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..

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..

Cyclohexyl Analogues of Ethylenediamine Dipropanoic Acid Induce Caspase-Independent Mitochondrial Apoptosis in Human Leukemic Cells

We investigated the cytotoxicity of recently synthesized (S,S)-ethyleridiamine-N,N'-di-2-(3-cyclohexyl)propanoic acid esters toward human leukemic cell lines and healthy blood mononuclear cells. Cell viability was assessed by acid phosphatase assay, apoptosis, and differentiation were analyzed by flow cytometry and electron microscopy, while intracellular localization of apoptosis-inducing factor (AIF) was determined by immunoblotting. It was demonstrated that methyl, ethyl, and n-propyl esters were toxic to HL-60, REH, MOLT-4, KG-1, JVM-2, and K-562 leukemic cell lines, while the nonesterified parental compound and n-butyl ester were devoid of cytotoxic action. The ethyl ester exhibited the highest cytotoxic activity (IC50 10.7 mu M-45.4 mu M), which was comparable to that of the prototypical anticancer drug cisplatin. The observed cytotoxic effect in HL-60 cells was associated with an increase in superoxide production and mitochondrial membrane depolarization, leading to apoptotic cell death characterized by phosphatidylserine externalization and DNA fragmentation in the absence of autophagic response. DNA fragmentation preceded caspase activation and followed AIF translocation from mitochondria to nucleus, which was indicative of caspase-independent apoptotic cell death. HL-60 cells treated with subtoxic concentration of the compound displayed morphological signs of granulocytic differentiation (nuclear indentations and presence of cytoplasmic primary granules), as well as an increased expression of differentiation markers CD11b and CD15. The cyclohexyl analogues of ethylenediamine dipropanoic acid were also toxic to peripheral blood mononuclear cells of both healthy controls and leukemic patients, the latter being more sensitive. Our data demonstrate that the toxicity of the investigated cyclohexyl compounds against leukemic cell lines is mediated by caspase-independent apoptosis associated with oxidative stress, mitochondrial dysfunction, and AIF translocation.Ministry of Science of the Republic of Serbia [41025, 172035

Cyclohexyl Analogues of Ethylenediamine Dipropanoic Acid Induce Caspase-Independent Mitochondrial Apoptosis in Human Leukemic Cells

We investigated the cytotoxicity of recently synthesized (S,S)-ethyleridiamine-N,N'-di-2-(3-cyclohexyl)propanoic acid esters toward human leukemic cell lines and healthy blood mononuclear cells. Cell viability was assessed by acid phosphatase assay, apoptosis, and differentiation were analyzed by flow cytometry and electron microscopy, while intracellular localization of apoptosis-inducing factor (AIF) was determined by immunoblotting. It was demonstrated that methyl, ethyl, and n-propyl esters were toxic to HL-60, REH, MOLT-4, KG-1, JVM-2, and K-562 leukemic cell lines, while the nonesterified parental compound and n-butyl ester were devoid of cytotoxic action. The ethyl ester exhibited the highest cytotoxic activity (IC50 10.7 mu M-45.4 mu M), which was comparable to that of the prototypical anticancer drug cisplatin. The observed cytotoxic effect in HL-60 cells was associated with an increase in superoxide production and mitochondrial membrane depolarization, leading to apoptotic cell death characterized by phosphatidylserine externalization and DNA fragmentation in the absence of autophagic response. DNA fragmentation preceded caspase activation and followed AIF translocation from mitochondria to nucleus, which was indicative of caspase-independent apoptotic cell death. HL-60 cells treated with subtoxic concentration of the compound displayed morphological signs of granulocytic differentiation (nuclear indentations and presence of cytoplasmic primary granules), as well as an increased expression of differentiation markers CD11b and CD15. The cyclohexyl analogues of ethylenediamine dipropanoic acid were also toxic to peripheral blood mononuclear cells of both healthy controls and leukemic patients, the latter being more sensitive. Our data demonstrate that the toxicity of the investigated cyclohexyl compounds against leukemic cell lines is mediated by caspase-independent apoptosis associated with oxidative stress, mitochondrial dysfunction, and AIF translocation.Ministry of Science of the Republic of Serbia [41025, 172035