The role of adenosine monophosphate-activated protein kinase in the neurotoxic effect of alpha-synuclein in vitro

Parkinsonova bolest (PB) se karakteriše progresivnom degeneracijom i izumiranjem dopaminergičkih neurona u pars compacta substantiae nigrae mezencefalona, a osnovni patološki supstrat bolesti je nakupljanje proteina alfasinukleina (ASYN) u zahvaćenim neuronima, i formiranje Levijevih tela. Cilj ovog istraživanja je bio da ispita uticaj najvažnijeg unutarćelijskog energetskog senzora, protein kinaze aktivirane adenozin monofosfatom (AMPK) u neurotoksičnostom delovanju ASYN. Istraživanje je sprovedeno na modelu in vitro, ćelijskoj liniji humanog neuroblastoma SH-SY5Y koja prekomerno eksprimira nemutirani (engl. wild type, wt) ASYN i koja je delovanjem retinoične kiseline diferentovana u neuronski fenotip. Prekomerna ekspresija ASYN dovela smanjenog preživljavanja ćelija humanog neuroblastoma, što je bilo praćeno morfološkim i ultrastrukturnim promenama koje odgovaraju apoptozi, uz aktivaciju kaspaza i fragmentaciju DNK. Nakupljanje ASYN u ćelijama uzrokovalo je smanjenje aktivnosti AMPK i njenog najvažnijeg aktivatora, ushodne kinaze LKB1 (engl. liver kinase B1, LKB1), što je za posledicu imalo i smanjenje fosforilacije nishodne kinaze Raptor; kako u diferentovanim ćelijama humanog neuroblastoma, tako i u primarnim neuronima izolovanim iz kore velikog mozga pacova. Nije pokazana promena u ekspresiji informacionih RNK (iRNK) drugih regulatora aktivnosti AMPK: kalcijum/kalmodulin-zavisne kinaze, sestrina 1 i 2, i protein fosfataze 2A. Primena farmakoloških aktivatora AMPK, 5-aminoimidazol- 4-karboksiamid ribonukleotida (AICAR) i N,N-dimetilimidodikarbonimidičnog diamid hidrohlorida (metformin), dovela je do značajnog poboljšanja preživljavanja ćelija u uslovima prekomerne ekspresije ASYN, što je bilo praćeno porastom aktivnosti signalnog puta LKB1/AMPK/Raptor, bez uticaja na smanjenje nivoa ASYN u ćelijama. U ovoj studiji je pokazano i da prekomerna ekspresija ASYN smanjuje aktivnost signalnog puta protein kinaze B(Akt)/Src, kao i da u ćelijama humanog neuroblastoma SH-SY5Y diferentovanim u neuronski fenotip indukuje autofagiju, uz porast autofagnog fluksa u ćelijama...In the present study, we investigated the role of the main intracellular energy sensor, adenosine monophosphate-activated protein kinase (AMPK), in the neurotoxicity of alpha-synuclein (ASYN), one of the key culprits in the pathogenesis of Parkinson’s disease. All experiments were conducted in retinoic acid-differentiated human neuroblastoma SH-SY5Y cells stably expressing wild type (wt) ASYN, as the in vitro model of Parkinson’s disease. The overexpression of ASYN caused loss of viability in retinoic aciddifferentiated SH-SY5Y human neuroblastoma cells accompanied by caspase activation, DNA fragmentation, and the morphological and ultrastructural changes characteristic for the apoptotic cell death. The mechanisms underlying the pro-apoptotic action of ASYN was associated with the reduced activity of both AMPK and its activator LKB1 (liver kinase B1), resulting in reduced phosphorylation of the downstream kinase Raptor. ASYNoverexpressing rat primary neurons also displayed decreased activity of LKB1/AMPK/Raptor pathway. However, mRNA expression of the upstream AMPK regulators CaMKKβ (Ca2+/calmodulin-dependent protein kinase), PP2A (protein phosphatase 2A), sestrin 1 and sestrin 2 was not significantly altered in ASYNoverexpressing cells. Restoration of AMPK activity by pharmacological AMPK activators, metformin (N, N-dimethylimidodicarbonimidic diamide) or AICAR (5- aminoimidazole-4-carboxamide ribonucleotide), reduced the in vitro neurotoxicity of ASYN overexpression. Of note, the pharmacological AMPK activators were apparently more efficient in improving the viability of ASYN-overexpressing cells than known neuroprotective agents, tyrosine hydroxylase inhibitor or antioxidant ascorbic acid. The pharmacological activation of AMPK was not accompanied by changes in ASYN levels in human neuroblastoma cells. The overproduction of ASYN diminished activation of phosphatidylinositol- 4,5-bisphosphate 3-kinase (PI3K)-activated Src/Akt signalling pathway, and resulted in autophagy induction and increased autophagic flux..

Supplementary data for the article: Filipović, N.; Polović, N.; Rašković, B.; Misirlić-Denčić, S.; Dulović, M.; Savić, M.; Nikšić, M.; Mitić, D.; Ancrossed D Signelković, K.; Todorović, T. Biological Activity of Two Isomeric N-Heteroaromatic Selenosemicarbazones and Their Metal Complexes. Monatshefte fur Chemie 2014, 145 (7), 1089–1099. https://doi.org/10.1007/s00706-014-1197-6

Supplementary material for: [https://doi.org/10.1007/s00706-014-1197-6]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1787

Supplementary data for the article: Filipović, N. R.; Marković, I.; Mitić, D.; Polović, N.; Milčić, M.; Dulović, M.; Jovanović, M.; Savić, M.; Nikšić, M.; Andelković, K.; et al. A Comparative Study of In Vitro Cytotoxic, Antioxidant, and Antimicrobial Activity of Pt(II), Zn(II), Cu(II), and Co(III) Complexes with N-Heteroaromatic Schiff Base (E)-2-[N’-(1-Pyridin-2-Yl-Ethylidene)Hydrazino]Acetate. Journal of Biochemical and Molecular Toxicology 2014, 28 (3), 99–110. https://doi.org/10.1002/jbt.21541

Supplementary material for: [https://doi.org/10.1002/jbt.21541]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1509

Supplementary data for the article: Filipović, N.; Polović, N.; Rašković, B.; Misirlić-Denčić, S.; Dulović, M.; Savić, M.; Nikšić, M.; Mitić, D.; Ancrossed D Signelković, K.; Todorović, T. Biological Activity of Two Isomeric N-Heteroaromatic Selenosemicarbazones and Their Metal Complexes. Monatshefte fur Chemie 2014, 145 (7), 1089–1099. https://doi.org/10.1007/s00706-014-1197-6

Supplementary material for: [https://doi.org/10.1007/s00706-014-1197-6]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1787

Supplementary data for the article: Filipović, N. R.; Marković, I.; Mitić, D.; Polović, N.; Milčić, M.; Dulović, M.; Jovanović, M.; Savić, M.; Nikšić, M.; Andelković, K.; et al. A Comparative Study of In Vitro Cytotoxic, Antioxidant, and Antimicrobial Activity of Pt(II), Zn(II), Cu(II), and Co(III) Complexes with N-Heteroaromatic Schiff Base (E)-2-[N’-(1-Pyridin-2-Yl-Ethylidene)Hydrazino]Acetate. Journal of Biochemical and Molecular Toxicology 2014, 28 (3), 99–110. https://doi.org/10.1002/jbt.21541

Supplementary material for: [https://doi.org/10.1002/jbt.21541]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1509

ニホン ジジョウ コース ニオケル プロジェクト ワーク ノ ココロミ : リュウガクセイ ノ タメ ノ セイカツ ハンドブック トクシマ ダイガク エ ヨウコソ ノ サクセイ オ トオシテ

共通教育の「日本事情I」では、グループ学習として効果のあるプロジェクト・ワークを取り上げ、「留学生のための生活ハンドブックー徳島大学へようこそ」を作成した。その作成を通してプロジェクトワークを振り返り、プロジェクトワークとは何か、どのような効果があるかなど考察し、次へと繋げて行きたい

The role of adenosine monophosphate-activated protein kinase in the neurotoxic effect of alpha-synuclein in vitro

Parkinsonova bolest (PB) se karakteriše progresivnom degeneracijom i izumiranjem dopaminergičkih neurona u pars compacta substantiae nigrae mezencefalona, a osnovni patološki supstrat bolesti je nakupljanje proteina alfasinukleina (ASYN) u zahvaćenim neuronima, i formiranje Levijevih tela. Cilj ovog istraživanja je bio da ispita uticaj najvažnijeg unutarćelijskog energetskog senzora, protein kinaze aktivirane adenozin monofosfatom (AMPK) u neurotoksičnostom delovanju ASYN. Istraživanje je sprovedeno na modelu in vitro, ćelijskoj liniji humanog neuroblastoma SH-SY5Y koja prekomerno eksprimira nemutirani (engl. wild type, wt) ASYN i koja je delovanjem retinoične kiseline diferentovana u neuronski fenotip. Prekomerna ekspresija ASYN dovela smanjenog preživljavanja ćelija humanog neuroblastoma, što je bilo praćeno morfološkim i ultrastrukturnim promenama koje odgovaraju apoptozi, uz aktivaciju kaspaza i fragmentaciju DNK. Nakupljanje ASYN u ćelijama uzrokovalo je smanjenje aktivnosti AMPK i njenog najvažnijeg aktivatora, ushodne kinaze LKB1 (engl. liver kinase B1, LKB1), što je za posledicu imalo i smanjenje fosforilacije nishodne kinaze Raptor; kako u diferentovanim ćelijama humanog neuroblastoma, tako i u primarnim neuronima izolovanim iz kore velikog mozga pacova. Nije pokazana promena u ekspresiji informacionih RNK (iRNK) drugih regulatora aktivnosti AMPK: kalcijum/kalmodulin-zavisne kinaze, sestrina 1 i 2, i protein fosfataze 2A. Primena farmakoloških aktivatora AMPK, 5-aminoimidazol- 4-karboksiamid ribonukleotida (AICAR) i N,N-dimetilimidodikarbonimidičnog diamid hidrohlorida (metformin), dovela je do značajnog poboljšanja preživljavanja ćelija u uslovima prekomerne ekspresije ASYN, što je bilo praćeno porastom aktivnosti signalnog puta LKB1/AMPK/Raptor, bez uticaja na smanjenje nivoa ASYN u ćelijama. U ovoj studiji je pokazano i da prekomerna ekspresija ASYN smanjuje aktivnost signalnog puta protein kinaze B(Akt)/Src, kao i da u ćelijama humanog neuroblastoma SH-SY5Y diferentovanim u neuronski fenotip indukuje autofagiju, uz porast autofagnog fluksa u ćelijama...In the present study, we investigated the role of the main intracellular energy sensor, adenosine monophosphate-activated protein kinase (AMPK), in the neurotoxicity of alpha-synuclein (ASYN), one of the key culprits in the pathogenesis of Parkinson’s disease. All experiments were conducted in retinoic acid-differentiated human neuroblastoma SH-SY5Y cells stably expressing wild type (wt) ASYN, as the in vitro model of Parkinson’s disease. The overexpression of ASYN caused loss of viability in retinoic aciddifferentiated SH-SY5Y human neuroblastoma cells accompanied by caspase activation, DNA fragmentation, and the morphological and ultrastructural changes characteristic for the apoptotic cell death. The mechanisms underlying the pro-apoptotic action of ASYN was associated with the reduced activity of both AMPK and its activator LKB1 (liver kinase B1), resulting in reduced phosphorylation of the downstream kinase Raptor. ASYNoverexpressing rat primary neurons also displayed decreased activity of LKB1/AMPK/Raptor pathway. However, mRNA expression of the upstream AMPK regulators CaMKKβ (Ca2+/calmodulin-dependent protein kinase), PP2A (protein phosphatase 2A), sestrin 1 and sestrin 2 was not significantly altered in ASYNoverexpressing cells. Restoration of AMPK activity by pharmacological AMPK activators, metformin (N, N-dimethylimidodicarbonimidic diamide) or AICAR (5- aminoimidazole-4-carboxamide ribonucleotide), reduced the in vitro neurotoxicity of ASYN overexpression. Of note, the pharmacological AMPK activators were apparently more efficient in improving the viability of ASYN-overexpressing cells than known neuroprotective agents, tyrosine hydroxylase inhibitor or antioxidant ascorbic acid. The pharmacological activation of AMPK was not accompanied by changes in ASYN levels in human neuroblastoma cells. The overproduction of ASYN diminished activation of phosphatidylinositol- 4,5-bisphosphate 3-kinase (PI3K)-activated Src/Akt signalling pathway, and resulted in autophagy induction and increased autophagic flux..

AMP-activated protein kinase inhibits MPP+-induced oxidative stress and apoptotic death of SH-SY5Y cells through sequential stimulation of Akt and autophagy.

We investigated the interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK), prosurvival kinase Akt, oxidative stress, and autophagy in the cytotoxicity of parkinsonian neurotoxin 1-methyl-4-phenyl piridinium (MPP+) towards SH-SY5Y human neuroblastoma cells. MPP+-mediated oxidative stress, mitochondrial depolarization, and apoptotic cell death were associated with rapid (within 2 h) activation of AMPK, its target Raptor, and prosurvival kinase Akt. Antioxidants N-acetylcysteine and butylated hydroxyanisole suppressed MPP+-induced cytotoxicity, AMPK, and Akt activation. A genetic or pharmacological inhibition of AMPK increased MPP+-triggered production of reactive oxygen species and cell death, and diminished Akt phosphorylation, while AMPK activation protected SH-SY5Y cells from MPP+. On the other hand, genetic or pharmacological inactivation of Akt stimulated MPP+-triggered oxidative stress and neurotoxicity, but did not affect AMPK activation. At later time-points (16-24 h), MPP+ inhibited the main autophagy repressor mammalian target of rapamycin, which coincided with the increase in the levels of autophagy marker microtubule-associated protein 1 light-chain 3B. MPP+ also increased the concentration of a selective autophagic target sequestosome-1/p62 and reduced the levels of lysosomal-associated membrane protein 1 and cytoplasmic acidification, suggesting that MPP+-induced autophagy was coupled with a decrease in autophagic flux. Nevertheless, further pharmacological inhibition of autophagy sensitized SH-SY5Y cells to MPP+-induced death. Antioxidants and AMPK knockdown reduced, whereas genetic inactivation of Akt potentiated neurotoxin-triggered autophagy. These results suggest that MPP+-induced oxidative stress stimulates AMPK, which protects SH-SY5Y cells through early activation of antioxidative Akt and late induction of cytoprotective autophagy

Palladium(II) Complexes with N-Heteroaromatic Bidentate Hydrazone Ligands: The Effect of the Chelate Ring Size and Lipophilicity on in vitro Cytotoxic Activity

Novel Pd(II) complex with N-heteroaromatic Schiff base ligand, derived from 8-quinolinecarboxaldehyde (q8a) and ethyl hydrazinoacetate (haOEt), was synthesized and characterized by analytical and spectroscopy methods. The structure of novel complex, as well as structures of its quinoline and pyridine analogues, was optimized by density functional theory calculations, and theoretical data show good agreement with experimental results. A cytotoxic action of the complexes was evaluated on cultures of human promyelocytic leukemia (HL-60), human glioma (U251), rat glioma (C6), and mouse fibrosarcoma (L929) cell lines. Among investigated compounds, only complexes with quinoline-based ligands reduce the cell numbers in a dose-dependent manner in investigated cell lines. The observed cytotoxic effect of two isomeric quinoline-based complexes is predominantly mediated through the induction of apoptotic cell death in HL-60 cell line. The cytotoxicity of most efficient novel Pd(II) complex is comparable to the activity of cisplatin, in all cell lines investigated

Synthesis, characterization and ROS-mediated cytotoxic action of novel (S, S)-1,3-propanediamine-N,N '-di-2-(3-cyclohexyl)propanoic acid and corresponding esters

This study involves the synthesis and characterization of novel cyclohexyl 1,3-propanediamine-N, N'-diacetate molecules as well as investigation of their cytotoxic action. New acid 1a was synthesized by reaction between (S)-2-amino-3-cyclohexylpropanoic acid and 1,3-dibromopropane, while the esters (1b-1e) derived from this acid were obtained by reaction of the corresponding absolute alcohol, thionyl chloride and synthesized acid. All compounds were characterized by IR, ESI-MS, (H-1, C-13 and HSQC) NMR spectroscopy and elemental analysis. The cytotoxic activity of all compounds was tested on several tumour cell lines: human (U251) and rat (C6) glioma, human promyelocytic leukaemia (HL-60), human neuroblastoma (SHSY-5Y) and mouse fibrosarcoma (L929) as well as primary rat astrocytes. The present study reveals potent antitumour activity of novel purely organic compounds (1a-1e), which was most pronounced in human glioma (U251) cells. The esterification is required for the novel compounds' cytotoxic action since the n-butyl ester 1e was the most efficient compound. Importantly, n-butyl ester 1e was more toxic to glioma cells in comparison to rat astrocytes, with 24-h IC50 values lower than those for cisplatin. n-Butyl ester 1e induced production of reactive oxygen species (ROS) and caused an oxidative- stress-derived accumulation of glioma cells in the G(0)/G(1) phase of the cell cycle, as well as caspase activation and DNA fragmentation, suggesting that apoptosis induction plays an important role in the novel compounds' antiglioma action. (C) 2014 Elsevier Inc. All rights reserved