Vrednotenje večfunkcionalnih spojin z uporabo celičnega modela Alzheimerjeve bolezni in vitro

Češek, Tjaša (avtor), Pišlar, Anja (mentor), Meden, Anže (komentor)

Alzheimerjeva bolezen (AB) je progresivna nevrodegenerativna bolezen, za katero še ne poznamo učinkovitega zdravljenja. Glede na amiloidogeno hipotezo je glavni vzrok nastanka bolezni prekomerno kopičenje peptida amiloida beta (Aβ) v zunajceličnih plakih. Oligomeri Aβ lahko preko vezave na alosterično mesto adrenergičnega receptorja α2A (α2AAR) ojačajo z noradrenalinom posredovano aktivacijo glikogen sintaze kinaze 3β (GSK3β) in posledično hiperfosforilacijo proteina tau, ki tvori znotrajcelične nevrofibrilarne pentlje. Holinergična hipoteza medtem predpostavlja, da je upad kognitivnih funkcij posledica okvare holinergičnih nevronov in zmanjšane koncentracije acetilholina. V sklopu magistrske naloge smo ovrednotili delovanje novih večfunkcionalnih spojin na celičnem modelu AB, ki so hkrati zaviralci encima butirilholin esteraze in antagonisti α2AAR, in sicer spojine A1112, A1130, A1134C, MSG-16 in A527-2. Na podlagi rezultatov citotoksičnosti spojin, ki smo jo vrednotili s pomočjo spektrofotometrije in pretočne citometrije na celicah SH-SY5Y, smo morebitno zaščitno delovanje spojin vrednotili pri koncentracijah, ki niso izkazovale značilnega vpliva na zmanjšano preživetje celic. Z uporabo Aβ v obliki fibril smo na nevroblastomski celični liniji SH-SY5Y postavili celični model AB, pri čemer se kot optimalna izkazala 5 µM koncentracija Aβ. Z metodama SDS-PAGE in prenosom western smo potrdili, da peptid spodbudi aktivacijo kinaze GSK3β in fosforilacijo proteina tau. Na postavljenem celičnem modelu nevrodegeneracije smo nato pokazali, da po stimulaciji z Aβ celično preživetje delno izboljšajo 5 in 10 µM koncentracije MSG-16 ter 1 in 2,5 µM koncentracije A527-2. Hkrati 10 µM MSG-16 in 1 µM A527-2 znižata z Aβ povečano aktivnost kaspaze-3. Spojini A1112 in A1130 medtem nista izkazali vpliva na aktivnost kaspaze-3 in preživetje celic SH-SY5Y po izpostavitvi Aβ. S prenosom western smo določili, da večfunkcionalne spojine zavirajo aktivacijo GSK3β ter znižajo raven fosforilacije proteina tau, spodbujeno s peptidom Aβ. Za boljše vrednotenje antagonističnega delovanja spojin na α2AAR smo celice SH-SY5Y z reagentom dbcAMP diferencirali v noradrenergični fenotip. Tudi na diferenciranih celicah smo pokazali, da vse vrednotene spojine znižajo raven fosforilacije proteina tau, ki jo preko vezave na α2AAR in posledične aktivacije kinaze GSK3β spodbudi Aβ. Naše eksperimentalne ugotovitve nakazujejo, da večfunkcionalni spojini MSG-16 in A527-2 zavirata nevrodegenerativne procese spodbujene s peptidom Aβ, pri čemer spojina MSG-16 zaradi manjše citotoksičnosti in močnejšega vpliva na raven fosforilacije proteina tau predstavlja najboljše izhodišče za razvoj novih terapevtsko uporabnejših učinkovin za zdravljenje AB.

• Jezik: Slovenski jezik
• Ključne besede: Alzheimerjeva bolezen, amiloid beta, zaviralci butirilholin esteraze, antagonisti α2A adrenergičnih receptorjev, nevroprotektivno delovanje
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-143850
• Datum objave v RUL: 14.01.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Evaluation of multifunctional compounds using an in vitro cell-based model of Alzheimer's disease

Izvleček

Alzheimer's disease (AD) is a progressive neurodegenerative disease without known effective treatment. According to amyloid hypothesis, the main cause of AD is the excessive accumulation of the amyloid beta peptide (Aβ) in extracellular plaques. By binding to an allosteric site on α2A adrenergic receptor (α2AAR), Aβ oligomers redirect norepinephrine signaling to glycogen synthase kinase 3β (GSK3β) activation and tau hyperphosphorylation, resulting in formation of neurofibrillary tangles. Meanwhile, cholinergic hypothesis proposes that cognitive decline is caused by degeneration of the cholinergic neurons and reduced acetylcholine concentrations. In the scope of master's thesis, we evaluated the activity of novel multifunctional compounds A1112, A1130, A1134C, MSG-16 and A527-2, that are both butyrylcholinesterase inhibitors and α2AAR antagonists using the in vitro cell model of AD. Following the cytotoxicity evaluation of multifunctional compounds, determined by the spectrophotometry and flow cytometry on SH-SY5Y cells, the potential protective effect of the compounds was evaluated at concentrations that did not significantly reduce cell survival. An in vitro cell model of AB was established on the SH-SY5Y neuroblastoma cell line using the fibrillar form of Aβ peptide, where 5 µM concentration demonstrated an optimal toxic effect. By SDS-PAGE and western blot, we confirmed that Aβ induces GSK3β activation and tau phosphorylation. On the established cell model of AB, we showed that pre-treatment of SH-SY5Y cells with 5 and 10 µM concentrations of MSG-16 as well as 1 and 2,5 µM concentrations of A527-2 partially improved cell viability compared to Aβ-treated cells. Furthermore, 10 µM MSG-16 and 1 µM A527-2 reduced the increased activity of caspase-3 in Aβ-stimulated SH-SY5Y cells. Meanwhile, compounds A1112 and A1130 did not improve cell viability and caspase-3 activity in Aβ-treated cells. By western blot method, we determined that multifunctional compounds inhibit GSK3β activation and reduce the level of tau protein phosphorylation in Aβ-stimulated cells. To better evaluate the antagonistic effect of compounds on α2AAR, SH-SY5Y cells were differentiated into a noradrenergic phenotype using dbcAMP. All compounds reduced the level of protein tau phosphorylation, which is induced by Aβ binding to α2AAR and consequent GSK3β activation. Our experimental findings indicate that multifunctional compounds MSG-16 and A527-2 inhibit Aβ-promoted neurodegenerative processes, with MSG-16 representing the best potential for the development of new therapeutically useful agents for the treatment of AD due to its lower cytotoxicity and more potent effect on tau phosphorylation.

• Ključne besede: Alzheimer's disease, amyloid beta, butyrylcholinesterase inhibitors, α2A adrenergic receptor antagonists, neuroprotection