Role of adenosine monophosphateactivated protein kinase and protein kinase B (PKB) in neurotoxic damage induced by 1-methyl-4-phenylpyridinium in vitro

Parkinsonova bolest (PB) je drugo po učestalosti neurodegenerativno oboljenje, koje karakteriše progresivna degeneracija i smrt dopaminergičkih (DA) neurona substantiae nigrae (SN) pars compacta, koji učestvuje u kontroli pokreta. Iako je etiologija PB u velikoj meri i dalje nedovoljno razjašnjena, rezultati brojnih eksperimentalnih studija, ukazali su da bi oksidativni stres, disfunkcija mitohondrija i ćelijskih sistema uključenih u homeostazu proteina, mogli biti odgovorni za degeneraciju i smrt DA neurona u SN. Ispitivanje uticaja neurotoksina MPP+ na neurone, je do danas ostao jedan od najznačajnijih modela za proučavanje molekularnih mehanizama oštećenja i smrti neurona. Cilj ovog istraživanja je bio da se ispita uloga oksidativnog stresa, kao i signalnih puteva unutarćelijsog energetskog senzora, adenozin monofosfatom aktivirane kinaze (AMPK), protein kinaze B/Akt - medijatora rasta i preživljavanja ćelija, autofagije, kao i njihovih interakcija, u modelu oštećenja neurona delovanjem MPP+. Istraživanje je sprovedeno na modelu ćelijske linije humanog neuroblastoma SH-SY5Y. Primena MPP+ je dovela do dozno i vremenski-zavisnog pada vijabiliteta i fragmentacije DNK, kome su prethodili porast oslobađanja superoksidnog anjona i depolarizacija unutrašnje mitohondrijalne membrane, kao i rana aktivacija signalnih puteva AMPK i Akt kinaze. Primena antioksidanasa, N-acetil cisteina ili butiliranog hidroksianizola, dovela je do delimičnog smanjenja citotoksičnog delovanja MPP+, koji je bio praćen padom nivoa aktivacije AMPK i Akt kinaze. Genska ili farmakološka inhibicija AMPK kinaze rezultirala je povećanom osetljivošću ćelija na MPP+, uz smanjenje aktivacije Akt kinaze, dok je farmakološka aktivacija AMPK rezultirala poboljšanim preživljavanjem ćelija tretiranih MPP+ toksinom. U uslovima farmakološke ili genske inhibicije Akt kinaze, ćelije su ispoljavale veću osetljivost na štetno dejstvo MPP+ toksina, uz povećanu produkciju superoksidnog anjona. Imunoblot analiza uticaja MPP+ na aktivnost mTOR signalnog puta, najvažijeg regulatora autofagije u ćeliji, pokazala je da MPP+ indukuje kasnu inhibiciju mTOR kompleksa 1 (mTORC1), kao i porast prevođenja proteina LC3-I (engl. Microtubule associated protein 1 light chain 3, LC3) u oblik vezan za autofagozome, LC3-II, što ukazuje na indukciju mTORC1-zavisne autofagije...characterized by the progressive degeneration and demise of the dopaminergic (DA) neurons in the substantia nigra (SN) pars compacta, part of the midbrain. Even though the etiology of PD is still insufficiently elucidated, research results implicate that oxidative stress, mitochondrial dysfunction and impairment of proein homeostasis cellular play an important role in DA neurons' degeneration and cell death. The MPP+ neurotoxic model has been widely used in study of molecular mechanismsm of neuronal demise in PD. The aim of this study was to assess the interaction between the signaling pathways of the main intracellular energy sensor, adenosine monophosphate activated kinase (AMPK), prosurvival protein kinase B/Akt, oxidative stress and autophagy in the effect of parkinsonian neurotoxin 1-methyl-4-phenyl piridinium (MPP+) The study was conducted on SH-SY5Y human neuroblastoma cell line. MPP+ caused the dose- and time- dependent decrease in cell viability and DNA fragmentation, preceded by increase in superoxide production (2 h) and subsequent inner mitochondrial membrane depolarization (8 h), accompanied by early activation of AMPK and Akt signaling pathways. The reactive oxygen species (ROS) scavengers, Nacetyl- L-cysteine and butylated hydroxyanisole partially alleviated the MPP+-induced cell death, causing decrease in phosphorylation levels of both AMPK and protein kinase B/Akt. Pharmacological or genetic AMPK inhibition further potentiated MPP+-induced ROS production and cell death, and diminished Akt phosphorylation, whereas AMPK activation exerted protective effects against MPP+-induced toxicity. Nevertheless, pharmacological or genetic inactivation of Akt caused an increase in MPP+-initiated oxidative stress and neurotoxicity, but failed to affect AMPK activation. Furthernore, MPP+ treatment at later time-points (16-24 h) inhibited the main autophagy repressor, mammalian target of rapamycin (mTOR), which was accompanied by the increased levels of the autophagy marker, microtubule-associated protein 1 light-chain 3B. On the other hand, the concentration of a selective autophagic target, sequestosome-1/p62, were increased in MPP+-treated cells, while levels of lysosomal-associated membrane protein 1 and cytoplasmic acidification were reduced, which suggested that MPP+- induced autophagy was associated with a decrease in 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