Lomerizine inhibits LPS-mediated neuroinflammation and tau hyperphosphorylation by modulating NLRP3, DYRK1A, and GSK3α/β

IntroductionLomerizine is a calcium channel blocker that crosses the blood–brain barrier and is used clinically in the treatment of migraines. However, whether lomerizine is beneficial in modulating neuroinflammatory responses has not been tested yet.MethodsTo assess the potential of lomerizine for repurposing as a treatment for neuroinflammation, we investigated the effects of lomerizine on LPS-induced proinflammatory responses in BV2 microglial cells, Alzheimer’s disease (AD) excitatory neurons differentiated from induced pluripotent stem cells (iPSCs), and in LPS-treated wild type mice.ResultsIn BV2 microglial cells, lomerizine pretreatment significantly reduced LPS-evoked proinflammatory cytokine and NLRP3 mRNA levels. Similarly, lomerizine pretreatment significantly suppressed the increases in Iba-1, GFAP, proinflammatory cytokine and NLRP3 expression induced by LPS in wild-type mice. In addition, lomerizine posttreatment significantly decreased LPS-stimulated proinflammatory cytokine and SOD2 mRNA levels in BV2 microglial cells and/or wild-type mice. In LPS-treated wild-type mice and AD excitatory neurons differentiated from iPSCs, lomerizine pretreatment ameliorated tau hyperphosphorylation. Finally, lomerizine abolished the LPS-mediated activation of GSK3α/β and upregulation of DYRK1A, which is responsible for tau hyperphosphorylation, in wild-type mice.DiscussionThese data suggest that lomerizine attenuates LPS-mediated neuroinflammatory responses and tau hyperphosphorylation and is a potential drug for neuroinflammation- or tauopathy-associated diseases

ApoE4 로 유도되는 알츠하이머 질병에서 ApoE Christchurch 변이의 이로운 영향

Alzheimer’s Disease (AD), APOE Christchurch variant, Tauopathy, Autophagy, Endo-lysosomal pathwayPSEN1 유전자에 E280A 돌연변이를 보유하고 있음에도 불구하고 70세까지 인지능력 저하가 나타나지 않은 사례가 보고되었다. 보고 된 논문에서APOE3유전자에 동형 접합체로 존재하는 Christchurch 변이를 밝혔으며, 해당 변이가 타우 병리학에 보호적인 기전으로 작용한다고 보고하였다. 비록 해당 보고가 가족력 알츠하이머 사례에서 타우 병리학과 인지기능 관련하여 Christchurch 변이의 보호적인 기전을 강력하게 주장하고 있지만, 알츠하이머 종류의 95% 이상을 차지하는 만발성 알츠하이머에서 또한 보호적인 기전이 재현되는지 연구가 필요하다. 더불어, Christchurch 변이의 보호적인 기전의 메커니즘 분석이 다뤄져야 한다. 해당 부분을 다루기위해, 인간 유도만능줄기세포를 이용하여 3가지의 동질 유전자 세포주를 사용하였다. 이는 각 APOE3, APOE4, 또는 APOE4ch 유전자형을 가지고 있으며 흥분성 신경세포로 분화되었다. 나는 APOE3, APOE4, 그리고 APOE4ch 흥분성 신경세포에서 외부에 존재하는 타우 단백질이 유입되는 양의 차이가 없음을 관찰했다. 하지만, 유입 이후 타우 단백질의 위치가 다르게 나타났다. APOE3 그리고 APOE4ch 흥분성 신경세포와 비교했을 때, APOE4 흥분성 신경세포에서는 초기 엔도솜과 재생 엔도솜에서 유입된 타우 단백질이 많이 관찰되었다. 더불어 APOE4 신경세포의 재생엔도솜에서는 많은 양의 누출이 관찰되었고, 이것은 재생 엔도솜에 존재하는 유입 된 타우 단백질이 세포질로 내뿜어질 수 있을 가능성을 제시했다. APOE4 흥분성 신경세포에 Christchurch 변이가 있는 경우, 타우 단백질이 재생 엔도솜에 존재하는 것이 감소하였고, 후기 엔도좀에 많은양의 유입된 타우가 존재하는 것을 관찰했다. 그리고 이는 리소좀 의존 자가포식 단백질 분해가 증가했을 가능성을 제시하였다. 재생 엔도솜에서의 누수 또한 Christchurch 변이로 인해 회복되었다. 타우 단백질 유입 이후, APOE4 흥분성 신경세포와 비교하여 APOE4ch 흥분성 신경세포에서 리소좀 의존 자가포식 타우 단백질 분해가 증가하고 타우 병리학이 개선된 것을 관찰하였다. APOE4ch 흥분성 신경세포에서Thr181 그리고 Ser202/Thr205가 인산화 된 타우 단백질이 APOE4신경세포와 비교해 보았을 때, 타우 유입 이후 증가하지 않는다는 것을 관찰했다. 이 결과는 Christchurch 변이가 있을 때, 초기 단계의 타우 인산화가 완화되고, 이는 Christchurch 변이가 초기 단계의 타우 병리학에 보호적인 역할을 함을 제시했다. 요약하자면, 나는 Christchurch 변이가 APOE4 신경세포에서 망가져 있는 리소좀 의존 자가포식 단백질 분해 능력을 회복시키고, 초기 단계의 타우 인산화를 완화시킨다는 결과를 얻었다. 이 연구는 APOE4로 유발되는 알츠하이머 질병에Christchurch 변이가 보호적인 역할을 하는 실마리를 제시하며, 타우 병리학에 대항하는 Christchurch변이의 보호적 메커니즘을 제안한다.An individual who did not display cognitive impairment until 70 despite carrying the E280A mutation in the PSEN1 gene is reported. The study identified a homozygote variant in the APOE3 gene (R136S, named the Christchurch variant) as a protective genetic factor against AD pathology. Although this case report strongly suggests the protective effect of the Christchurch variant on tau pathology and cognitive impairment in familial AD, it has to be evaluated whether the variant is also effective in late-onset AD, which accounts for more than 95% of AD forms. It is of great interest to determine the protective function of a variant against APOE4-associated AD that is a strong genetic risk factor for late-onset AD. Furthermore, the underlying mechanisms of its protective function should be investigated. To address these points, I used isogenic human-induced pluripotent stem cells (hiPSCs) that consist of either APOE3, APOE4, or APOE4+Christchurch variant (APOE4ch) and differentiated these hiPSCs to excitatory neurons. I compared the internalization activity of extracellular tau protein between isogenic neurons and found no differences. However, the location of tau protein after internalization was different between genotypes. Most of the internalized tau protein was in the early endosomes and recycling endosomes in APOE4 neurons compared to other genotypes. And I discovered a lot of leakage in the recycling endosomes that could facilitate the cytosol escape of tau in APOE4 neurons. It seems that the Christchurch variant attenuates tau translocation toward recycling endosomes and facilitates its movement to the late endosome suggesting a potential increase in lysosomal-dependent autophagic protein degradation. The leakage of recycling endosomes was also restored by the Christchurch variant. After tau proteins internalization, the lysosomal-dependent autophagic degradation was increased and the tau pathology was ameliorated in APOE4ch neurons compared to APOE4 neurons. The phosphorylated tau, especially p-TAU(Thr181) and p-TAU(Ser202/Thr205) was not increased in APOE4ch neurons after tau internalization compared to APOE4 neurons. That suggests the early stage of tau phosphorylation was ameliorated by the Christchurch variant, and the variant has a more protective effect in the early phase of tau pathology. To summarize, I discovered that the Christchurch variant restores the impaired autophagy activity in APOE4 neurons and ameliorates the early stage of tau phosphorylation. Taken together, this investigation provides evidence of protective roles for the Christchurch variant in APOE4-associated AD and insight into the mechanisms of its resistance against tau pathology.I. Introduction 1 1.1 APOE3 Christchurch homozygote variant 1 1.2 Phosphorylation of tau protein in AD 2 1.3 Tau protein internalization and spreading 3 1.4 Disrupted endo-lysosomal pathway in AD 4 II. Materials and Methods 6 2.1 Reagents and antibodies 6 2.2 Cell culture 7 2.2.1 Human-induced pluripotent stem cell maintenance 7 2.2.2 Excitatory neuron differentiation 7 2.3 Tau 441 protein aggregation 8 2.4 Alexa-594 fluorescence labeling 8 2.5 Immunocytochemistry 8 2.6 Immunoblotting 9 2.7 Human TAU ELISA 9 2.8 GAL3 infection 10 2.9 Statistical analysis 10 III. Results 11 3.1 Generation of isogenic APOE4ch hiPSCs using CRISPR-Cas9 system 11 3.2 Generation of excitatory neurons from hiPSCs 13 3.3 Confirmation of tau protein aggregation and Alexa-594 fluorescence labeling 15 3.4 Endosomal distribution of tau proteins after internalization and increased tau proteins translocation into the late endosomes by Christchurch variant 17 3.5 Potential ruptures of recycling endosomes in APOE4 neurons which was restored by the Christchurch variant 22 3.6 Increased autophagy-dependent tau degradation in APOE4ch neurons compared to APOE4 neurons 31 3.7 Autophagy activator mimics the protective function of the Christchurch variant in APOE4 neurons 37 3.8 The amelioration of tau pathology in APOE4ch neurons 40 3.9 The protective function of the Christchurch variant against tau protein aggregation 45 IV. Discussion 49 V. Reference 52 Abstract in Korea 58MasterdCollectio

Relationship of FDG PET/CT Textural Features with the Tumor Microenvironment and Recurrence Risks in Patients with Advanced Gastric Cancers

The relationship between 2-deoxy-2-[18F]fluoro-D-glucose (FDG) positron emission tomography/computed tomography (PET/CT) textural features and histopathological findings in gastric cancer has not been fully evaluated. We investigated the relationship between the textural features of primary tumors on FDG PET/CT with histopathological findings and recurrence-free survival (RFS) in patients with advanced gastric cancer (AGC). Fifty-six patients with AGC who underwent FDG PET/CT for staging work-ups were retrospectively enrolled. Conventional parameters and the first- and second-order textural features of AGC were extracted using PET textural analysis. Upon histopathological analysis, along with histopathological classification and staging, the degree of CD4, CD8, and CD163 cell infiltrations and expressions of interleukin-6 and matrix-metalloproteinase-11 (MMP-11) in the primary tumor were assessed. The histopathological classification, Lauren classification, lymph node metastasis, CD8 T lymphocyte and CD163 macrophage infiltrations, and MMP-11 expression were significantly associated with the textural features of AGC. The multivariate survival analysis showed that increased FDG uptake and intra-tumoral metabolic heterogeneity were significantly associated with an increased risk of recurrence after curative surgery. Textural features of AGC on FDG PET/CT showed significant correlations with the inflammatory response in the tumor microenvironment and histopathological features of AGC, and they showed significant prognostic values for predicting RFS

PD-L1 Expression Correlated with Clinicopathological Factors and Akt/Stat3 Pathway in Oral SCC

Programmed cell death ligand 1 (PD-L1) is an immune checkpoint molecule that inhibits immune responses. The physiological and prognostic role of the PD-L1 signaling pathway in the oral maxillofacial region is unclear. This study aimed to investigate the role of PD-L1 in the progression of oral squamous cell carcinoma (OSCC). Furthermore, clinicopathological factors related to PD-L1 expression were examined in patients with OSCC through immunohistochemistry (IHC) of tissue sections and through an in vitro study in OSCC cells. The medical records, radiographic findings, and mortality referrals of 81 patients obtained from the National Statistical Office were reviewed. IHC was performed on tissue specimens of these patients to determine the expression levels of PD-L1, which showed significant statistical differences based on age, tumor size, TNM stage, cervical lymph node metastasis, and locoregional recurrence. Patients with a high PD-L1 expression had significantly poorer survival rates. Multivariate analysis using the Cox proportional model confirmed the high relative risk ratio for high PD-L1 expression, TNM stage, and neck node metastasis, all of which were significantly associated with a poor prognosis in patients with OSCC. The in vitro study showed that SAS and YD38 cells transfected with PD-L1 siRNA had significantly increased apoptosis, reduced proliferative capacity, and tumorigenicity

Lomerizine inhibits LPS-mediated neuroinflammation and tau hyperphosphorylation by modulating NLRP3, DYRK1A, and GSK3α/β

Introduction: Lomerizine is a calcium channel blocker that crosses the blood–brain barrier and is used clinically in the treatment of migraines. However, whether lomerizine is beneficial in modulating neuroinflammatory responses has not been tested yet. Methods: To assess the potential of lomerizine for repurposing as a treatment for neuroinflammation, we investigated the effects of lomerizine on LPS-induced proinflammatory responses in BV2 microglial cells, Alzheimer’s disease (AD) excitatory neurons differentiated from induced pluripotent stem cells (iPSCs), and in LPS-treated wild type mice. Results: In BV2 microglial cells, lomerizine pretreatment significantly reduced LPS-evoked proinflammatory cytokine and NLRP3 mRNA levels. Similarly, lomerizine pretreatment significantly suppressed the increases in Iba-1, GFAP, proinflammatory cytokine and NLRP3 expression induced by LPS in wild-type mice. In addition, lomerizine posttreatment significantly decreased LPS-stimulated proinflammatory cytokine and SOD2 mRNA levels in BV2 microglial cells and/or wild-type mice. In LPS-treated wild-type mice and AD excitatory neurons differentiated from iPSCs, lomerizine pretreatment ameliorated tau hyperphosphorylation. Finally, lomerizine abolished the LPS-mediated activation of GSK3α/β and upregulation of DYRK1A, which is responsible for tau hyperphosphorylation, in wild-type mice. Discussion: These data suggest that lomerizine attenuates LPS-mediated neuroinflammatory responses and tau hyperphosphorylation and is a potential drug for neuroinflammation- or tauopathy-associated diseases.TRU

Predicting the Recurrence of Gastric Cancer Using the Textural Features of Perigastric Adipose Tissue on [¹⁸F]FDG PET/CT

This study aimed to assess the relationship between the histopathological and textural features of perigastric adipose tissue (AT) on 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography/computed tomography (PET/CT) and to evaluate the prognostic significance of perigastric AT textural features in predicting recurrence-free survival (RFS) in patients with gastric cancer. Sixty-nine patients with gastric cancer who underwent staging [18F]FDG PET/CT and subsequent curative surgery were retrospectively reviewed. Textural features of perigastric AT were extracted from PET images. On histopathological analysis, CD4, CD8, and CD163 cell infiltration and matrix metalloproteinase-11 and interleukin-6 (IL-6) expression in perigastric AT were graded. The degree of CD163 cell infiltration in perigastric AT was significantly correlated with the mean standardized uptake value (SUV), SUV histogram entropy, grey-level co-occurrence matrix (GLCM) energy, and GLCM entropy of perigastric AT. The degree of IL-6 expression in the perigastric AT was significantly correlated with the mean and median SUVs of perigastric AT. In multivariate survival analysis, GLCM entropy, GLCM dissimilarity, and GLCM homogeneity of perigastric AT were significant predictors of RFS. The textural features of perigastric AT on [18F]FDG PET/CT significantly correlated with inflammatory response in perigastric AT and were significant prognostic factors for predicting RFS in patients with gastric cancer

<i>Sasa borealis</i> Ethanol Extract Protects PC12 Neuronal Cells against Oxidative Stress

The overproduction of reactive oxygen species (ROS) can cause oxidative stress to biomolecules such as nucleic acids, proteins, and lipids, leading to neurodegenerative disorders. Sasa borealis (SB) has antioxidant, anti-inflammatory, antidiabetic, and anti-obesity effects. We evaluated the neuroprotective activity of SB on hydrogen peroxide (H2O2)-induced oxidative stress. We investigated the antioxidant and neuroprotective effects of SB water extract (SBW) and SB ethanol extract (SBE) by measuring the radical scavenging activities and intracellular ROS production. SBE, which had a high level of isoorientin, had higher antioxidative activities than SBW in 2,2′-azino-bis-(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt (ABTS+) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. It also reduced ROS generation in pheochromocytoma 12 (PC12) cells more significantly than SBW. It increased the translation of heme oxygenase-1 (HO-1), superoxide dismutase 2 (SOD2), catalase (CAT), and glutathione peroxidase (GPx) with a corresponding increase in the translation of NF-E2-related factor-2 (Nrf-2). In conclusion, SBE with high levels of phenolic compounds such as isoorientin shows promise for preventing neurodegenerative diseases

Hypoxia-inducible adrenomedullin accelerates hepatocellular carcinoma cell growth

Adrenomedullin is implicated in tumor progression and induced by hypoxia. We evaluated if adrenomedullin signaling is active in hepatocellular carcinoma (HCC), especially under hypoxic conditions, and to analyze its prognostic implication in HCC patients. HCC cells expressed adrenomedullin and its receptor, and hypoxia induced adrenomedullin expression. Adrenomedullin stimulated HCC cell growth via Akt activation, which was prevented by adrenomedullin peptide inhibitor. Clinico-pathological analysis revealed adrenomedullin extent was related to vascular invasion and N-cadherin intensity, which were reported to indicate a poor prognosis. In conclusion, adrenomedullin signaling is hypoxia-inducible and functionally active in HCCs, and its expression may be a prognostic factor