TY  - CONF
AU  - Jeremić, Marija
AU  - Jovanović, Maja
AU  - Tovilović-Kovačević, Gordana
AU  - Harhaji-Trajković, Ljubica
AU  - Zogović, Nevena
AU  - Vukojević, Milica
AU  - Kostić, Vladimir
AU  - Marković, Ivanka D.
AU  - Trajković, Vladimir
PY  - 2021
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/4450
AB  - Alpha-synuclein (ASYN) is regarded as one of the key culprits in
pathogenesis of synucleinopathies, including Parkinson’s disease,
and impaired regulation of autophagy is associated with the
ASYN aggregation. Autophagy is regulated by complex mechanisms,
including AMP activated protein kinase (AMPK), a key
energy sensor regulating cellular metabolism to maintain energy
homeostasis. The aim of our study was to investigate the role of
AMPK and autophagy in neurotoxic effect of secreted ASYN, as
well as dopamine-modified and nitrated recombinant wild-type
ASYN oligomers, on retinoic acid (RA)-differentiated SH-SY5Y
cells. The culture supernatant from neuroblastoma cells stably
expressing wt ASYN was collected and used as conditioned medium (CM). The presence of wt ASYN in CM was confirmed
by immunoblot, following lyophilisation. The CM, as well as
recombinant dopamine-modified or nitrated ASYN, all reduced
viability in differentiated SH-SY5Y cells. This decrease in viability
was accompanied by reduced AMPK activation, increased
conversion of LC3-I to LC3-II and increase in Beclin-1 level, as
demonstrated by immunoblot. Pharmacological activators of
AMPK and autophagy (metformin and AICAR) significantly
increased the cells’ viability in the presence of CM and modified
ASYN forms. Level of AMPK-activated pULK was reduced in
presence of CM, but pharmacological activators of AMPK
reversed that effect. Pharmacological inhibitors of autophagy,
further reduced cell viability in the presence of extracellular
ASYN. The shRNA-mediated LC3 downregulation, as well as
the RNA interference-mediated knockdown of ATG7 gene, both
important for autophagosome biogenesis/maturation, increased
sensitivity of SH-SY5Y cells to the extracellular ASYN-induced
toxicity. These data demonstrate the protective role of AMPK
and autophagy against the toxicity of extracellular ASYN, suggesting
that their modulation may be a promising neuroprotective
strategy in Parkinson’s disease.
PB  - Hoboken: Wiley
C3  - FEBS OpenBio
T1  - Neurotoxic effect of extracellular alpha-synuclein can be alleviated by AMPK and autophagy
IS  - Supplement 1
VL  - 11
SP  - 463
UR  - https://hdl.handle.net/21.15107/rcub_ibiss_4450
ER  - 

@conference{
author = "Jeremić, Marija and Jovanović, Maja and Tovilović-Kovačević, Gordana and Harhaji-Trajković, Ljubica and Zogović, Nevena and Vukojević, Milica and Kostić, Vladimir and Marković, Ivanka D. and Trajković, Vladimir",
year = "2021",
abstract = "Alpha-synuclein (ASYN) is regarded as one of the key culprits in
pathogenesis of synucleinopathies, including Parkinson’s disease,
and impaired regulation of autophagy is associated with the
ASYN aggregation. Autophagy is regulated by complex mechanisms,
including AMP activated protein kinase (AMPK), a key
energy sensor regulating cellular metabolism to maintain energy
homeostasis. The aim of our study was to investigate the role of
AMPK and autophagy in neurotoxic effect of secreted ASYN, as
well as dopamine-modified and nitrated recombinant wild-type
ASYN oligomers, on retinoic acid (RA)-differentiated SH-SY5Y
cells. The culture supernatant from neuroblastoma cells stably
expressing wt ASYN was collected and used as conditioned medium (CM). The presence of wt ASYN in CM was confirmed
by immunoblot, following lyophilisation. The CM, as well as
recombinant dopamine-modified or nitrated ASYN, all reduced
viability in differentiated SH-SY5Y cells. This decrease in viability
was accompanied by reduced AMPK activation, increased
conversion of LC3-I to LC3-II and increase in Beclin-1 level, as
demonstrated by immunoblot. Pharmacological activators of
AMPK and autophagy (metformin and AICAR) significantly
increased the cells’ viability in the presence of CM and modified
ASYN forms. Level of AMPK-activated pULK was reduced in
presence of CM, but pharmacological activators of AMPK
reversed that effect. Pharmacological inhibitors of autophagy,
further reduced cell viability in the presence of extracellular
ASYN. The shRNA-mediated LC3 downregulation, as well as
the RNA interference-mediated knockdown of ATG7 gene, both
important for autophagosome biogenesis/maturation, increased
sensitivity of SH-SY5Y cells to the extracellular ASYN-induced
toxicity. These data demonstrate the protective role of AMPK
and autophagy against the toxicity of extracellular ASYN, suggesting
that their modulation may be a promising neuroprotective
strategy in Parkinson’s disease.",
publisher = "Hoboken: Wiley",
journal = "FEBS OpenBio",
title = "Neurotoxic effect of extracellular alpha-synuclein can be alleviated by AMPK and autophagy",
number = "Supplement 1",
volume = "11",
pages = "463",
url = "https://hdl.handle.net/21.15107/rcub_ibiss_4450"
}

Jeremić, M., Jovanović, M., Tovilović-Kovačević, G., Harhaji-Trajković, L., Zogović, N., Vukojević, M., Kostić, V., Marković, I. D.,& Trajković, V.. (2021). Neurotoxic effect of extracellular alpha-synuclein can be alleviated by AMPK and autophagy. in FEBS OpenBio
Hoboken: Wiley., 11(Supplement 1), 463.
https://hdl.handle.net/21.15107/rcub_ibiss_4450

Jeremić M, Jovanović M, Tovilović-Kovačević G, Harhaji-Trajković L, Zogović N, Vukojević M, Kostić V, Marković ID, Trajković V. Neurotoxic effect of extracellular alpha-synuclein can be alleviated by AMPK and autophagy. in FEBS OpenBio. 2021;11(Supplement 1):463.
https://hdl.handle.net/21.15107/rcub_ibiss_4450 .

Jeremić, Marija, Jovanović, Maja, Tovilović-Kovačević, Gordana, Harhaji-Trajković, Ljubica, Zogović, Nevena, Vukojević, Milica, Kostić, Vladimir, Marković, Ivanka D., Trajković, Vladimir, "Neurotoxic effect of extracellular alpha-synuclein can be alleviated by AMPK and autophagy" in FEBS OpenBio, 11, no. Supplement 1 (2021):463,
https://hdl.handle.net/21.15107/rcub_ibiss_4450 .

TY  - JOUR
AU  - Đuranović, Andrija
AU  - Jeremić, Marija
AU  - Zogović, Nevena
AU  - Tovilović-Kovačević, Gordana
AU  - Dulović, Marija
PY  - 2016
UR  - http://radar.ibiss.bg.ac.rs/handle/123456789/6339
AB  - Uvod. U osnovi patogenetskog mehanizma Parkinsonove bolesti leži povećano nakupljanje proteina α-sinukleina (ASYN) u dopaminergičkim neuronima substantiae nigrae, što, između ostalog, dovodi i do smrti ćelija. Iako je ranije smatran isključivo unutarćelijskim proteinom, novijim istraživanjima je pokazano da se ASYN nakuplja i u vanćelijskom prostoru. Do nakupljanja ASYN može da dođe usled oštećenja sistema za razgradnju proteina, od kojih je najznačajniji mehanizam autofagija. Autofagija je regulisana proizvodima ATG gena (engl. autophagy-related genes). Jedan od njih, ATG7, ima bitnu ulogu u formiranju i sazrevanju autofagozoma. Cilj. Cilj ovog rada je bio da se ispita uloga autofagije u neurotoksičnosti vanćelijskog ASYN na SH-SY5Y ćelije humanog neuroblastoma, diferentovanih u neuronski fenotip. Materijali i metode. Svi eksperimenti su rađeni na SH-SY5Y ćelijskoj liniji humanog neuroblastoma. Ćelije su diferentovane 'all-trans' retinoičnom kiselinom i tretirane medijumom koji sadrži vanćelijski ASYN. Imunoblot metodom je potvrđeno prisustvo vanćelijskog ASYN i praćena promena eskpresije markera autofagije, proteina LC3-II i beklin-1. Metodom transfekcije sa malom interferirajućom RNK inhibirana je ekspresija ATG7 proteina. Vijabilitet i broj ćelija određeni su kristal violet testom. Rezultati. Vanćelijski ASYN dovodi do značajnog smanjenja vijabiliteta SH-SY5Y ćelija, što je praćeno povećanjem nivoa markera autofagije, proteina LC3-II i beklin-1. Analizom ćelijskog vijabiliteta primećen je značajan pad u broju živih ćelija kod kojih je utišan gen za ATG7, a koje su gajene u prisustvu vanćelijskog ASYN. Zaključak. Vanćelijski ASYN dovodi do smanjenog preživljavanja SH-SY5Y ćelija diferentovanih u neuronski fenotip, što je praćeno indukcijom autofagije. Inhibicija autofagije preko utišavanja ATG7 gena dovela je do povećane osetljivosti ćelija gajenih u prisustvu vanćelijskog ASYN, što ukazuje na moguću protektivnu ulogu autofagije u neurotoksičnosti, izazvanoj nagomilavanjem ASYN u vanćelijskom prostoru.
AB  - Introduction: The accumulation of alpha-synuclein (ASYN) in susceptible neurons is considered to be a major contributing factor in pathogenesis of Parkinson’s disease. Although ASYN was considered as an intracellular protein, recent data suggest that it can be detected extracellularly. Autophagy plays an important role in ASYN degradation; therefore, impairment of autophagy could be an important contributor to ASYN accumulation. ATG (autophagy-related genes) proteins function at several physiologically continuous steps in autophagy, and ATG7 is considered as essential in autophagosome formation and maturation.
Aim: The aim of this study was to investigate the role of autophagy in neurotoxicity, caused by extracellular ASYN.
Material and methods: All experiments were conducted in all-trans retinoic acid-differentiated human neuroblastoma SH-SY5Y cells, that were exposed to extracellular ASYN. The presence of extracellular ASYN and the expression of autophagy markers, beclin-1 and LC3-II, were monitored using immunoblotting. Transfection, with small interfering RNA (siRNA), was used to knock down ATG7 gene. Cell viability was assessed using crystal violet dye exclusion assay.
Results: Extracellular ASYN caused significant loss of viability in differentiated SH-SY5Y cells, accompanied by the increase in expression of beclin-1 and in conversion of LC3-I to autophagosome-associated LC3-II. The RNA interference-mediated knock-down of ATG7 increased the sensitivity of SH-SY5Y cells to the extracellular ASYN-induced toxicity.
Conclusion: Extracellular ASYN caused loss of viability in differentiated SH-SY5Y cells accompanied by autophagy induction. Having in mind that inhibition of autophagy, through ATG7 knock-down increased cell death, we can conclude that autophagy could have a protective role in the harmful effect of extracellular ASYN.
PB  - Belgrade: University of Belgrade, Faculty of Medicine
T2  - Medicinski podmladak
T1  - Uloga autofagije u neurotoksičnom delovanju vanćelijskog alfa sinukleina na SH-SY5Y ćelije humanog neuroblastoma
T1  - The role of autophagy in neurotoxicity caused by extracellular ASYN
IS  - 4
VL  - 67
DO  - 10.5937/mp67-12608
SP  - 47
EP  - 53
ER  - 

@article{
author = "Đuranović, Andrija and Jeremić, Marija and Zogović, Nevena and Tovilović-Kovačević, Gordana and Dulović, Marija",
year = "2016",
abstract = "Uvod. U osnovi patogenetskog mehanizma Parkinsonove bolesti leži povećano nakupljanje proteina α-sinukleina (ASYN) u dopaminergičkim neuronima substantiae nigrae, što, između ostalog, dovodi i do smrti ćelija. Iako je ranije smatran isključivo unutarćelijskim proteinom, novijim istraživanjima je pokazano da se ASYN nakuplja i u vanćelijskom prostoru. Do nakupljanja ASYN može da dođe usled oštećenja sistema za razgradnju proteina, od kojih je najznačajniji mehanizam autofagija. Autofagija je regulisana proizvodima ATG gena (engl. autophagy-related genes). Jedan od njih, ATG7, ima bitnu ulogu u formiranju i sazrevanju autofagozoma. Cilj. Cilj ovog rada je bio da se ispita uloga autofagije u neurotoksičnosti vanćelijskog ASYN na SH-SY5Y ćelije humanog neuroblastoma, diferentovanih u neuronski fenotip. Materijali i metode. Svi eksperimenti su rađeni na SH-SY5Y ćelijskoj liniji humanog neuroblastoma. Ćelije su diferentovane 'all-trans' retinoičnom kiselinom i tretirane medijumom koji sadrži vanćelijski ASYN. Imunoblot metodom je potvrđeno prisustvo vanćelijskog ASYN i praćena promena eskpresije markera autofagije, proteina LC3-II i beklin-1. Metodom transfekcije sa malom interferirajućom RNK inhibirana je ekspresija ATG7 proteina. Vijabilitet i broj ćelija određeni su kristal violet testom. Rezultati. Vanćelijski ASYN dovodi do značajnog smanjenja vijabiliteta SH-SY5Y ćelija, što je praćeno povećanjem nivoa markera autofagije, proteina LC3-II i beklin-1. Analizom ćelijskog vijabiliteta primećen je značajan pad u broju živih ćelija kod kojih je utišan gen za ATG7, a koje su gajene u prisustvu vanćelijskog ASYN. Zaključak. Vanćelijski ASYN dovodi do smanjenog preživljavanja SH-SY5Y ćelija diferentovanih u neuronski fenotip, što je praćeno indukcijom autofagije. Inhibicija autofagije preko utišavanja ATG7 gena dovela je do povećane osetljivosti ćelija gajenih u prisustvu vanćelijskog ASYN, što ukazuje na moguću protektivnu ulogu autofagije u neurotoksičnosti, izazvanoj nagomilavanjem ASYN u vanćelijskom prostoru., Introduction: The accumulation of alpha-synuclein (ASYN) in susceptible neurons is considered to be a major contributing factor in pathogenesis of Parkinson’s disease. Although ASYN was considered as an intracellular protein, recent data suggest that it can be detected extracellularly. Autophagy plays an important role in ASYN degradation; therefore, impairment of autophagy could be an important contributor to ASYN accumulation. ATG (autophagy-related genes) proteins function at several physiologically continuous steps in autophagy, and ATG7 is considered as essential in autophagosome formation and maturation.
Aim: The aim of this study was to investigate the role of autophagy in neurotoxicity, caused by extracellular ASYN.
Material and methods: All experiments were conducted in all-trans retinoic acid-differentiated human neuroblastoma SH-SY5Y cells, that were exposed to extracellular ASYN. The presence of extracellular ASYN and the expression of autophagy markers, beclin-1 and LC3-II, were monitored using immunoblotting. Transfection, with small interfering RNA (siRNA), was used to knock down ATG7 gene. Cell viability was assessed using crystal violet dye exclusion assay.
Results: Extracellular ASYN caused significant loss of viability in differentiated SH-SY5Y cells, accompanied by the increase in expression of beclin-1 and in conversion of LC3-I to autophagosome-associated LC3-II. The RNA interference-mediated knock-down of ATG7 increased the sensitivity of SH-SY5Y cells to the extracellular ASYN-induced toxicity.
Conclusion: Extracellular ASYN caused loss of viability in differentiated SH-SY5Y cells accompanied by autophagy induction. Having in mind that inhibition of autophagy, through ATG7 knock-down increased cell death, we can conclude that autophagy could have a protective role in the harmful effect of extracellular ASYN.",
publisher = "Belgrade: University of Belgrade, Faculty of Medicine",
journal = "Medicinski podmladak",
title = "Uloga autofagije u neurotoksičnom delovanju vanćelijskog alfa sinukleina na SH-SY5Y ćelije humanog neuroblastoma, The role of autophagy in neurotoxicity caused by extracellular ASYN",
number = "4",
volume = "67",
doi = "10.5937/mp67-12608",
pages = "47-53"
}

Đuranović, A., Jeremić, M., Zogović, N., Tovilović-Kovačević, G.,& Dulović, M.. (2016). Uloga autofagije u neurotoksičnom delovanju vanćelijskog alfa sinukleina na SH-SY5Y ćelije humanog neuroblastoma. in Medicinski podmladak
Belgrade: University of Belgrade, Faculty of Medicine., 67(4), 47-53.
https://doi.org/10.5937/mp67-12608

Đuranović A, Jeremić M, Zogović N, Tovilović-Kovačević G, Dulović M. Uloga autofagije u neurotoksičnom delovanju vanćelijskog alfa sinukleina na SH-SY5Y ćelije humanog neuroblastoma. in Medicinski podmladak. 2016;67(4):47-53.
doi:10.5937/mp67-12608 .

Đuranović, Andrija, Jeremić, Marija, Zogović, Nevena, Tovilović-Kovačević, Gordana, Dulović, Marija, "Uloga autofagije u neurotoksičnom delovanju vanćelijskog alfa sinukleina na SH-SY5Y ćelije humanog neuroblastoma" in Medicinski podmladak, 67, no. 4 (2016):47-53,
https://doi.org/10.5937/mp67-12608 . .