Progranulin-deficient macrophages cause cardiotoxicity under hypoxic conditions.

Myocardial infarction (MI) induces structural and electrical cardiac remodeling in response to ischemic insult, causing lethal arrhythmias and sudden death. Progranulin (PGRN) is a glycoprotein mainly expressed in macrophages that modulates the immune responses. In this study, we investigated the direct influence of PGRN knockout (Grn-/-) macrophages on post-MI pathophysiology. An MI mouse model was established by ligating the left coronary artery for RNA sequencing and electrocardiographic analysis. Bone marrow-derived macrophages (BMDMs) were injected into mice and supernatant was collected for the measurement of reactive oxygen species (ROS) levels and extracellular flux analysis. Administration of Grn-/- BMDMs prolonged the QT intervals in the MI mouse model. Moreover, genes highly expressed in macrophages were upregulated in Grn-/- heart after MI. Post-hypoxic supernatant of Grn-/- BMDMs increased the oxygen-glucose deprivation-induced cardiomyocyte death. Grn-/- BMDMs exhibited increased ROS production, oxygen consumption, and extracellular acidification under hypoxia and inflammatory conditions. These findings suggest that PGRN deficiency causes cardiotoxicity via secretory components of macrophages that exhibit metabolic abnormalities under hypoxia.

Linagliptin decreased the tumor progression on glioblastoma model.

PURPOSE: Dipeptidyl peptidase-4 (DPP-4) inhibitors are oral hypoglycemic drugs and are used for type II diabetes. Previous studies showed that DPP-4 expression is observed in several tumor types and DPP-4 inhibitors suppress the tumor progression on murine tumor models. In this study, we evaluated the role of DPP-4 and the antitumor effect of a DPP-4 inhibitor, linagliptin, on glioblastoma (GBM). METHODS: We analyzed DPP-4 expression in glioma patients by the public database. We also analyzed DPP-4 expression in GBM cells and the murine GBM model. Then, we evaluated the cell viability, cell proliferation, cell migration, and expression of some proteins on GBM cells with linagliptin. Furthermore, we evaluated the antitumor effect of linagliptin in the murine GBM model. RESULTS: The upregulation of DPP-4 expression were observed in human GBM tissue and murine GBM model. In addition, DPP-4 expression levels were found to positively correlate with the grade of glioma patients. Linagliptin suppressed cell viability, cell proliferation, and cell migration in GBM cells. Linagliptin changed the expression of phosphorylated NF-kB, cell cycle, and cell adhesion-related proteins. Furthermore, oral administration of linagliptin decreases the tumor progression in the murine GBM model. CONCLUSION: Inhibition of DPP-4 by linagliptin showed the antitumor effect on GBM cells and the murine GBM model. The antitumor effects of linagliptin is suggested to be based on the changes in the expression of several proteins related to cell cycle and cell adhesion via the regulation of phosphorylated NF-kB. This study suggested that DPP-4 inhibitors could be a new therapeutic strategy for GBM.

The role of progranulin in macrophages of a glioblastoma model.

PURPOSE: Glioblastoma (GBM), characterized by astrocytic tumorigenesis, remains one of the most prognostically challenging tumor types. Targeting entire GBM microenvironment using novel therapeutic factors is currently desired investigation approach. In this study, we focused on progranulin (PGRN), a regulator of diverse cellular functions. Recent studies implicated PGRN in the poor prognostics of GBM patients. However, the specific role of PGRN in the GBM microenvironment remains elusive. METHODS: We utilized public databases of GBM patient and previous single-cell RNA sequence to examine association between PGRN expression and patient survival/grade, and expression levels of PGRN in each cell constituting the tumor microenvironment. To clarify the role of PGRN in Tumor-associated macrophage (TAM), we examined cell proliferation and expression of some proteins in murine GBM cells when cell supernatants derived from TAM of PGRN knockout (Grn-/-) or wild type mice were treated with murine GBM cells. RESULTS: Our results reveal significant PGRN expression in macrophages within the GBM environment, suggesting an association between increased PGRN expression in macrophages and tumor malignancy. TAM induction led to PGRN expression enhancement. Treatment with Grn-/- mouse -derived bone marrow-derived macrophage (BMDM) supernatant resulted in diminished GBM cell proliferation and cell cycle- and mesenchymal GBM subtype-associated reduced protein expression. Furthermore, the Grn-/- mouse-derived BMDM supernatant treatment reduced the phosphorylated STAT3 expression in GBM cells, while the expression of IL-6 and IL-10, known STAT3 pathway activators, diminished in Grn-/- mouse-derived BMDMs. CONCLUSION: Our results suggest that macrophage-derived PGRN is pivotal for fostering malignant transformations within the tumor microenvironment.

BCL6B Contributes to Ocular Vascular Diseases via Notch Signal Silencing.

BACKGROUND: Endothelial cell activation is tightly controlled by the balance between VEGF (vascular endothelial cell growth factor) and Notch signaling pathway. VEGF destabilizes blood vessels and promotes neovascularization, which are common features of sight-threatening ocular vascular disorders. Here, we show that BCL6B (B-cell CLL/lymphoma 6 member B protein), also known as BAZF, ZBTB28, and ZNF62, plays a pivotal role in the development of retinal edema and neovascularization. METHODS: The pathophysiological physiological role of BCL6B was investigated in cellular and animal models mimicking 2 pathological conditions: retinal vein occlusion and choroidal neovascularization. An in vitro experimental system was used in which human retinal microvascular endothelial cells were supplemented with VEGF. Choroidal neovascularization cynomolgus monkey model was generated to investigate the involvement of BCL6B in the pathogenesis. Mice lacking BCL6B or treated with BCL6B-targeting small-interfering ribose nucleic acid were examined for histological and molecular phenotypes. RESULTS: In retinal endothelial cells, the BCL6B expression level was increased by VEGF. BCL6B-deficient endothelial cells showed Notch signal activation and attenuated cord formation via blockage of the VEGF-VEGFR2 signaling pathway. Optical coherence tomography images showed that choroidal neovascularization lesions were decreased by BCL6B-targeting small-interfering ribose nucleic acid. Although BCL6B mRNA expression was significantly increased in the retina, BCL6B-targeting small-interfering ribose nucleic acid suppressed ocular edema in the neuroretina. The increase in proangiogenic cytokines and breakdown of the inner blood-retinal barrier were abrogated in BCL6B knockout (KO) mice via Notch transcriptional activation by CBF1 (C promotor-binding factor 1) and its activator, the NICD (notch intracellular domain). Immunostaining showed that Müller cell activation, a source of VEGF, was diminished in BCL6B-KO retinas. CONCLUSIONS: These data indicate that BCL6B may be a novel therapeutic target for ocular vascular diseases characterized by ocular neovascularization and edema.

Investigation into the usefulness of cynomolgus monkeys with spontaneously elevated intraocular pressure as a model for glaucoma treatment research.

Many glaucoma treatments focus on lowering intraocular pressure (IOP), with novel drugs continuing to be developed. One widely used model involves raising IOP by applying a laser to the trabecular iris angle (TIA) of cynomolgus monkeys to damage the trabecular meshwork. This model, however, presents challenges such as varying IOP values, potential trabecular meshwork damage, and risk of animal distress. This study investigated whether animals with naturally high IOP (>25 mmHg) could be used to effectively evaluate IOP-lowering drugs, thereby possibly replacing laser-induced models. Relationships between TIA size, IOP, and pupil diameter were also examined. Three representative IOP-lowering drugs (latanoprost, timolol, ripasudil) were administered, followed by multiple IOP measurements and assessment of corneal thickness, TIA, and pupil diameter via anterior segment optical coherence tomography (AS-OCT). There was a positive correlation was noted between IOP and corneal thickness before instillation, and a negative correlation between IOP and TIA before instillation. Our findings suggest animals with naturally high IOP could be beneficial for glaucoma research and development as a viable replacement for the laser-induced model and that measuring TIA using AS-OCT along with IOP yields a more detailed evaluation.

Both hemoglobin and hemin cause damage to retinal pigment epithelium through the iron ion accumulation.

Subretinal hemorrhages result in poor vision and visual field defects. During hemorrhage, several potentially toxic substances are released from iron-based hemoglobin and hemin, inducing cellular damage, the detailed mechanisms of which remain unknown. We examined the effects of excess intracellular iron on retinal pigment epithelial (RPE) cells. A Fe2+ probe, SiRhoNox-1 was used to investigate Fe2+ accumulation after treatment with hemoglobin or hemin in the human RPE cell line ARPE-19. We also evaluated the production of reactive oxygen species (ROS) and lipid peroxidation. Furthermore, the protective effect of-an iron chelator, 2,2'-bipyridyl (BP), and ferrostatin-1 (Fer-1) on the cell damage, was evaluated. Fe2+ accumulation increased in the hemoglobin- or hemin-treated groups, as well as intracellular ROS production and lipid peroxidation. In contrast, BP treatment suppressed RPE cell death, ROS production, and lipid peroxidation. Pretreatment with Fer-1 ameliorated cell death in a concentration-dependent manner and suppressed ROS production and lipid peroxidation. Taken together, these findings indicate that hemoglobin and hemin, as well as subretinal hemorrhage, may induce RPE cell damage and visual dysfunction via intracellular iron accumulation.

Timolol maleate, a ß blocker eye drop, improved edema in a retinal vein occlusion model.

Purpose: To investigate the therapeutic effects of eye drops, namely, timolol maleate, a ß-adrenergic receptor antagonist, and latanoprost, a prostaglandin F2α analog, on retinal edema in a murine retinal vein occlusion (RVO) model. Methods: An RVO model was established using laser-induced RVO in mice, which were administered timolol maleate and latanoprost eye drops several times after venous occlusion. Subsequently, the thickness of the inner nuclear layer (INL) and the expression levels of such genes as Vegf and Atf4, which are stress markers of the endoplasmic reticulum, were examined. Primary human cultured retinal microvascular endothelial cells (HRMECs) were treated with timolol under hypoxic conditions, after which the gene expression pattern was investigated. Importantly, an integrated stress response inhibitor (ISRIB) was used in the RVO model, he known ISRIB, which suppresses the expression of ATF4 in retinal edema. Results: Increased INL thickness was suppressed by timolol eye drops, as were the expressions of Vegf and Atf4, in the RVO model. However, latanoprost eye drops did not induce any change in INL thickness. In HRMECs, hypoxic stress and serum deprivation increased the Vegf and Atf4 expressions; in response, treatment with timolol suppressed the Vegf expression. Furthermore, the ISRIB decreased the Vegf expression pattern and edema formation, which are associated with RVO. Conclusions: These results indicate that timolol eye drops may be a potential option for RVO treatment.

ALK5 inhibitor acts on trabecular meshwork cell and reduces intraocular pressure.

Intraocular pressure (IOP) is the most important risk factor for the onset and progression of glaucoma. IOP reduction has been proven effective in the treatment of glaucoma. IOP is controlled by the production and outflow of the aqueous humor (AH), and the trabecular meshwork (TM) is the main pathway for AH drainage from the eye. However, there are few conventional IOP-lowering treatments that target TM, and there is a need for such treatments. In this study, we screened for the expression level of fibronectin as an indicator and identified an activin receptor-like kinase (ALK) 5 inhibitor. Western blot analysis showed that SB431542, an ALK 5 inhibitor, reduced fibronectin and α-SMA expression. Moreover, a single dose of the ALK5 inhibitor SB431542 reduced IOP in mice, and the IOP-lowering effect of the ALK5 inhibitor was greater than that of a Rho-associated coiled-coil-containing protein kinase inhibitor (Y-27632). Repeated dosing with ALK5 inhibitor eye drops (once daily) enhanced the murine IOP-lowering effect. Furthermore, ALK5 inhibition decreased the expression of extracellular matrix (ECM) mRNA and suppressed ECM production. These findings suggest that ALK5 inhibitors may contribute to the development of new treatments for glaucoma that target the TM.

TRPV4 channels promote vascular permeability in retinal vascular disease.

This study aimed to determine the role of transient receptor potential vanilloid 4 (TRPV4), a calcium (Ca2+)-permeable cation channel, in the pathophysiology of retinal vascular disease. The retinal vein occlusion (RVO) murine model was created by irradiating retinal veins using lasers. TRPV4 expression and localization were evaluated in RVO mice retinas. In addition, we examined the effects of TRPV4 antagonists (RQ-00317310, HC-067047, GSK2193874, and GSK2798745) on retinal edema, blood flow, and ischemic areas in RVO mice. Furthermore, changes in the retinal expression of tumor necrosis factor (TNF)-α and aquaporin4 (AQP4) by RQ-00317310 were analyzed using Western blot. We also assessed the barrier integrity of epithelial cell monolayers using trans-endothelial electrical resistance (TEER) in Human Retinal Microvascular Endothelial Cells (HRMECs). The expression of TRPV4 was significantly increased and co-localized with glutamine synthetase (GS), a Müller glial marker, in the ganglion cell layer (GCL) of the RVO mice. Moreover, RQ-00317310 administration ameliorated the development of retinal edema and ischemia in RVO mice. In addition, the up regulation of TNF-α and down-regulation of AQP4 were lessened by the treatment with RQ-00317310. Treatment with GSK1016790A, a TRPV4 agonist, increased vascular permeability, while RQ-00317310 treatment decreased vascular endothelial growth factor (VEGF)- or TRPV4-induced retinal vascular hyperpermeability in HRMECs. These findings suggest that TRPV4 plays a role in the development of retinal edema and ischemia. Thus, TRPV4 could be a new therapeutic target against the pathological symptoms of retinal vascular diseases.

Canagliflozin Inhibits Glioblastoma Growth and Proliferation by Activating AMPK.

Sodium-glucose transporter 2 (SGLT2) inhibitors are antidiabetic drugs affecting SGLT2. Recent studies have shown various cancers expressing SGLT2, and SGLT2 inhibitors attenuating tumor proliferation. We evaluated the antitumor activities of canagliflozin, a SGLT2 inhibitor, on glioblastoma (GBM). Three GBM cell lines, U251MG (human), U87MG (human), and GL261 (murine), were used. We assessed the expression of SGLT2 of GBM through immunoblotting, specimen-use, cell viability assays, and glucose uptake assay with canagliflozin. Then, we assessed phosphorylation of AMP-activated protein kinase (AMPK), p70 S6 kinase, and S6 ribosomal protein by immunoblotting. Concentrations of 5, 10, 20, and 40 µM canagliflozin were used in these tests. We also evaluated cell viability and immunoblotting using U251MG with siRNA knockdown of SGLT2. Furthermore, we divided the mice into vehicle group and canagliflozin group. The canagliflozin group was administrated with 100 mg/kg of canagliflozin orally for 10 days starting from the third days post-GBM transplant. The brains were removed and the tumor volume was evaluated using sections. SGLT2 was expressed in GBM cell and GBM allograft mouse. Canagliflozin administration at 40 µM significantly inhibited cell proliferation and glucose uptake into the cell. Additionally, canagliflozin at 40 µM significantly increased the phosphorylation of AMPK and suppressed that of p70 S6 kinase and S6 ribosomal protein. Similar results of cell viability assays and immunoblotting were obtained using siRNA SGLT2. Furthermore, although less effective than in vitro, the canagliflozin group significantly suppressed tumor growth in GBM-transplanted mice. This suggests that canagliflozin can be used as a potential treatment for GBM.

The pathological association between the anterior eye segment and the retina in a murine model of neovascular glaucoma.

Neovascular glaucoma (NVG) is caused by the formation of new blood vessels in the angle, iris, and cornea in retinal ischemic disease, such as proliferative diabetic retinopathy (PDR) and retinal vein occlusion (RVO), which can reduce the visual acuity. However, the pathophysiological symptoms of NVG are still not well understood because there is no model for the formation of NVG in the angle, iris, and cornea. The aim of this study was to investigate the involvement of NVG during ischemic disease, in a murine model of retinal ischemia. We evaluated the changes of the intraocular pressure (IOP) and pathological symptoms in the anterior eye segment and retina in this model, and the changes in the RNA or protein expression of vascular endothelial growth factor (VEGF) and fibrosis-related factors were analyzed in the retina and cornea by quantitative real-time polymerase chain reaction or western blot, respectively. Furthermore, we examined the changes in IOP after intravitreal injection of an anti-VEGF antibody. First, NVG formed in the retinal ischemic murine model, and the IOP was elevated in mice with NVG formation. Interestingly, VEGF expression was decreased in the retina but increased in the cornea in the murine model of NVG. On the other hand, fibrosis-related factors were increased in the retina and also significantly increased in the cornea in NVG. Moreover, the administration of anti-VEGF antibody immediately after vessel occlusion suppressed the increase in IOP, but administration at 7 days after vessel occlusion accelerated the increase in IOP. These findings suggest that the formation of NVG may be correlated with the pathological symptoms of retinal ischemic disease, via changes in VEGF and fibrosis-related factor expression.

3-Nitropropionic Acid Enhances Ferroptotic Cell Death via NOX2-Mediated ROS Generation in STHdhQ111 Striatal Cells Carrying Mutant Huntingtin.

Huntington's disease (HD) is a hereditary neurodegenerative disease that involves an expansion of the CAG repeats of the Huntingtin (HTT) gene, but the disease onset and progression do not necessarily correspond to the extent of CAG repeats. Decreased mitochondrial complex II activity has also been reported to be closely associated with disease pathogenesis. Here, we examined the mechanism of cell death induced by 3-nitropropionic acid (3-NP), a mitochondrial complex II inhibitor, using striatal cells (STHdhQ111 cells) derived from HD model mice with mutant HTT carrying the CAG repeat extended. Treatment with 3-NP (5 mM) enhanced cell death in STHdhQ111 compared to STHdhQ7 cells with normal HTT. Ferrostatin-1, an inhibitor of ferroptosis, and deferoxamine, an iron chelator, markedly inhibited 3-NP-induced cell death in both the STHdh cell lines. On the other hands, cell death was not abrogated by a broad-spectrum caspase inhibitor, Z-VAD-FMK, indicating that this cell death was caspase-independent. Cell death caused by 3-NP is suggested to be due to ferroptosis. Furthermore, 3-NP-induced cell death was markedly inhibited by GSK2795039, a reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) inhibitor, suggesting that cell death is mainly mediated by intracellular superoxide anion (O2-) production through NOX2. Furthermore, a mitochondria-targeted superoxide dismutase mimetic (Mito-TEMPO), partially inhibited 3-NP-induced cell death, suggesting that O2- production in the mitochondria is partially responsible for cell death. These results indicate that 3-NP-induced cell death in the STHdhQ111 cells is caspase-independent, non-apoptotic, and that ferroptotic cell death is mainly induced via NOX2 activation.

The Involvement of Progranulin for α-Synuclein Reduction through Autolysosome Formation.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor symptoms and neuropathological features, such as loss of dopaminergic neurons in the substantia nigra pars compacta and accumulation of alpha-synuclein (α-Syn). Progranulin (PGRN) is a secreted growth factor that exhibits anti-inflammatory properties and regulates lysosomal function. Although autophagy-lysosome pathway is the main degradative pathway for α-Syn, the molecular mechanistic relationship between PD and PGRN remains unclear. In this study, we investigated the role of PGRN in PD pathology. PGRN protein expression in striatum was increased in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model mice. Intracerebroventricular (i.c.v.) administration of PGRN ameliorated the decrease in expression of tyrosine hydroxylase, a dopaminergic neuron marker, in MPTP-treated mice. Furthermore, i.c.v. administration of PGRN ameliorated 6-hydroxydopamine-induced motor deficits. In SH-SY5Y human neuroblastoma cells, 1-methyl-4-phenylpyridinium ion (MPP+), an active metabolite of MPTP, increased α-Syn expression. In contrast, PGRN ameliorated MPP+-induced increase in α-Syn expression. Although PGRN decreased the levels of autophagy-related proteins Sequestosome-1 (p62) and MAP1LC3 (LC3)-II, PGRN did not influence the phosphorylation of AMP-activated protein kinase and mechanistic target of rapamycin, which are also proteins that regulate autophagy. Immunostaining analysis showed that PGRN ameliorated MPP+-induced increase of LC3 puncta, indicator of autophagosome, and co-localization of LC3 and α-Syn. The DALGreen assay showed that PGRN ameliorated MPP+-induced decreasing trend of autolysosomes. These results suggest that PGRN participates in α-Syn degradation via acceleration of the autophagy-lysosome pathway and is a potential therapeutic target for PD.

Arctigenin Prevents Retinal Edema in a Murine Retinal Vein Occlusion Model.

Macular edema causes vision loss in patients with retinal vein occlusion (RVO) and diabetic macular edema (DME). The intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents is used for treatment; however, this therapy is invasive, and recurrence occurs in some cases. The establishment of a non-invasive treatment would help to solve these problems. Here, we focused on arctigenin, a lignan polyphenol found in burdock sprout, and has effects on inflammatory and microcirculatory when taken orally. We hypothesized that oral intake of arctigenin could be effective against retinal edema in RVO and DME. In this study, the degree of retinal edema by measuring the total retinal thickness using optical coherence tomography (OCT) and the thickness of the inner nuclear layer (INL) by hematoxylin-eosin (H&E) staining were investigated. Oral administration of arctigenin ameliorated retinal edema in an RVO murine model by inhibiting the decrease in occludin and vascular endothelial (VE)-cadherin. Moreover, in retinas with edema, arctigenin suppressed the induction of VEGF, tumor necrosis factor α (TNFα), and matrix metallopeptidase 9 (MMP9). Next, the effects of arctigenin on barrier function were assessed in human retinal microvascular endothelial cells (HRMECs) by measuring the trans-endothelial electrical resistance (TEER) and conducting fluorescein isothiocyanate (FITC)-dextran permeability assays. Arctigenin showed a protective effect against VEGF-induced barrier dysfunction. In addition, arctigenin inhibited the TNFα-mediated activation of the nuclear factor-kappaB (NF-κB)/p38 mitogen-activated protein kinase (MAPK) pathway. These results suggested that oral administration of arctigenin has beneficial effects on retinal edema by inhibiting vascular hyperpermeability in endothelial cells.

Piezo1 activation induces fibronectin reduction and PGF2α secretion via arachidonic acid cascade.

Glaucoma is a neurodegenerative disease that leads to blindness, and lowering intraocular pressure (IOP) is very important in glaucoma treatment. The trabecular meshwork is responsible for aqueous humor outflow, and the accumulation of fibronectin in trabecular meshwork is known to cause ocular hypertension. We have already shown that Piezo1 activation has an IOP lowering effect in mice and suppresses fibronectin expression level in human trabecular meshwork cells (HTMC). In this study, we report the mechanism of the reduction of fibronectin caused by Piezo1 activation. Activation of Piezo1 in HTMC showed increased expression of matrix metalloproteinase-2 (MMP-2) and cyclooxygenase (COX)-2, and decreased fibronectin expression. In addition, Piezo1 activation enhanced phosphorylation of cytosolic phospholipase A2 (cPLA2), and inhibitors targeting cPLA2 and COX-2 suppressed Yoda 1, a Piezo1 agonist, induced fibronectin reduction. These results indicate that the arachidonic acid cascade underlies this reaction, and, in support of this hypothesis, activation of Piezo1 promoted secretion of prostaglandin F2α (PGF2α) in HTMC. These results indicate that the activation of Piezo1 in HTMC promotes the degrading of fibronectin by promoting the arachidonic acid cascade and increasing the expression of PGF2α and MMP-2.

Excess adiponectin in eyes with progressive ocular vascular diseases.

Anti-vascular endothelial growth factor (VEGF) therapies are now the first-line treatment for many ocular diseases, but some patients are non-responders to these therapies. The purpose of this study was to determine whether the level of adiponectin increased the pathogenesis of retinal edema and neovascularization in the retina of progressive ocular vascular diseases. We examined the role played by adiponectin in two types of cells and animal models which are retinal vein occlusion (RVO) and oxygen-induced retinopathy (OIR) mice. Our results showed that an injection of anti-adiponectin antibody ameliorated the retinal edema and ischemia through the depression of the expression level of VEGF-related factors and tight junction-related proteins in the retina of RVO mice. The intravitreal injection of anti-adiponectin antibody also decreased the degree of retinal neovascularization in an OIR mice. In addition, exposure of human retinal microvascular endothelial cells and human brain microvascular pericytes in culture to adiponectin increased both the vascular permeability and neovascularization through the increase of inflammatory factor and the dropout of the pericytes. These findings indicate that adiponectin plays a critical role in retinal edema and neovascularization, and adiponectin is a potential therapeutic target for the treatment of diabetic macular edema, proliferative diabetic retinopathy, and RVO.

NMDA receptor signaling induces the chemoresistance of temozolomide via upregulation of MGMT expression in glioblastoma cells.

PURPOSE: The alkylating agent temozolomide (TMZ) has a significant impact on the prognosis of glioblastoma (GBM) patients. Therefore, maximizing TMZ efficacy is important for GBM treatment. Many reports have shown that glutamate signaling promotes GBM progression via glutamate receptors, including N-methyl-D-aspartate receptors (NMDARs). Although NMDARs promote cell migration and invasion of GBM cells, their role in TMZ resistance remains unclear. Therefore, we focused on NMDAR signaling and investigated its effects on TMZ resistance. METHODS: We investigated the effect of NMDAR signaling on O6-methylguanine DNA methyltransferase (MGMT), a DNA repair enzyme that induces chemoresistance to TMZ, using quantitative real-time polymerase chain reaction and western blotting in human GBM T98G cells. In addition, we used memantine (MEM), an NMDAR antagonist, to investigate the cytotoxic effect of TMZ/MEM combination and its detailed mechanism. RESULTS: Activation of NMDAR by N-methyl-D-aspartate (NMDA) elevated MGMT expression and suppressed the effect of TMZ in T98G cells. In contrast, knockdown of NMDAR by NMDAR1 shRNA decreased MGMT expression and enhanced the effect of TMZ in T98G cells. The cytotoxic effect of TMZ was enhanced by MEM in T98G cells. Inhibition of NMDAR by MEM decreased MGMT expression and increased DNA alkylation by TMZ. CONCLUSION: NMDAR signaling induced chemoresistance of TMZ via the upregulation of MGMT expression in GBM cells. Furthermore, MEM inhibited TMZ-induced MGMT upregulation and increased the cytotoxic effect of TMZ on MGMT-positive cells. This study demonstrates that the combination of TMZ and MEM could be a new therapeutic strategy for MGMT-positive GBM. Overview of this study. NMDAR signaling controls the expression of MGMT and the cytotoxic effect of TMZ.

Glutamatergic dysfunction is associated with phenotypes of VGF-overexpressing mice.

VGF nerve growth factor inducible (VGF) is a neuropeptide precursor, which is induced by several neurotrophic factors, including nerve growth factor and brain-derived neurotrophic factor. Clinically, an upregulation of VGF levels has been reported in the cerebrospinal fluid and prefrontal cortex of patients with schizophrenia. In our previous study, mice overexpressing VGF exhibited schizophrenia-related behaviors. In the current study, we characterized the biochemical changes in the brains of VGF-overexpressing mice. Metabolomics analysis of neurotransmitters revealed that glutamic acid and N-acetyl-L-aspartic acid were increased in the striatum of VGF-overexpressing mice. Additionally, the present study revealed that MK-801, which causes the disturbance in glutamic acid metabolism, increased the expression level of VGF-derived peptide (NAPP129, named VGF20), and VGF-overexpressing mice had higher sensitivity to MK-801. These results suggest that VGF may modulate the regulation of glutamic acid levels and the degree of glutamic acid signaling.

Crocetin protects against mitochondrial damage induced by UV-A irradiation in corneal epithelial cell line HCE-T cells.

The corneal epithelium is located at the outermost layer of the ocular surface and continuously exposed to environmental factors, such as ultraviolet (UV) radiation from sunlight. UV irradiation causes excessive production of reactive oxygen species (ROS) in cells, which results in oxidative damage to membrane-bound organelles such as mitochondria, eventually leading to cell death. Crocetin, a natural carotenoid found in plants, has various biological properties including antioxidant activity. In this study, we investigated the effects of crocetin on UV-A-induced cell injury in the corneal epithelium. Using an in vitro system with the human corneal epithelial cell-transformed (HCE-T) cell line, pretreatment with 10 µM crocetin suppressed the reduction of cell viability induced by UV-A exposure. Crocetin ameliorated the decrease in oxygen consumption rates and the mitochondrial fragmentation that occurred following UV-A irradiation. Crocetin inhibited both ROS production and the activation of the apoptosis pathway; it also preserved the defects of epithelial cell polarity and barrier function in UV-A-irradiated HCE-T cells. The reduction in apical Mucin-16 expression was partially recovered in the presence of crocetin. Taking these findings together, we conclude that crocetin has a protective effect against UV-A irradiation-induced mitochondrial injury in corneal epithelial cells.

The protective effect of Centella asiatica and its constituent, araliadiol on neuronal cell damage and cognitive impairment.

Alzheimer's disease (AD) is characterized by progressive cognitive decline, and the number of affected individuals has increased worldwide. However, there are no effective treatments for AD. Therefore, it is important to prevent the onset of dementia. Oxidative stress and endoplasmic reticulum (ER) stress are increased in the brains of AD patients, and are postulated to induce neuronal cell death and cognitive dysfunction. In this study, Centella asiatica, a traditional Indian medicinal herb, were fractionated and compared for their protective effects against glutamate and tunicamycin damage. Araliadiol was identified as a component from the fraction with the highest activity. Further, murine hippocampal cells (HT22) were damaged by glutamate, an oxidative stress inducer. C. asiatica and araliadiol suppressed cell death and reactive oxygen species production. HT22 cells were also injured by tunicamycin, an ER stress inducer. C. asiatica and araliadiol prevented cell death by mainly inhibiting PERK phosphorylation; additionally, C. asiatica also suppressed the expression levels of GRP94 and BiP. In Y-maze test, oral administration of araliadiol (10 mg/kg/day) for 7 days ameliorated the arm alternation ratio in mice with scopolamine-induced cognitive impairment. These results suggest that C. asiatica and its active component, araliadiol, have neuroprotective effects, which may prevent cognitive dysfunction.