Tauopathy models in Neuro2a and C6 cell-lines, regarding Alzheimer's disease
(2023) MOBN03 20231Degree Projects in Molecular Biology
- Abstract
- Alzheimer’s disease is classified as a neurodegenerative disease and have the misfolding and aggregation of the protein Tau as an underlying cause. Tau loses its regular function to support the cytoskeleton and to protect DNA and RNA. The loss of function results in prion-like spread where regular Tau can bind and add to the formation of fibrils or neurofibrillary tangles.
The aim with this thesis was to create cell models in Neuro2a and C6 cells, respectively, which were transfected with Tau fibrils. These fibrils were observed utilizing flow cytometry and complemented with immunoimaging in which Tau and GFP antibodies were utilised. The analysis method “flow cytometric analysis of inclusions and trafficking” or FloIT was adapted and... (More) - Alzheimer’s disease is classified as a neurodegenerative disease and have the misfolding and aggregation of the protein Tau as an underlying cause. Tau loses its regular function to support the cytoskeleton and to protect DNA and RNA. The loss of function results in prion-like spread where regular Tau can bind and add to the formation of fibrils or neurofibrillary tangles.
The aim with this thesis was to create cell models in Neuro2a and C6 cells, respectively, which were transfected with Tau fibrils. These fibrils were observed utilizing flow cytometry and complemented with immunoimaging in which Tau and GFP antibodies were utilised. The analysis method “flow cytometric analysis of inclusions and trafficking” or FloIT was adapted and utilised for aggregates found with flow cytometry.
The aim was accomplished by utilising lentiviral vectors adapted to each cell-line through a titration experiment to transduce cells with GFP and Tau-GFP, respectively. Optimisation experiments were adapted for each cell-line in a single biological replicate setup were verified with a biological triplicate sample setup experiment before finalisation of the cell models.
FloIT proved to be a sufficient analysis method of aggregates, even with autofluorescence problems. These problems turned up as a streak-like pattern in both cell-lines and could be due to under-compensation of lasers. The cell model in Neuro2a cells was successful in both FloIT and immunoimaging. However, C6 cells were overseeded for immunoimaging and this resulted in difficulties for the analysis program to define cells and aggregates, which showed the need for further optimisation of the method. (Less) - Popular Abstract (Swedish)
- Cellmodell för analys av Tau klumpar vid Alzheimers sjukdom
Sjukdomar som drabbar hjärnan kan klassas som neurodegenerativa och ge olika symptom, exempelvis demens, svårt med finmotorik och svårt att finna ord. Både Alzheimers sjukdom och Parkinsons sjukdom delar dessa symptom, fastän det finns olika bakomliggande orsaker. Denna sammankoppling mellan olika neurodegenerativa sjukdomars symptom pekar mot att det finns ett större sammanhang mellan de bakomliggande orsakerna. Detta bidrar till en komplexitet som utmanar nutida forskning.
Insjuknandet i Alzheimers sjukdom sker oftast vid 60-85 år. En identifierad bakomliggande orsak är klumpbildningen av proteinet Tau. Tau är viktigt för cellers interna skelett och skyddandet av... (More) - Cellmodell för analys av Tau klumpar vid Alzheimers sjukdom
Sjukdomar som drabbar hjärnan kan klassas som neurodegenerativa och ge olika symptom, exempelvis demens, svårt med finmotorik och svårt att finna ord. Både Alzheimers sjukdom och Parkinsons sjukdom delar dessa symptom, fastän det finns olika bakomliggande orsaker. Denna sammankoppling mellan olika neurodegenerativa sjukdomars symptom pekar mot att det finns ett större sammanhang mellan de bakomliggande orsakerna. Detta bidrar till en komplexitet som utmanar nutida forskning.
Insjuknandet i Alzheimers sjukdom sker oftast vid 60-85 år. En identifierad bakomliggande orsak är klumpbildningen av proteinet Tau. Tau är viktigt för cellers interna skelett och skyddandet av arvsmassa. Klumpbildningen av Tau leder till att nervceller som vanligtvis liknar cypresser med hjälp av sitt interna skelett, nu liknar fotbollar och avlider för att de inte har kontakt med andra nervceller. I det långa loppet leder detta till förtvining av hjärnan.
Denna studie fokuserade på att skapa en cellbaserad modell för att kunna analysera klumpar av Tau genom två metoder. Flödescytometri och immuncytokemi analyserade Tau klumpar med olika lasrar där immuncytokemi visualiserar var klumparna befinner sig i cellen (Bild 1). Metoderna kompletterar varandra då båda kan analysera Tau klumpar i olika utsträckning.
För vidare utveckling av modellen kan man aktivera ett system i cellerna som städar bort Tau klumparna och annat ”skräp” som cellen inte har nytta av. Om systemet fungerar öppnas ett forskningsområde som möjliggör skapandet av läkemedel som kan aktivera systemet, vilket kan leda till en broms av Alzheimers sjukdomsförlopp.
Masterexamensprojekt i Molekylärbiologi 60 hp 2023
Biologiska institutionen, Lunds universitet
Handledare: Luís Quintino & Cecilia Lundberg
CNS Genterapi, BMC (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9145273
- author
- Gustavsson, Linnéa
- supervisor
- organization
- course
- MOBN03 20231
- year
- 2023
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 9145273
- date added to LUP
- 2024-01-16 14:27:19
- date last changed
- 2024-01-16 14:27:19
@misc{9145273,
abstract = {{Alzheimer’s disease is classified as a neurodegenerative disease and have the misfolding and aggregation of the protein Tau as an underlying cause. Tau loses its regular function to support the cytoskeleton and to protect DNA and RNA. The loss of function results in prion-like spread where regular Tau can bind and add to the formation of fibrils or neurofibrillary tangles.
The aim with this thesis was to create cell models in Neuro2a and C6 cells, respectively, which were transfected with Tau fibrils. These fibrils were observed utilizing flow cytometry and complemented with immunoimaging in which Tau and GFP antibodies were utilised. The analysis method “flow cytometric analysis of inclusions and trafficking” or FloIT was adapted and utilised for aggregates found with flow cytometry.
The aim was accomplished by utilising lentiviral vectors adapted to each cell-line through a titration experiment to transduce cells with GFP and Tau-GFP, respectively. Optimisation experiments were adapted for each cell-line in a single biological replicate setup were verified with a biological triplicate sample setup experiment before finalisation of the cell models.
FloIT proved to be a sufficient analysis method of aggregates, even with autofluorescence problems. These problems turned up as a streak-like pattern in both cell-lines and could be due to under-compensation of lasers. The cell model in Neuro2a cells was successful in both FloIT and immunoimaging. However, C6 cells were overseeded for immunoimaging and this resulted in difficulties for the analysis program to define cells and aggregates, which showed the need for further optimisation of the method.}},
author = {{Gustavsson, Linnéa}},
language = {{eng}},
note = {{Student Paper}},
title = {{Tauopathy models in Neuro2a and C6 cell-lines, regarding Alzheimer's disease}},
year = {{2023}},
}