Proteome analysis of APPPS1-21 and wild-type mice

Abstract

Formål: Demens er et stigende problem, specielt i den industrielle verden, da den forventede levetid stiger. Lige nu er 10-30 % af verdens befolkning over 65 år ramt af Alzheimers sygdom, som i langt de fleste tilfælde er sporadisk. Der er lavet mange studier omkring Alzheimers sygdom, men indtil videre har det ikke været muligt fuldtud at forklare de forskellige patologiske kendetegn, hvordan forskellige faktorer påvirker hinanden, for eksempel stress og søvnmangel, eller hvorfor nogle mennesker udvikler sygdommen. Dette projekts formål er at undersøge proteomet af mus med Alzheimers sygdom (APPPS1-21 mus) og vildtype mus, med og uden kronisk stress. Metoder: For at skabe overblik over proteinerne i de forskellige test dyr blev der lavet SDS-page. Efterfølgende blev hippocampus prøverne klargjort til label free quantification på en Thermo-Electron QExactive HF-X mass spectrometer (ThermoFisher Scientific) ved brug af filter-aided samples preparation (FASP). PERSEUS (Max Planck Institute of biochemistry, v1.6.5.0) og PEAKS Studio X (Bioinformatics Solutions Inc.) blev brugt til at bearbejde data, lave figurer og statistiske analyser, herunder volcano plots, Hawaii plots og Principal Component Analysis (PCA). Resultater: Resultaterne fra SDS-page forsøgene viste ikke nogle tydelige forskelle på de forskellige mus eller mellem de forskellige testgrupper. Både volcano plots og Hawaii plots viste et stort antal proteiner som enten var opregulerede eller nedregulerede i de forskellige testgrupper, inklusiv proteiner involveret i at styre døgnrytmen, samt ApoE, APP og GFAP, som alle er mistænkt for at være involveret i Alzheimers sygdom. PCA undersøgelser viste ikke nogle grupper af proteiner som kunne være årsag til at resultaterne blev grupperet på den måde de gjorde bortset fra de proteiner der er involveret i døgnrytmen. Konklusion: Resultaterne af disse forsøg viste flere interessante ting i forhold til forskningen i Alzheimers sygdom. ApoE, som er en risikofaktor for at udvikle familiær Alzheimers, var signifikant opreguleret i APPPS1-21, men var nedreguleret i de mus som også have været udsat for kronisk stress, og videre analyser viste at niveauet i APPPS1-21 + CMS var det samme som i vildtype musene. Flere af de proteiner som er involveret i døgnrytmen var signifikant ændret i alle test grupper i sammenligningen med vildtype musene, hvilket også kan relateres til kliniske forsøg af søvnmangels effekt på akkumuleringen af -amyloid, hvilket også sker i ellers raske mennesker.

Objective: Dementia is a growing problem, especially in the industrial world, because the life expectancy is increasing. At the moment 10-30 % of the world’s population above 65 years of age are effected of Alzheimer’s disease, with most cases being sporadic. Many studies of Alzheimer’s disease have been made, but to date no one have been able to fully explain the different pathological hallmarks, how different factor affect the disease, e.g. stress and sleep deprivation, or why the disease develops in some people. This project aims to investigate the proteomes in hippocampus of both mice with Alzheimer’s disease (APPPS1-21 mice) and wild-type mice with and without exposure to chronic stress. Methods: To create an overview of the proteins in the different test subjects SDS-page was performed. Afterwards was filter-aided samples preparation (FASP) used to prepare the hippocampus samples for label free quantification using a Thermo-Electron QExactive HF-X mass spectrometer (ThermoFisher Scientific). PERSEUS (Max Planck Institute of biochemistry, v1.6.5.0) and PEAKS Studio X (Bioinformatics Solutions Inc.) were used in order to work with the data, make figures, and any statistical analyses, e.g. volcano plots, Hawaii plots, and Principal Component Analysis (PCA). Results: The results of the SDS-page did not show any differences between the different mice or between test groups. Both volcano plots and the Hawaii plots showed a significant number proteins that have been either upregulated or downregulated in the different test groups, including proteins involved in the circadian cycle, as well as ApoE, APP, and GFAP, which all are suspected as being involved with Alzheimer’s disease. The PCA showed no clear groups of proteins involved in the way the results grouped except those involved in the circadian cycle. Conclusion: The experiments resulted in several interesting results for the research in Alzheimer’s disease. ApoE, which has been implicated in the risk of developing familiar Alzheimer’s disease, was found significantly increased in APPPS1-21, whereas it was decreased in mice that have been exposed to stress, and in APPPS1-21 + CMS to an extend that it similar to the wild-type mice. Several proteins involved in the circadian cycle was also found to significantly altered in all test groups compared to the healthy wild-type mice, which can be related to the studies of the effect of sleep deprivation on the accumulation of -amyloid, also in healthy test subjects.

A master thesis from Aalborg University

Proteome analysis of APPPS1-21 and wild-type mice - A novel chronic stress model