Uloga kinaze aktivirane adenozin monofosfatom u neurotoksičnom delovanju alfa-sinukleina in vitro
The role of adenosine monophosphate-activated protein kinase in the neurotoxic effect of alpha-synuclein in vitro
Author
Dulović, Marija Z.Mentor
Marković, Ivanka
Committee members
Kostić, VladimirIsaković, Aleksandra

Trajković, Vladimir

Andrić, Silvana

Metadata
Show full item recordAbstract
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 ac...tivity 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...
Faculty:
Универзитет у Београду, Медицински факултетDate:
09-09-2015Projects:
- Motor and non-motor symptoms and signs in parkinsonism: clinical, morphological and molecular-genetic correlates (RS-175090)
- Modulation of intracellular energy balance-controlling signalling pathways in therapy of cancer and neuro-immuno-endocrine disorders (RS-41025)