Vpliv oblike in dolžine peptida amiloid beta na preživetje nevroblastomske celične linije SH-SY5Y in na vsebnost profilina 1 v njej

Gaber, Tjaša (author), Pečar Fonović, Urša (mentor)

Alzheimerjeva bolezen (AB) je progresivna nevrodegenerativna bolezen, ki pretežno pesti starejši del populacije. Najbolj znana hipoteza, ki opisuje nastanek bolezni je amiloidogena hipoteza, ki pravi, da so zunajcelični plaki, ki nastanejo zaradi prekomernega odlaganja peptida amiloid beta (Aβ), glavni razlog za nastanek AB. Aβ je peptid, ki se v plakih nahaja pretežno v dolžinah 1–40 in 1–42, pa tudi v dolžini 25–35. Za Aβ 1–40 in 1–42 je bila pokazana specifična vezava s profilinom 1, ki je življenjsko pomemben protein. Sodeluje pri procesu polimerizacije aktina, v nevronih pa je pomemben regulator morfologije in sinaptične plastičnosti. V okviru magistrske naloge smo želeli preveriti citotoksični vpliv peptida Aβ različnih dolžin (1–40, 1–42, 25–35) in oblik (monomeri in fibrile), ter določiti subletalno koncentracijo za nevroblastomsko celično linijo SH-SY5Y v treh časovnih intervalih (24, 48 in 72 ur). Viabilnost celic smo določali s kolorimetrično metodo MTS. Ugotovili smo, da je peptid Aβ 1–42 tako v obliki monomerov kot fibril za celice najbolj toksičen. Monomeri že pri koncentraciji 1 µM po 48 urah povzročijo statistično značilno zmanjšanje viabilnosti celic, fibrile pa pri 2 µM koncentraciji. Prav tako posnetki celic v prisotnosti peptida 1–42 kažejo največ morfoloških sprememb. Za vse tri peptide smo pokazali, da je preživetje celic koncentracijsko in časovno odvisno, kjer se z večanjem koncentracije in daljšo časovno izpostavljenostjo peptidu manjša viabilnost celic. S peptidi v določeni subletalni koncentraciji smo nadalje preverili njihov vpliv na vsebnost proflina 1 v celicah. Celice smo za 24 ur izpostavili dvema različnima koncentracijama obeh oblik posameznega peptida in v celičnih lizatih z metodama SDS-PAGE ter prenos western določili vsebnost profilina 1 in aktina. Vsebnost profilina 1 v celicah je pri vseh peptidih v obliki monomerov večja kot pri kontrolnih vzorcih. Prav tako tudi pri fibrilah, razen pri peptidu 1–42 100 nM koncentracije, medtem ko fibrile peptida 25–35 povzročijo zmanjšano vsebnost profilina 1 v celičnih lizatih. Aβ bi lahko imel vpliv na procese transkripcije in translacije profilina 1, kar bo predmet nadaljnjih raziskav.

• Language: Slovenian
• Keywords: Alzheimerjeva bolezen, amiloid beta, citotoksičnost, profilin 1, celična linija SH-SY5Y.
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-135416
• Publication date in RUL: 12.03.2022

Secondary language

• Language: English
• Title: Effect of amyloid beta peptide form and length on the survival of neuroblastoma cell line SH-SY5Y and its profilin 1 content

Abstract

Alzheimer's disease is a progressive neurodegenerative disease that predominantly affects the elder population. Leading hypothesis describing the cause of the disease is the amyloid hypothesis, according to which the main cause of Alzheimer’s disease are the extracellular plaques formed in excessive deposition of the amyloid beta peptide (Aβ). Aβ is a peptid, predominantly found in plaques with lengths 1–40 and 1–42, but also in length 25–35. Aβ 1–40 and 1–42 have proved to bind specifically to profilin 1, a vital protein. Profilin 1 regulates actin polymerisation and is crucial for morphology and synaptic plasticity regulation in neurons. The goal of our master’s thesis was to verify the cytotoxic effect of Aβ peptide of different lengths (1–40, 1–42, 25–35) and forms (monomers and fibrils), and to determine the sublethal concentration for the SH-SY5Y neuroblastoma cell line at three time intervals (24, 48 and 72 hours). Cell viability was determined by the MTS colorimetric method. Results showed that Aβ 1–42 peptide is most toxic to cells in both monomeric and fibril forms. Monomers at a concentration as low as 1 µM caused a statistically significant decrease in cell viability after 48 hours, whereas fibrils reacted in the same manner at 2 µM. Similarly, cell microscopy showed the biggest morphological changes in the presence of peptide 1–42. For all three peptides the results have proved that cell survival is concentration- and time-dependent, where cell viability decreases with increasing concentration and longer exposure to the peptide. At given sublethal concentration further analyses were performed to observe the effect on profilin 1 content in cells. Cells were exposed to two different concentrations of both forms of each peptide for 24 hours and profilin 1 and actin content in cell lysates were determined by SDS-PAGE and western blot method. The cellular profilin 1 content was higher for all peptides in monomeric form compared to control samples. Through observation of fibrils, a similar conclusion was formed, with the exception of peptide 1–42 at 100 nM concentration, while fibrils of peptide 25–35 showed a reduced profilin 1 content in cell lysates. Therefore, Aβ could have an effect on the transcription and translation processes of profilin 1, which poses a possibility for further investigation.

• Keywords: Alzheimer’s disease, amyloid beta, cytotoxicity, profilin 1, SH-SY5Y cell line.