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1.
Molecules ; 25(3)2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-32028607

RESUMO

Accumulation of ß-amyloid (Aß) in the brain has been implicated in the pathology of Alzheimer's disease (AD). Aß is produced from the Aß precursor protein (APP) through the amyloidogenic pathway by ß-, and γ-secretase. Alternatively, APP can be cleaved by α-, and γ-secretase, precluding the production of Aß. Thus, stimulating α-secretase mediated APP processing is considered a therapeutic option not only for decreasing Aß production but for increasing neuroprotective sAPPα. We have previously reported that 7-deoxy-trans-dihydronarciclasine (E144), the active component of Lycoris chejuensis, decreases Aß production by attenuating APP level, and retarding APP maturation. It can also improve cognitive function in the AD model mouse. In this study, we further analyzed the activating effect of E144 on α-secretase. Treatment of E144 increased sAPPα, but decreased ß-secretase products from HeLa cells stably transfected with APP. E144 directly activated ADAM10 and ADAM17 in a substrate-specific manner both in cell-based and in cell-free assays. The Lineweaver-Burk plot analysis revealed that E144 enhanced the affinities of A Disintegrin and Metalloproteinases (ADAMs) towards the substrate. Consistent with this result, immunoprecipitation analysis showed that interactions of APP with ADAM10 and ADAM17 were increased by E144. Our results indicate that E144 might be a novel agent for AD treatment as a substrate-specific activator of α-secretase.

2.
Int J Mol Sci ; 21(3)2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-32050555

RESUMO

Cholesterol is a critical component of eukaryotic membranes, where it contributes to regulating transmembrane signaling, cell-cell interaction, and ion transport. Dysregulation of cholesterol levels in the brain may induce neurodegenerative diseases, such as Alzheimer's disease, Parkinson disease, and Huntington disease. We previously reported that augmenting membrane cholesterol level regulates ion channels by decreasing the level of phosphatidylinositol 4,5-bisphosphate (PIP2), which is closely related to ß-amyloid (Aß) production. In addition, cholesterol enrichment decreased PIP2 levels by increasing the expression of the ß1 isoform of phospholipase C (PLC) in cultured cells. In this study, we examined the effect of a high-cholesterol diet on phospholipase C (PLCß1) expression and PIP2 levels in rat brain. PIP2 levels were decreased in the cerebral cortex in rats on a high-cholesterol diet. Levels of PLCß1 expression correlated with PIP2 levels. However, cholesterol and PIP2 levels were not correlated, suggesting that PIP2 level is regulated by cholesterol via PLCß1 expression in the brain. Thus, there exists cross talk between cholesterol and PIP2 that could contribute to the pathogenesis of neurodegenerative diseases.

3.
Ann Surg ; 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31469747

RESUMO

OBJECTIVE: The primary aim of this study was to compare patient-reported outcomes (PROs) of women who underwent immediate implant-based breast reconstruction (IBBR) after receiving either: (1) nipple-sparing mastectomy (NSM) or (2) simple mastectomy with subsequent nipple reconstruction (SNR). BACKGROUND: The goal of post-mastectomy breast reconstruction is to restore body image and quality-of-life after mastectomy, but removal of the nipple-areolar complex may have its own negative psychosocial consequences. NSM may have increased in popularity due to its perceived oncologic safety in many women and to reports of superior aesthetic results with this technique. Despite the increased utilization of NSM, few studies have evaluated the impact of NSM on PROs and complications in women undergoing immediate IBBR. METHODS: We performed a secondary analysis of the Mastectomy Reconstruction Outcomes Consortium study, a multicenter, prospective cohort study that recruited patients between February 2012 and July 2015. The primary aim of this study was to compare PROs as measured by BREAST-Q scores between women who underwent IBBR after NSM versus SNR. The secondary aim was to compare complication rates. Mixed-effects regression models controlled for demographic and clinical covariates. RESULTS: Of the 600 women in the study, 286 (47.7%) underwent NSM. After controlling for baseline patient characteristics, we found no significant differences in satisfaction with breast, psychosocial well-being, physical well-being or sexual well-being between women undergoing NSM versus SNR. Mastectomy type was not a significant predictor of complications or reconstructive failure. CONCLUSIONS: Despite reports of superior aesthetics for NSM over simple mastectomy with nipple reconstruction in immediate IBBR, we found no significant differences at 2 years after reconstruction in patient satisfaction, quality-of-life, or complication rates.

4.
Mol Nutr Food Res ; 63(3): e1800474, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30365228

RESUMO

SCOPE: In the previous study, Glycyrrhiza uralensis Fisch extract (GUE) inhibited Aß secretion by inhibiting ß-site APP-cleaving enzyme 1 (BACE1) transcription, and the active compounds semilicoisoflavone B (SB) and licoflavonol (LF) inhibited Aß secretion. SB corresponds to the same mechanism as GUE, but LF has a different mechanism. In this study, the mechanism underlying inhibition of Aß by LF is investigated. METHODS AND RESULTS: The effects of LF on Aß, sAPPα, and sAPPß secretion are evaluated by ELISA, and the effect of LF on BACE1 expression is detected by western blotting. It is found that the effect of LF on Aß secretion is due to promotion of BACE1 protein degradation, and that the effect of LF on Aß and BACE1 expression is attenuated after cotreatment with the lysosome inhibitor chloroquine. In a subsequent mechanistic study, it is found that LF increases BACE1 phosphorylation to increase its interactions with ADP ribosylation factor-binding proteins 1 and 3 (GGA1 and GGA3, respectively) and eventually facilitate BACE1 delivery to lysosomes for degradation. CONCLUSION: This study is the first to demonstrate that the BACE1 phosphorylation inducer LF can modulate BACE1 trafficking and lead to facilitating degradation of BACE1, eventually decreasing Aß secretion.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Flavonoides/farmacologia , Glycyrrhiza uralensis/química , Precursor de Proteína beta-Amiloide/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células HeLa , Humanos , Fosforilação , Transporte Proteico
5.
J Ethnopharmacol ; 231: 363-373, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30496841

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Twenty South African medicinal plant species were selected by conducting a literature review based on the relevant information of their reported traditional medicinal uses and scientific reports against Alzheimer's disease, dementia, anxiety, mental illness, depression, acetylcholinesterase inhibition, headache, epilepsy, convulsion, hysteria, and sedative effects. AIM OF STUDY: The goal of this study was to investigate the biological activity of the traditionally used medicinal plant extracts against Alzheimer's disease by in vitro screening of the extracts to determine their potential to decrease levels of Aß42 protein. MATERIAL AND METHODS: Different plant parts (leaves, stem, bark, and stalks) of twenty selected plants were collected from the Manie van der Schijff Botanical Garden, University of Pretoria. Plant parts were dried, ground and then extracted using DCM:MeOH (1:1). We measured the levels of ß-amyloid precursor protein proteolytic products in HeLa cells stably transfected with APP carrying the Swedish mutation using ELISA. RESULTS: Of 33 plant extract 10 (30.3%) were found active based on the potential to significantly reduce the production of Aß42. Amongst them extracts of leaves of Xysmalobium undulatum (Apocynaceae), leaves of Cussonia paniculata (Araliaceae) and leaves of Schotia brachypetala (Fabaceae) potently decreased the production of Aß42 by 77.3 ±â€¯0.5%, 57.5 ±â€¯1.3%, and 44.8 ±â€¯0.1%, respectively. X. undulatum and S. brachypetala enhanced non-amyloidogenic processing of ß-amyloid precursor protein, thereby decreasing Aß42 level. We also showed that C. paniculata induced the decrease of Aß42 level through inhibiting APP processing. In addition, we isolated two cardenolides, compound [A] and [B], from X. undulatum and found that they potently decreased the Aß42 production. CONCLUSION: These data suggest that the extract of X. undulatum, C. paniculata, and S. brachypetala have potential to be developed for Alzheimer's disease treatment. These active extracts and compounds are considered for further studies which examine their efficacy towards the reduction of Aß42 through inhibiting APP process.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Magnoliopsida , Extratos Vegetais/farmacologia , Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/genética , Células HeLa , Humanos , Plantas Medicinais , África do Sul
6.
Biomol Ther (Seoul) ; 27(3): 276-282, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30332887

RESUMO

ß-amyloid precursor protein (APP) can be cleaved by α-, and γ-secretase at plasma membrane producing soluble ectodomain fragment (sAPPα). Alternatively, following endocytosis, APP is cleaved by ß-, and γ-secretase at early endosomes generating ß-amyloid (Aß), the main culprit in Alzheimer's disease (AD). Thus, APP endocytosis is critical for Aß production. Recently, we reported that Monsonia angustifolia, the indigenous vegetables consumed in Tanzania, improved cognitive function and decreased Aß production. In this study, we examined the underlying mechanism of justicidin A, the active compound of M. angustifolia, on Aß production. We found that justicidin A reduced endocytosis of APP, increasing sAPPα level, while decreasing Aß level in HeLa cells overexpressing human APP with the Swedish mutation. The effect of justicidin A on Aß production was blocked by endocytosis inhibitors, indicating that the decreased APP endocytosis by justicidin A is the underlying mechanism. Thus, justicidin A, the active compound of M. angustifolia, may be a novel agent for AD treatment.

7.
Mol Neurobiol ; 55(12): 8953-8964, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29619739

RESUMO

The critical pathological feature of Alzheimer's disease (AD) is the accumulation of ß-amyloid (Aß), the main constituent of amyloid plaques. ß-amyloid precursor protein (APP) undergoes amyloidogenic cleavage by ß- and γ-secretase generating Aß at endosomes or non-amyloidogenic processing by α-secretase precluding the production of Aß at the plasma membrane. Recently, several natural products have been widely researched on the prevention of Aß accumulation for AD treatment. We previously reported that Lycoris chejuensis K. Tae et S. Ko (CJ), which originated from Jeju Island in Korea, improved the disrupted memory functions and reduced Aß production in vivo. Here, we further explored the effect of its active component, 7-deoxy-trans-dihydronarciclasine (coded as E144), on Aß generation and the underlying mechanism. Our results showed that E144 reduced the level of APP, especially its mature form, in HeLa cells overexpressing human APP with the Swedish mutation. Concomitantly, E144 decreased the levels of Aß, sAPPß, sAPPα, and C-terminal fragment. In addition, administration of E144 normalized the behavioral deficits in Tg2576 mice, an APP transgenic mouse model of AD. E144 also decreased the Aß and APP levels in the cerebral cortex of Tg2576 mice. Thus, we propose that E144 could be a potential drug candidate for an anti-amyloid disease-modifying AD therapy.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Isoquinolinas/uso terapêutico , Transtornos da Memória/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Células HeLa , Humanos , Isoquinolinas/química , Isoquinolinas/farmacologia , Aprendizagem , Masculino , Transtornos da Memória/patologia , Camundongos Transgênicos , Solubilidade
8.
Mol Nutr Food Res ; 62(6): e1700633, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29143445

RESUMO

SCOPE: Glycyrrhiza uralensis extract (GUE) has been reported to improve amyloid beta (Aß)-induced cognitive deficits in mice. However, the mechanisms underlying this effect and the components involved have not been previously explored. Extracellular Aß plaques are one of the major pathological hallmarks of Alzheimer's disease (AD). Therefore, decreasing Aß levels is one strategy for preventing the etiology of AD. This study aims to test the effect of GUE and semilicoisoflavone B (SB) on Aß secretion and investigates the mechanism underlying this effect. METHODS AND RESULTS: GUE and its bio-activated compound SB reduce Aß secretion. We find that this effect contribute to the downregulation of the ß-secretase-1 (BACE1) protein and mRNA. In a subsequent mechanism study, we find that GUE and SB regulate BACE1 transcription factors by inducing the expression of peroxisome proliferator activated receptor γ (PPARγ) and inhibiting the phosphorylation of signal transducer and activator of transcription 3. In addition, the effect of GUE and SB on BACE1 expression and Aß secretion are attenuated by treatment with PPARγ-siRNA or its antagonist, GW9662. CONCLUSION: These findings indicate that GUE and SB may function as PPARγ agonists, thereby inhibiting BACE1 expression and ultimately reducing the secretion of Aß.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Flavonoides/farmacologia , Glycyrrhiza uralensis , PPAR gama/genética , Extratos Vegetais/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Células HeLa , Humanos , PPAR gama/agonistas , Fosforilação , Fator de Transcrição STAT3/metabolismo
9.
Biochem Biophys Res Commun ; 490(2): 486-491, 2017 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-28624365

RESUMO

The pathological hallmark of Alzheimer's disease (AD) is associated with the accumulation of amyloid-ß (Aß) derived from proteolytic processing of amyloid-ß precursor protein (APP). APP undergoes post-translational modification including N- and O-glycosylation. O-GlcNAcylation is a novel type of O-glycosylation, mediated by O-GlcNAc transferase attaching O-ß-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of the target proteins. O-GlcNAc is removed by O-GlcNAcase. We have previously reported that increasing O-GlcNAcylated APP using the O-GlcNAcase inhibitor, PUGNAc, increases its trafficking rate to the plasma membrane and decreases its endocytosis rate, resulting in decreased Aß production. However, O-GlcNAc modification sites in APP are unknown. In this study, we mutated three predicted O-GlcNAc modification threonine residues of APP into alanines (T291A, T292A, and T576A) and expressed them in HeLa cells. These APP mutants showed reduced O-GlcNAcylation levels, indicating that these sites were endogenously O-GlcNAcylated. Thr 576 was the major O-GlcNAcylation site when cell was treated with PUGNAc. We also showed that the effects of PUGNAc on APP trafficking to the plasma membrane and Aß production were prevented in the T576A mutant. These results implicate Thr 576 as the major O-GlcNAcylation site in APP and indicate that O-GlcNAcylation of this residue regulates its trafficking and processing. Thus, specific O-GlcNAcylation of APP at Thr 576 may be a novel and promising drug target for AD therapeutics.


Assuntos
Acetilglucosamina/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Acilação , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/análise , Precursor de Proteína beta-Amiloide/genética , Membrana Celular/metabolismo , Glicosilação , Células HeLa , Humanos , Mutação Puntual , Processamento de Proteína Pós-Traducional , Transporte Proteico , Treonina/análise , Treonina/genética , Treonina/metabolismo , Rede trans-Golgi/metabolismo
10.
J Agric Food Chem ; 65(15): 3133-3140, 2017 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-28378593

RESUMO

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is characterized by the accumulation of neurotoxic ß-amyloid (Aß) peptides, which consequently affects cognitive decline and memory impairment. Current research on AD treatment is actively focusing on the prevention of neurotoxic Aß peptide accumulation. Monsonia angustifolia is reported to be consumed as an indigenous vegetable in Tanzania. In this study, we investigated the effect of the ethanol (EtOH) extract of M. angustifolia dried ground material on Aß production and spatial learning ability as protection against AD. The formation of Aß peptides was significantly reduced in HeLa cells stably transfected with the Swedish mutant form of ß-amyloid precursor protein (APPsw) after treatment with a 60% EtOH extract of M. angustifolia. We next examined the cognitive-improving effects of the EtOH extract in vivo. Tg2576 mice were treated with extract for 6 months and subjected to Morris water maze and novel object recognition tests. The results showed that the 60% EtOH extract of M. angustifolia significantly ameliorated behavioral deficits of the AD transgenic mice and reduced the level of insoluble Aß42 in the cerebral cortex and hippocampus. We further found that the 60% EtOH extract was effective for memory function recovery after shorter treatment (4 months). In addition, we isolated and identified several single compounds, justicidin A, 5-methoxyjusticidin A, chinensinaphthol, retrochinensinaphthol methyl ether, and suchilactone, from M. angustifolia and tested these compounds. Among them, justicidin A potently decreased the formation of Aß in APPsw-transfected cells. These data suggest that the 60% EtOH extract of M. angustifolia has the potential to be developed as a treatment of AD. Furthermore, justicidin A may contribute, at least partially, to the Aß alteration observed with the extract treatment.


Assuntos
Doença de Alzheimer/prevenção & controle , Geraniaceae/química , Extratos Vegetais/administração & dosagem , Doença de Alzheimer/metabolismo , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Cognição/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Extratos Vegetais/química
11.
Neurochem Res ; 42(2): 667-677, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27933547

RESUMO

Microglia play a critical role in controlling the homeostasis of the brain, but over-activated microglia secrete pro-inflammatory mediators and cytokines, which induce neuronal cell death. Fucoxanthin (Fx), a marine carotenoid, has demonstrated a variety of beneficial health effects. Despite accumulating evidence supporting the immune-modulating effects of Fx in vitro, the underlying signaling pathways remain unknown. In the present study, Fx dose-dependently inhibited the secretion of lipopolysaccharide (LPS)-induced pro-inflammatory mediators including interleukin (IL)-6, tumor necrosis factor (TNF)-α, reactive oxygen species (ROS), prostaglandin (PG) E2, and nitric oxide (NO) productions, and also suppressed the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 enzymes. Further, the reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated IL-6, TNF-α, iNOS, and COX-2 mRNA expression were suppressed by treatment with Fx in a dose-dependently manner. The mechanism studies indicated that Fx blocks protein kinase B (Akt)/nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPKs)/transcription factor (AP)-1 pathways. In addition, we demonstrated that Fx increases nuclear factor erythroid 2-related factor (Nrf)-2 activation and heme oxygenase (HO)-1 expression in LPS-activated BV-2 microglia. Subsequently, we found that Fx also mediates the reactive oxygen species (ROS) by activating protein kinase A (PKA)/cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) pathway, and promotes the production of brain-derived neurotrophic factor (BDNF). These results indicate that Fx may be more effective and potential than other candidates via either decreasing the pro-inflammatory factors production or increasing the neuroprotective molecules expression for therapy of neurodegenerative diseases.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição AP-1/metabolismo , Xantofilas/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Lipopolissacarídeos/toxicidade , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , NF-kappa B/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Fator de Transcrição AP-1/antagonistas & inibidores
12.
Biochem Biophys Res Commun ; 467(4): 955-60, 2015 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-26471307

RESUMO

Deposition of amyloid-ß (Aß) in the brain is the main culprit of Alzheimer's disease (AD). Aß is derived from sequential proteolytic cleavage of amyloid-ß precursor protein (APP). Newly synthesized APP is transported from endoplasmic reticulum to the plasma membrane via trans-Golgi network (TGN) after post-translational modification including N- and O-glycosylation. APP is internalized through clathrin-dependent endocytosis from the plasma membrane to the early endosomes. In this study, we investigated the regulation of APP trafficking and processing by mutating three threonine residues known as O-glycosylation sites. We separately mutated three threonine residues of APP695 into alanines (T291A, T292A, and T576A) and expressed them in HeLa cells. Among these APP mutants, only T576A mutant showed reduced cell surface levels, indicating this residue regulates its trafficking. We also confirmed that trafficking from TGN to the plasma membrane was decreased in T576A mutant. Consistent with these observations, T576A mutant accumulated in the early endosomes, and the secreted Aß level was increased. Thus, these results indicate that threonine 576 residue of APP regulates its trafficking and processing.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Processamento de Proteína Pós-Traducional , Treonina/metabolismo , Glicosilação , Células HeLa , Humanos , Mutação , Organelas/metabolismo , Transporte Proteico , Treonina/química
13.
J Agric Food Chem ; 63(31): 6979-88, 2015 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-26218758

RESUMO

We found that an extract of Lycoris chejuensis and its three isolated active components, narciclasine, 7-deoxynarciclasine, and 7-deoxy-trans-dihydronarciclasine, each significantly reduced the formation of amyloid-ß peptides in HeLa cells transfected with an amyloid precursor protein carrying the Swedish mutation up to 45 ± 3.6%. The extract down-regulated amyloid precursor protein, especially the mature form by up to 88%, and reduced the ability of secretases to generate toxic amyloid-ß. Double-transgenic mice treated with the extract for 4 months also showed significantly reduced levels of amyloid-ß and plaques while exhibiting improved memory functions in the Morris water maze and novel object recognition tests. In conclusion, the extract and isolated active components of L. chejuensis decreased the production of amyloid-ß by attenuating amyloid precursor protein levels. Furthermore, the extract improved the disrupted memory functions in animals while inhibiting amyloid plaque formation. Thus, this extract, as well as its active components, could prove beneficial in the treatment of Alzheimer's disease.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Lycoris/química , Extratos Vegetais/administração & dosagem , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Transgênicos
14.
Biochem Biophys Res Commun ; 463(1-2): 7-12, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25983327

RESUMO

Macroautophagy (hereafter referred to as autophagy) is a catabolic process for the degradation and recycling of cellular components. Autophagy digests intracellular components, recycling material subsequently used for new protein synthesis. The Ca(2+)- and Mg(2+)-permeable transient receptor potential melastatin 7 (TRPM7) channel underlies the constitutive Ca(2+) influx in some cells. Since autophagy is regulated by cytosolic Ca(2+) level, we set out to determine whether Ca(2+) influx through the TRPM7 channel regulates basal autophagy. When TRPM7 channel expression was induced from HEK293 cells in a nutrient-rich condition, LC3-II level increased indicating the increased level of basal autophagy. The effect of TRPM7 channel on basal autophagy was via Ca(2+)/calmodulin-dependent protein kinase kinase ß, and AMP-activated protein kinase pathway. In contrast, the level of basal autophagy was decreased when the endogenous TRPM7 channel in SH-SY5Y cells was down-regulated using short hairpin RNA. Similarly, an inhibitor for TRPM7 channel decreased the level of basal autophagy. In addition, the inhibitory effect of channel inhibitor on basal autophagy was reversed by increasing extracellular Ca(2+)concentration, suggesting that Ca(2+) influx through TRPM7 channel directly links to basal autophagy. Thus, our studies demonstrate the new role of TRPM7 channel-mediated Ca(2+) entry in the regulation of basal autophagy.


Assuntos
Autofagia/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Canais de Cátion TRPM/fisiologia , Proteínas Quinases Ativadas por AMP/metabolismo , Sinalização do Cálcio , Linhagem Celular , Regulação para Baixo , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutagênese , Técnicas de Patch-Clamp , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genética
15.
J Alzheimers Dis ; 44(1): 261-75, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25208619

RESUMO

Amyloid-ß protein precursor (AßPP) is transported to the plasma membrane, where it is sequentially cleaved by α-secretase and γ-secretase. This is called non-amyloidogenic pathway since it precludes the production of amyloid-ß (Aß), the main culprit of Alzheimer's disease (AD). Alternatively, once AßPP undergoes clathrin-dependent endocytosis, it can be sequentially cleaved by ß-secretase and γ-secretase at endosomes, producing Aß (amyloidogenic pathway). ß-N-acetylglucosamine (GlcNAc) can be attached to serine/threonine residues of the target proteins. This novel type of O-linked glycosylation is called O-GlcNAcylation mediated by O-GlcNAc transferase (OGT). The removal of GlcNAc is mediated by O-GlcNAcase (OGN). Recently, it is shown that O-GlcNAcylation of AßPP increases the non-amyloidogenic pathway. To investigate the regulatory role for O-GlcNAcylation in AßPP processing, we first tested the effects of inhibitor for OGN, PUGNAc, on AßPP metabolism in HeLa cells stably transfected with Swedish mutant form of AßPP. Increasing O-GlcNAcylated AßPP level increased α-secretase product while decreased ß-secretase products. We found that PUGNAc increased the trafficking rate of AßPP from the trans-Golgi network to the plasma membrane, and selectively decreased the endocytosis rate of AßPP. These events may contribute to the increased AßPP level in the plasma membrane by PUGNAc. Inhibiting clathrin-dependent endocytosis prevented the effect of PUGNAc on Aß, suggesting that the effect of PUGNAc was mainly mediated by decreasing AßPP endocytosis. These results strongly indicate that O-GlcNAcylation promotes the plasma membrane localization of AßPP, which enhances the non-amyloidogenic processing of AßPP. Thus, O-GlcNAcylation of AßPP can be a potential therapeutic strategy for AD.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Membrana Celular/metabolismo , Endocitose/genética , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Células CHO , Membrana Celular/efeitos dos fármacos , Clatrina/metabolismo , Cricetulus , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Células HeLa , Humanos , Mutação/genética , N-Acetilglucosaminiltransferases/metabolismo , Oximas/farmacologia , Fenilcarbamatos/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Fatores de Tempo , Transfecção
16.
Channels (Austin) ; 7(4): 275-87, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23793622

RESUMO

Human ether-a-go-go-related gene (HERG) K(+) channel underlies the rapidly activating delayed rectifier K(+) conductance (IKr) during normal cardiac repolarization. Also, it may regulate excitability in many neuronal cells. Recently, we showed that enrichment of cell membrane with cholesterol inhibits HERG channels by reducing the levels of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] due to the activation of phospholipase C (PLC). In this study, we further explored the effect of cholesterol enrichment on HERG channel kinetics. When membrane cholesterol level was mildly increased in human embryonic kidney (HEK) 293 cells expressing HERG channel, the inactivation and deactivation kinetics of HERG current were not affected, but the activation rate was significantly decelerated at all voltages tested. The application of PtdIns(4,5)P2 or inhibitor for PLC prevented the effect of cholesterol enrichment, while the presence of antibody against PtdIns(4,5)P2 in pipette solution mimicked the effect of cholesterol enrichment. These results indicate that the effect of cholesterol enrichment on HERG channel is due to the depletion of PtdIns(4,5)P2. We also found that cholesterol enrichment significantly increases the expression of ß1 and ß3 isoforms of PLC (PLCß1, PLCß3) in the membrane. Since the effects of cholesterol enrichment on HERG channel were prevented by inhibiting transcription or by inhibiting PLCß1 expression, we conclude that increased PLCß1 expression leads to the deceleration of HERG channel activation rate via downregulation of PtdIns(4,5)P2. These results confirm a crosstalk between two plasma membrane-enriched lipids, cholesterol and PtdIns(4,5)P2, in the regulation of HERG channels.


Assuntos
Colesterol/farmacologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fosfolipase C beta/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Cinética , Fosfatidilinositol 4,5-Difosfato/deficiência , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase C beta/metabolismo
17.
J Biol Chem ; 288(29): 20868-82, 2013 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-23723072

RESUMO

Amyloid ß-peptide (Aß) pathology is an invariant feature of Alzheimer disease, preceding any detectable clinical symptoms by more than a decade. To this end, we seek to identify agents that can reduce Aß levels in the brain via novel mechanisms. We found that (20S)-Rg3, a triterpene natural compound known as ginsenoside, reduced Aß levels in cultured primary neurons and in the brains of a mouse model of Alzheimer disease. The (20S)-Rg3 treatment induced a decrease in the association of presenilin 1 (PS1) fragments with lipid rafts where catalytic components of the γ-secretase complex are enriched. The Aß-lowering activity of (20S)-Rg3 directly correlated with increased activity of phosphatidylinositol 4-kinase IIα (PI4KIIα), a lipid kinase that mediates the rate-limiting step in phosphatidylinositol 4,5-bisphosphate synthesis. PI4KIIα overexpression recapitulated the effects of (20S)-Rg3, whereas reduced expression of PI4KIIα abolished the Aß-reducing activity of (20S)-Rg3 in neurons. Our results substantiate an important role for PI4KIIα and phosphoinositide modulation in γ-secretase activity and Aß biogenesis.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ginsenosídeos/farmacologia , Microdomínios da Membrana/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Presenilina-1/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Ativação Enzimática/efeitos dos fármacos , Ginsenosídeos/química , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Receptores de Fator de Crescimento Neural/química , Receptores de Fator de Crescimento Neural/metabolismo , Receptores Notch/química , Receptores Notch/metabolismo , Relação Estrutura-Atividade
18.
J Neurodegener Dis ; 2013: 407903, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-26316988

RESUMO

Cerebral elevation of 42-residue amyloid ß-peptide (Aß42) triggers neuronal dysfunction in Alzheimer's disease (AD). Even though a number of cholesterol modulating agents have been shown to affect Aß generation, the role of cholesterol in the pathogenesis of AD is not clear yet. Recently, we have shown that increased membrane cholesterol levels downregulates phosphatidylinositol 4,5-bisphosphate (PIP2) via activation of phospholipase C (PLC). In this study, we tested whether membrane cholesterol levels may affect the Aß42 production via changing PIP2 levels. Increasing membrane cholesterol levels decreased PIP2 and increased secreted Aß42. Supplying PIP2, by using a PIP2-carrier system, blocked the effect of cholesterol on Aß42. We also found that cholesterol increased the expressions of ß1 and ß3 PLC isoforms (PLCß1, PLCß3). Silencing the expression of PLCß1 prevented the effects of cholesterol on PIP2 levels as well as on Aß42 production, suggesting that increased membrane cholesterol levels increased secreted Aß42 by downregulating PIP2 via enhancing the expression of PLCß1. Thus, cholesterol metabolism may be linked to Aß42 levels via PLCß1 expression and subsequent changes in PIP2 metabolism.

19.
Dev Neurobiol ; 72(6): 865-77, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22102510

RESUMO

Presenilins (PS1 and PS2) are multifunctional proteins involved in a diverse array of molecular and cellular functions, including proteolysis, development, neurogenesis, synaptic plasticity, ion channel regulation and phospholipid metabolism. Mutations in presenilin genes are responsible for the majority of Familial Alzheimer disease (FAD). Consequently, FAD-associated mutations in genes encoding PS1 or PS2 lead to several key cellular phenotypes, including alterations in proteolysis of ß-amyloid precursor protein (APP) and Ca(2+) entry. The mechanism underlying presenilin (PS)-mediated modulation of Ca(2+) entry remains to be determined. Our previous studies showed that the PS-dependent down-regulation of phosphatidylinositol-4,5-bisphosphate (PIP2) is attributable to the observed Ca(2+) deficits. In this study, we attempted to identify the ion channel that is subject to the PIP2 and PS-dependent modulation. We found that Ca(2+) or Zn(2+) entry via the transient receptor potential melastatin 7 (TRPM7) channel was attenuated by the presence of FAD-associated PS1 mutants, such as ΔE9 and L286V. TRPM7 has been implicated in Mg(2+) homeostasis and embryonic development. The intracellular delivery of PIP2 restored TRPM7-mediated Ca(2+) influx, indicating that the observed deficits in Ca(2+) entry are due to downregulation of PIP2. Conversely, PS1 and PS2 deficiency, previously shown to upregulate PIP2 levels, potentiated TRPM7-mediated Ca(2+) influx. PS-dependent changes in Ca(2+) influx could be neutralized by a TRPM7 channel blocker. Collectively, these results indicate that TRPM7 may underlie the Ca(2+) entry deficits observed in FAD-associated PS mutants and suggest that the normal function of PS involves regulation of TRPM7 through a PIP2-dependent mechanism.


Assuntos
Fosfoinositídeo Fosfolipase C/metabolismo , Presenilina-1/genética , Presenilinas/genética , Canais de Cátion TRPM/genética , Animais , Células CHO , Cálcio/metabolismo , Células Cultivadas , Cricetinae , Regulação para Baixo , Células HEK293 , Humanos , Presenilina-1/metabolismo , Presenilinas/metabolismo , Proteínas Serina-Treonina Quinases , RNA Interferente Pequeno , Canais de Cátion TRPM/metabolismo , Zinco/metabolismo
20.
J Neurochem ; 112(5): 1286-94, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20015154

RESUMO

Ubiquitously expressed Mg(2+)-inhibitory cation (MIC) channels are permeable to Ca2+ and Mg2+ and are essential for cell viability. When membrane cholesterol level was increased by pre-incubating cells with a water-soluble form of cholesterol, the endogenous MIC current in HEK293 cells was negatively regulated. The application of phosphatidylinositol 4,5-bisphosphate (PIP2) recovered MIC current from cholesterol effect. As PIP2 is the direct modulator for MIC channels, high cholesterol content may cause down-regulation of PIP2. To test this possibility, we examined the effect of cholesterol on two exogenously expressed PIP2-sensitive K+ channels: human Ether-a-go-go related gene (HERG) and KCNQ. Enrichment with cholesterol inhibited HERG currents, while inclusion of PIP2 in the pipette solution blocked the cholesterol effect. KCNQ channel was also inhibited by cholesterol. The effects of cholesterol on these channels were blocked by pre-incubating cells with inhibitors for phospholipase C, which may indicate that cholesterol enrichment induces the depletion of PIP2 via phospholipase C activation. Lipid analysis showed that cholesterol enrichment reduced gamma-(32)P incorporation into PIP2 by approximately 35%. Our results suggest that cholesterol may modulate ion channels by changing the levels of PIP2. Thus, an important cross-talk exists among two plasma membrane-enriched lipids, cholesterol and PIP2.


Assuntos
Colesterol/farmacologia , Regulação para Baixo/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go/fisiologia , Canais de Potássio KCNQ/fisiologia , Fosfatos de Fosfatidilinositol/farmacologia , Biofísica , Cálcio/metabolismo , Linhagem Celular Transformada , Sobrevivência Celular/fisiologia , Colesterol/metabolismo , Cromatografia em Camada Delgada/métodos , Regulação para Baixo/genética , Canal de Potássio ERG1 , Estimulação Elétrica , Ensaio de Imunoadsorção Enzimática/métodos , Canais de Potássio Éter-A-Go-Go/genética , Proteínas de Fluorescência Verde/genética , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Canais de Potássio KCNQ/genética , Magnésio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Técnicas de Patch-Clamp/métodos , Fosfatidilinositol 4,5-Difosfato , Fosfatos de Fosfatidilinositol/metabolismo , Fatores de Tempo , Transfecção/métodos , Fosfolipases Tipo C/metabolismo
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