Toll-like Receptor 2 Exerts Oligodendrocyte Protection in White Matter Stroke Through Downstream NF-κB-cIAP2

Toll-like Receptor 2 Exerts Oligodendrocyte Protection in White Matter Stroke Through Downstream NF-κB-cIAP2 | 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 Article Toll-like Receptor 2 Exerts Oligodendrocyte Protection in White Matter Stroke Through Downstream NF-κB-cIAP2 Jun Young Choi, Hanki Kim, Xuelian Jin, Samma Chowdhury, Yue-Xian Cui, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8957590/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 Ischemic white matter stroke (WMS) is a substantial proportion of ischemic stroke, a major contributor to vascular dementia and a common pathologic finding in neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease. Despite its significance, disease-specific treatment strategies for WMS are not available as of now, making WMS a major unmet medical need. The principal pathomechanism of WMS is the death of oligodendrocytes (OLs) and ensuing demyelination, therefore the prevention of ischemic OL death is crucial to overcoming WMS. In the present article, we delineate the signaling pathway of an OL-protective mechanism that rescues ischemic OL death, which is initiated by Toll-like receptor 2 (TLR2). Through a series of in vitro experiments with primary OLs, utilizing the TLR2 agonist, oxygen-glucose deprivation, chemical inhibitors, siRNA, and overexpression vectors for candidate signaling proteins, we uncovered that the mitogen-activated protein kinase p38 pathway and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway are necessary and sufficient factors for the TLR2-mediated OL protection. In detail, the p38 pathway took action through the NF-κB pathway to exert its protective functions, and the final downstream effector was cellular inhibitor of apoptosis protein 2 (cIAP2). We validated the presence of the TLR2-NF-κB-cIAP2 axis in an unbiased search of the OL transcriptome, in an animal model of internal capsule WMS, and in human OLs. In conclusion, we demonstrate that the TLR2-NF-κB-cIAP2 axis is an OL-protective mechanism against WMS, which may be a valuable therapeutic target for drug development/repositioning to address the unmet medical need. Biological sciences/Neuroscience/Regeneration and repair in the nervous system Biological sciences/Neuroscience/Diseases of the nervous system Biological sciences/Neuroscience/Molecular neuroscience Biological sciences/Neuroscience/Cell death in the nervous system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Ischemic stroke is a major cause of morbidity and mortality worldwide. Humans are unique among mammals for having a larger proportion of white matter (WM) than gray matter (GM), which is reflected in stroke pathology: approximately twenty-five percent of ischemic strokes are located exclusively in the WM, and more than half of the damaged regions in large-vessel occlusive strokes involve the WM. 1,2 Furthermore, white matter stroke (WMS) is an important prognostic factor for recovery after a large vessel occlusive stroke, and when repeated, is a major contributor to vascular dementia, the second most common form of senile dementia. 3 , 4 In contrast with such human pathophysiology, many rodent studies on ischemic stroke have focused on GM pathology, utilizing the middle cerebral artery occlusion (MCAO) model, given that rodents have only ten to fifteen percent of WM in their brains. 5 The lack of consideration for WMS in rodent models is one of the potential reasons behind the limited progress in trials for neuroprotective agents for ischemic stroke. The implications and significance of WMS are not confined to ischemic brain diseases. WMS may contribute to the pathogenesis or progression of a variety of neurodegenerative diseases. For example, WM pathology is a common feature in Alzheimer’s disease (AD), hinting at a potential association between WM ischemia and Alzheimer’s disease. 6 Recent studies report a correlation between extensive WMS and early deterioration in AD. Therefore, it is conceivable that preventing or alleviating the WMS may delay clinical deterioration in AD patients. Another example of WMS involvement in neurodegenerative diseases can be found with Parkinson’s disease (PD). WMS is closely associated with the progression of cognition and even with L-dopa responsiveness in patients with PD. 7,8 In brief summary, WMS is an important underlying feature of neurodegenerative diseases and closely associated with disease progression. Principal pathologic findings in WMS include ischemic oligodendrocyte (OL) death and subsequent demyelination. OLs are intrinsically vulnerable to ischemic insult, a fact reflected in WM often sustaining greater damage than GM following an ischemic event. 9 Therefore, protecting OLs from ischemic cell death or promoting oligodendrogenesis and remyelination after WMS are crucial therapeutic strategies to mitigate the detrimental effects of WMS and slow down the progression of neurodegenerative diseases. A notable characteristic of WMS is its slow progression, suggesting an extended therapeutic window. Unfortunately, despite the wide time frame for treatment, there are no WMS-specific therapeutic strategies currently available, making WMS a critical unmet medical need both in itself and for other vascular and neurodegenerative diseases. In prior studies, our group published the cell-autonomous role of toll-like receptor 2 (TLR2) in ischemic oligodendrocyte death and demyelination in a focal internal capsule infarction model of WMS, and reported that high mobility group box 1 (HMGB1) acts as an endogenous autocrine activator of TLR2, and that this axis is necessary, though not sufficient, to protect OLs from ischemic insult. 10 , 11 More recently, we have demonstrated that HMGB1 acts through TLR2 as an autocrine chemoattractant for oligodendrocyte precursor cells (OPCs) to facilitate OPC recruitment in WMS lesions. 12 Despite such key findings, the OL-survival signaling pathways downstream of TLR2 has not yet been fully elucidated. The characterization of this downstream intracellular survival-signaling pathway is essential for the establishment of WMS-specific druggable targets and novel therapeutic strategies. In the current study, we aim to discover the detailed signaling pathways involved in the TLR2-mediated OL survival from ischemic insult. Results TLR2 downstream signals ERK1/2, p38 and NF-κB, but not AKT and JNK were activated in OLs TLR2 is a transmembrane TLR which is known to act primarily through the MyD88 pathway and not the TIR domain-containing adaptor inducing interferon-β (TRIF) pathway. 13 Therefore, candidates for the OL-protective signaling pathways downstream of TLR2 include; the mitogen-activated protein kinase (MAP kinase) pathway (such as ERK1/2, JNK, and p38), the nuclear factor-kappa-light-chain-enhancer of activated B (NF-κB) pathway, and the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) pathway 13 . The first step in delineating the OL-protective signaling pathway of TLR2 was to observe which of these candidates would be activated in OLs by the TLR2 agonist Pam3CSK4 (Pam3). We applied Pam3 to primary cultured OLs, which we have shown to protect OLs from oxygen-glucose deprivation (OGD) in our previous report. We measured the activation of candidate signal proteins at 15, 30, and 60 minutes after Pam3 application ( Fig. 1 a ) . Increased phosphorylation of p38 ( p =.0005), ERK1/2 ( p <.0001), and CREB ( p <.0001) (downstream effector of MAP kinase pathways) after Pam3 application on OLs were observed, along with the increased degradation of IκB-α ( p <.0001), indicating the activation of NF-κB. However, there was no phosphorylation of AKT and JNK in the same circumstances ( Fig. 1 b,c ) . These findings were suggestive that activation of TLR2 did not cause the activation of AKT and JNK in OLs. Thus, AKT and JNK were exempted from potential candidates and excluded from further experiments ( Fig. 1 d ) . We measured the activation of p38, ERK1/2, CREB, and NF-κB following OGD, with or without Pam3 application ( Fig. 1 e ) . An increase in the phosphorylation of p38 ( p =.03), ERK1/2 ( p =.02), and CREB ( p =.02) in the combined OGD + Pam3 group compared to the OGD-alone group was observed, and OGD + Pam3 showed the highest expression of the three signaling proteins ( Fig. 1 f, g ) . In case of the NF-κB pathway, both OGD ( p <.0001) and Pam3 ( p =.003) caused increased IκB-α degradation, and the OGD + Pam3 group exhibited the lowest expression of IκB-α ( Fig. 1 g ) . p38 and NF-κB, but not ERK1/2, are necessary for TLR2-mediated OL protection from ischemia Given that p38, ERK1/2, CREB and NF-κB showed activation after TLR2 activation in oligodendrocytes, we performed further examinations to test which signaling proteins were essential to TLR2-mediated OL protection from ischemia. First, we applied inhibitors to each signaling protein to observe the reversal of Pam3-mediated protection of OLs from OGD; SB202190 for p38 inhibition, U0126 for ERK1/2 inhibition, and BAY 11-7085 for NF-κB inhibition. In concordance with our prior studies, TLR2 agonism by Pam3 reduced OGD-induced OL death, measured as LDH release from dead OLs ( Fig. 2 a,b ) . Both p38 and NF-κB inhibition by SB202190 and BAY 11-7085, respectively, resulted in the reversal of TLR2-mediated OL protection ( p <.0001). However, intriguingly, ERK1/2 inhibition with U0126 was not sufficient to overturn TLR2-mediated OL protection ( Fig. 2 a ) . We validated the results through siRNA knock-down of each signal protein, p38, ERK1/2, CREB, and NF-κB, with siRNA transfection through electroporation. The siRNA successfully reduced the expression of each targeted protein compared to the control siRNA ( p <.0001) (Fig. S1 a-d) . The knock-down of p38 and NF-κB reversed TLR2-mediated OL protection from OGD ( p <.0001, p =.0002), which was in concordance with the chemical inhibitors. The NF-κB knock-down showed increased OL death after OGD even in the absence of Pam3 ( p =.008). Additionally, also in concordance with the chemical inhibitors, neither ERK1/2 nor CREB knock-down was capable of reversing TLR2-mediated OL protection ( Fig. 2 b ) . In summary, we deduced that the ERK1/2 pathway, although activated in OLs by TLR2 agonism, does not reduce ischemic OL death. The ERK1/2 pathway was exempted from subsequent experiments, leaving only the p38 and NF-κB pathway as candidate mechanisms necessary for TLR2-mediated OL protection ( Fig. 2 c ) . For further confirmation, we evaluated cell death with fluorescence-associated cell sorting (FACS) by propidium iodide (PI) and Annexin V after siRNA transfection to knock down either p38 or NF-κB. The results demonstrated that the knock-down of either p38 or NF-κB reversed TLR2-mediated OL protection, specifically in terms of apoptotic cell death (Fig. S1 e-g) . The OL protection of TLR2-mediated p38 activation is exerted through NF-κB Through the previous experiments, we narrowed down the candidate mechanisms to p38 and NF-κB, both of which were necessary factors for TLR2-mediated OL protection. We asked whether these two factors were also sufficient for TLR2-mediated OL protection. To clarify, we introduced the constitutive activation of p38 and NF-κB through the overexpression of MKK6 for p38 and IKK2 for NF-κB. We measured OL death after OGD, comparing the OL-protective effect of Pam3, MKK6 and IKK2. The activation of p38 by MKK6 showed protective effects comparable to that of Pam3. The activation of NF-κB by IKK2 had a higher protective effect compared with that of both Pam3 application ( p =.03) and p38 activation by MKK6 ( p =.01) ( Fig. 3 a ) . To confirm whether the overexpression of each kinase properly activated their corresponding target protein, we performed immunoblotting ( Fig. 3 b ) . As expected from prior tests ( Fig. 1 ) , Pam3 application successfully phosphorylated both p38 ( p =.005) and p65 ( p =.001), a subunit of NF-κB. IKK2 overexpression resulted in a significantly increased phosphorylation of p65 ( p <.0001) but not p38. Interestingly, MKK6 overexpression caused the increased phosphorylation of not only p38 ( p <.0001), but also p65 ( p =.02) ( Fig. 3 c ) . We also observed the change in expression of the effector anti-apoptotic proteins B-cell leukemia/lymphoma 2 (Bcl2) and cellular inhibition of apoptosis protein 2 (cIAP2); while the distinction is not absolute, Bcl2 is more closely associated with p38 and cIAP2 is strongly related with NF-κB. 14 The expression of cIAP2 was upregulated by Pam3 ( p =.03), IKK2 ( p <.0001) and MKK6 ( p =.03), and the degree of upregulation showed the same pattern of that of p65 phosphorylation caused by the same agents ( Fig. 3 c,d ) . Conversely, Bcl2 expression was not different between all groups ( Fig. 3 d ) . These findings suggested that NF-κB was more likely to be a sufficient factor of TLR2-mediated OL protection, and that the protective effects of p38 were more likely to be exerted through the NF-κB pathway. To decipher the potential crosstalk between p38 and NF-κB, we crossly overexpressed the activating kinase while inhibiting the other through siRNA knock-down; IKK2 overexpression with p38 siRNA knockdown, and MKK6 overexpression with p65 siRNA knockdown. NF-κB activation by IKK2 overexpression was sufficient to protect OLs from death by OGD even in the presence of p38 siRNA knock-down ( p <.0001). However, p38 activation by MKK6 overexpression was not sufficient to prevent OGD-induced OL death when p65 was knocked down ( Fig. 3 e ) . Immunoblotting of each protein showed successful activation and knock-down of targeted proteins ( Fig. 3 f-h ) . The expression of the anti-apoptotic effector cIAP2 was reflective of these results; NF-κB activation upregulated cIAP2 even with p38 knock-down ( p =.02) ( Fig. 3 i ) , while p38 activation, which was previously shown to upregulate cIAP2 ( Fig. 3 d ) , could not replicate cIAP2 upregulation when the knock-down of p65 was in place. Through these findings, we concluded that NF-κB is the pathway that is both necessary and sufficient for TLR2-mediated OL protection from ischemia, and that the OL protection of p38 took effect via NF-κB. cIAP2 is a necessary and sufficient factor to protect OL from ischemic insult through NF-κB activation Given that cIAP2 was closely correlated with NF-κB activation and OL protection ( Fig. 3 d,i ) , we further probed the role of cIAP2 as an essential effector for NF-κB signaling in OLs and its protective effect. To this end, we transfected siRNA for cIAP2 knock-down in OLs with or without IKK2 overexpression. cIAP2 knock-down reversed the OL protection by NF-κB activation via IKK2 ( p =.02) ( Fig. 4 a ) . cIAP2 expression levels were downregulated by cIAP2 siRNA, reversing upregulation caused by IKK2-mediated NF-κB activation ( p =.003) ( Fig. 4 b,c ) . Notably, NF-κB activation could raise cIAP2 levels ( p =.01) and exert OL protection ( p =.0001) even in the presence of cIAP2 knock-down ( Fig. 4 a-c ) . The degree of OL death showed a pattern that was the inverse of the cIAP2 expression levels in corresponding conditions ( Fig. 4 a-c ) . Thus, we demonstrated that cIAP2 is the downstream effector of NF-κB necessary for its OL-protective function. After uncovering cIAP2 as a necessary factor of the TLR2-NF-κB axis in OL protection, we further tested whether cIAP2 was sufficient for OL protection. To test this hypothesis, we overexpressed cIAP2 with or without the knock-down of NF-κB with p65 siRNA. The knock-down of p65 exacerbated OL death after OGD ( p =.0008), and cIAP2 overexpression could mitigate this effect ( p <.0001) ( Fig. 4 d ) . cIAP2 overexpression was also sufficient to induce OL protection independently of NF-κB inhibition ( p <.0001) ( Fig. 4 d ) . Similarly with the previous experiment, NF-κB inhibition with p65 siRNA could lower cIAP2 levels ( p =.01) and exacerbate OGD-induced OL death ( p =.006) even in the presence of cIAP2 overexpression ( Fig. 4 d-f ) . The pattern of OGD-induced OL death was reflective of cIAP2 levels of corresponding conditions ( Fig. 4 d,f ) . In summary, we concluded that cIAP2 is the downstream effector of NF-κB, necessary and sufficient for its OL-protective function. The presence of the TLR2-NF-κB-cIAP2 axis in the in vitro OL transcriptome To validate our findings in an unbiased manner, we performed bulk RNA sequencing comparing OLs exposed to OGD, with or without Pam3. The addition of Pam3 induced a change in transcriptional profile visible upon principle component analysis (PCA) ( Fig. 5 a ) . Differentially expressed genes (DEGs) were mostly upregulated in the OGD+Pam3 group ( Fig. 5 b,c ) . Seventeen DEGs, including Birc3 (the gene name for cIAP2), were associated with the NF-κB pathway (KEGG rno04064), and all of them were upregulated in the OGD+Pam3 group except for Traf6 ( Fig. 5 c ) . Enriched Gene Ontology (GO) terms including Birc3 were; Negative regulation of apoptotic process, Regulation of apoptotic process and Canonical NF-kappaB signal transduction ( Fig. 5 d ) . Gene set enrichment analysis (GSEA) of the bulk RNA seq data for KEGG pathways showed that KEGG rno04064 (NF-κB pathway) was enriched in the OGD+Pam3 group (Normalized enrichment score (NES) = 2.09, adjusted p =. 2.28e-05), and that Birc3 ranked second among pathway-associated genes in terms of fold change ( Fig. 5 e ) . For a more comprehensive insight into the significance of the DEGs, we devised a composite score based on the KEGG GSEA results; The fold change of a specific gene multiplied by the number of enriched KEGG pathways the gene is involved in. When genes were ranked according to this score, Birc3 was among the highest ranking genes in the dataset, third in rank after chemokines ( Fig. 5 f ) . Collectively, we detected evidence of the TLR2-NF-κB-cIAP2 axis in a comprehensive and unbiased searching of the OL transcriptome. Intriguingly, a number of cyclin-dependent kinase inhibitors, namely Cdkn1a (log 2 (fold change) = 0.58, adjusted p = 3.0 × 10 − 6 ), Cdkn2b (log 2 (fold change) = 0.46, adjusted p= .001), and Cdkn1b (log 2 (fold change) = 0.29, adjusted p =.03), were upregulated in the OGD+Pam3 group (Table.S1) . In vivo confirmation in an animal model of WMS to see the protective effect of NF-κB activation To validate our findings in vivo , we devised an animal experiment utilizing a rodent model of WMS. A focal infarction in the internal capsule of C57BL/6 mice was induced by administration of the nitric oxide synthase (NOS) inhibitor N5-(1-Iminoethyl)-L-ornithine (L-NIO). Three weeks before L-NIO administration, control (AAV9-mMBP-mCherry) and IKK2 overexpression (AAV9-mMBP-IKK2) adeno-associated virus (AAV) vectors, given OL specificity through the myelin basic protein (MBP) promoter, were administered ( Fig. 6 a,b ) . IKK2 was chosen over cIAP2 as the gene to deliver, given that cIAP2 siRNA couldn’t completely reverse the protective effects of IKK2 overexpression ( p =.04) ( Fig. 4 a ) . The specificity of the AAV vector to OLs could be measured by assessing colocalization of mCherry with the OL marker Olig2, which showed that ~ 96.7% of mCherry+ cells were Olig2+, indicating successful OL-selective gene delivery (Fig. S2 a-c) . Motor-sensory function was measured by the tape removal test, at one day before and three, seven days after L-NIO injection, after which brain samples were collected for histology ( Fig. 6 a ) . Demyelinating lesion volume, measured by Eriochrome cyanine staining, was smaller in IKK2 group animals ( p =.002), which also showed better motor-sensory function (lower latency for tape removal from affected paw, p =.03) that was linearly correlated to lesion volume (r = 0.78, p =.003) ( Fig. 6 c-f ) . p65 phosphorylation ( p =.04), cIAP2 upregulation ( p =.002), and decreased caspase 3 cleavage ( p =.02) in OLs was observed in the perilesional area of IKK2 group animals, suggesting that the OL protection, mainly anti-apoptotic effect of NF-κB through cIAP2 was taking effect in vivo ( Fig. 6 g-l ) . Astroglial and microglial activation was not different between groups (Fig.S3a-e) . Our past studies have documented the exacerbation of L-NIO-induced demyelination in TLR2(-/-) mice 10 , 11 ; we hypothesized that the activation of downstream NF-κB by IKK2 would rescue this effect, regardless of TLR2 deficiency. In concordance with prior studies, the baseline lesion volume was larger, motor-sensory function was worse, and cIAP2 immunoreactivity was lower in TLR2(-/-) animals compared to wildtype animals (Fig.S4b,c,f) . The linear correlation between lesion volume and motor-sensory function was preserved in TLR2(-/-) animals (r = 0.62, p =.03) (Fig.S4d) . As we hypothesized, the OL protection by NF-κB activation could indeed be observed in TLR2(-/-) animals; lesion volume was reduced in TLR2(-/-)-IKK2 animals ( p =.02), which also exhibit improved motor-sensory function ( p =.0001) (Fig.S4b,c) . More OLs were positive for cIAP2 in the perilesional area of TLR2(-/-)-IKK2 animals ( p =.04) (Fig.S4e,f) . Astroglial, microglial was not different between groups (Fig.S4g-k) . In conclusion, the OL-protective effect of NF-κB activation could be observed in an in vivo animal model of WMS, without regard to TLR2 functionality. Evidence of the TLR2-NFkB-cIAP2 axis in human brain diseases Through our animal experiments, we observed OL protection against WMS through NF-κB-cIAP2. TLR2-deficient animals showed larger lesions, impaired neurological function, and lower cIAP2 immunoreactivity, which could be rescued by activation of NF-κB-cIAP2. To observe the existence of the TLR2-NF-κB-cIAP2 axis in human brain OLs, we re-analyzed two single-nucleus transcriptome datasets; a dataset of human vascular dementia (VaD) (Mitroi et al., 2022) and multiple sclerosis (MS) (Macnair et al., 2025) to observe the correlation between TLR2 and BIRC3 (cIAP2) in mature OLs, OPCs/committed OL progenitors (COPs), and microglia in the brain. In the VaD dataset, mature OLs showed a robust linear correlation between TLR2-BIRC3 (r = 0.81, p<.0001), TLR2-RELA (NF-κB) (r = 0.86, p<.0001), and RELA-BIRC3 (r = 0.71, p<.0001) once cells with zero values for the genes were removed ( Fig. 7 a ) . Microglia also showed a significant correlation between the three genes, but the magnitude of linear correlation was lower compared to mature OLs. OPCs/COPs did not have a sufficient expression of TLR2, RELA, and BIRC3 to enable an analysis of correlation. The MS dataset showed a generally similar pattern, with mature OLs showing a robust linear correlation between components of the TLR2-NF-κB-cIAP2 axis ( Fig. 7 b ) . However, the mature OLs in the MS dataset did not have a sufficient number of cells that had non-zero values for TLR2 and BIRC3 , rendering correlation analysis in accurate. To overcome the significant zero-inflation, which is characteristic of single-cell level transcriptome analyses, we generated “metacells”, which are aggregates of cells showing similar gene expression, by K means clustering using the UMAP plot coordinates of individual cells (Fig.S5b) . Through this strategy, the correlation between TLR2 and BIRC3 could be assessed (Fig.S5c) . Collectively, we have demonstrated the presence of the TLR2-NF-κB-cIAP2 axis in the human brain OL transcriptome. Discussion The detailed signaling pathway for TLR2-mediated OL protection, from ischemic cell death, has been delineated in the present study. While microglia are the cells in the central nervous system (CNS) which express the widest range of TLRs, OLs and also astrocytes are known to express TLR2 and TLR3. 15 The existence of a cell-autonomous protective mechanism for OLs, acting through TLR2 upon autocrine damage signals such as HMGB1, has been demonstrated in our previous studies. 10 – 12 As TLR2 signaling has numerous downstream pathways and functions, some of which are associated with inflammation, characterization of the precise pathway through which TLR2 signaling exerts its OL protection is essential for drug development/repositioning exploiting TLR2-mediated OL protection. Using the TLR2 agonist Pam3, chemical inhibitors, siRNA knock-down and overexpression for TLR2 downstream signals, we have identified the NF-κB pathway and cIAP2 as both necessary and sufficient factors for TLR2-mediated OL protection. TLR2 and other TLRs are pattern recognition receptors (PRRs), initially demonstrated to recognize pathogen-associated molecular patterns (PAMPs) to orchestrate innate immune responses, and later reported to also recognize damage-associated molecular patterns (DAMPs) which are also necessary for regulating immune responses. 13 , 16 In contradiction to predictions that TLR2 signaling would result in neuroinflammation and CNS degeneration, numerous studies have shown that TLR2 signaling is beneficial for axon growth and glial survival, and that blocking the TLR2 pathway results in exacerbated CNS pathologies. 17 More specifically, TLR2 agonists such as Zymosan or Pam3 have been shown to promote axonal growth and alleviate WM damage in the brain and spinal cord, while blockage of TLR2 has been reported to induce multiple abnormalties in the brain and exacerbate CNS pathologies. 18 – 21 In a prior study, the sterile stimulation of microglia with Pam3 resulted in a mixed manifestation of both the pro-inflammatory M1 and pro-regenerative M2 phenotype rather than an exclusive M1 activation, which collectively induced a neuroprotective response to laser-induced spinal cord injury (SCI). 19 Other studies have shown that constitutive TLR2 deletion resulted in detrimental glial activation leading to WM damage in a transgenic AD mouse model, leading to an amyloid β(Aβ)-independent exacerbation of AD pathology. 17 In addition to such prior knowledge, our study elucidates a cell autonomous OL-protection mechanism that takes action through TLR2 and downstream NF-κB-cIAP2. TLR agonists such as monophosphoryl lipid A and CpG 1018, although not TLR2-specific, are currently in clinical use, serving as evidence of the efficacy and safety of this class of drugs, therefore making them a plausible target for drug repositioning. 22 , 23 The role of NF-κB in OL physiology and pathophysiology has been explored in previous studies, although such prior research is scarce compared to the bulk of knowledge on NF-κB in general. In vitro and animal level studies demonstrate that while the inactivation of IKK2 in mouse CNS cells (achieved by a nestin promoter) or astrocytes resulted in alleviation of toxic demyelination induced with cuprizone, its specific inactivation in OLs resulted in elevated myelin loss and remyelination failure in an animal model combining cuprizone and interferon-gamma (IFN-γ) administration. 24 – 28 Such studies provide further evidence that the protective function of NF-κB for OLs is strictly cell-autonomous, and suggests that non-cell-autonomous activation of NF-κB may actually be disadvantageous for OL survival, emphasizing the necessity for meticulous cell type targeting in order for potential OL therapeutic strategies based on NF-κB to be effective. NF-κB is also known as a essential factor for the survival of Schwann cells, the peripheral nervous system (PNS) counterpart of OLs, in an animal model of sciatic nerve injury. 29 – 31 Indirect clues on the role of NF-κB for OL survival can be found in research studies investigating tumor necrosis factor alpha (TNF-α), another activator of the NF-κB pathway, in multiple sclerosis (MS); while TNF-α is a well known contributor to MS and experimental autoimmune encephalitis (EAE) pathology, the use of anti-TNF-α agents resulted in a paradoxical exacerbation of MS shown by an increased number of demyelinating lesions. 32 – 34 Comprehensively, such findings warrant caution with the use of NF-κB inhibitors, many of which are ongoing clinical trial for the treatment of cancer. 35 , 36 Many broad NF-κB inhibitors have failed to become clinical drugs due to their inability to control the downside associated with suppressing beneficial functions tied to the NF-κB pathway. The present study shows that the resistance of OLs and CNS myelin to ischemic demyelination and WMS is another crucial benefit of the NF-κB pathway, further emphasizing the importance of precision medicine for this double-edged sword. The correlation with WMS may be another area in which NF-κB inhibitors should be evaluated, especially for patients with or at high risk of WMS, such as old age and hypertension. Unlike with demyelination, evidence on the role of NF-κB for developmental myelination or myelination under normal conditions devoid of injury, both for OLs and Schwann cells, is conflicting, with some studies demonstrating that NF-κB is necessary for myelin formation while others showing it to be dispensable. A human case study has shown CNS myelin defects in three of five male patients with Xq28 duplication encompassing Methyl-CpG binding protein 2 (MECP2), which represses interleukin-1 receptor-associated kinase 1 (IRAK1). While IRAK1 is not specifically upstream of NF-κB exclusively, the myelin defects were more precisely correlated with the duplication of NEMO (IKKγ), which has inhibitory effects on the NF-κB pathway. 37 , 38 In our study, NF-κB or p38 inhibition did not promote OL cell death in the absence of OGD ( Fig. 2 a,b ) . Moreover, siRNA knock-down of cIAP2 itself did not lower cIAP2 levels without it being increased by the overexpression of IKK2 ( Fig. 4 c ) , and p65 siRNA knock-down could decrease cIAP2 levels even in the presence of direct cIAP2 overexpression ( p =.01) ( Fig. 4 f ) , indicating that NF-κB is a very potent regulator of cIAP2 expression in OLs, and that cIAP2 has low basal levels in the absence of activating stimuli. Constitutive activation of IKK/NF-κB has been shown to induce OL senescence and subsequent myelin loss, and the participance of NF-κB in general cellular senescence has been reported in different fields of research such as cancer biology. 39 , 40 The upregulation of cyclin-dependent kinase inhibitors in Pam3-treated OLs further validates the senescence-inducing effect of prolonged NF-κB activation in OLs. This provides additional clarification that the NF-κB pathway is a survival signal for OLs in detrimental circumstances rather than a constitutively active promoter of myelination, and that setting an appropriate timepoint of application is another key factor necessary to harness and utilize NF-κB-mediated OL protection. The function of cIAP2 in OLs is a topic that is further elusive than that of NF-κB. The significance of cIAP2 in protecting murine kidneys and human tubular epithelial cells from ischemia/reperfusion injury from apoptosis has been documented. 41 As for the CNS and OLs, a prior study reported that cIAP2(-/-) mice exhibited increased OL death and demyelination in response to EAE, but primarily focused on the impact of cIAP2 deletion in microglia, and not the cell-autonomous action of cIAP2 in OLs. 42 Another study demonstrated that Leukemia inhibitory factor (LIF), a cytokine growth factor known to promote OL survival in vitro , could arrest OL death and demyelination in an animal model of spinal cord injury through survival pathways including cIAP2. 43 In our in vitro/in vivo experiments, cIAP2 functioned as the end effector of the TLR2-NF-κB axis to promote OL survival in the face of ischemic damage. Re-analysis of the human brain transcriptome showed that mature OLs exhibit a higher proportion of cells expressing BIRC3 compared to OPC/COPs (Fig.S5a) , which may serve as one of the factors behind the higher vulnerability of OPC/COPs to ischemic damage. 44 The significance of cIAP2 as an OL-protective mechanism in our experiments and as a potential mechanism behind maturation-dependent ischemia resistance collectively suggests that cIAP2 is a core component of OL resistance to ischemia. Additionally, as with NF-κB, second mitochondria-derived activator of caspases (SMAC) mimetics, which suppress IAP action, are showing promise as effective anti-tumor agents for various cancers including glioblastoma, and such drugs may also benefit from evaluation for potential side effects related to WM injury. 45 During the delineation of the TLR2-NF-κB-cIAP2 pathway, we excluded a number of potential alternatives. The PI3k-Akt pathway and the MAP kinase JNK was dropped at the early stages of the sequence of in vitro experiments, as they showed no response to Pam3. While ERK1/2 and downstream CREB (which may also serve as downstream for p38) were activated by Pam3, they did not participate in the OL-protection response to OGD. While ERK1/2 did not promote OL survival, our previous studies demonstrate its crucial role as the mediator of a TLR2-mediated autocrine chemotactic response to HMGB1, facilitating OPC migration and recruitment into WMS lesions. In our bulk RNA-seq results, we were able to see a substantial upregulation of various chemokines and chemotaxis-related GO terms ( Fig. 5 d,f ) in the Pam3-treated OLs, which may be attributable to ERK1/2 activity. p38 was the one signal which also prevented OL death by OGD, despite its protection being dependent on NF-κB. A few prior studies show that p38 regulates OPC proliferation and differentiation into myelinating OLs, which has not been reported for NF-κB. 46–48 Future research demonstrating that p38 is OL-protective against WMS in vivo may further potentiate its value as a therapeutic target. Here, we describe the cell-autonomous OL-protective mechanism in WMS, initiated by TLR2 and taking action through downstream NF-κB and cIAP2. While meticulous targeting for cell type and timing is needed, this mechanism may serve as a valuable therapeutic target for WMS, which is a major unmet medical need in the CNS. Methods and materials Primary oligodendrocyte culture Primary OPCs were isolated from cerebral cortices of postnatal day 1 Sprague-Dawley rats, following a method utilizing differential centrifugation with density gradient-forming agent Optiprep™, developed in this laboratory. 49 Details are described in the Supplementary Information section. Oxygen-glucose deprivation (OGD) and application of chemical agents For in vitro OGD, OLs cultured for 24 hours in OL differentiation medium were washed with sterile PBS and transferred to an anaerobic chamber (95% N2 and 5% CO2). For the OGD group, the culture media was exchanged to a glucose-free OL differentiation media, and glucose (4.5 g/L) was added for the control. OLs were exposed to OGD for 6 hours, transferred back to normoxia with the addition of 4.5 g/L glucose and reoxygenation for another 18 hours. Chemical inhibitors for designated signaling pathways, such as SB202190 (Cell signaling, #8158, 1 µM) for p38, U0126 (Cell Signaling Technology, #9930S, 1 µM) for ERK1/2, and BAY 11-7085 (Santa Cruz, #sc202490, 1 µM) for NF-κB and the TLR2 agonist Pam3 (Invivogen, #tlrl-pms, 1 µg/mL) were added accordingly during the total 24-hour OGD/reoxygenation process. Lactate dehydrogenase(LDH) cell death assay At the endpoint of OGD/reoxygenation (24 hours after OGD onset), cell death was quantified by LDH assays using an colorimetric LDH assay kit (Takara Bio, #MK401) in accordance to the manufacturer’s protocol. LDH levels were measured as the optical density (OD) value at 490 nm. The LDH level corresponding to complete cell death was determined in sister cultures exposed to 1.5% Triton X-100 (high control, HC). Baseline LDH levels were determined in a medium-alone condition without cells (low control, LC). The percentage of cell death in each experiment were calculated using the following formula: % of OL death = (experimental value – LC)/(HC-LC) × 100. Transfection of siRNA and overexpression plasmids to primary OLs The Amaxa Nucleofector® electroporation system and basic Nucleofector® kit for primary mammalian glial cells (Lonza, #VPI-1006) was used to transfect siRNAs and overexpression plasmids for each candidate protein. Briefly, OPCs, detached and pelleted after five days of proliferation, were resuspended with Nucleofector® solution with the designated siRNA/plasmid or corresponding control siRNA (Santa Cruz, #sc37007)/control plasmid (Origene, #PCMV-6XL4). Resuspended OPCs were transferred into an electroporation cuvette and were electroporated using the Nucleofector® II machine (program number for OLs/glia: A-033 or O-017). OL differentiation medium was added immediately after electroporation. OPCs were seeded onto designated culture surfaces at 3 × 10 4 cells/cm 2 in OL differentiation medium and cultured for 24 hours before OGD exposure. Detailed siRNA and plasmids are listed in the Supplementary Information section. Immunoblotting Cultured OLs were washed twice with PBS and harvested with a lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM MgCl 2 , 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and a protease/phosphatase inhibitor cocktail. Cell lysates were centrifuged at 20,000 × g/20 min/4°C. Protein concentration was quantified by the Bradford assay (Bio-Rad, #5000205). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was done with 10% or 4–20% gradient gels (Bio-Rad), and the proteins on the gels were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk or 5% bovine serum albumin for 1 hours at room temperature and then incubated with primary antibodies. After repeated washing with 0.05% TBST, the membranes were incubated at RT with horse radish peroxidase-conjugated secondary antibodies. Finally, the membranes were visualized using enhanced chemiluminescence detection (Pierce). Detailed antibodies are listed in the Supplementary Information section. Isolation of RNA and RNA-seq data processing and analysis RNA from primary OLs, three samples per group, was extracted using RNeasy Plus Micro Kit (Qiagen), following the manufacturer’s protocol. Purified RNA samples at 150–250 ng/µL concentration, 20 µL/sample and RNA Integrity Number (RIN) above 9.8 were processed for library preparation and RNA-sequencing at Novogene (Seoul, Korea) using the Novaseq Xplus PE150 platform with 6Gb sequencing depth. The resulting raw .fastq files were quality-controlled with FastQC. kallisto-quant was used to quantify transcript abundances, using the Rattus Norvegicus genome/transcriptome (Rnor 6.0) as reference. The count matrix of transcript abundances was preprocessed and analyzed in R/Bioconductor (version 4.5.1/3.21); Non-integer values (characteristic of kallisto-quant ) were rounded to integers and counts of different transcripts for the same gene were summed, and the preprocessed count matrix was normalized and analyzed with DESeq2 . Subsequent pathway enrichment analyses and gene set enrichment analysis (GSEA) was performed with clusterProfiler . Preparation and injection of recombinant adeno-associated virus (AAV) A recombinant AAV vector expressing IKK2 was constructed by inserting the full-length cDNA for human IKK2 into the pAAV-CAGGFP (origene). Then, AAV serotype 9 (AAV9) viral particles with mouse MBP promoter were produced by the custom AAV production service at the virus facility of Research Animal Resource Center at the Korea Institute of Science and Technology. Premade control AAV9-mMBP-mCherry viral particles were purchased from the same facility. The titer of both viral particles was 5 x 10 12 genome copies/mL. Animals and surgical procedures C57BL/6 male mice (Orient Bio, Republic of Korea) or TLR2 (-/-) male mice (on a C57BL/6 background; 25–28 g, originally generated by Takeuchi, O., et al.), were used. Mice were housed four or five per cage in a temperature- and humidity-controlled facility under a 12-hour light/dark cycle with ad libitum access to food and water. All procedures adhered to ethical guidelines to minimize animal suffering and the number of animals used, reviewed and approved by the Institutional Animal Care And Use Committee of Ajou University. The optimal number of animals was determined based on prior studies. 10 – 12 Details are described in the Supplementary Information section. Tape removal test To quantify motor-sensory deficits following right internal capsular infarction, we performed a tape removal test, according to a previously described protocol with minor changes. 50 Details are described in the Supplementary Information section. Tissue processing and immunohistochemistry After transcardiac perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), brains were removed, post-fixed for 2 hours, and then immersed into a graded series of sucrose solutions for cryopreservation. Coronal sections (30µm thick) of the brain were obtained with a cryostat (Leica). To quantify the area of demyelination, coronal brain sections were stained with eriochrome cyanine, a dye that stains myelinated white matter. The eriochrome cyanine solution was composed as such; 24:1 (volume:volume) of 0.2% eriochrome cyanine RC (Sigma Aldrich):10% FeCl3·6H2O (Sigma Aldrich) in 3% HCl. For immunofluorescent staining, brain sections were incubated overnight at 4°C with primary antibodies. Sections were washed with PBS and then incubated with appropriate Alexa Fluor 488-, 594-, 647, and 680-tagged secondary antibodies (Invitrogen). Slides were mounted with an aqueous mounting medium (Biomeda, #M01) and visualized with a confocal microscope (Carl Zeiss, LSM800). Detailed antibodies are listed in the Supplementary Information section. Re-analysis of human brain transcriptome data Two human single-nucleus transcriptome datasets; datasets of human vascular dementia (VaD) (Mitroi et al., 2022) and multiple sclerosis (MS) (Macnair et al., 2025) were acquired from publically available databaseses. The datasets were imported to R/Bioconductor (version 4.5.1/3.21); the packages Seurat and harmony were used for data processing and integration. After Louvain clustering ( FindClusters in Seurat ) and determination of cell type, the correlation between the three genes TLR2, RELA, and BIRC3 was analyzed. Cells with no expression for the genes being analyzed for correlation were exempted from the corresponding analysis. Statistical analysis and image quantification Statistical analyses used in the article were conducted with GraphPad Prism, version 10.4.1 (GraphPad Software, Boston, Massachusetts, United States). One-way analysis of variance (ANOVA) and Kruskal–Wallis tests with Dunn’s multiple comparison tests, Welch’s t-tests, Mann-Whitney U tests, Pearson’s correlation tests, and simple linear regression were performed. Statistical analyses were two-sided except for one case, and statistical significance was defined as p < 0.05. Cell counting, fluorescence area & intensity quantification was performed with QuPath. Additional details on statistical methods for each experiment are provided in the corresponding figures and legends. Declarations Data availability The bulk-RNA sequencing results in this article have been deposited in the Gene Expression Omnibus (GEO) database at the National Center for Biotechnology (NCBI), under the ascension number GSE318814. The human vascular dementia dataset (Mitroi et al., 2022) can be found in the GEO database under the ascension number GSE213897, and the multiple sclerosis dataset (Macnair et al., 2025) can be found at Zenodo ( https://zenodo.org/records/8338963 ). Author information These authors contributed equally: Hanki Kim, Xuelian Jin, Jun Young Choi These authors jointly supervised this work: Jun Young Choi, Byung Gon Kim Authors and affiliations Department of Brain Science, Ajou University School of Medicine, Suwon, 16499, Republic of Korea Hanki Kim, Xuelian Jin, Jun Young Choi, Samma Tasneem Chowdhury, Yue-Xian Cui, Hyo Jin Cho, Seungyon Koh, Byung Gon Kim Department of Anatomy, Ajou University School of Medicine, Suwon 16499, Republic of Korea Hanki Kim Department of Neurology, Ajou University School of Medicine, Suwon, 16499, Republic of Korea Jun Young Choi, Byung Gon Kim Division of Emergency Neurology, Department of Emergency Medicine, Ajou University School of Medicine, Suwon, 16499, Republic of Korea Seungyon Koh Department of Biochemistry & Molecular Biology, Ajou University School of Medicine, Suwon, 16499, Republic of Korea Su Bin Lim Center for Convergence Research of Neurological Disorders, Ajou University School of Medicine, Suwon, 16499, Republic of Korea Hanki Kim, Jun Young Choi, Seungyon Koh, Byung Gon Kim Research Institute for Basic Sciences, Ajou University, Suwon, 16499, Republic of Korea Hanki Kim Department of Geriatrics, The Affiliated Suqian First People’s Hospital of Nanjing Medical University, Suqian, Jiangsu Province, 223800, China Xuelian Jin Department of Neurology, Yanbian University Hospital, Yanbian University, Yanjii, Jilin Province, 133002, China Yue-Xian Cui Research Animal Resource Center, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea Seung Eun Lee Competing interests The authors declare no competing interests Supplementary information Supplementary materials Uncropped raw immunoblot images Author contributions H.K : Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. X.J : Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – review & editing. J.Y.C : Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. S.T.C : Formal analysis, Investigation, Validation, Writing – review & editing. Y.C : Formal analysis, Methodology, Validation, Writing – review & editing. H.J.C : Data curation, Validation, Writing – review & editing. B.J.K : Data curation, Validation, Writing – review & editing. S.K : Formal analysis, Validation, Writing – review & editing. S.E.L : Methodology, Validation, Writing – review & editing. S.B.L : Data curation, Formal Analysis, Validation, Writing – review & editing. B.G.K : Conceptualization, Funding acquisition, Methodology, Supervision, Validation, Writing – review & editing. All authors approved the final version of the article, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS-2021-NR060141). This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (RS-2024-00335969, RS-2019-NR040055) This research was supported by the Korean Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare (RS-2025-2549402, RS-2025-02215497, RS-2021-KH113820) References Wang, Y. et al. White Matter Injury in Ischemic Stroke. Prog. Neurobiol. 141, 45 (2016). Nowak, B. et al. Animal models of focal ischemic stroke: brain size matters. Frontiers in stroke 2, (2023). 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Additional Declarations There is no conflict of interest Supplementary Files TLR2NFkBSupplementaryMaterialsFinaal20260224.docx Supplementary materials TLR2NFkBUncroppedBlotImagesFinal20260224.docx Uncropped raw immunoblot images Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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-8957590","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":601725458,"identity":"9bdf4743-3ce5-42d5-bb16-9e76e2ad3ded","order_by":0,"name":"Jun Young 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12:52:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8957590/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8957590/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104780033,"identity":"b9ffae3e-8dea-4215-8111-b6074a507b07","added_by":"auto","created_at":"2026-03-17 07:49:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":413666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUncovering TLR2-mediated changes of candidate signaling proteins in primary oligodendrocytes. a \u003c/strong\u003eImmunoblots of signaling proteins in primary OLs stimulated by the TLR2 agonist Pam3. \u003cstrong\u003eb-c\u003c/strong\u003eQuantification of signaling proteins in \u003cstrong\u003ea\u003c/strong\u003e (n=5 biologically independent samples/experiments. One-way ANOVA and Tukey’s post hoc test, two-sided. Data presented as mean values ± SEM). \u003cstrong\u003ed\u003c/strong\u003e Schematic of potential candidate pathways for TLR2-mediated OL protection, before and after exclusion. Generated with the assistance of Biorender.com. \u003cstrong\u003ee \u003c/strong\u003eImmunoblots of signaling proteins in primary OLs exposed to combinations of the following variables; OGD and Pam3. \u003cstrong\u003ef-g\u003c/strong\u003e Quantification of signaling proteins in \u003cstrong\u003ee\u003c/strong\u003e (n=3 biologically independent samples/experiments. One-way ANOVA and Tukey’s post hoc test, two-sided. Data presented as mean values ± SEM). AKT = Protein Kinase B; CREB = cAMP Response Element-Binding protein; ERK = Extracellular signal-Regulated Kinase; IKK = IκB Kinase; IRAK = Interleukin-1 Receptor-Associated Kinase; IκB-α = Inhibitor of NF-κB alpha; JNK = c-Jun N-terminal Kinase; MEK = Mitogen-activated protein Kinase Kinase; MKK = Mitogen-activated protein Kinase Kinase; MyD88 = Myeloid differentiation primary response protein 88; NEMO = NF-κB Essential Modulator; NF-κB = Nuclear Factor kappa-light-chain-enhancer of activated B cells; OGD = Oxygen-Glucose Deprivation; Pam3 = Pam3CSK4; PI3K = Phosphoinositide 3-Kinase; TAB2 = TAK1-Binding Protein 2; TAK1 = TGFβ Activated Kinase 1; TIRAP = Toll/Interleukin-1 Receptor domain-containing Adaptor Protein; TLR = Toll-like Receptor; TRAF = TNF Receptor-Associated Factor.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8957590/v1/c4448774d18c7cd5cb67fce1.png"},{"id":104406140,"identity":"740c8da0-6f8d-42e4-a063-7e328263e0ba","added_by":"auto","created_at":"2026-03-11 12:24:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":118411,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNecessity of candidate signaling proteins for TLR2-mediated oligodendrocyte protection. a \u003c/strong\u003eLDH cell death assays in primary OLs, exposed to combinations of the following variables; OGD, Pam3 and chemical inhibitors for candidate signaling proteins (n=5 biologically independent samples/experiments. One-way ANOVA and Tukey’s post hoc test, two-sided. Data presented as mean values ± SEM). \u003cstrong\u003eb \u003c/strong\u003eLDH cell death assays in primary OLs, exposed to combinations of the following variables; OGD, Pam3 and siRNA knock-down for candidate signaling proteins s (n=3 biologically independent samples/experiments. One-way ANOVA and Tukey’s post hoc test, two-sided. Data presented as mean values ± SEM). \u003cstrong\u003ec\u003c/strong\u003eSchematic of potential candidate pathways for TLR2-mediated OL protection, before and after exclusion. Generated with the assistance of Biorender.com. AKT = Protein Kinase B; CREB = cAMP Response Element-Binding protein; ERK = Extracellular signal-Regulated Kinase; IKK = IκB Kinase; IκB-α = Inhibitor of NF-κB alpha; JNK = c-Jun N-terminal Kinase; LDH = Lactate Dehydrogenase; MEK = Mitogen-activated protein Kinase Kinase; MKK = Mitogen-activated protein Kinase Kinase; NEMO = NF-κB Essential Modulator; NF-κB = Nuclear Factor kappa-light-chain-enhancer of activated B cells; OGD = Oxygen-Glucose Deprivation; Pam3 = Pam3CSK4; PI3K = Phosphoinositide 3-Kinase; TAK1 = TGFβ Activated Kinase 1; TLR = Toll-like Receptor.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8957590/v1/6ed2eae55cdb0b6b466c309d.png"},{"id":104371709,"identity":"1c240786-fc60-4b74-8fdf-037b0eeebf11","added_by":"auto","created_at":"2026-03-11 05:15:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":252426,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSufficiency of candidate signaling proteins for TLR2-mediated oligodendrocyte protection. a \u003c/strong\u003eLDH cell death assays in primary OLs, exposed to combinations of the following variables; OGD, Pam3 and overexpression vectors for candidate signaling proteins (n=3 biologically independent samples/experiments. One-way ANOVA and Tukey’s post hoc test, two-sided. Data presented as mean values ± SEM). \u003cstrong\u003eb \u003c/strong\u003eImmunoblots of signaling proteins in primary OLs exposed to combinations of the following variables; Pam3, and overexpression vectors for candidate signaling proteins. \u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003eQuantification of signaling proteins in \u003cstrong\u003eb\u003c/strong\u003e (n=3 biologically independent samples/experiments. One-way ANOVA and Tukey’s post hoc test, two-sided. Data presented as mean values ± SEM).\u003cstrong\u003e e \u003c/strong\u003eLDH cell death assays in primary OLs, exposed to combinations of the following variables; OGD, overexpression vectors and siRNA knock-down for candidate signaling proteins s (n=3 biologically independent samples/experiments. One-way ANOVA and Tukey’s post hoc test, two-sided. Data presented as mean values ± SEM). \u003cstrong\u003ef \u003c/strong\u003eImmunoblots of signaling proteins in primary OLs exposed to combinations of the following variables; Overexpression vectors and siRNA knock-down for candidate signaling proteins. \u003cstrong\u003eg-i\u003c/strong\u003e Quantification of signaling proteins in \u003cstrong\u003ef\u003c/strong\u003e (n=3 biologically independent samples/experiments. One-way ANOVA and Tukey’s post hoc test, two-sided. Data presented as mean values ± SEM). \u003cstrong\u003ej\u003c/strong\u003e Schematic of TLR2-mediated OL protection, reflecting experiment results of \u003cstrong\u003eFig.3\u003c/strong\u003e. Generated with the assistance of Biorender.com. Bcl2 = B-cell Lymphoma 2; cIAP2 = Cellular Inhibitor of Apoptosis Protein 2; Cyt = Cytoplasm; IKK = IκB Kinase; LDH = Lactate Dehydrogenase; MKK = Mitogen-activated protein Kinase Kinase; NEMO = NF-κB Essential Modulator; NF-κB = Nuclear Factor kappa-light-chain-enhancer of activated B cells; Nuc = Nucleus; OGD = Oxygen-Glucose Deprivation; Pam3 = Pam3CSK4; TAK1 = TGFβ Activated Kinase 1; TLR = Toll-like Receptor.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8957590/v1/0e097116e6be02976c8253c2.png"},{"id":104405992,"identity":"bc2b51b7-eae6-43c5-8316-94877270b1a2","added_by":"auto","created_at":"2026-03-11 12:24:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":153517,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe NF-κB-cIAP2 axis in TLR2-mediated oligodendrocyte protection. a \u003c/strong\u003eLDH cell death assays in primary OLs, exposed to combinations of the following variables; OGD, overexpression vector for IKK2 and siRNA knock-down of cIAP2 (n=3 biologically independent samples/experiments. One-way ANOVA and Tukey’s post hoc test, two-sided. Data presented as mean values ± SEM). \u003cstrong\u003eb \u003c/strong\u003eImmunoblots of signaling proteins in primary OLs exposed to combinations of the following variables; Overexpression vector for IKK2 and siRNA knock-down of cIAP2. \u003cstrong\u003ec\u003c/strong\u003eQuantification of signaling proteins in \u003cstrong\u003eb\u003c/strong\u003e (n=3 biologically independent samples/experiments. One-way ANOVA and Tukey’s post hoc test, two-sided. Data presented as mean values ± SEM). \u003cstrong\u003ed \u003c/strong\u003eLDH cell death assays in primary OLs, exposed to combinations of the following variables; OGD, overexpression vector for cIAP2 and siRNA knock-down of p65 (n=3 biologically independent samples/experiments. One-way ANOVA and Tukey’s post hoc test, two-sided. Data presented as mean values ± SEM). \u003cstrong\u003ee \u003c/strong\u003eImmunoblots of signaling proteins in primary OLs exposed to combinations of the following variables; Overexpression vector for cIAP2 and siRNA knock-down of p65. \u003cstrong\u003ef\u003c/strong\u003e Quantification of signaling proteins in \u003cstrong\u003ee\u003c/strong\u003e (n=3 biologically independent samples/experiments. One-way ANOVA and Tukey’s post hoc test, two-sided. Data presented as mean values ± SEM). \u003cstrong\u003eg\u003c/strong\u003e Schematic of TLR2-mediated OL protection, reflecting experiment results of \u003cstrong\u003eFig.4\u003c/strong\u003e. Generated with the assistance of Biorender.com. cIAP2 = Cellular Inhibitor of Apoptosis Protein 2; Cyt = Cytoplasm; IKK = IκB Kinase; LDH = Lactate Dehydrogenase; MKK = Mitogen-activated protein Kinase Kinase; NEMO = NF-κB Essential Modulator; NF-κB = Nuclear Factor kappa-light-chain-enhancer of activated B cells; Nuc = Nucleus; Myc = Myelocytomatosis; OGD = Oxygen-Glucose Deprivation; TAK1 = TGFβ Activated Kinase 1; TLR = Toll-like Receptor\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8957590/v1/c942459629a4afad5ecd00a0.png"},{"id":104371711,"identity":"febd8ed5-b0e7-46f8-b07e-b9b248c3e12b","added_by":"auto","created_at":"2026-03-11 05:15:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":238132,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvidence of the TLR2-NF-κB-cIAP2 axis in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e oligodendrocyte transcriptome. a \u003c/strong\u003ePCA of Bulk-RNA sequencing results of primary OLs exposed to OGD, with or without Pam3 (n=3 biologically independent samples/experiments). \u003cstrong\u003eb\u003c/strong\u003e Volcano plot of differentially expressed genes (DEGs), comparing the OGD+Pam3 group to the OGD-only control group. \u003cstrong\u003ec\u003c/strong\u003e Heatmap of DEGs, comparing the OGD+Pam3 group to the OGD-only control group. \u003cstrong\u003ed\u003c/strong\u003e Dot plot of ten upregulated GO terms in the OGD+Pam3 group in comparison to the OGD-only control group. \u003cstrong\u003ee\u003c/strong\u003e GSEA for KEGG pathway rno04064 (NF-κB pathway for \u003cem\u003eRattus Norvegicus\u003c/em\u003e), comparing the OGD+Pam3 group to the OGD-only control group. \u003cstrong\u003ef\u003c/strong\u003e Heatmap of upregulated KEGG pathways and associated genes, comparing the OGD+Pam3 group to the OGD-only control group. Birc3 = Baculoviral IAP Repeat-Containing 3; cIAP2 = Cellular Inhibitor of Apoptosis Protein 2; Cyt = Cytoplasm; DEG = Differentially Expressed Gene; GO = Gene Ontology; GSEA = Gene Set Enrichment Analysis; IKK = IκB Kinase; KEGG = Kyoto Encyclopedia of Genes and Genomes; NF-κB = Nuclear Factor kappa-light-chain-enhancer of activated B cells; OGD = Oxygen-Glucose Deprivation; Pam3 = Pam3CSK4; PCA = Principal Component Analysis; TLR = Toll-like Receptor\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8957590/v1/66424f5a1149bed56308a374.png"},{"id":104406128,"identity":"86f59896-4064-40e7-9b38-2792d170ba5d","added_by":"auto","created_at":"2026-03-11 12:24:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2094860,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e oligodendrocyte protection by the NF-κB -cIAP2 axis in the L-NIO demyelination model. a \u003c/strong\u003eSchematic of the animal experiment. Generated with the assistance of Biorender.com. \u003cstrong\u003eb\u003c/strong\u003eConfiguration of viral vectors. Generated with the assistance of Biorender.com. \u003cstrong\u003ec\u003c/strong\u003e Representative images of L-NIO-induced demyelinating lesions in the right internal capsule of the murine brain, stained with eriochrome cyanine or anti-MBP antibodies. \u003cstrong\u003ed\u003c/strong\u003e Quantification of lesion volume based on eriochrome cyanine staining in \u003cstrong\u003ec\u003c/strong\u003e (n=6 biologically independent samples/experiments. Welch’s t-test, two-sided. Data presented as mean values ± SEM). \u003cstrong\u003ee\u003c/strong\u003e Quantification of tape removal test results (removal time) by days after L-NIO-induced demyelination (n=6 biologically independent samples/experiments. Two-way ANOVA with Šídák's multiple comparisons test, two-sided). \u003cstrong\u003ef\u003c/strong\u003e Correlation between lesion volume and tape removal time (n=6 biologically independent samples/experiments, Pearson’s correlation). \u003cstrong\u003eg-l\u003c/strong\u003eRepresentative images of immunostaining for cCas3, CC1, phoshpho-p65, Olig2, and cIAP2 with corresponding quantification graphs (n=6 biologically independent samples/experiments. Welch’s t-test, two-sided. Data presented as mean values ± SEM). AAV = Adeno-associated Virus; CC1 = Adenomatous Polyposis Coli (APC) clone CC1 antibody; cCas3 = Cleaved Caspase 3; cIAP2 = Cellular Inhibitor of Apoptosis Protein 2; DAPI = 4′,6-diamidino-2-phenylindole; dpi = Days Post-injection; IKK = IκB Kinase; ITR = Inverted Terminal Repeat; L-NIO = N5-(1-Iminoethyl)-L-ornithine; MBP = Myelin Basic Protein; mCh = mCherry; NF-κB = Nuclear Factor kappa-light-chain-enhancer of activated B cells; NOS = Nitric Oxide Synthase; TLR = Toll-like Receptor; WT = Wildtype\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8957590/v1/aec09f78a01a0b9a99ffe60a.png"},{"id":104371713,"identity":"b8123ddc-6288-44e6-a36e-9db3ae2c9414","added_by":"auto","created_at":"2026-03-11 05:15:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":459790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation between TLR2, RELA, and BIRC3 in the human brain transcriptome. a \u003c/strong\u003eUMAP plot of the human vascular dementia dataset (Mitroi et al., 2022) and Pearson’s correlation between the three genes \u003cem\u003eTLR2\u003c/em\u003e, \u003cem\u003eRELA\u003c/em\u003e, and \u003cem\u003eBIRC3\u003c/em\u003efor three cell types; mature OLs, OPCs/COPs, and microglia. \u003cstrong\u003eb\u003c/strong\u003e UMAP plot of the human multiple sclerosis dataset (Macnair et al., 2025) and Pearson’s correlation between the three genes \u003cem\u003eTLR2\u003c/em\u003e, \u003cem\u003eRELA\u003c/em\u003e, and \u003cem\u003eBIRC3\u003c/em\u003efor three cell types; mature OLs, OPCs/COPs, and microglia. BCAS1 = Breast Carcinoma Amplified Sequence 1; BIRC3 = Baculoviral IAP Repeat Containing 3; CSF1R = Colony stimulating factor 1 receptor; MBP = Myelin basic protein; OPC/COP = Oligodendrocyte progenitor cell/committed oligodendrocyte progenitor; RELA = RELA proto-oncogene, NF-kB subunit; TLR = Toll-like receptor\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-8957590/v1/8b81d2af2e92f8718d734106.png"},{"id":107869735,"identity":"0d86dfc9-069f-4630-b6c6-356fe15bd0dc","added_by":"auto","created_at":"2026-04-27 07:38:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4039002,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8957590/v1/652a0afa-d3dc-4121-8731-d2ce33c3e6b8.pdf"},{"id":104406081,"identity":"60038f10-b0d8-4bee-b174-0b266cac23c4","added_by":"auto","created_at":"2026-03-11 12:24:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2908601,"visible":true,"origin":"","legend":"Supplementary materials","description":"","filename":"TLR2NFkBSupplementaryMaterialsFinaal20260224.docx","url":"https://assets-eu.researchsquare.com/files/rs-8957590/v1/7d1d9745e44f5bef250ce6cc.docx"},{"id":104371715,"identity":"0f3f6395-aa56-43ba-818e-83a76df6f26f","added_by":"auto","created_at":"2026-03-11 05:15:16","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":31929840,"visible":true,"origin":"","legend":"Uncropped raw immunoblot images","description":"","filename":"TLR2NFkBUncroppedBlotImagesFinal20260224.docx","url":"https://assets-eu.researchsquare.com/files/rs-8957590/v1/e1707b833e0b14d8aa1a9d2e.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Toll-like Receptor 2 Exerts Oligodendrocyte Protection in White Matter Stroke Through Downstream NF-κB-cIAP2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIschemic stroke is a major cause of morbidity and mortality worldwide. Humans are unique among mammals for having a larger proportion of white matter (WM) than gray matter (GM), which is reflected in stroke pathology: approximately twenty-five percent of ischemic strokes are located exclusively in the WM, and more than half of the damaged regions in large-vessel occlusive strokes involve the WM.\u003csup\u003e1,2\u003c/sup\u003e Furthermore, white matter stroke (WMS) is an important prognostic factor for recovery after a large vessel occlusive stroke, and when repeated, is a major contributor to vascular dementia, the second most common form of senile dementia.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e In contrast with such human pathophysiology, many rodent studies on ischemic stroke have focused on GM pathology, utilizing the middle cerebral artery occlusion (MCAO) model, given that rodents have only ten to fifteen percent of WM in their brains.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e The lack of consideration for WMS in rodent models is one of the potential reasons behind the limited progress in trials for neuroprotective agents for ischemic stroke.\u003c/p\u003e \u003cp\u003eThe implications and significance of WMS are not confined to ischemic brain diseases. WMS may contribute to the pathogenesis or progression of a variety of neurodegenerative diseases. For example, WM pathology is a common feature in Alzheimer\u0026rsquo;s disease (AD), hinting at a potential association between WM ischemia and Alzheimer\u0026rsquo;s disease.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Recent studies report a correlation between extensive WMS and early deterioration in AD. Therefore, it is conceivable that preventing or alleviating the WMS may delay clinical deterioration in AD patients. Another example of WMS involvement in neurodegenerative diseases can be found with Parkinson\u0026rsquo;s disease (PD). WMS is closely associated with the progression of cognition and even with L-dopa responsiveness in patients with PD.\u003csup\u003e7,8\u003c/sup\u003e In brief summary, WMS is an important underlying feature of neurodegenerative diseases and closely associated with disease progression.\u003c/p\u003e \u003cp\u003ePrincipal pathologic findings in WMS include ischemic oligodendrocyte (OL) death and subsequent demyelination. OLs are intrinsically vulnerable to ischemic insult, a fact reflected in WM often sustaining greater damage than GM following an ischemic event.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Therefore, protecting OLs from ischemic cell death or promoting oligodendrogenesis and remyelination after WMS are crucial therapeutic strategies to mitigate the detrimental effects of WMS and slow down the progression of neurodegenerative diseases. A notable characteristic of WMS is its slow progression, suggesting an extended therapeutic window. Unfortunately, despite the wide time frame for treatment, there are no WMS-specific therapeutic strategies currently available, making WMS a critical unmet medical need both in itself and for other vascular and neurodegenerative diseases.\u003c/p\u003e \u003cp\u003eIn prior studies, our group published the cell-autonomous role of toll-like receptor 2 (TLR2) in ischemic oligodendrocyte death and demyelination in a focal internal capsule infarction model of WMS, and reported that high mobility group box 1 (HMGB1) acts as an endogenous autocrine activator of TLR2, and that this axis is necessary, though not sufficient, to protect OLs from ischemic insult.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e More recently, we have demonstrated that HMGB1 acts through TLR2 as an autocrine chemoattractant for oligodendrocyte precursor cells (OPCs) to facilitate OPC recruitment in WMS lesions.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Despite such key findings, the OL-survival signaling pathways downstream of TLR2 has not yet been fully elucidated. The characterization of this downstream intracellular survival-signaling pathway is essential for the establishment of WMS-specific druggable targets and novel therapeutic strategies. In the current study, we aim to discover the detailed signaling pathways involved in the TLR2-mediated OL survival from ischemic insult.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eTLR2 downstream signals ERK1/2, p38 and NF-κB, but not AKT and JNK were activated in OLs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTLR2 is a transmembrane TLR which is known to act primarily through the MyD88 pathway and not the TIR domain-containing adaptor inducing interferon-β (TRIF) pathway.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Therefore, candidates for the OL-protective signaling pathways downstream of TLR2 include; the mitogen-activated protein kinase (MAP kinase) pathway (such as ERK1/2, JNK, and p38), the nuclear factor-kappa-light-chain-enhancer of activated B (NF-κB) pathway, and the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) pathway\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The first step in delineating the OL-protective signaling pathway of TLR2 was to observe which of these candidates would be activated in OLs by the TLR2 agonist Pam3CSK4 (Pam3). We applied Pam3 to primary cultured OLs, which we have shown to protect OLs from oxygen-glucose deprivation (OGD) in our previous report. We measured the activation of candidate signal proteins at 15, 30, and 60 minutes after Pam3 application \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Increased phosphorylation of p38 (\u003cem\u003ep\u003c/em\u003e=.0005), ERK1/2 (\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001), and CREB (\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001) (downstream effector of MAP kinase pathways) after Pam3 application on OLs were observed, along with the increased degradation of IκB-α (\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001), indicating the activation of NF-κB. However, there was no phosphorylation of AKT and JNK in the same circumstances \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb,c\u003cb\u003e)\u003c/b\u003e. These findings were suggestive that activation of TLR2 did not cause the activation of AKT and JNK in OLs. Thus, AKT and JNK were exempted from potential candidates and excluded from further experiments \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe measured the activation of p38, ERK1/2, CREB, and NF-κB following OGD, with or without Pam3 application \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. An increase in the phosphorylation of p38 (\u003cem\u003ep\u003c/em\u003e=.03), ERK1/2 (\u003cem\u003ep\u003c/em\u003e=.02), and CREB (\u003cem\u003ep\u003c/em\u003e=.02) in the combined OGD\u0026thinsp;+\u0026thinsp;Pam3 group compared to the OGD-alone group was observed, and OGD\u0026thinsp;+\u0026thinsp;Pam3 showed the highest expression of the three signaling proteins \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, g\u003cb\u003e)\u003c/b\u003e. In case of the NF-κB pathway, both OGD (\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001) and Pam3 (\u003cem\u003ep\u003c/em\u003e=.003) caused increased IκB-α degradation, and the OGD\u0026thinsp;+\u0026thinsp;Pam3 group exhibited the lowest expression of IκB-α \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ep38 and NF-κB, but not ERK1/2, are necessary for TLR2-mediated OL protection from ischemia\u003c/h2\u003e \u003cp\u003eGiven that p38, ERK1/2, CREB and NF-κB showed activation after TLR2 activation in oligodendrocytes, we performed further examinations to test which signaling proteins were essential to TLR2-mediated OL protection from ischemia. First, we applied inhibitors to each signaling protein to observe the reversal of Pam3-mediated protection of OLs from OGD; SB202190 for p38 inhibition, U0126 for ERK1/2 inhibition, and BAY 11-7085 for NF-κB inhibition. In concordance with our prior studies, TLR2 agonism by Pam3 reduced OGD-induced OL death, measured as LDH release from dead OLs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b\u003cb\u003e)\u003c/b\u003e. Both p38 and NF-κB inhibition by SB202190 and BAY 11-7085, respectively, resulted in the reversal of TLR2-mediated OL protection (\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001). However, intriguingly, ERK1/2 inhibition with U0126 was not sufficient to overturn TLR2-mediated OL protection \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe validated the results through siRNA knock-down of each signal protein, p38, ERK1/2, CREB, and NF-κB, with siRNA transfection through electroporation. The siRNA successfully reduced the expression of each targeted protein compared to the control siRNA (\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001) \u003cb\u003e(Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea-d)\u003c/b\u003e. The knock-down of p38 and NF-κB reversed TLR2-mediated OL protection from OGD (\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001, \u003cem\u003ep\u003c/em\u003e=.0002), which was in concordance with the chemical inhibitors. The NF-κB knock-down showed increased OL death after OGD even in the absence of Pam3 (\u003cem\u003ep\u003c/em\u003e=.008). Additionally, also in concordance with the chemical inhibitors, neither ERK1/2 nor CREB knock-down was capable of reversing TLR2-mediated OL protection \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. In summary, we deduced that the ERK1/2 pathway, although activated in OLs by TLR2 agonism, does not reduce ischemic OL death. The ERK1/2 pathway was exempted from subsequent experiments, leaving only the p38 and NF-κB pathway as candidate mechanisms necessary for TLR2-mediated OL protection \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eFor further confirmation, we evaluated cell death with fluorescence-associated cell sorting (FACS) by propidium iodide (PI) and Annexin V after siRNA transfection to knock down either p38 or NF-κB. The results demonstrated that the knock-down of either p38 or NF-κB reversed TLR2-mediated OL protection, specifically in terms of apoptotic cell death \u003cb\u003e(Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ee-g)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe OL protection of TLR2-mediated p38 activation is exerted through NF-κB\u003c/h3\u003e\n\u003cp\u003eThrough the previous experiments, we narrowed down the candidate mechanisms to p38 and NF-κB, both of which were necessary factors for TLR2-mediated OL protection. We asked whether these two factors were also sufficient for TLR2-mediated OL protection.\u003c/p\u003e \u003cp\u003eTo clarify, we introduced the constitutive activation of p38 and NF-κB through the overexpression of MKK6 for p38 and IKK2 for NF-κB. We measured OL death after OGD, comparing the OL-protective effect of Pam3, MKK6 and IKK2. The activation of p38 by MKK6 showed protective effects comparable to that of Pam3. The activation of NF-κB by IKK2 had a higher protective effect compared with that of both Pam3 application (\u003cem\u003ep\u003c/em\u003e=.03) and p38 activation by MKK6 (\u003cem\u003ep\u003c/em\u003e=.01) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. To confirm whether the overexpression of each kinase properly activated their corresponding target protein, we performed immunoblotting \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. As expected from prior tests \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, Pam3 application successfully phosphorylated both p38 (\u003cem\u003ep\u003c/em\u003e=.005) and p65 (\u003cem\u003ep\u003c/em\u003e=.001), a subunit of NF-κB. IKK2 overexpression resulted in a significantly increased phosphorylation of p65 (\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001) but not p38. Interestingly, MKK6 overexpression caused the increased phosphorylation of not only p38 (\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001), but also p65 (\u003cem\u003ep\u003c/em\u003e=.02) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. We also observed the change in expression of the effector anti-apoptotic proteins B-cell leukemia/lymphoma 2 (Bcl2) and cellular inhibition of apoptosis protein 2 (cIAP2); while the distinction is not absolute, Bcl2 is more closely associated with p38 and cIAP2 is strongly related with NF-κB.\u003csup\u003e14\u003c/sup\u003e The expression of cIAP2 was upregulated by Pam3 (\u003cem\u003ep\u003c/em\u003e=.03), IKK2 (\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001) and MKK6 (\u003cem\u003ep\u003c/em\u003e=.03), and the degree of upregulation showed the same pattern of that of p65 phosphorylation caused by the same agents \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec,d\u003cb\u003e)\u003c/b\u003e. Conversely, Bcl2 expression was not different between all groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. These findings suggested that NF-κB was more likely to be a sufficient factor of TLR2-mediated OL protection, and that the protective effects of p38 were more likely to be exerted through the NF-κB pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo decipher the potential crosstalk between p38 and NF-κB, we crossly overexpressed the activating kinase while inhibiting the other through siRNA knock-down; IKK2 overexpression with p38 siRNA knockdown, and MKK6 overexpression with p65 siRNA knockdown. NF-κB activation by IKK2 overexpression was sufficient to protect OLs from death by OGD even in the presence of p38 siRNA knock-down (\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001). However, p38 activation by MKK6 overexpression was not sufficient to prevent OGD-induced OL death when p65 was knocked down \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. Immunoblotting of each protein showed successful activation and knock-down of targeted proteins \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef-h\u003cb\u003e)\u003c/b\u003e. The expression of the anti-apoptotic effector cIAP2 was reflective of these results; NF-κB activation upregulated cIAP2 even with p38 knock-down (\u003cem\u003ep\u003c/em\u003e=.02) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei\u003cb\u003e)\u003c/b\u003e, while p38 activation, which was previously shown to upregulate cIAP2 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e, could not replicate cIAP2 upregulation when the knock-down of p65 was in place. Through these findings, we concluded that NF-κB is the pathway that is both necessary and sufficient for TLR2-mediated OL protection from ischemia, and that the OL protection of p38 took effect via NF-κB.\u003c/p\u003e \u003cp\u003e \u003cb\u003ecIAP2 is a necessary and sufficient factor to protect OL from ischemic insult through NF-κB activation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven that cIAP2 was closely correlated with NF-κB activation and OL protection \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed,i\u003cb\u003e)\u003c/b\u003e, we further probed the role of cIAP2 as an essential effector for NF-κB signaling in OLs and its protective effect. To this end, we transfected siRNA for cIAP2 knock-down in OLs with or without IKK2 overexpression. cIAP2 knock-down reversed the OL protection by NF-κB activation via IKK2 (\u003cem\u003ep\u003c/em\u003e=.02) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. cIAP2 expression levels were downregulated by cIAP2 siRNA, reversing upregulation caused by IKK2-mediated NF-κB activation (\u003cem\u003ep\u003c/em\u003e=.003) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb,c\u003cb\u003e)\u003c/b\u003e. Notably, NF-κB activation could raise cIAP2 levels (\u003cem\u003ep\u003c/em\u003e=.01) and exert OL protection (\u003cem\u003ep\u003c/em\u003e=.0001) even in the presence of cIAP2 knock-down \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c\u003cb\u003e)\u003c/b\u003e. The degree of OL death showed a pattern that was the inverse of the cIAP2 expression levels in corresponding conditions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c\u003cb\u003e)\u003c/b\u003e. Thus, we demonstrated that cIAP2 is the downstream effector of NF-κB necessary for its OL-protective function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter uncovering cIAP2 as a necessary factor of the TLR2-NF-κB axis in OL protection, we further tested whether cIAP2 was sufficient for OL protection. To test this hypothesis, we overexpressed cIAP2 with or without the knock-down of NF-κB with p65 siRNA. The knock-down of p65 exacerbated OL death after OGD (\u003cem\u003ep\u003c/em\u003e=.0008), and cIAP2 overexpression could mitigate this effect (\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. cIAP2 overexpression was also sufficient to induce OL protection independently of NF-κB inhibition (\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. Similarly with the previous experiment, NF-κB inhibition with p65 siRNA could lower cIAP2 levels (\u003cem\u003ep\u003c/em\u003e=.01) and exacerbate OGD-induced OL death (\u003cem\u003ep\u003c/em\u003e=.006) even in the presence of cIAP2 overexpression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-f\u003cb\u003e)\u003c/b\u003e. The pattern of OGD-induced OL death was reflective of cIAP2 levels of corresponding conditions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed,f\u003cb\u003e)\u003c/b\u003e. In summary, we concluded that cIAP2 is the downstream effector of NF-κB, necessary and sufficient for its OL-protective function.\u003c/p\u003e\n\u003ch3\u003eThe presence of the TLR2-NF-κB-cIAP2 axis in the in vitro OL transcriptome\u003c/h3\u003e\n\u003cp\u003eTo validate our findings in an unbiased manner, we performed bulk RNA sequencing comparing OLs exposed to OGD, with or without Pam3. The addition of Pam3 induced a change in transcriptional profile visible upon principle component analysis (PCA) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Differentially expressed genes (DEGs) were mostly upregulated in the OGD+Pam3 group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb,c\u003cb\u003e)\u003c/b\u003e. Seventeen DEGs, including \u003cem\u003eBirc3\u003c/em\u003e (the gene name for cIAP2), were associated with the NF-κB pathway (KEGG rno04064), and all of them were upregulated in the OGD+Pam3 group except for \u003cem\u003eTraf6\u003c/em\u003e\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. Enriched Gene Ontology (GO) terms including \u003cem\u003eBirc3\u003c/em\u003e were; Negative regulation of apoptotic process, Regulation of apoptotic process and Canonical NF-kappaB signal transduction \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. Gene set enrichment analysis (GSEA) of the bulk RNA seq data for KEGG pathways showed that KEGG rno04064 (NF-κB pathway) was enriched in the OGD+Pam3 group (Normalized enrichment score (NES)\u0026thinsp;=\u0026thinsp;2.09, adjusted \u003cem\u003ep\u003c/em\u003e=. 2.28e-05), and that \u003cem\u003eBirc3\u003c/em\u003e ranked second among pathway-associated genes in terms of fold change \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. For a more comprehensive insight into the significance of the DEGs, we devised a composite score based on the KEGG GSEA results; The fold change of a specific gene multiplied by the number of enriched KEGG pathways the gene is involved in. When genes were ranked according to this score, \u003cem\u003eBirc3\u003c/em\u003e was among the highest ranking genes in the dataset, third in rank after chemokines \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e. Collectively, we detected evidence of the TLR2-NF-κB-cIAP2 axis in a comprehensive and unbiased searching of the OL transcriptome. Intriguingly, a number of cyclin-dependent kinase inhibitors, namely \u003cem\u003eCdkn1a\u003c/em\u003e (log\u003csub\u003e2\u003c/sub\u003e(fold change)\u0026thinsp;=\u0026thinsp;0.58, adjusted \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;3.0 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), \u003cem\u003eCdkn2b\u003c/em\u003e (log\u003csub\u003e2\u003c/sub\u003e(fold change)\u0026thinsp;=\u0026thinsp;0.46, adjusted \u003cem\u003ep=\u003c/em\u003e.001), and \u003cem\u003eCdkn1b\u003c/em\u003e (log\u003csub\u003e2\u003c/sub\u003e(fold change)\u0026thinsp;=\u0026thinsp;0.29, adjusted \u003cem\u003ep\u003c/em\u003e=.03), were upregulated in the OGD+Pam3 group \u003cb\u003e(Table.S1)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo confirmation in an animal model of WMS to see the protective effect of NF-κB activation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo validate our findings \u003cem\u003ein vivo\u003c/em\u003e, we devised an animal experiment utilizing a rodent model of WMS. A focal infarction in the internal capsule of C57BL/6 mice was induced by administration of the nitric oxide synthase (NOS) inhibitor N5-(1-Iminoethyl)-L-ornithine (L-NIO). Three weeks before L-NIO administration, control (AAV9-mMBP-mCherry) and IKK2 overexpression (AAV9-mMBP-IKK2) adeno-associated virus (AAV) vectors, given OL specificity through the myelin basic protein (MBP) promoter, were administered \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea,b\u003cb\u003e)\u003c/b\u003e. IKK2 was chosen over cIAP2 as the gene to deliver, given that cIAP2 siRNA couldn\u0026rsquo;t completely reverse the protective effects of IKK2 overexpression (\u003cem\u003ep\u003c/em\u003e=.04) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. The specificity of the AAV vector to OLs could be measured by assessing colocalization of mCherry with the OL marker Olig2, which showed that ~\u0026thinsp;96.7% of mCherry+ cells were Olig2+, indicating successful OL-selective gene delivery \u003cb\u003e(Fig.\u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea-c)\u003c/b\u003e. Motor-sensory function was measured by the tape removal test, at one day before and three, seven days after L-NIO injection, after which brain samples were collected for histology \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDemyelinating lesion volume, measured by Eriochrome cyanine staining, was smaller in IKK2 group animals (\u003cem\u003ep\u003c/em\u003e=.002), which also showed better motor-sensory function (lower latency for tape removal from affected paw, \u003cem\u003ep\u003c/em\u003e=.03) that was linearly correlated to lesion volume (r\u0026thinsp;=\u0026thinsp;0.78, \u003cem\u003ep\u003c/em\u003e=.003) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-f\u003cb\u003e)\u003c/b\u003e. p65 phosphorylation (\u003cem\u003ep\u003c/em\u003e=.04), cIAP2 upregulation (\u003cem\u003ep\u003c/em\u003e=.002), and decreased caspase 3 cleavage (\u003cem\u003ep\u003c/em\u003e=.02) in OLs was observed in the perilesional area of IKK2 group animals, suggesting that the OL protection, mainly anti-apoptotic effect of NF-κB through cIAP2 was taking effect \u003cem\u003ein vivo\u003c/em\u003e \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg-l\u003cb\u003e)\u003c/b\u003e. Astroglial and microglial activation was not different between groups \u003cb\u003e(Fig.S3a-e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eOur past studies have documented the exacerbation of L-NIO-induced demyelination in TLR2(-/-) mice\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e; we hypothesized that the activation of downstream NF-κB by IKK2 would rescue this effect, regardless of TLR2 deficiency. In concordance with prior studies, the baseline lesion volume was larger, motor-sensory function was worse, and cIAP2 immunoreactivity was lower in TLR2(-/-) animals compared to wildtype animals \u003cb\u003e(Fig.S4b,c,f)\u003c/b\u003e. The linear correlation between lesion volume and motor-sensory function was preserved in TLR2(-/-) animals (r\u0026thinsp;=\u0026thinsp;0.62, \u003cem\u003ep\u003c/em\u003e=.03) \u003cb\u003e(Fig.S4d)\u003c/b\u003e. As we hypothesized, the OL protection by NF-κB activation could indeed be observed in TLR2(-/-) animals; lesion volume was reduced in TLR2(-/-)-IKK2 animals (\u003cem\u003ep\u003c/em\u003e=.02), which also exhibit improved motor-sensory function (\u003cem\u003ep\u003c/em\u003e=.0001) \u003cb\u003e(Fig.S4b,c)\u003c/b\u003e. More OLs were positive for cIAP2 in the perilesional area of TLR2(-/-)-IKK2 animals (\u003cem\u003ep\u003c/em\u003e=.04) \u003cb\u003e(Fig.S4e,f)\u003c/b\u003e. Astroglial, microglial was not different between groups \u003cb\u003e(Fig.S4g-k)\u003c/b\u003e. In conclusion, the OL-protective effect of NF-κB activation could be observed in an \u003cem\u003ein vivo\u003c/em\u003e animal model of WMS, without regard to TLR2 functionality.\u003c/p\u003e\n\u003ch3\u003eEvidence of the TLR2-NFkB-cIAP2 axis in human brain diseases\u003c/h3\u003e\n\u003cp\u003eThrough our animal experiments, we observed OL protection against WMS through NF-κB-cIAP2. TLR2-deficient animals showed larger lesions, impaired neurological function, and lower cIAP2 immunoreactivity, which could be rescued by activation of NF-κB-cIAP2. To observe the existence of the TLR2-NF-κB-cIAP2 axis in human brain OLs, we re-analyzed two single-nucleus transcriptome datasets; a dataset of human vascular dementia (VaD) (Mitroi et al., 2022) and multiple sclerosis (MS) (Macnair et al., 2025) to observe the correlation between \u003cem\u003eTLR2\u003c/em\u003e and \u003cem\u003eBIRC3\u003c/em\u003e (cIAP2) in mature OLs, OPCs/committed OL progenitors (COPs), and microglia in the brain. In the VaD dataset, mature OLs showed a robust linear correlation between \u003cem\u003eTLR2-BIRC3\u003c/em\u003e (r\u0026thinsp;=\u0026thinsp;0.81, p\u0026lt;.0001), \u003cem\u003eTLR2-RELA\u003c/em\u003e (NF-κB) (r\u0026thinsp;=\u0026thinsp;0.86, p\u0026lt;.0001), and \u003cem\u003eRELA-BIRC3\u003c/em\u003e (r\u0026thinsp;=\u0026thinsp;0.71, p\u0026lt;.0001) once cells with zero values for the genes were removed \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Microglia also showed a significant correlation between the three genes, but the magnitude of linear correlation was lower compared to mature OLs. OPCs/COPs did not have a sufficient expression of TLR2, RELA, and BIRC3 to enable an analysis of correlation. The MS dataset showed a generally similar pattern, with mature OLs showing a robust linear correlation between components of the TLR2-NF-κB-cIAP2 axis \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. However, the mature OLs in the MS dataset did not have a sufficient number of cells that had non-zero values for \u003cem\u003eTLR2\u003c/em\u003e and \u003cem\u003eBIRC3\u003c/em\u003e, rendering correlation analysis in accurate. To overcome the significant zero-inflation, which is characteristic of single-cell level transcriptome analyses, we generated \u0026ldquo;metacells\u0026rdquo;, which are aggregates of cells showing similar gene expression, by K means clustering using the UMAP plot coordinates of individual cells \u003cb\u003e(Fig.S5b)\u003c/b\u003e. Through this strategy, the correlation between \u003cem\u003eTLR2\u003c/em\u003e and \u003cem\u003eBIRC3\u003c/em\u003e could be assessed \u003cb\u003e(Fig.S5c)\u003c/b\u003e. Collectively, we have demonstrated the presence of the TLR2-NF-κB-cIAP2 axis in the human brain OL transcriptome.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe detailed signaling pathway for TLR2-mediated OL protection, from ischemic cell death, has been delineated in the present study. While microglia are the cells in the central nervous system (CNS) which express the widest range of TLRs, OLs and also astrocytes are known to express TLR2 and TLR3.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e The existence of a cell-autonomous protective mechanism for OLs, acting through TLR2 upon autocrine damage signals such as HMGB1, has been demonstrated in our previous studies.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e As TLR2 signaling has numerous downstream pathways and functions, some of which are associated with inflammation, characterization of the precise pathway through which TLR2 signaling exerts its OL protection is essential for drug development/repositioning exploiting TLR2-mediated OL protection. Using the TLR2 agonist Pam3, chemical inhibitors, siRNA knock-down and overexpression for TLR2 downstream signals, we have identified the NF-κB pathway and cIAP2 as both necessary and sufficient factors for TLR2-mediated OL protection.\u003c/p\u003e \u003cp\u003eTLR2 and other TLRs are pattern recognition receptors (PRRs), initially demonstrated to recognize pathogen-associated molecular patterns (PAMPs) to orchestrate innate immune responses, and later reported to also recognize damage-associated molecular patterns (DAMPs) which are also necessary for regulating immune responses.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e In contradiction to predictions that TLR2 signaling would result in neuroinflammation and CNS degeneration, numerous studies have shown that TLR2 signaling is beneficial for axon growth and glial survival, and that blocking the TLR2 pathway results in exacerbated CNS pathologies.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e More specifically, TLR2 agonists such as Zymosan or Pam3 have been shown to promote axonal growth and alleviate WM damage in the brain and spinal cord, while blockage of TLR2 has been reported to induce multiple abnormalties in the brain and exacerbate CNS pathologies.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e In a prior study, the sterile stimulation of microglia with Pam3 resulted in a mixed manifestation of both the pro-inflammatory M1 and pro-regenerative M2 phenotype rather than an exclusive M1 activation, which collectively induced a neuroprotective response to laser-induced spinal cord injury (SCI).\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Other studies have shown that constitutive TLR2 deletion resulted in detrimental glial activation leading to WM damage in a transgenic AD mouse model, leading to an amyloid β(Aβ)-independent exacerbation of AD pathology.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e In addition to such prior knowledge, our study elucidates a cell autonomous OL-protection mechanism that takes action through TLR2 and downstream NF-κB-cIAP2. TLR agonists such as monophosphoryl lipid A and CpG 1018, although not TLR2-specific, are currently in clinical use, serving as evidence of the efficacy and safety of this class of drugs, therefore making them a plausible target for drug repositioning.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe role of NF-κB in OL physiology and pathophysiology has been explored in previous studies, although such prior research is scarce compared to the bulk of knowledge on NF-κB in general. \u003cem\u003eIn vitro\u003c/em\u003e and animal level studies demonstrate that while the inactivation of IKK2 in mouse CNS cells (achieved by a nestin promoter) or astrocytes resulted in alleviation of toxic demyelination induced with cuprizone, its specific inactivation in OLs resulted in elevated myelin loss and remyelination failure in an animal model combining cuprizone and interferon-gamma (IFN-γ) administration.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Such studies provide further evidence that the protective function of NF-κB for OLs is strictly cell-autonomous, and suggests that non-cell-autonomous activation of NF-κB may actually be disadvantageous for OL survival, emphasizing the necessity for meticulous cell type targeting in order for potential OL therapeutic strategies based on NF-κB to be effective. NF-κB is also known as a essential factor for the survival of Schwann cells, the peripheral nervous system (PNS) counterpart of OLs, in an animal model of sciatic nerve injury.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Indirect clues on the role of NF-κB for OL survival can be found in research studies investigating tumor necrosis factor alpha (TNF-α), another activator of the NF-κB pathway, in multiple sclerosis (MS); while TNF-α is a well known contributor to MS and experimental autoimmune encephalitis (EAE) pathology, the use of anti-TNF-α agents resulted in a paradoxical exacerbation of MS shown by an increased number of demyelinating lesions.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Comprehensively, such findings warrant caution with the use of NF-κB inhibitors, many of which are ongoing clinical trial for the treatment of cancer.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Many broad NF-κB inhibitors have failed to become clinical drugs due to their inability to control the downside associated with suppressing beneficial functions tied to the NF-κB pathway. The present study shows that the resistance of OLs and CNS myelin to ischemic demyelination and WMS is another crucial benefit of the NF-κB pathway, further emphasizing the importance of precision medicine for this double-edged sword. The correlation with WMS may be another area in which NF-κB inhibitors should be evaluated, especially for patients with or at high risk of WMS, such as old age and hypertension.\u003c/p\u003e \u003cp\u003eUnlike with demyelination, evidence on the role of NF-κB for developmental myelination or myelination under normal conditions devoid of injury, both for OLs and Schwann cells, is conflicting, with some studies demonstrating that NF-κB is necessary for myelin formation while others showing it to be dispensable. A human case study has shown CNS myelin defects in three of five male patients with Xq28 duplication encompassing Methyl-CpG binding protein 2 (MECP2), which represses interleukin-1 receptor-associated kinase 1 (IRAK1). While IRAK1 is not specifically upstream of NF-κB exclusively, the myelin defects were more precisely correlated with the duplication of NEMO (IKKγ), which has inhibitory effects on the NF-κB pathway.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e In our study, NF-κB or p38 inhibition did not promote OL cell death in the absence of OGD \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea,b\u003cb\u003e)\u003c/b\u003e. Moreover, siRNA knock-down of cIAP2 itself did not lower cIAP2 levels without it being increased by the overexpression of IKK2 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e, and p65 siRNA knock-down could decrease cIAP2 levels even in the presence of direct cIAP2 overexpression (\u003cem\u003ep\u003c/em\u003e=.01) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e, indicating that NF-κB is a very potent regulator of cIAP2 expression in OLs, and that cIAP2 has low basal levels in the absence of activating stimuli. Constitutive activation of IKK/NF-κB has been shown to induce OL senescence and subsequent myelin loss, and the participance of NF-κB in general cellular senescence has been reported in different fields of research such as cancer biology.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e The upregulation of cyclin-dependent kinase inhibitors in Pam3-treated OLs further validates the senescence-inducing effect of prolonged NF-κB activation in OLs. This provides additional clarification that the NF-κB pathway is a survival signal for OLs in detrimental circumstances rather than a constitutively active promoter of myelination, and that setting an appropriate timepoint of application is another key factor necessary to harness and utilize NF-κB-mediated OL protection.\u003c/p\u003e \u003cp\u003eThe function of cIAP2 in OLs is a topic that is further elusive than that of NF-κB. The significance of cIAP2 in protecting murine kidneys and human tubular epithelial cells from ischemia/reperfusion injury from apoptosis has been documented.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e As for the CNS and OLs, a prior study reported that cIAP2(-/-) mice exhibited increased OL death and demyelination in response to EAE, but primarily focused on the impact of cIAP2 deletion in microglia, and not the cell-autonomous action of cIAP2 in OLs.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e Another study demonstrated that Leukemia inhibitory factor (LIF), a cytokine growth factor known to promote OL survival \u003cem\u003ein vitro\u003c/em\u003e, could arrest OL death and demyelination in an animal model of spinal cord injury through survival pathways including cIAP2.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e In our \u003cem\u003ein vitro/in vivo\u003c/em\u003e experiments, cIAP2 functioned as the end effector of the TLR2-NF-κB axis to promote OL survival in the face of ischemic damage. Re-analysis of the human brain transcriptome showed that mature OLs exhibit a higher proportion of cells expressing \u003cem\u003eBIRC3\u003c/em\u003e compared to OPC/COPs \u003cb\u003e(Fig.S5a)\u003c/b\u003e, which may serve as one of the factors behind the higher vulnerability of OPC/COPs to ischemic damage.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e The significance of cIAP2 as an OL-protective mechanism in our experiments and as a potential mechanism behind maturation-dependent ischemia resistance collectively suggests that cIAP2 is a core component of OL resistance to ischemia. Additionally, as with NF-κB, second mitochondria-derived activator of caspases (SMAC) mimetics, which suppress IAP action, are showing promise as effective anti-tumor agents for various cancers including glioblastoma, and such drugs may also benefit from evaluation for potential side effects related to WM injury.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eDuring the delineation of the TLR2-NF-κB-cIAP2 pathway, we excluded a number of potential alternatives. The PI3k-Akt pathway and the MAP kinase JNK was dropped at the early stages of the sequence of \u003cem\u003ein vitro\u003c/em\u003e experiments, as they showed no response to Pam3. While ERK1/2 and downstream CREB (which may also serve as downstream for p38) were activated by Pam3, they did not participate in the OL-protection response to OGD. While ERK1/2 did not promote OL survival, our previous studies demonstrate its crucial role as the mediator of a TLR2-mediated autocrine chemotactic response to HMGB1, facilitating OPC migration and recruitment into WMS lesions. In our bulk RNA-seq results, we were able to see a substantial upregulation of various chemokines and chemotaxis-related GO terms \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed,f\u003cb\u003e)\u003c/b\u003e in the Pam3-treated OLs, which may be attributable to ERK1/2 activity. p38 was the one signal which also prevented OL death by OGD, despite its protection being dependent on NF-κB. A few prior studies show that p38 regulates OPC proliferation and differentiation into myelinating OLs, which has not been reported for NF-κB.\u003csup\u003e46–48\u003c/sup\u003e Future research demonstrating that p38 is OL-protective against WMS \u003cem\u003ein vivo\u003c/em\u003e may further potentiate its value as a therapeutic target.\u003c/p\u003e \u003cp\u003eHere, we describe the cell-autonomous OL-protective mechanism in WMS, initiated by TLR2 and taking action through downstream NF-κB and cIAP2. While meticulous targeting for cell type and timing is needed, this mechanism may serve as a valuable therapeutic target for WMS, which is a major unmet medical need in the CNS.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e\n\n "},{"header":"Methods and materials","content":"\u003ch2\u003ePrimary oligodendrocyte culture\u003c/h2\u003e\u003cp\u003ePrimary OPCs were isolated from cerebral cortices of postnatal day 1 Sprague-Dawley rats, following a method utilizing differential centrifugation with density gradient-forming agent Optiprep™, developed in this laboratory.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e Details are described in the \u003cb\u003eSupplementary Information\u003c/b\u003e section.\u003c/p\u003e\u003ch3\u003eOxygen-glucose deprivation (OGD) and application of chemical agents\u003c/h3\u003e\u003cp\u003eFor \u003cem\u003ein vitro\u003c/em\u003e OGD, OLs cultured for 24 hours in OL differentiation medium were washed with sterile PBS and transferred to an anaerobic chamber (95% N2 and 5% CO2). For the OGD group, the culture media was exchanged to a glucose-free OL differentiation media, and glucose (4.5 g/L) was added for the control. OLs were exposed to OGD for 6 hours, transferred back to normoxia with the addition of 4.5 g/L glucose and reoxygenation for another 18 hours.\u003c/p\u003e\u003cp\u003eChemical inhibitors for designated signaling pathways, such as SB202190 (Cell signaling, #8158, 1 µM) for p38, U0126 (Cell Signaling Technology, #9930S, 1 µM) for ERK1/2, and BAY 11-7085 (Santa Cruz, #sc202490, 1 µM) for NF-κB and the TLR2 agonist Pam3 (Invivogen, #tlrl-pms, 1 µg/mL) were added accordingly during the total 24-hour OGD/reoxygenation process.\u003c/p\u003e\u003ch2\u003eLactate dehydrogenase(LDH) cell death assay\u003c/h2\u003e\u003cp\u003eAt the endpoint of OGD/reoxygenation (24 hours after OGD onset), cell death was quantified by LDH assays using an colorimetric LDH assay kit (Takara Bio, #MK401) in accordance to the manufacturer’s protocol. LDH levels were measured as the optical density (OD) value at 490 nm. The LDH level corresponding to complete cell death was determined in sister cultures exposed to 1.5% Triton X-100 (high control, HC). Baseline LDH levels were determined in a medium-alone condition without cells (low control, LC). The percentage of cell death in each experiment were calculated using the following formula: % of OL death = (experimental value – LC)/(HC-LC) × 100.\u003c/p\u003e\u003ch2\u003eTransfection of siRNA and overexpression plasmids to primary OLs\u003c/h2\u003e\u003cp\u003eThe Amaxa Nucleofector® electroporation system and basic Nucleofector® kit for primary mammalian glial cells (Lonza, #VPI-1006) was used to transfect siRNAs and overexpression plasmids for each candidate protein. Briefly, OPCs, detached and pelleted after five days of proliferation, were resuspended with Nucleofector® solution with the designated siRNA/plasmid or corresponding control siRNA (Santa Cruz, #sc37007)/control plasmid (Origene, #PCMV-6XL4). Resuspended OPCs were transferred into an electroporation cuvette and were electroporated using the Nucleofector® II machine (program number for OLs/glia: A-033 or O-017). OL differentiation medium was added immediately after electroporation. OPCs were seeded onto designated culture surfaces at 3 × 10\u003csup\u003e4\u003c/sup\u003e cells/cm\u003csup\u003e2\u003c/sup\u003e in OL differentiation medium and cultured for 24 hours before OGD exposure. Detailed siRNA and plasmids are listed in the \u003cb\u003eSupplementary Information\u003c/b\u003e section.\u003c/p\u003e\u003ch2\u003eImmunoblotting\u003c/h2\u003e\u003cp\u003eCultured OLs were washed twice with PBS and harvested with a lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and a protease/phosphatase inhibitor cocktail. Cell lysates were centrifuged at 20,000 × g/20 min/4°C. Protein concentration was quantified by the Bradford assay (Bio-Rad, #5000205). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was done with 10% or 4–20% gradient gels (Bio-Rad), and the proteins on the gels were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk or 5% bovine serum albumin for 1 hours at room temperature and then incubated with primary antibodies. After repeated washing with 0.05% TBST, the membranes were incubated at RT with horse radish peroxidase-conjugated secondary antibodies. Finally, the membranes were visualized using enhanced chemiluminescence detection (Pierce). Detailed antibodies are listed in the \u003cb\u003eSupplementary Information\u003c/b\u003e section.\u003c/p\u003e\u003ch2\u003eIsolation of RNA and RNA-seq data processing and analysis\u003c/h2\u003e\u003cp\u003eRNA from primary OLs, three samples per group, was extracted using RNeasy Plus Micro Kit (Qiagen), following the manufacturer’s protocol. Purified RNA samples at 150–250 ng/µL concentration, 20 µL/sample and RNA Integrity Number (RIN) above 9.8 were processed for library preparation and RNA-sequencing at Novogene (Seoul, Korea) using the Novaseq Xplus PE150 platform with 6Gb sequencing depth. The resulting raw .fastq files were quality-controlled with FastQC. \u003cem\u003ekallisto-quant\u003c/em\u003e was used to quantify transcript abundances, using the Rattus Norvegicus genome/transcriptome (Rnor 6.0) as reference. The count matrix of transcript abundances was preprocessed and analyzed in R/Bioconductor (version 4.5.1/3.21); Non-integer values (characteristic of \u003cem\u003ekallisto-quant\u003c/em\u003e) were rounded to integers and counts of different transcripts for the same gene were summed, and the preprocessed count matrix was normalized and analyzed with \u003cem\u003eDESeq2\u003c/em\u003e. Subsequent pathway enrichment analyses and gene set enrichment analysis (GSEA) was performed with \u003cem\u003eclusterProfiler\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003ePreparation and injection of recombinant adeno-associated virus (AAV)\u003c/h2\u003e\u003cp\u003eA recombinant AAV vector expressing IKK2 was constructed by inserting the full-length cDNA for human IKK2 into the pAAV-CAGGFP (origene). Then, AAV serotype 9 (AAV9) viral particles with mouse MBP promoter were produced by the custom AAV production service at the virus facility of Research Animal Resource Center at the Korea Institute of Science and Technology. Premade control AAV9-mMBP-mCherry viral particles were purchased from the same facility. The titer of both viral particles was 5 x 10\u003csup\u003e12\u003c/sup\u003e genome copies/mL.\u003c/p\u003e\u003ch2\u003eAnimals and surgical procedures\u003c/h2\u003e\u003cp\u003eC57BL/6 male mice (Orient Bio, Republic of Korea) or TLR2 (-/-) male mice (on a C57BL/6 background; 25–28 g, originally generated by Takeuchi, O., et al.), were used. Mice were housed four or five per cage in a temperature- and humidity-controlled facility under a 12-hour light/dark cycle with ad libitum access to food and water. All procedures adhered to ethical guidelines to minimize animal suffering and the number of animals used, reviewed and approved by the Institutional Animal Care And Use Committee of Ajou University. The optimal number of animals was determined based on prior studies.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Details are described in the \u003cb\u003eSupplementary Information\u003c/b\u003e section.\u003c/p\u003e\u003ch2\u003eTape removal test\u003c/h2\u003e\u003cp\u003eTo quantify motor-sensory deficits following right internal capsular infarction, we performed a tape removal test, according to a previously described protocol with minor changes.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e Details are described in the \u003cb\u003eSupplementary Information\u003c/b\u003e section.\u003c/p\u003e\u003ch2\u003eTissue processing and immunohistochemistry\u003c/h2\u003e\u003cp\u003eAfter transcardiac perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), brains were removed, post-fixed for 2 hours, and then immersed into a graded series of sucrose solutions for cryopreservation. Coronal sections (30µm thick) of the brain were obtained with a cryostat (Leica). To quantify the area of demyelination, coronal brain sections were stained with eriochrome cyanine, a dye that stains myelinated white matter. The eriochrome cyanine solution was composed as such; 24:1 (volume:volume) of 0.2% eriochrome cyanine RC (Sigma Aldrich):10% FeCl3·6H2O (Sigma Aldrich) in 3% HCl. For immunofluorescent staining, brain sections were incubated overnight at 4°C with primary antibodies. Sections were washed with PBS and then incubated with appropriate Alexa Fluor 488-, 594-, 647, and 680-tagged secondary antibodies (Invitrogen). Slides were mounted with an aqueous mounting medium (Biomeda, #M01) and visualized with a confocal microscope (Carl Zeiss, LSM800). Detailed antibodies are listed in the \u003cb\u003eSupplementary Information\u003c/b\u003e section.\u003c/p\u003e\u003ch2\u003eRe-analysis of human brain transcriptome data\u003c/h2\u003e\u003cp\u003eTwo human single-nucleus transcriptome datasets; datasets of human vascular dementia (VaD) (Mitroi et al., 2022) and multiple sclerosis (MS) (Macnair et al., 2025) were acquired from publically available databaseses. The datasets were imported to R/Bioconductor (version 4.5.1/3.21); the packages \u003cem\u003eSeurat\u003c/em\u003e and \u003cem\u003eharmony\u003c/em\u003e were used for data processing and integration. After Louvain clustering (\u003cem\u003eFindClusters\u003c/em\u003e in \u003cem\u003eSeurat\u003c/em\u003e) and determination of cell type, the correlation between the three genes TLR2, RELA, and BIRC3 was analyzed. Cells with no expression for the genes being analyzed for correlation were exempted from the corresponding analysis.\u003c/p\u003e\u003ch2\u003eStatistical analysis and image quantification\u003c/h2\u003e\u003cp\u003eStatistical analyses used in the article were conducted with GraphPad Prism, version 10.4.1 (GraphPad Software, Boston, Massachusetts, United States). One-way analysis of variance (ANOVA) and Kruskal–Wallis tests with Dunn’s multiple comparison tests, Welch’s t-tests, Mann-Whitney U tests, Pearson’s correlation tests, and simple linear regression were performed. Statistical analyses were two-sided except for one case, and statistical significance was defined as \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. Cell counting, fluorescence area \u0026amp; intensity quantification was performed with QuPath. Additional details on statistical methods for each experiment are provided in the corresponding figures and legends.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eThe bulk-RNA sequencing results in this article have been deposited in the Gene Expression Omnibus (GEO) database at the National Center for Biotechnology (NCBI), under the ascension number GSE318814. The human vascular dementia dataset (Mitroi et al., 2022) can be found in the GEO database under the ascension number GSE213897, and the multiple sclerosis dataset (Macnair et al., 2025) can be found at Zenodo (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://zenodo.org/records/8338963\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch2\u003eAuthor information\u003c/h2\u003e\n\u003cp\u003eThese authors contributed equally: Hanki Kim, Xuelian Jin, Jun Young Choi\u003c/p\u003e\n\u003cp\u003eThese authors jointly supervised this work: Jun Young Choi, Byung Gon Kim\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors and affiliations\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Brain Science, Ajou University School of Medicine, Suwon, 16499, Republic of Korea\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHanki Kim, Xuelian Jin, Jun Young Choi, Samma Tasneem Chowdhury, Yue-Xian Cui, Hyo Jin Cho, Seungyon Koh, Byung Gon Kim\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Anatomy, Ajou University School of Medicine, Suwon 16499, Republic of Korea\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHanki Kim\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Neurology, Ajou University School of Medicine, Suwon, 16499, Republic of Korea\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJun Young Choi, Byung Gon Kim\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDivision of Emergency Neurology, Department of Emergency Medicine, Ajou University School of Medicine, Suwon, 16499, Republic of Korea\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeungyon Koh\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Biochemistry \u0026amp; Molecular Biology, Ajou University School of Medicine, Suwon, 16499, Republic of Korea\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSu Bin Lim\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCenter for Convergence Research of Neurological Disorders, Ajou University School of Medicine, Suwon, 16499, Republic of Korea\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHanki Kim, Jun Young Choi, Seungyon Koh, Byung Gon Kim\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch Institute for Basic Sciences, Ajou University, Suwon, 16499, Republic of Korea\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHanki Kim\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Geriatrics, The Affiliated Suqian First People\u0026rsquo;s Hospital of Nanjing Medical University, Suqian, Jiangsu Province, 223800, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXuelian Jin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Neurology, Yanbian University Hospital, Yanbian University, Yanjii, Jilin Province, 133002, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYue-Xian Cui\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch Animal Resource Center, Korea Institute of Science and Technology, Seoul, 02792, Republic of Korea\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeung Eun Lee\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003ch2\u003eSupplementary information\u003c/h2\u003e\n\u003cp\u003eSupplementary materials\u003c/p\u003e\n\u003cp\u003eUncropped raw immunoblot images\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eH.K\u003c/strong\u003e: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Software, Validation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eX.J\u003c/strong\u003e: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eJ.Y.C\u003c/strong\u003e: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eS.T.C\u003c/strong\u003e: Formal analysis, Investigation, Validation, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eY.C\u003c/strong\u003e: Formal analysis, Methodology, Validation, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eH.J.C\u003c/strong\u003e: Data curation, Validation, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eB.J.K\u003c/strong\u003e: Data curation, Validation, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eS.K\u003c/strong\u003e: Formal analysis, Validation, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eS.E.L\u003c/strong\u003e: Methodology, Validation, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eS.B.L\u003c/strong\u003e: Data curation, Formal Analysis, Validation, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eB.G.K\u003c/strong\u003e: Conceptualization, Funding acquisition, Methodology, Supervision, Validation, Writing \u0026ndash; review \u0026amp; editing. All authors approved the final version of the article, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS-2021-NR060141).\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (RS-2024-00335969, RS-2019-NR040055)\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Korean Health Industry Development Institute (KHIDI) funded by the Ministry of Health \u0026amp; Welfare (RS-2025-2549402, RS-2025-02215497, RS-2021-KH113820)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang, Y. \u003cem\u003eet al.\u003c/em\u003e White Matter Injury in Ischemic Stroke. \u003cem\u003eProg. 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Protoc.\u003c/em\u003e 4, 1560\u0026ndash;1564 (2009).\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":"","lastPublishedDoi":"10.21203/rs.3.rs-8957590/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8957590/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Ischemic white matter stroke (WMS) is a substantial proportion of ischemic stroke, a major contributor to vascular dementia and a common pathologic finding in neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease. Despite its significance, disease-specific treatment strategies for WMS are not available as of now, making WMS a major unmet medical need. The principal pathomechanism of WMS is the death of oligodendrocytes (OLs) and ensuing demyelination, therefore the prevention of ischemic OL death is crucial to overcoming WMS. In the present article, we delineate the signaling pathway of an OL-protective mechanism that rescues ischemic OL death, which is initiated by Toll-like receptor 2 (TLR2). Through a series of in vitro experiments with primary OLs, utilizing the TLR2 agonist, oxygen-glucose deprivation, chemical inhibitors, siRNA, and overexpression vectors for candidate signaling proteins, we uncovered that the mitogen-activated protein kinase p38 pathway and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway are necessary and sufficient factors for the TLR2-mediated OL protection. In detail, the p38 pathway took action through the NF-κB pathway to exert its protective functions, and the final downstream effector was cellular inhibitor of apoptosis protein 2 (cIAP2). We validated the presence of the TLR2-NF-κB-cIAP2 axis in an unbiased search of the OL transcriptome, in an animal model of internal capsule WMS, and in human OLs. In conclusion, we demonstrate that the TLR2-NF-κB-cIAP2 axis is an OL-protective mechanism against WMS, which may be a valuable therapeutic target for drug development/repositioning to address the unmet medical need.","manuscriptTitle":"Toll-like Receptor 2 Exerts Oligodendrocyte Protection in White Matter Stroke Through Downstream NF-κB-cIAP2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-11 05:15:11","doi":"10.21203/rs.3.rs-8957590/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":"ddd7a94d-eb12-458e-b60f-e4aabfca78f5","owner":[],"postedDate":"March 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":64036837,"name":"Biological sciences/Neuroscience/Regeneration and repair in the nervous system"},{"id":64036838,"name":"Biological sciences/Neuroscience/Diseases of the nervous system"},{"id":64036839,"name":"Biological sciences/Neuroscience/Molecular neuroscience"},{"id":64036840,"name":"Biological sciences/Neuroscience/Cell death in the nervous system"}],"tags":[],"updatedAt":"2026-04-24T09:49:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-11 05:15:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8957590","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8957590","identity":"rs-8957590","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}