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1.
Cancer Immunol Immunother ; 73(8): 157, 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38834889

ABSTRACT

Interleukin-2 (IL-2), a cytokine with pleiotropic immune effects, was the first approved cancer immunotherapy agent. However, IL-2 is associated with systemic toxicity due to binding with its ligand IL-2Rα, such as vascular leakage syndrome, limiting its clinical applications. Despite efforts to extend the half-life of IL-2 and abolish IL-2Rα interactions, the risk of toxicity remains unresolved. In this study, we developed the bispecific fusion protein MB2033, comprising a novel IL-2 variant (IL-2v) connected to anti-programmed death ligand 1 (PD-L1) via a silenced Fc domain. The IL-2v of MB2033 exhibits attenuated affinity for IL-2Rßγ without binding to IL-2Rα. The binding affinity of MB2033 for PD-L1 is greater than that for IL-2Rßγ, indicating its preferential targeting of PD-L1+ tumor cells to induce tumor-specific immune activation. Accordingly, MB2033 exhibited significantly reduced regulatory T cell activation, while inducing comparable CD8+ T cell activation to recombinant human IL-2 (rhIL-2). MB2033 induced lower immune cell expansion and reduced cytokine levels compared with rhIL-2 in human peripheral blood mononuclear cells, indicating a decreased risk of peripheral toxicity. MB2033 exhibited superior anti-tumor efficacy, including tumor growth inhibition and complete responses, compared with avelumab monotherapy in an MC38 syngeneic mouse model. In normal mice, MB2033 was safer than non-α IL-2v and tolerable up to 30 mg/kg. These preclinical results provide evidence of the dual advantages of MB2033 with an enhanced safety and potent clinical efficacy for cancer treatment.


Subject(s)
B7-H1 Antigen , Interleukin-2 , Recombinant Fusion Proteins , Animals , Mice , Humans , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/genetics , B7-H1 Antigen/metabolism , B7-H1 Antigen/antagonists & inhibitors , Female , Mice, Inbred C57BL , Immunotherapy/methods , Cell Line, Tumor , Melanoma, Experimental/drug therapy , Melanoma, Experimental/immunology
2.
Stem Cell Res ; 75: 103303, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38211411

ABSTRACT

Human induced pluripotent stem cells (hiPSCs) have potential use in regerenrative medicine for disease modeling and drug screening studies. The AAVS1 locus has been validated as a stable transgene expression and safe genomic location. Therefore, we inserted the enhanced green fluorescent protein (EGFP) gene into the AAVS1 locus of hiPSCs, using CRISPR/Cas9 genome editing. The results showed that the hiPSCs stably expressed EGFP in pluripotency and differentiated into three germ lineages. Our results strongly indicate that the EGFP-tagged cell line has potential for use in in vivo and in vitro experiments for monitoring cell location and type.


Subject(s)
Induced Pluripotent Stem Cells , Humans , Induced Pluripotent Stem Cells/metabolism , CRISPR-Cas Systems/genetics , Cell Line , Green Fluorescent Proteins/metabolism
3.
J Pers Med ; 12(5)2022 May 19.
Article in English | MEDLINE | ID: mdl-35629249

ABSTRACT

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a component of the extracellular environment and is suggested to play an indirect role in regulating Aß production and the pathophysiology of Aß deposition in brains. However, studies on the amount of TIMP-3 in bodily fluids of Alzheimer's disease (AD) patients have not been conducted. Here, we investigated the relationship between fluid TIMP-3 levels and AD pathology. We first showed that the fluid levels of TIMP-3 were lower in AD dementia patients compared with in non-AD patients. ELISA results revealed that plasma levels of TIMP-3 in 65 patients with AD were significantly lower than those in 115 healthy control subjects and 71 mild cognitive impairment (MCI) subjects. Furthermore, we found that cerebrospinal fluid (CSF) level of TIMP-3 was decreased in AD compared with that in healthy control. These data suggest that fluid TIMP-3 levels negatively correlated with progress of cognitive decline. Collectively, our study suggests that alterations of fluid TIMP-3 levels might be associated with AD pathology.

4.
Elife ; 102021 01 28.
Article in English | MEDLINE | ID: mdl-33507149

ABSTRACT

Derived from a common precursor cell, the balance between Th17 and Treg cells must be maintained within immune system to prevent autoimmune diseases. IL-1ß-mediated IL-1 receptor (IL-1R) signaling is essential for Th17-cell biology. Fine-tuning of IL-1R signaling is controlled by two receptors, IL-1RI and IL-RII, IL-1R accessory protein, and IL-1R antagonist. We demonstrate that the decoy receptor, IL-1RII, is important for regulating IL-17 responses in TCR-stimulated CD4+ T cells expressing functional IL-1RI via limiting IL-1ß responsiveness. IL-1RII expression is regulated by NFAT via its interaction with Foxp3. The NFAT/FOXP3 complex binds to the IL-1RII promoter and is critical for its transcription. Additionally, IL-1RII expression is dysregulated in CD4+ T cells from patients with rheumatoid arthritis. Thus, differential expression of IL-1Rs on activated CD4+ T cells defines unique immunological features and a novel molecular mechanism underlies IL-1RII expression. These findings shed light on the modulatory effects of IL-1RII on Th17 responses.


Subject(s)
Forkhead Transcription Factors/genetics , NFATC Transcription Factors/genetics , Receptors, Interleukin-1 Type II/genetics , Th17 Cells/metabolism , Forkhead Transcription Factors/metabolism , Humans , Interleukin-18/metabolism , NFATC Transcription Factors/metabolism , Receptors, Interleukin-1 Type II/metabolism
5.
Biochem Biophys Res Commun ; 528(3): 413-419, 2020 07 30.
Article in English | MEDLINE | ID: mdl-32513532

ABSTRACT

Coronavirus disease 2019 (COVID-19) is a worldwide pandemic. It has a high transmission rate among humans, and is a threat to global public health. However, there are no effective prophylactics or therapeutics available. It is necessary to identify vulnerable and susceptible groups for adequate protection and care against this disease. Recent studies have reported that COVID-19 has angiotensin-converting enzyme 2 (ACE2) as a functional receptor, which may lead to the development of severe cerebrovascular diseases (CVD), including strokes, in patients with risk factors for CVD such as diabetes and smoking. Thus, the World Health Organization (WHO) advised caution against COVID-19 for smokers and patients with underlying clinical symptoms, including cardiovascular diseases. Here, we observed ACE2 expression in the brain of rat middle cerebral artery occlusion (MCAO) model and evaluated the effects of cigarette smoke extract (CSE) and diabetes on ACE2 expression in vessels. We showed that the levels of ACE2 expression was increased in the cortex penumbra after ischemic injuries. CSE treatment significantly elevated ACE2 expression in human brain vessels. We found that ACE2 expression was upregulated in primary cultured human blood vessels with diabetes compared to healthy controls. This study demonstrates that ACE2 expression is increased in ischemic brains and vessels exposed to diabetes or smoking, makes them vulnerable to COVID-19 infection.


Subject(s)
Betacoronavirus/metabolism , Brain Ischemia/virology , Brain/blood supply , Diabetes Mellitus , Peptidyl-Dipeptidase A/biosynthesis , Receptors, Virus/biosynthesis , Smokers , Stroke/virology , Up-Regulation , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/pathogenicity , Brain/drug effects , Brain Ischemia/genetics , Brain Ischemia/metabolism , COVID-19 , Coronavirus Infections/genetics , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Diabetes Mellitus/genetics , Diabetes Mellitus/metabolism , Disease Models, Animal , Disease Susceptibility , Infarction, Middle Cerebral Artery/complications , Male , Mice , Mice, Inbred C57BL , Pandemics , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/genetics , Pneumonia, Viral/metabolism , Pneumonia, Viral/virology , Rats , Rats, Sprague-Dawley , Receptors, Virus/genetics , SARS-CoV-2 , Smoke/adverse effects , Stroke/genetics , Stroke/metabolism , Up-Regulation/drug effects
6.
J Neuropathol Exp Neurol ; 79(5): 484-492, 2020 05 01.
Article in English | MEDLINE | ID: mdl-32296844

ABSTRACT

N-cadherin is a synaptic adhesion molecule stabilizing synaptic cell structure and function. Cleavage of N-cadherin by γ-secretase produces a C-terminal fragment, which is increased in the brains of Alzheimer disease (AD) patients. Here, we investigated the relationship between fluid N-cadherin levels and AD pathology. We first showed that the cleaved levels of N-cadherin were increased in homogenates of postmortem brain from AD patients compared with that in non-AD patients. We found that cleaved N-cadherin levels in the cerebrospinal fluid were increased in AD dementia compared with that in healthy control. ELISA results revealed that plasma levels of N-cadherin in 76 patients with AD were higher than those in 133 healthy control subjects. The N-cadherin levels in the brains of an AD mouse model, APP Swedish/PS1delE9 Tg (APP Tg) were reduced compared with that in control. The N-terminal fragment of N-cadherin produced by cleavage at a plasma membrane was detected extravascularly, accumulated in senile plaques in the cortex of an APP Tg mouse. In addition, N-cadherin plasma levels were increased in APP Tg mice. Collectively, our study suggests that alteration of N-cadherin levels might be associated with AD pathology.


Subject(s)
Alzheimer Disease/cerebrospinal fluid , Antigens, CD/blood , Antigens, CD/cerebrospinal fluid , Brain Chemistry , Cadherins/blood , Cadherins/cerebrospinal fluid , Aged , Aged, 80 and over , Amyloid beta-Peptides/administration & dosage , Animals , Brain/blood supply , Cells, Cultured , Disease Models, Animal , Female , Humans , Male , Mice, Transgenic , Microglia/drug effects , Microglia/metabolism
7.
Arch Gerontol Geriatr ; 87: 103995, 2020.
Article in English | MEDLINE | ID: mdl-31874328

ABSTRACT

INTRODUCTION: Beta-amyloid is considered to be a pathophysiological marker in Alzheimer's disease (AD). Soluble amyloid precursor proteins (sAPPs) -α (sAPPα) and -ß (sAPPß), which are the byproducts of non-amyloidogenic and amyloidogenic process of APP, respectively, have been repeatedly observed in the cerebrospinal fluids (CSF) of AD patients. The present study focused on the determination of sAPP levels in peripheral blood. METHODS: The plasma protein levels of sAPPα and sAPPß were measured with ELISA. Plasma from 52 AD patients, 98 amnestic mild cognitive impairment (MCI) patients, and 114 cognitively normal controls were compared. RESULTS: The plasma level of sAPPß was significantly increased in AD patients than in cognitively healthy controls. However, no significant change in plasma sAPPα was observed among the three groups. Furthermore, the plasma sAPPß levels significantly correlated with cognitive assessment scales, such as clinical dementia rating (CDR), and mini-mental status examination (MMSE). Interestingly, sAPPα and sAPPß had a positive correlation with each other in blood plasma, similar to previous studies on CSF sAPP. This correlation was stronger in the MCI and AD groups than in the cognitively healthy controls. CONCLUSIONS: These results suggest that individuals with elevated plasma sAPPß levels are at an increased risk of AD; elevation in these levels may reflect the progression of disease.


Subject(s)
Alzheimer Disease/blood , Amyloid beta-Protein Precursor/blood , Cognitive Dysfunction/blood , Aged , Biomarkers/blood , Case-Control Studies , Disease Progression , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Peptide Fragments/blood
8.
PLoS One ; 14(11): e0224941, 2019.
Article in English | MEDLINE | ID: mdl-31770379

ABSTRACT

Notch signaling is an evolutionarily conserved pathway that regulates cell-cell interactions through binding of Notch family receptors to their cognate ligands. Notch signaling has an essential role in vascular development and angiogenesis. Recent studies have reported that Notch may be implicated in Alzheimer's disease (AD) pathophysiology. We measured the levels of soluble Notch1 (sNotch1) in the plasma samples from 72 dementia patients (average age 75.1 y), 89 subjects with amnestic mild cognitive impairment (MCI) (average age 73.72 y), and 150 cognitively normal controls (average age 72.34 y). Plasma levels of sNotch1 were 25.27% lower in dementia patients as compared to healthy control subjects. However, the level of Notch1 protein was significantly increased in human brain microvascular endothelial cells (HBMECs) after amyloid-beta treatment. Also, Notch1 mRNA level was significantly increased in HBMECs and iPSC-derived neuronal cells from AD patient compared to normal control. These results indicate that altered expression of Notch1 might be associated with the risk of Alzheimer's disease.


Subject(s)
Alzheimer Disease/metabolism , Receptor, Notch1/metabolism , ADAM10 Protein/genetics , ADAM10 Protein/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/blood , Amyloid beta-Peptides/metabolism , Case-Control Studies , Dementia/metabolism , Female , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Male , Middle Aged , Neurons/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, Notch1/blood , Receptor, Notch1/genetics
9.
Sci Rep ; 9(1): 4741, 2019 03 18.
Article in English | MEDLINE | ID: mdl-30894637

ABSTRACT

Alzheimer's disease (AD) is a major cause of dementia. Growing evidence suggests that dysregulation of autophagy, a cellular mechanism essential for self-digestion of damaged proteins and organelles, is involved in neurological degenerative diseases including AD. Previously, we reported that autophagosomes are increased in the brains of AD mouse model. However, the plasma levels of autophagic markers have not yet been investigated in patients with AD. In this study, we investigated the expression of autophagy-related genes 5 and 12 (ATG5 and ATG12, respectively) in cells in vitro upon amyloid-beta (Aß) treatment and in the plasma of AD patients. ATG5-ATG12 complex levels were increased in primary rat cortical neurons and human umbilical vein endothelial cells after Aß treatment. Furthermore, we compared plasma from 69 patients with dementia, 82 patients with mild cognitive impairment (MCI), and 127 cognitively normal control participants. Plasma levels of ATG5 were significantly elevated in patients with dementia (149.3 ± 7.5 ng/mL) or MCI (152.9 ± 6.9 ng/mL) compared with the control subjects (129.0 ± 4.1 ng/mL) (p = 0.034, p = 0.016, respectively). Our results indicate that alterations in the plasma ATG5 levels might be a potential biomarker in patients at risk for AD.


Subject(s)
Alzheimer Disease/blood , Autophagy-Related Protein 5/blood , Aged , Amyloid beta-Peptides/pharmacology , Animals , Autophagy-Related Protein 12/metabolism , Autophagy-Related Protein 5/metabolism , Biomarkers/blood , Case-Control Studies , Cells, Cultured , Female , Humans , Male , Mice , Rats
10.
Sci Rep ; 7(1): 17713, 2017 12 18.
Article in English | MEDLINE | ID: mdl-29255164

ABSTRACT

Alzheimer's disease (AD) is a common disorder of progressive cognitive decline among elderly subjects. Angiogenesis-related factors including vascular endothelial growth factor (VEGF) might be involved in the pathogenesis of AD. Soluble form of the VEGF receptor is likely to be an intrinsic negative counterpart of VEGF. We measured the plasma levels of VEGF and its two soluble receptors (sVEGFR1 and sVEGFR2) in 120 control subjects, 75 patients with mild cognitive impairment, and 76 patients with AD using ELISA. Plasma levels of VEGF in patients with AD were higher than those in healthy control subjects. However, plasma levels of sVEGFR1 and sVEGFR2 were lower in patients with AD than in healthy control subjects. Levels of VEGFR2 mRNA were significantly decreased in human umbilical vein endothelial cells after amyloid-beta treatment. Further, protein levels of VEGFR2 were also decreased in the brains of AD model mice. In addition, we show that the expression of sVEGFR2 and VEGFR2 was also decreased by the transfection with the Notch intracellular domain. These results indicate that the alterations of VEGF and its two receptors levels might be associated with those at risk for Alzheimer's disease.


Subject(s)
Alzheimer Disease/genetics , Vascular Endothelial Growth Factor Receptor-1/physiology , Vascular Endothelial Growth Factor Receptor-2/physiology , Aged , Alzheimer Disease/metabolism , Angiogenesis Inducing Agents , Animals , Brain/metabolism , Cognitive Dysfunction/genetics , Cognitive Dysfunction/metabolism , Disease Models, Animal , Female , Human Umbilical Vein Endothelial Cells , Humans , Male , Mice , Vascular Endothelial Growth Factor A/analysis , Vascular Endothelial Growth Factor A/blood , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-1/analysis , Vascular Endothelial Growth Factor Receptor-1/blood , Vascular Endothelial Growth Factor Receptor-2/analysis , Vascular Endothelial Growth Factor Receptor-2/blood , Vascular Endothelial Growth Factor Receptor-2/metabolism , Vascular Endothelial Growth Factors/metabolism
11.
Mol Cell Biochem ; 430(1-2): 1-9, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28239754

ABSTRACT

Neprilysin (NEP) is a zinc metallopeptidase that cleaves a number of small peptides into inactive forms. Despite the recent evidence of a significant correlation between the levels of NEP in plasma and the severity of obesity in humans, a cause-and-effect relationship or a functional role of NEP in obesity has remained uncertain. In this study, we show that NEP has a positive regulatory effect on fat cell formation from precursor cells. NEP increases the accumulation of cytoplasmic triglycerides in 3T3-L1 preadipocytes or the C3H10T1/2 mesenchymal stem cell line in differentiation conditions. Consistently, cells expressing NEP showed an increase in mRNA expression of adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), and the adipocyte markers aP2 and adipsin. Furthermore, this NEP-enhanced induction of adipogenesis was found to require the enzymatic activity of NEP, leading to augmentation of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway. In summary, our results indicate that NEP accelerates adipogenesis through enhancement of insulin-mediated PI3K-Akt activation and imply a high therapeutic value of NEP in treating obesity and obesity-related disorders.


Subject(s)
Adipogenesis , Neprilysin/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , 3T3-L1 Cells , Animals , CCAAT-Enhancer-Binding Proteins/metabolism , Mice , PPAR gamma/metabolism , Proto-Oncogene Proteins c-akt/metabolism
12.
Sci Rep ; 6: 24933, 2016 04 26.
Article in English | MEDLINE | ID: mdl-27112200

ABSTRACT

The neuronal accumulation of phosphorylated tau plays a critical role in the pathogenesis of Alzheimer's disease (AD). Here, we examined the effect of fisetin, a flavonol, on tau levels. Treatment of cortical cells or primary neurons with fisetin resulted in significant decreases in the levels of phosphorylated tau. In addition, fisetin decreased the levels of sarkosyl-insoluble tau in an active GSK-3ß-induced tau aggregation model. However, there was no difference in activities of tau kinases and phosphatases such as protein phosphatase 2A, irrespective of fisetin treatment. Fisetin activated autophagy together with the activation of transcription factor EB (TFEB) and Nrf2 transcriptional factors. The activation of autophagy including TFEB is likely due to fisetin-mediated mammalian target of rapamycin complex 1 (mTORC1) inhibition, since the phosphorylation levels of p70S6 kinase and 4E-BP1 were decreased in the presence of fisetin. Indeed, fisetin-induced phosphorylated tau degradation was attenuated by chemical inhibitors of the autophagy-lysosome pathway. Together the results indicate that fisetin reduces levels of phosphorylated tau through the autophagy pathway activated by TFEB and Nrf2. Our result suggests fisetin should be evaluated further as a potential preventive and therapeutic drug candidate for AD.


Subject(s)
Autophagy , Flavonoids , NF-E2-Related Factor 2 , tau Proteins , Animals , Rats , Alzheimer Disease/metabolism , Autophagy/drug effects , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Carrier Proteins/metabolism , Cell Culture Techniques , Flavonoids/pharmacology , Flavonols , Intracellular Signaling Peptides and Proteins , Neurons/metabolism , NF-E2-Related Factor 2/metabolism , Phosphoproteins/metabolism , Phosphorylation , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , tau Proteins/metabolism
13.
Immunology ; 146(4): 645-56, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26407164

ABSTRACT

Zinc is an essential trace element that plays pivotal roles in multiple facets of the immune system. Besides its catalytic and structural roles, zinc also functions as an intracellular signalling molecule, and changes in zinc levels can cause both direct and indirect modulation of immune responses. Further, cytoplasmic levels of bioavailable zinc in immune cells are largely influenced by many extracellular stimuli. Here we provide evidence that zinc represses memory T helper type 17 responses in humans by inhibiting interleukin-1ß (IL-1ß)-mediated signal. In vitro zinc treatment of CD4(+) T cells in the presence of activated monocytes inhibited interferon-γ-producing cells and IL-17-producing cells, but not IL-4-producing cells. Of note, production of IL-17(+) cells from memory CD4(+) T cells, which is significantly up-regulated by lipopolysaccharide-stimulated monocytes, was preferentially repressed by zinc. Increased cytoplasmic zinc in T cells suppressed IL-1ß signalling through repression of phosphorylation of IL-1 receptor-associated kinase 4 (IRAK4), so leading to an inhibitory effect on T helper type 17 responses facilitated by monocyte-derived IL-1ß in humans. These findings suggest that extracellular zinc bioavailability may affect memory CD4(+) T-cell responses by modulating the zinc-mediated signalling pathway.


Subject(s)
Immunologic Memory , Interleukin-1 Receptor-Associated Kinases/metabolism , Interleukin-1beta/metabolism , Th17 Cells/immunology , Th17 Cells/metabolism , Zinc/metabolism , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cells, Cultured , Cytokines/metabolism , Humans , Immunologic Memory/drug effects , Lymphocyte Activation/immunology , Models, Biological , Monocytes/immunology , Monocytes/metabolism , Phosphorylation , Signal Transduction/drug effects , Th17 Cells/drug effects , Zinc/pharmacology
14.
J Alzheimers Dis ; 47(3): 639-43, 2015.
Article in English | MEDLINE | ID: mdl-26401699

ABSTRACT

Alzheimer's disease (AD) is the most common type of dementia in the elderly. The accumulation of amyloid-ß peptides and tau proteins is the major pathogenic event of AD. There is accumulating evidence that both tau and amyloid-ß linked to the small ubiquitin-like modifier (SUMO), which is increased in the brain of AD model mouse. The present study focused on the determination of SUMO1 protein level in AD blood plasma by the ELISA methods. We compared plasma from 80 dementia patients (average age 75.3 y), 89 persons with amnestic mild cognitive impairment (MCI) (average age 73.71 y),and 133 cognitively normal controls (average age 71.97 y). The plasma level of SUMO1 was significantly increased in dementia patients, as compared to control groups. The levels of SUMO1 correlated to decreased Mini-Mental State Examination (r =-0.123, p = 0.029). These results suggest that elevated plasma SUMO1 levels may be associated with AD.


Subject(s)
Alzheimer Disease/blood , Cognitive Dysfunction/blood , SUMO-1 Protein/blood , Aged , Amnesia/blood , Biomarkers/blood , Blood Chemical Analysis , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Mental Status Schedule
15.
Autophagy ; 11(1): 100-12, 2015.
Article in English | MEDLINE | ID: mdl-25484073

ABSTRACT

Autophagy is one of the main mechanisms in the pathophysiology of neurodegenerative disease. The accumulation of autophagic vacuoles (AVs) in affected neurons is responsible for amyloid-ß (Aß) production. Previously, we reported that SUMO1 (small ubiquitin-like modifier 1) increases Aß levels. In this study, we explored the mechanisms underlying this. We investigated whether AV formation is necessary for Aß production by SUMO1. Overexpression of SUMO1 increased autophagic activation, inducing the formation of LC3-II-positive AVs in neuroglioma H4 cells. Consistently, autophagic activation was decreased by the depletion of SUMO1 with small hairpin RNA (shRNA) in H4 cells. The SUMO1-mediated increase in Aß was reduced by the autophagy inhibitors (3-methyladenine or wortmannin) or genetic inhibitors (siRNA targeting ATG5, ATG7, ATG12, or HIF1A), respectively. Accumulation of SUMO1, ATG12, and LC3 was seen in amyloid precursor protein transgenic mice. Our results suggest that SUMO1 accelerates the accumulation of AVs and promotes Aß production, which is a key mechanism for understanding the AV-mediated pathophysiology of Alzheimer disease.


Subject(s)
Amyloid beta-Peptides/metabolism , Autophagy , SUMO-1 Protein/metabolism , Amyloid beta-Peptides/ultrastructure , Animals , Apoptosis Regulatory Proteins/metabolism , Beclin-1 , Brain/metabolism , Cell Line, Tumor , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice, Transgenic , Microtubule-Associated Proteins/metabolism , Plaque, Amyloid/metabolism , RNA, Small Interfering/metabolism , Transfection , Up-Regulation
16.
Biochem Biophys Res Commun ; 454(1): 196-201, 2014 Nov 07.
Article in English | MEDLINE | ID: mdl-25450380

ABSTRACT

We previously showed that NDP52 (also known as calcoco2) plays a role as an autophagic receptor for phosphorylated tau facilitating its clearance via autophagy. Here, we examined the expression and association of NDP52 with autophagy-regulated gene (ATG) proteins including LC3, as well as phosphorylated tau and amyloid-beta (Aß) in brains of an AD mouse model. NDP52 was expressed not only in neurons, but also in microglia and astrocytes. NDP52 co-localized with ATGs and phosphorylated tau as expected since it functions as an autophagy receptor for phosphorylated tau in brain. Compared to wild-type mice, the number of autophagic vesicles (AVs) containing NDP52 in both cortex and hippocampal regions was significantly greater in AD model mice. Moreover, the protein levels of NDP52 and phosphorylated tau together with LC3-II were also significantly increased in AD model mice, reflecting autophagy impairment in the AD mouse model. By contrast, a significant change in p62/SQSTM1 level was not observed in this AD mouse model. NDP52 was also associated with intracellular Aß, but not with the extracellular Aß of amyloid plaques. We conclude that NDP52 is a key autophagy receptor for phosphorylated tau in brain. Further our data provide clear evidence for autophagy impairment in brains of AD mouse model, and thus strategies that result in enhancement of autophagic flux in AD are likely to be beneficial.


Subject(s)
Alzheimer Disease/metabolism , Brain/metabolism , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , tau Proteins/metabolism , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Animals , Autophagy/physiology , Brain/pathology , Disease Models, Animal , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microtubule-Associated Proteins/metabolism , Neuroglia/metabolism , Neurons/metabolism , Phosphorylation , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Tissue Distribution , tau Proteins/chemistry
17.
FEBS Lett ; 588(17): 3081-8, 2014 Aug 25.
Article in English | MEDLINE | ID: mdl-24952354

ABSTRACT

Sulforaphane (SFN), an activator of nuclear factor E2-related factor 2 (Nrf2), has been reported to induce autophagy in several cells. However, little is known about its signaling mechanism of autophagic induction. Here, we provide evidence that SFN induces autophagy with increased levels of LC3-II through extracellular signal-regulated kinase (ERK) activation in neuronal cells. Pretreatment with NAC (N-acetyl-l-cysteine), a well-known antioxidant, completely blocked the SFN-induced increase in LC3-II levels and activation of ERK. Knockdown or overexpression of Nrf2 did not affect autophagy. Together, the results suggest that SFN-mediated generation of reactive oxygen species (ROS) induces autophagy via ERK activation, independent of Nrf2 activity in neuronal cells.


Subject(s)
Autophagy/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Isothiocyanates/pharmacology , Neurons/cytology , Neurons/drug effects , Animals , Cell Line, Tumor , Enzyme Activation/drug effects , Humans , Mice , Neurons/metabolism , Reactive Oxygen Species/metabolism , Sulfoxides
18.
Mol Cells ; 37(3): 248-56, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24625575

ABSTRACT

N-acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) is a N-acetylhexosamine kinase that belong to the sugar kinase/heat shock protein 70/actin superfamily. In this study, we investigated both the expression and function of NAGK in neurons. Immunohistochemistry of rat brain sections showed that NAGK was expressed at high levels in neurons but at low levels in astrocytes. Immunocytochemistry of rat hippocampal dissociate cultures confirmed these findings and showed that NAGK was also expressed at low levels in oligodendrocytes. Furthermore, several NAGK clusters were observed in the nucleoplasm of both neuron and glia. The overexpression of EGFP- or RFP (DsRed2)-tagged NAGK in rat hippocampal neurons (DIV 5-9) increased the complexity of dendritic architecture by increasing the numbers of primary dendrites and dendritic branches. In contrast, knockdown of NAGK by shRNA resulted in dendrite degeneration, and this was prevented by the co-expression of RFP-tagged NAGK. These results suggest that the upregulation of dendritic complexity is a non-canonical function of NAGK.


Subject(s)
Dendrites/enzymology , Phosphotransferases (Alcohol Group Acceptor)/physiology , Animals , Cell Shape , Gene Expression , HEK293 Cells , Hippocampus/cytology , Humans , Mice , NIH 3T3 Cells , Rats, Sprague-Dawley
19.
Biochem Biophys Res Commun ; 445(3): 584-90, 2014 Mar 14.
Article in English | MEDLINE | ID: mdl-24561245

ABSTRACT

Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as (51)Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.


Subject(s)
Colorectal Neoplasms/blood , High-Throughput Screening Assays/methods , Interferon-gamma/blood , Killer Cells, Natural/immunology , Adult , Aged , Aged, 80 and over , Colorectal Neoplasms/immunology , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Interferon-gamma/immunology , Male , Middle Aged
20.
J Histochem Cytochem ; 61(7): 522-8, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23525569

ABSTRACT

The expression of the Crlz-1 gene in mouse testis, where it was found to be expressed most highly among the tested mouse organs, was analyzed spatiotemporally by employing RT-PCR and in situ hybridization techniques with the aid of immunohistochemistry and/or immunofluorescence methods. In 1-week-old neonatal testis, Crlz-1 was strongly expressed in the spermatogonia and Sertoli cells in its seminiferous cord. In 2- to 3-week-old prepubertal testis, where Sertoli cells cease to proliferate, Crlz-1 expression dropped and remained weakly at the rim layer of seminiferous cords and/or tubules, where spermatogonia are present. In the adult testis at 12 weeks after birth, Crlz-1 was expressed mainly in the spermatids near the lumen of seminiferous tubules. In a further in situ hybridization of Crlz-1 in the 12-week-old adult testis with hematoxylin nuclear counterstaining, Crlz-1 was mainly expressed at step 16 of spermatids between stages VII and VIII of seminiferous tubules as well as in their residual bodies at stage IX of seminiferous tubules.


Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Developmental , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Sertoli Cells/metabolism , Spermatids/metabolism , Spermatogenesis , Spermatogonia/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Animals , Cell Proliferation , Core Binding Factor alpha Subunits/metabolism , Male , Mice , Seminiferous Tubules/growth & development , Seminiferous Tubules/metabolism , Sertoli Cells/cytology , Signal Transduction , Spermatids/cytology , Spermatogonia/cytology , Time Factors , Wnt Proteins/metabolism , beta Catenin/metabolism
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