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Investigating the effect of Aβ overexpression on tau phosphorylation

Palm, Lina (2020) MOBN03 20192
Degree Projects in Molecular Biology
Abstract
The breakdown of neuronal networks and synaptic degeneration are responsible for the memory impairment associated to Alzheimer’s disease (AD). Amyloid-β (Aβ) and tau are the two proteins that have been identified as the hallmarks for pathological changes found in the brain of Alzheimer’s patients. These two hallmarks have been extensively studied separately, but how they relate to each other and their possible synergistic effect is less understood. Therefore, the aim of this project was to determine the effect of Aβ overexpression on tau phosphorylation by measuring changes in fluorescence intensity of phospho-tau epitopes. This question was addressed in one approach with two comparisons. Two amyloid precursor protein (APP) transfected... (More)
The breakdown of neuronal networks and synaptic degeneration are responsible for the memory impairment associated to Alzheimer’s disease (AD). Amyloid-β (Aβ) and tau are the two proteins that have been identified as the hallmarks for pathological changes found in the brain of Alzheimer’s patients. These two hallmarks have been extensively studied separately, but how they relate to each other and their possible synergistic effect is less understood. Therefore, the aim of this project was to determine the effect of Aβ overexpression on tau phosphorylation by measuring changes in fluorescence intensity of phospho-tau epitopes. This question was addressed in one approach with two comparisons. Two amyloid precursor protein (APP) transfected Neuroblastoma-2a (N2a) cell lines (WT or Swedish mutation APP) were co-transfected with two different tau constructs (WT or P301L mutation tau).

The first comparison was measuring the fluorescence intensity of tau antibodies between constructs when expressed in the APP transfected cell lines to evaluate the constructs behaviour in Aβ overexpressing cells. The second comparison investigated the effect of Aβ overexpression by measuring the tau antibody fluorescence intensity of APP WT neurons compared to APPswe neurons (expressing the tau constructs). In our results we found a trend indicating that there was no significant difference in tau intensity when comparing the Tau WT and Tau P301L constructs in either APP cell line. In addition to this, it could also be observed that both APP cell lines without any transfected tau constructs showed an overall lower tau antibody fluorescence intensity compared to cells co-transfected with either construct. These observations indicated that the constructs were functional and that the transfections had been successfully performed. However, when observing the results of APP overexpression and its effect on tau phosphorylation, there was no clear difference in phospho-tau antibody fluorescence intensity comparing the two cell lines.

Due to lack of semi-quantitative data obtained in this project it was not feasible to draw any conclusions on how Aβ overexpression effects tau phosphorylation. However, this project provided an insight into the behaviour of the tau constructs when expressed in APP transfected cells, which could be used as a foundation for further experiments and contributes to a higher understanding of AD pathology. (Less)
Popular Abstract
The interplay between Amyloid-β and tau in Alzheimer’s Disease

Dementia is characterised by a progressive cognitive decline and Alzheimer’s Disease (AD) comprise 60-80% of all dementia cases. Environmental as well as genetic factors are believed to increase the risk of AD, but age is considered to be the principle risk factor. The degeneration of neurons in AD is characterised by the build-up of two proteins in the brain; amyloid-β-containing plaques and neurofibrillary tangles (NFTs), composed of hyperphosphorylated tau.

In this project the aim was to determine the effect that Aβ has on tau phosphorylation when either is being overexpressed. This was performed by comparing the levels of tau protein in two mouse Neuroblastoma-2a... (More)
The interplay between Amyloid-β and tau in Alzheimer’s Disease

Dementia is characterised by a progressive cognitive decline and Alzheimer’s Disease (AD) comprise 60-80% of all dementia cases. Environmental as well as genetic factors are believed to increase the risk of AD, but age is considered to be the principle risk factor. The degeneration of neurons in AD is characterised by the build-up of two proteins in the brain; amyloid-β-containing plaques and neurofibrillary tangles (NFTs), composed of hyperphosphorylated tau.

In this project the aim was to determine the effect that Aβ has on tau phosphorylation when either is being overexpressed. This was performed by comparing the levels of tau protein in two mouse Neuroblastoma-2a (N2a) cell lines as they were transfected with different tau constructs. The cell lines (APP WT and mutant APPswe) expressed different levels of Aβ and they were transfected with either tau WT, mutant tau P301L or no additional tau at all.

These comparisons were performed by measuring the fluorescence intensity of the tau protein in cells. Tau was stained with different antibodies that emits a fluorescence light when they bind to tau and this was captured by taking images through a fluorescence microscope (as can be seen in the figure). The amount of light detected from the cells are correlated to the amount of tau present in the cells. An imaging analysis software ‘CellProfiler’ was used to transform the fluorescent light into semi- quantitative data that could be compared through statistical analyses.

The effects of Aβ overproduction on tau levels
No significant difference in tau intensity when comparing the Tau WT and Tau P301L constructs in either cell line could be noted. As expected, it was observed that both cell lines without any transfected tau constructs, showed an overall lower tau fluorescence intensity compared to the cells that were transfected with either tau construct. However, when observing the results of Aβ overproduction, it showed no clear difference in tau levels when the two different APP cell lines were compared to one another.

It is clear that Aβ and tau are biologically linked, but it was not feasible to draw any exact conclusions on how Aβ overexpression effects tau phosphorylation, owing to a low sample size and the lack of quantitative biochemical data. Even though the overarching goal for this project could not be met it still offered an insight into the differences between Tau constructs when expressed in cells overproducing Aβ. The tau constructs could be used for future studies and this project was a step in the direction of setting up tools to further our understanding of the link between tau and Aβ.

Master’s Degree Project in Molecular Biology, 60 credits, 2020
Department of Biology, Lund University

Advisors: Gunnar Gouras and Laura Torres-Garcia
Department of Experimental Medical Science, BMC, Lund University (Less)
Please use this url to cite or link to this publication:
author
Palm, Lina
supervisor
organization
course
MOBN03 20192
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9012687
date added to LUP
2020-06-04 09:19:09
date last changed
2020-06-04 09:19:09
@misc{9012687,
  abstract     = {The breakdown of neuronal networks and synaptic degeneration are responsible for the memory impairment associated to Alzheimer’s disease (AD). Amyloid-β (Aβ) and tau are the two proteins that have been identified as the hallmarks for pathological changes found in the brain of Alzheimer’s patients. These two hallmarks have been extensively studied separately, but how they relate to each other and their possible synergistic effect is less understood. Therefore, the aim of this project was to determine the effect of Aβ overexpression on tau phosphorylation by measuring changes in fluorescence intensity of phospho-tau epitopes. This question was addressed in one approach with two comparisons. Two amyloid precursor protein (APP) transfected Neuroblastoma-2a (N2a) cell lines (WT or Swedish mutation APP) were co-transfected with two different tau constructs (WT or P301L mutation tau). 

The first comparison was measuring the fluorescence intensity of tau antibodies between constructs when expressed in the APP transfected cell lines to evaluate the constructs behaviour in Aβ overexpressing cells. The second comparison investigated the effect of Aβ overexpression by measuring the tau antibody fluorescence intensity of APP WT neurons compared to APPswe neurons (expressing the tau constructs). In our results we found a trend indicating that there was no significant difference in tau intensity when comparing the Tau WT and Tau P301L constructs in either APP cell line. In addition to this, it could also be observed that both APP cell lines without any transfected tau constructs showed an overall lower tau antibody fluorescence intensity compared to cells co-transfected with either construct. These observations indicated that the constructs were functional and that the transfections had been successfully performed. However, when observing the results of APP overexpression and its effect on tau phosphorylation, there was no clear difference in phospho-tau antibody fluorescence intensity comparing the two cell lines.

Due to lack of semi-quantitative data obtained in this project it was not feasible to draw any conclusions on how Aβ overexpression effects tau phosphorylation. However, this project provided an insight into the behaviour of the tau constructs when expressed in APP transfected cells, which could be used as a foundation for further experiments and contributes to a higher understanding of AD pathology.},
  author       = {Palm, Lina},
  language     = {eng},
  note         = {Student Paper},
  title        = {Investigating the effect of Aβ overexpression on tau phosphorylation},
  year         = {2020},
}