Implementation of the neuro-glia-vascular unit through co-culture of adult neural stem cells and vascular cells and transcriptomic analysis of diverse Aß assembly types.

BACKGROUND: The blood-brain barrier (BBB) is a specialized layer between blood vessels and tissue in the brain, which is comprised of a neuro-glia-vascular (NGV) unit, thus play a vital role in various brain diseases. NEW METHOD: We developed the in vitro NGV units by co-culturing brain microvascular endothelial cells (BMECs; bEnd.3) and primary neural stem cells extracted from subventricular zone of adult mice. This approach was designed to mimic the RNA profile conditions found in the microvessels of a mouse brain and confirmed through various comparative transcriptome analyses. RESULTS: Optimal NGV unit development was achieved by adjusting cell density-dependent co-culture ratios. Specifically, the morphogenic development and neuronal association of astrocyte endfeet were well observed in the contact region with BMECs in the NGV unit. Through transcriptome analysis, we compared co-cultured bEnd.3/NSCs with monocultured bEnd.3 or NSCs and additionally compared them with previously reported mouse brain vascular tissue to show that this NGV unit model is a suitable in vitro model for neurological disease such as Alzheimer's disease (AD). COMPARISON WITH EXISTING METHOD(S): This in vitro NGV unit was formed from neural stem cells and vascular cells in the brain of adult mice, not embryos. It is very useful for studying brain disease mechanisms by identifying proteins and genes associated with diseases progress. CONCLUSIONS: We suggest that this simple in vitro NGV model is appropriate to investigate the relationship between BBB changes and pathological factors in the fields of neurovascular biology and cerebrovascular diseases including AD.

Targeting casein kinase 1 for cancer therapy: current strategies and future perspectives.

Casein Kinase 1 (CK1) is a family of serine/threonine protein kinases that play a crucial role in various cellular processes, including cell proliferation, survival, and metabolism. The dysregulation of CK1 expression has been implicated in the development and progression of several types of cancer, making it an attractive target for anticancer therapy. In this review, we provide an overview of the current strategies employed to target CK1 for cancer therapy and discuss the future perspectives in this field. We highlight the different approaches, including small molecule inhibitors, RNA interference, genome editing, and immunotherapies, which hold immense potential for targeted modulation of CK1 activity in cancer cells. Furthermore, we discuss the challenges associated with targeting CK1 and propose potential strategies to overcome these hurdles. Overall, targeting CK1 holds great promise as a therapeutic strategy for cancer treatment, and further research in this area is warranted.

Effects of Gryllus bimaculatus and Oxya chinensis sinuosa extracts on brain damage via blood-brain barrier control and apoptosis in mice with pentylenetetrazol-induced epilepsy.

The demand for environmentally friendly foods with high nutritional value and low carbon emissions is increasing with the aging of the global population and the crisis of food resources. Edible insects are becoming increasingly well-known as such foods. This study evaluated the effects and mechanisms of Gryllus bimaculatus (Cricket) (Gb) and Oxya chinensis sinuosa (Grasshopper) (Ocs) extracts on epilepsy. A pentylenetetrazol (PTZ)-induced seizure mouse model was used for the study, and Gb and Ocs extracts were administered for 29 days on alternate days at concentrations of 8 g/kg and 16 g/kg. The integrity of the blood-brain barrier (BBB) and brain edema was measured using the perfusion of Evans blue dye and brain water content. Gb and Ocs extracts prevented BBB permeabilization and cerebral edema through increasing the expression of tight junction-associated proteins in the endothelial cells and reducing water content in PTZ-treated mice. Additionally, Gb and Ocs extracts protected neurons from oxidative stress and apoptosis in different brain areas. These protective effects were demonstrated through the restoration of the expression of neuronal nuclear protein and postsynaptic density protein-95, thus increasing the levels of glutathione and superoxide dismutase, decreasing lipid peroxidation, and recovering apoptosis-associated proteins, such as Bax, cleaved PARP, and cleaved caspase-3, in epileptic mice. In addition, Gb and Ocs extracts rescued PTZ-induced hyperexcitable neurons to control mice level, as supported by the restored expression of gamma-aminobutyric acid (GABA) transporter 1, the metabotropic glutamate receptors-GRM2/3, and BDNF. This study suggested that Gb and Ocs extracts are novel medicinal candidates that can help ameliorate epilepsy by improving BBB health and preventing oxidative stress-mediated apoptosis.

Casein kinase 1 controls the shuttling of epidermal growth factor receptor and estrogen receptor in endometrial carcinoma induced by breast cancer hormonal therapy: Relevance of GPER1/Src.

Casein kinase 1 plays a crucial role in carcinogenesis. 4-Hydroxytamoxifen (4-OHT), which is widely used to treat breast cancer, often leads to the development of endometrial carcinoma with poor prognosis, particularly among women who receiving long-term treatment. This study was performed to elucidate whether specific inhibition of casein kinase 1 (CK1) controls 4-OHT-mediated Ishikawa cell carcinogenesis. 4-OHT significantly stimulated the activity of estrogen receptor alpha (ERα) and nuclear translocation and expression of epidermal growth factor receptor (EGFR) from the plasma membrane to perinuclear or nuclear regions, as well as the activities of G-protein-coupled estrogen receptor 1 (GPER1) and Src in Ishikawa cells. However, inhibition of EGFR by Gefitinib blocked all these events, and inhibition of GPER1 or Src produced a partial block. GPER1 and Src controlled Ishikawa cell carcinogenesis in different manners: GPER1 accelerated EGFR mobility without affecting ERα activity, while Src activated ERα and EGFR without any change in GPER1 expression. EGFR and GPER1 performed reciprocal regulation in endometrial cell carcinogenesis via direct interaction in 4-OHT-treated Ishikawa cells, implying a possible key role of GPER1 in these events. Inhibition of CK1 by CKI-7 and IC261, however, impeded all changes beginning with EGFR translocation and activity in 4-OHT-treated Ishikawa cells. These findings indicate that inhibition of CK1 could control 4-OHT-mediated activation and translocation of ER/EGFR and GPER1/Src expression, inhibiting 4-OHT-triggered endometrial carcinogenesis. Therefore, targeting of CK1 by CKI-7 and IC261 could be a prospective adjuvant therapy for breast cancer patients taking tamoxifen.

Ginsenoside protopanaxadiol protects adult retinal pigment epithelial-19 cells from chloroquine by modulating autophagy and apoptosis.

Chloroquine often causes serious eye and vision problems, which are mainly mediated by lysosomotropic alteration. In this study, we investigated whether the ginsenoside protopanaxadiol relieves chloroquine-induced retinopathy by restoring lysosomotropic abnormalities in human adult retinal pigment epithelial-19 cells. Cytotoxicity was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Morphological alterations in autophagosomes of adult retinal pigment epithelial-19 cells was detected using confocal microscopy. Apoptosis was examined using flow cytometry, whereas cellular reactive oxygen species levels were determined by measuring the fluorescence intensity of 5-(and-6)-carboxy-2'-7'-dichlorohydrofluorescein diacetate. Lysosomal function was assessed by measuring lysosomal pH and enzyme activity. Immunoprecipitation and western blotting analyses were performed. Adult retinal pigment epithelial-19 cells accumulated autophagosomes with fusion defects in lysosomes and reactive oxygen species formation following exposure to chloroquine. This effect trapped Beclin-1 and B-cell lymphoma 2 interfering with autophagy initiation and autophagosome development. Protopanaxadiol alleviated chloroquine-induced toxicity by modulating the interaction between Beclin-1 and Bcl-2, which was mediated by the AMP-activated protein kinase-mammalian target of rapamycin signal axis. Furthermore, autophagy and apoptosis were simultaneously controlled by protopanaxadiol via upregulation of autophagy flux and decreased reactive oxygen species formation and apoptotic protein expression. These findings suggest that protopanaxadiol is a promising treatment strategy for chloroquine-mediated retinopathy.

Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

The antimalarial drug chloroquine (CQ) induces retinopathy, a disorder characterized by lysosomotropic alteration. In this study, we examined whether D4476 (4-(4-(2,3-dihydrobenzo [1,4] dioxin-6-yl)-5-pyridin-2-yl-1H-imidazole-2-yl) benzamide), a specific casein kinase 1 inhibitor, alleviate CQ-induced retinopathy in adult retinal pigment epithelial (ARPE-19) cells. Cultured ARPE-19 cells were exposed to CQ with or without D4476 and cell death was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To examine autophagy flux, ARPE-19 cells were transfected with green fluorescence protein light chain 3 (GFP-LC3)-red fluorescence protein (RFP)-LC3ΔG plasmid DNA and co-stained with the lysosomal-associated membrane protein (LAMP)-1 antibody. Western blotting and fluorescence-activated cell sorting (FACS) showed apoptosis, whereas the fluorescence intensity of 2'-7'-dichlorofluorescein diacetate revealed levels of cellular oxidative stress. We then confirmed the effect of D4476 on the interaction between Beclin 1 and B-cell lymphoma-2 (Bcl-2) through immunoprecipitation with an anti-Bcl-2 antibody. Following CQ exposure, ARPE-19 cells accumulated autophagosomes because of defective lysosomal degradation. Furthermore, CQ trapped Beclin 1 with Bcl-2, disturbing autophagy initiation and autolysosome formation. However, D4476 alleviated CQ-induced effects by rescuing ARPE-19 cells from CQ-induced toxicity by modulating the association between Beclin 1 and Bcl-2. Therefore, D4476 controls autophagy and apoptosis simultaneously by upregulating autophagy flux, decreasing ROS formation, and triggering the expression of anti-apoptotic proteins through inhibition of mTOR, JNK, and p38 MAPK signals. We conclude that D4476 is a promising treatment strategy for CQ-mediated retinopathy.

CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells.

Tamoxifen (TAM) is a selective estrogen receptor modulator used for breast cancer patients. Prolonged use of tamoxifen is not recommended for some patients. In this study, we aimed to identify molecular targets sensitive to TAM using a genome-wide gene deletion library screening of fission yeast heterozygous mutants. From the screening, casein kinase 1 gamma 2 (CSNK1G2), a serine-/threonine protein kinase, was the most sensitive target to TAM with a significant cytotoxicity in estrogen receptor-positive (ER+) breast cancer cells but with only a slight toxicity in the case of ER- cells. In addition, tumor sphere formation and expression of breast stem cell marker genes such as CD44/CD2 were greatly inhibited by CSNK1G2 knockdown in ER+ breast cancer cells. Consistently, CSNK1G2 altered ERα activity via phosphorylation, specifically at serine (Ser)167, as well as the regulation of estrogen-responsive element (ERE) of estrogen-responsive genes such as CTSD and GREB1. However, ERα silencing almost completely blocked CSNK1G2-induced TAM sensitivity. In ER+ breast cancer cells, combined treatment with TAM and CSNK1G2 knockdown further enhanced the TAM-mediated decrease in phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (S6K) signaling but not extracellular signal-regulated kinase (ERK) signaling. Inversely, in ER- cells treated with TAM, only ERK and PI3K signaling was altered by CSNK1G2 knockdown. The CK1 inhibitor, D4476, partly mimicked the CSNK1G2 knockdown effect in ER+ breast cancer cells, but with a broader repression ranging from PI3K/AKT/mTOR/S6K to ERK signaling. Collectively, these results suggest that CSNK1G2 plays a key role in sensitizing TAM toxicity in ER+ and ER- breast cancer cells via differently regulating PI3K/AKT/mTOR/S6K and ERK signaling.

Knockdown of vps54 aggravates tamoxifen-induced cytotoxicity in fission yeast.

Tamoxifen (TAM) is an anticancer drug used to treat estrogen receptor (ER)‒positive breast cancer. However, its ER-independent cytotoxic and antifungal activities have prompted debates on its mechanism of action. To achieve a better understanding of the ER-independent antifungal action mechanisms of TAM, we systematically identified TAM-sensitive genes through microarray screening of the heterozygous gene deletion library in fission yeast (Schizosaccharomyces pombe). Secondary confirmation was followed by a spotting assay, finally yielding 13 TAM-sensitive genes under the drug-induced haploinsufficient condition. For these 13 TAM-sensitive genes, we conducted a comparative analysis of their Gene Ontology (GO) 'biological process' terms identified from other genome-wide screenings of the budding yeast deletion library and the MCF7 breast cancer cell line. Several TAM-sensitive genes overlapped between the yeast strains and MCF7 in GO terms including 'cell cycle' (cdc2, rik1, pas1, and leo1), 'signaling' (sck2, oga1, and cki3), and 'vesicle-mediated transport' (SPCC126.08c, vps54, sec72, and tvp15), suggesting their roles in the ER-independent cytotoxic effects of TAM. We recently reported that the cki3 gene with the 'signaling' GO term was related to the ER-independent antifungal action mechanisms of TAM in yeast. In this study, we report that haploinsufficiency of the essential vps54 gene, which encodes the GARP complex subunit, significantly aggravated TAM sensitivity and led to an enlarged vesicle structure in comparison with the SP286 control strain. These results strongly suggest that the vesicle-mediated transport process might be another action mechanism of the ER-independent antifungal or cytotoxic effects of TAM.

Bone remodeling effects of Korean Red Ginseng extracts for dental implant applications.

BACKGROUND: The formation of a nanotube layer on a titanium nanotube (N-Ti) plate facilitates an active reaction between bone cells and the material surface via efficient delivery of the surface materials of the dental implant into the tissues. Studies have reported that Korean Red Ginseng extracts (KRGEs) are involved in a variety of pharmacological activities: we investigated whether implantation with a KRGE-loaded N-Ti miniimplant affects osteogenesis and osseointegration. METHODS: KRGE-loaded nanotubes were constructed by fabrication on pure Ti via anodization, and MC3T3-E1 cells were cultured on the N-Ti. N-Ti implants were subsequently placed on a rat's edentulous mandibular site. New bone formation and bone mineral density were measured to analyze osteogenesis and osseointegration. RESULTS: KRGE-loaded N-Ti significantly increased the proliferation and differentiation of MC3T3-E1 cells compared with cells on pure Ti without any KRGE loading. After 1-4 weeks, the periimplant tissue in the edentulous mandibular of the healed rat showed a remarkable increase in new bone formation and bone mineral density. In addition, high levels of the bone morphogenesis protein-2 and bone morphogenesis protein-7, besides collagen, were expressed in the periimplant tissues. CONCLUSION: Our findings suggest that KRGE-induced osteogenesis and osseointegration around the miniimplant may facilitate the clinical application of dental implants.

Metallothionein-3 as a multifunctional player in the control of cellular processes and diseases.

Transition metals, such as iron, copper, and zinc, play a very important role in life as the regulators of various physiochemical reactions in cells. Abnormal distribution and concentration of these metals in the body are closely associated with various diseases including ischemic seizure, Alzheimer's disease, diabetes, and cancer. Iron and copper are known to be mainly involved in in vivo redox reaction. Zinc controls a variety of intracellular metabolism via binding to lots of proteins in cells and altering their structure and function. Metallothionein-3 (MT3) is a representative zinc binding protein predominant in the brain. Although the role of MT3 in other organs still needs to be elucidated, many reports have suggested critical roles for the protein in the control of a variety of cellular homeostasis. Here, we review various biological functions of MT3, focusing on different cellular molecules and diseases involving MT3 in the body.

A role of metallothionein-3 in radiation-induced autophagy in glioma cells.

Although metallothionein-3 (MT3), a brain-enriched form of metallothioneins, has been linked to Alzheimer's disease, little is known regarding the role of MT3 in glioma. As MT3 plays a role in autophagy in astrocytes, here, we investigated its role in irradiated glioma cells. Irradiation increased autophagy flux in GL261 glioma cells as evidenced by increased levels of LC3-II but decreased levels of p62 (SQSTM1). Indicating that autophagy plays a cytoprotective role in glioma cell survival following irradiation, measures inhibiting autophagy flux at various steps decreased their clonogenic survival of irradiated GL261 as well as SF295 and U251 glioma cells. Knockdown of MT3 with siRNA in irradiated glioma cells induced arrested autophagy, and decreased cell survival. At the same time, the accumulation of labile zinc in lysosomes was markedly attenuated by MT3 knockdown. Indicating that such zinc accumulation was important in autophagy flux, chelation of zinc with tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), induced arrested autophagy in and reduced survival of GL261 cells following irradiation. Suggesting a possible mechanism for arrested autophagy, MT3 knockdown and zinc chelation were found to impair lysosomal acidification. Since autophagy flux plays a cytoprotective role in irradiated glioma cells, present results suggest that MT3 and zinc may be regarded as possible therapeutic targets to sensitize glioma cells to ionizing radiation therapy.

Enhanced antibacterial activity of titanium by surface modification with polydopamine and silver for dental implant application.

BACKGROUND: Biofilm formation and microbial colonization on the surface of implant devices may cause dental caries and peri-implantitis. Therefore, various surface treatments have been developed to improve the antibacterial activity of titanium implant. METHODS: Silver-loaded polydopamine coating was formed by immersing pure titanium in dopamine hydrochloride/HCl buffer solution for 24 h in 50 mL silver nitrate solutions with different concentrations for 30 min. Microbial growth inhibition and microbial growth curve analyses for bacterial solutions of Streptococcus mutans and Porphyromonas gingivalis incubated with the specimens were respectively conducted by counting the numbers of colonies on agar solid medium and by measuring absorbance using enzyme-linked immunosorbent assay reader. RESULTS: Silver nanoparticles were uniformly distributed over the whole surface of the polydopamine and silver-coated titanium specimens. The numbers of microbial colonies for both bacteria cultured with surface-modified titanium were significantly lower than those cultured with uncoated titanium. When Streptococcus mutans and Porphyromonas gingivalis were cultured with surface-modified titanium, the lag phase of the growth curves for both bacteria was continually maintained, whereas the lag phase for Streptococcus mutans and Porphyromonas gingivalis changed to exponential phase after 9 and 15 h, respectively, when both bacteria were cultured with uncoated titanium. CONCLUSION: It was confirmed that the coating of polydopamine and silver on the surface of titanium effectively retards the microbial growth, which can cause the formation of biofilm and pathogenesis of gum disease in the mouth.

In Vitro and In Vivo Characterization of N-Acetyl-L-Cysteine Loaded Beta-Tricalcium Phosphate Scaffolds.

Beta-tricalcium phosphate bioceramics are widely used as bone replacement scaffolds in bone tissue engineering. The purpose of this study is to develop beta-tricalcium phosphate scaffold with the optimum mechanical properties and porosity and to identify the effect of N-acetyl-L-cysteine loaded to beta-tricalcium phosphate scaffold on the enhancement of biocompatibility. The various interconnected porous scaffolds were fabricated using slurries containing various concentrations of beta-tricalcium phosphate and different coating times by replica method using polyurethane foam as a passing material. It was confirmed that the scaffold of 40 w/v% beta-tricalcium phosphate with three coating times had optimum microstructure and mechanical properties for bone tissue engineering application. The various concentration of N-acetyl-L-cysteine was loaded on 40 w/v% beta-tricalcium phosphate scaffold. Scaffold group loaded 5 mM N-acetyl-L-cysteine showed the best viability of MC3T3-E1 preosteoblastic cells in the water-soluble tetrazolium salt assay test.

Editor's Highlight: A Genome-wide Screening of Target Genes Against Silver Nanoparticles in Fission Yeast.

To identify target genes against silver nanoparticles (AgNPs), we screened a genome-wide gene deletion library of 4843 fission yeast heterozygous mutants covering 96% of all protein encoding genes. A total of 33 targets were identified by a microarray and subsequent individual confirmation. The target pattern of AgNPs was more similar to those of AgNO3 and H2O2, followed by Cd and As. The toxic effect of AgNPs on fission yeast was attributed to the intracellular uptake of AgNPs, followed by the subsequent release of Ag+, leading to the generation of reactive oxygen species (ROS). Next, we focused on the top 10 sensitive targets for further studies. As described previously, 7 nonessential targets were associated with detoxification of ROS, because their heterozygous mutants showed elevated ROS levels. Three novel essential targets were related to folate metabolism or cellular component organization, resulting in cell cycle arrest and no induction in the transcriptional level of antioxidant enzymes such as Sod1 and Gpx1 when 1 of the 2 copies was deleted. Intriguingly, met9 played a key role in combating AgNP-induced ROS generation via NADPH production and was also conserved in a human cell line.

The effect of the cwf14 gene of fission yeast on cell wall integrity is associated with rho1.

In all eukaryotic organisms, a wide range of morphologies are responsible for critical cellular function and development. In particular, the Rho GTPases, which are highly conserved from yeast to mammals, are key molecules in signaling pathways that control cell polarity processes and cell wall biosynthesis, which are fundamental aspects of morphogenesis. Therefore, using haploinsufficiency deletion mutants of the fission yeast Schizosaccharomyces pombe, we screened the slow-growing mutants and their morphogenesis, specifically focusing on regulation of their Rho GTPases. Based on this screening, we found that the cwf14 mutant of S. pombe exhibited the slow growth and abnormal phenotypes with an elongated cell shape and thicker cell wall when compared with wild-type cells. In particular, cells with the cwf14 deletion showed excessive Rho1 expression. However, the wildtype strain with ectopically expressed Rho1 did not exhibited any significant change in the level of cwf14, suggesting that cwf14 may act on the upstream of Rho1. Furthermore, the cells with a cwf14 deletion also have increased sensitivity to ß-glucanase, a cell wall-digesting enzyme, which is also seen in Rho1-overexpressing cells. Overall, our results suggest that the cwf14 plays a key role in fission yeast morphogenesis and cell wall biosynthesis and/or degradation possibly via the regulation of Rho1 expression.

The effect of hydrothermal spark discharge anodization in the early integration of implants in sheep sinuses.

OBJECTIVES: Spark discharge anodic oxidation forms a porous oxide film on titanium implant surfaces, which may increase surface roughness and enhance early osseointegration. This study aimed to clinically and histomorphometric compare commercially-available sandblasted (RBM) implants, treated with hydrothermal anodization and placed into an animal maxillary sinus model. MATERIALS AND METHODS: Thirty 3.75 mm × 8.5 mm threaded titanium implants were placed into the maxillary sinuses of 10 sheep via an external approach, with three test groups and 10 implants per group: right side, Control = CP-titanium with RBM surface, Test group 1 = CP-titanium with RBM + anodized surface; left side, Test group 2 = Ti-6Al-7Nb with RBM + anodized surface. Schneiderian membranes were elevated but not bone grafted. Resonant frequency analysis (RFA) was measured at surgery. Animals were sacrificed after 1 month unloaded healing. Resin-embedded undemineralized ground-sections were digitised, and mean bone-implant contact (% BIC) was measured bilaterally for the best-three consecutive threads. RESULTS: Seven of 30 implants showed signs of failure. RFA was low at placement but did not differ between the groups (group mean ISQ values ranged from 23 to 35; χ(2)  = 0.37). RFA was not repeated at sacrifice due to implant instability. Histomorphometric analysis showed % BIC was highest for control (34.8 ± 15.7), followed by Test 1 (29.6 ± 18.1) and Test 2 implants (23.3 ± 22.7), but this difference was not statistically significant (χ(2)  = 0.3). DISCUSSION AND CONCLUSIONS: Early integration of RBM implants placed into thin maxillary sinus walls was not enhanced by hydrothermal anodization of implant surfaces. This may be related to the initial low stability of the implants and the relatively short healing period. However, non-anodized RBM surfaces showed promising results, with % BIC values comparable to the best estimates of other studies using sinus grafting. Whether the modification of the implant surfaces through anodization with simultaneous sinus grafting would promote enhanced early osseointegration, is a subject of future research.

Metallothionein-3 modulates the amyloid ß endocytosis of astrocytes through its effects on actin polymerization.

BACKGROUND: Astrocytes may play important roles in the pathogenesis of Alzheimer's disease (AD) by clearing extracellular amyloid beta (Aß) through endocytosis and degradation. We recently showed that metallothionein 3 (Mt3), a zinc-binding metallothionein that is enriched in the central nervous system, contributes to actin polymerization in astrocytes. Because actin is likely involved in the endocytosis of Aß, we investigated the possible role of Mt3 in Aß endocytosis by cortical astrocytes in this study. RESULTS: To assess the route of Aß uptake, we exposed cultured astrocytes to fluorescently labeled Aß1-40 or Aß1-42 together with chloropromazine (CP) or methyl-beta-cyclodextrin (MßCD), inhibitors of clathrin- and caveolin-dependent endocytosis, respectively. CP treatment almost completely blocked Aß1-40 and Aß1-42 endocytosis, whereas exposure to MßCD had no significant effect. Actin disruption with cytochalasin D (CytD) or latrunculin B also completely blocked Aß1-40 and Aß1-42 endocytosis. Because the absence of Mt3 also results in actin disruption, we examined Aß1-40 and Aß1-42 uptake and expression in Mt3 (-/-) astrocytes. Compared with wild-type (WT) cells, Mt3 (-/-) cells exhibited markedly reduced Aß1-40 and Aß1-42 endocytosis and expression of Aß1-42 monomers and oligomers. A similar reduction was observed in CytD-treated WT cells. Finally, actin disruption and Mt3 knockout each increased the overall levels of clathrin and the associated protein phosphatidylinositol-binding clathrin assembly protein (PICALM) in astrocytes. CONCLUSIONS: Our results suggest that the absence of Mt3 reduces Aß uptake in astrocytes through an abnormality in actin polymerization. In light of evidence that Mt3 is downregulated in AD, our findings indicate that this mechanism may contribute to the extracellular accumulation of Aß in this disease.

Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast.

Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that an rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs.

Targeting ERK1/2-bim signaling cascades by BH3-mimetic ABT-737 as an alternative therapeutic strategy for oral cancer.

To date, many different chemotherapeutic agents have been widely used as common treatments for oral cancers. However, their therapeutic effects have been disappointing, and these agents may have unwanted side effects. Among the many regulatory factors, overexpression of pro-survival Bcl-2 family members may promote resistance to chemotherapeutic drugs in many tumors. The BH3 domain-only proteins effectively antagonize their apoptotic activities. Therefore, there is substantial interest in developing chemotherapeutic drugs that directly target pro-survival Bcl-2 proteins by mimicking the BH3 domain and unleashing pro-apoptotic molecules in tumor cells. Among the numerous available small molecule BH3 mimetics, ABT-737, a potent small molecule that binds to Bcl-2/Bcl-xL with high affinity, has anti-tumor activity in a wide variety of cancer cells. However, the effects of ABT-737 on human oral cancers and the underlying molecular mechanisms have not previously been elucidated. In the present study, we observed that inactivation of the ERK1/2 signaling pathway using ABT-737 dramatically increased the expression of pro-apoptotic protein Bim via transcriptional and/or posttranslational regulation, in a cell type-dependent manner, inducing mitochondria-mediated apoptosis of human oral cancer cells. To the best of our knowledge, this is the first demonstration of the antitumor effects of ABT-737 on human oral cancers.

The zinc ionophore clioquinol reverses autophagy arrest in chloroquine-treated ARPE-19 cells and in APP/mutant presenilin-1-transfected Chinese hamster ovary cells.

Arrested autophagy may contribute to the pathogenesis of Alzheimer's disease. Because we found that chloroquine (CQ) causes arrested autophagy but clioquinol (ClioQ), a zinc ionophore, activates autophagic flux, in the present study, we examined whether ClioQ can overcome arrested autophagy induced by CQ or mutant presenilin-1 (mPS1). CQ induced vacuole formation and cell death in adult retinal pigment epithelial (ARPE-19) cells, but co-treatment with ClioQ attenuated CQ-associated toxicity in a zinc-dependent manner. Increases in lysosome dilation and blockage of autophagic flux by CQ were also markedly attenuated by ClioQ treatment. Interestingly, CQ increased lysosomal pH in amyloid precursor protein (APP)/mPS1-expressing Chinese hamster ovary 7WΔE9 (CHO-7WΔE9) cell line, and ClioQ partially re-acidified lysosomes. Furthermore, accumulation of amyloid-ß (Aß) oligomers in CHO-7WΔE9 cells was markedly attenuated by ClioQ. Moreover, intracellular accumulation of exogenously applied fluorescein isothiocyanate-conjugated Aß(1-42) was also increased by CQ but was returned to control levels by ClioQ. These results suggest that modulation of lysosomal functions by manipulating lysosomal zinc levels may be a useful strategy for clearing intracellular Aß oligomers.