ)-binding domain; MCAO, middle cerebral artery occlusion. *p<0.05, **p<0.01, ***p<0.001 vs. saline-treated MCAO controls, and ^#p<0.05, ^##p<0.01, ^###p<0.001 between indicated groups.

NBD peptide confers robust neuroprotection in the post-ischemic brain and ameliorates neurological and motor deficits

We then investigated whether the NBD peptide-mediated anti-inflammatory effects translated into neuroprotection in the post-ischemic brain. Intranasal administration of NBD peptide (10, 50, or 100 μg/kg) at 3 h post-MCAO significantly reduced mean infarct volume in a dose-dependent manner (Fig. 4A, B). Administration of 100 μg/kg of NBD peptide at 6 h post-MCAO also significantly reduced the mean infarct volume (Fig. 4A, B), suggesting a wide-therapeutic window for this peptide. The mean modified neurological severity scores (mNSSs) assessed at 24 h post-MCAO were also significantly reduced in a dose- and time-dependent manner (Fig. 4C). Edema in the ischemic hemisphere, measured 7 d post-MCAO, was significantly suppressed in the NBD peptide-administered group (100 μg/kg, 3 h post-MCAO). This suppression was accompanied by decreased vascular permeability, as evidenced by IgG staining (Fig. 4D~F), indicating a notable effect on edema resolution at a delayed time point (Fig. 4D, E). Assessment of impaired motor activity at 1, 3, and 7 d post-MCAO using the rota-rod, corner turn, and beam balance tests revealed a significant mitigation of sensorimotor deficits and recovery of motor function in the NBD peptide-administered group (Fig. 4G~I). In contrast, the mutant peptide (100 μg/kg, 3 h post-MCAO) showed no inhibitory effect (Fig. 4G~I). Collectively, these findings demonstrate that NBD peptide exerts robust neuroprotective effects in the post-ischemic brain with a broad therapeutic window. These effects were accompanied by significant recovery of sensorimotor function.

Fig. 4.

NBD peptide confers neuroprotection and functional recovery in the post-MCAO brain. (A~C) NBD peptide (NBDp, 10, 50, or 100 µg/kg) or mutant NBD peptide (mNBDp, 100 µg/kg) was administered intranasally 3 or 6 h post-MCAO, as indicated. Representative TTC-stained coronal brain sections at 24 h post-MCAO are shown (A). Quantification of mean infarct volumes (B) and modified neurological severity scores (mNSS) (C) were assessed at 1 d post-MCAO. (D~I) NBDp (100 µg/kg) or mNBDp (100 µg/kg) was administered intranasally 3 h post-MCAO. Brain edema (E) and vascular permeability (F) quantified as IgG-positive area (D) were assessed at 7 d post-MCAO and presented as the mean±SEM (n=4~5). Motor activities were assessed by the rota-rod test (G), corner turn test (H), and beam balance test (I) at 1, 3, and 7 d post-MCAO. Sham, sham-operated animals; MCAO, saline-treated MCAO control animals; MCAO+NBDp, NBD peptide-administered MCAO animals; MCAO+mNBDp, mutant NBD peptide-administered MCAO animals (n=4~5/group). NBD, NEMO (IKKγ)-binding domain; MCAO, middle cerebral artery occlusion. *p<0.05, **p<0.01, ***p<0.001 compared to the saline-treated MCAO group and ^#p<0.05, ^##p<0.01, ^###p<0.001 compared to the mNBDp-treated MCAO group.

DISCUSSION

This study demonstrates a robust neuroprotective effect of the NBD peptide in the post-ischemic brain. This neuroprotection is mediated by its anti-inflammatory action, achieved through the inhibition of NEMO-IKKα/IKKβ complex formation in microglia. Crucially, this is the first report to show that NBD peptide significantly reduces infarct formation concurrently improving neurological and motor functions after cerebral ischemia, along with elucidating its underlying molecular mechanism.

The activity of NF-κB following cerebral ischemia is known to be persistently elevated, as evidenced by sustained IκB-α phosphorylation from 6 to 24 h post-MCAO (Fig. 3C). NBD peptide significantly inhibits IκB-α phosphorylation observed at 24 h, suggesting prolonged anti-inflammatory effects. These results provide a plausible explanation for the extended therapeutic window observed for NBD peptide in MCAO-induced stroke (Fig. 4). While microglia are major contributors to inflammation in the ischemic brain, dysregulation of astrocytic NF-κB activation has been implicated in various neuroinflammatory and neurodegenerative diseases. Furthermore, interplay between astrocytes and microglia during these processes has also been reported [22, 23]. The effects of astrocytic NF-κB activation can be either protective or detrimental depending on the timing of NF-κB activation and the disease stage [22, 23]. Therefore, further investigation is warranted into the anti-inflammatory effects of NBD peptide in brain cells beyond microglia, and the interrelationship between them. Consistent with our findings, similar anti-inflammatory effects of the NBD peptide have been reported in various pathological conditions within the CNS. Given that NBD peptides are fused with a protein transduction domain (PTD) derived from the Antennapedia homeodomain, which enhances membrane translocation [10, 11], diverse administration routes are possible, including, intracerebrovascular, intravenous, and intranasal delivery. For instance, Rangasamy et al. [16] demonstrated that intranasal administration of NBD peptides significantly reduced Aβ accumulation and cytokine induction, accompanied by an attenuation of memory loss and motor function deficits in an Alzheimer’s disease model. Luo et al. [17] reported the alleviation of perihematomal inflammation in experimental intracerebral hemorrhage through intracerebrovascular administration of NBD peptide. Recently, NBD peptide-mediated alleviation of neuronal pyroptosis was reported in spinal cord injury by inhibiting acid sphingomyelinase-induced lysosome membrane permeabilization [24]. Desai et al. [18] examined beneficial effects of this peptide with a high dose (160 µg/kg) administered 2 h pre-treatment and showed protective effects using TUNNEL staining. However, the current study revealed the NBD peptide’s potent neuroprotective capacity at 10 or 50 ug/kg, even when administered up to 6 h post-MCAO. Furthermore, significant reduction in infarct volumes correlated with substantial recovery of neurological and motor deficits at delayed time points, highlighting its therapeutic promise.

Notably, the significant recovery from brain edema and motor deficits observed at 7 d post-MCAO (Fig. 4) suggests a critical role for the NBD peptide in the delayed phase following ischemic injury. This delayed improvement could be attributed to the sustained efficacy of the peptide or its specific molecular mechanism. While the primary beneficial effects observed in this study are likely derived from the inhibition of NEMO-IKKα/IKKβ complex formation, it is also plausible that NBD peptide binding could influence other functions of NEMO not directly related to this complex disruption. For instance, function of NEMO as a polyubiquitin-binding protein, including non-degradative types, has been documented [25]. Regarding this, recent report showed that linear polyubiquitin binding to NEMO induces NF-κB through a second interaction with IKKβ [26], however, the relationship between NBD and this second interaction remains to be studied. Furthermore, NF-κB-independent functions of NEMO have been reported, such as promoting autophagosomal clearance of protein aggregates by acting as an autophagy adapter [27]. Although, the specific amino-acid sequences involved in these alternative NEMO functions remain to be fully characterized, it is conceivable that NBD peptide binding might indirectly modulate them. Combined with its anti-inflammatory effects, which could contribute to vessel regeneration and antioxidant defense, these potential influences on other NEMO functions might collectively contribute to the improved outcomes observed following transient MCAO. Overall, these results provide novel preclinical insights into the therapeutic potential of NBD peptide in cerebral ischemia. We show acute effects on infarct size and subacute improvements in neurological and motor function. However, comprehensive evaluations of long-term functional recovery at delayed time points are necessary for clinical translation. Further investigation into the alternative mechanisms of action of NBD peptide aforementioned is also warranted.

Given the critical role of NF-kB signaling in numerous pathologies, significant research has focused on developing effective and safe NF-kB inhibitors. Conventional NF-kB inhibitors, which target elements such as IKK, the proteasome, or reactive oxygen species, exhibit potent anti-inflammatory effects and have facilitated the development of diverse drug candidates. However, their lack of specificity often leads to overly broad effects beyond NF-kB regulation, thereby disrupting cellular homeostasis. In contrast, the NBD peptide specifically targets a critical and non-redundant step in the canonical NF-κB activation pathway. By largely preserving basal NF-κB activity, which is essential for normal cellular functions, the NBD peptide may avoid adverse outcomes. Recently, extensive research efforts are underway to enhance the therapeutic potential of NBD peptide. These efforts primarily focus on modification to improve efficacy, strategies for enhanced bioavailability and delivery, and the development of novel related drugs. For instance, researchers are actively searching for novel ligand binding motifs to design new peptide inhibitors of NEMO [28] and developing NBD mimetic with improved pharmacological properties [29]. Concurrently, advancements in drug delivery, such as encapsulating the peptide in liposomes [30, 31] or extracellular vesicles [32], are being explored to improve targeting and stability. Future studies on the specific application of these enhanced NBD peptide strategies in the context of cerebral ischemia, particularly focusing on long-term outcomes and translational potential, are warranted.

Collectively, our findings demonstrate that NBD peptide exerts a potent neuroprotective effect in the post-ischemic brain. This occurs through the inhibition of NEMO-IKKα/IKKβ complex formation, a critical step in the activation of NF-κB signaling. By disrupting this interaction, NBD peptide effectively attenuates the downstream pro-inflammatory cascade, thereby mitigating brain damage and supporting recovery processes.