Optimization of an in vitro model to elucidate the role of Ciita levels on alpha-synuclein uptake, processing and spread
(2022) MOBN03 20212Degree Projects in Molecular Biology
- Abstract
- Parkinson's disease (PD) is a neurodegenerative disease that is characterized by alpha-synuclein (α-syn) aggregations and loss of dopaminergic neurons. The cause for PD is still being researched but neuroinflammation has gained increasing awareness. Polymorphism in class II, major histocompatibility complex, transactivator (Ciita), which regulates major histocompatibility complex II (MHCII) expression, has shown to affect the aggregation and spread of α-syn in vivo. However, it is unknown how different levels of MHCII affects α-syn uptake, processing and spread. This project aimed to optimize an in vitro setup to study the relation between MHCII levels to α-syn uptake, processing and spread. Interferon gamma (IFN-γ) was used to stimulate... (More)
- Parkinson's disease (PD) is a neurodegenerative disease that is characterized by alpha-synuclein (α-syn) aggregations and loss of dopaminergic neurons. The cause for PD is still being researched but neuroinflammation has gained increasing awareness. Polymorphism in class II, major histocompatibility complex, transactivator (Ciita), which regulates major histocompatibility complex II (MHCII) expression, has shown to affect the aggregation and spread of α-syn in vivo. However, it is unknown how different levels of MHCII affects α-syn uptake, processing and spread. This project aimed to optimize an in vitro setup to study the relation between MHCII levels to α-syn uptake, processing and spread. Interferon gamma (IFN-γ) was used to stimulate BV2 mouse microglia cells, a cell line often used in in vitro models. The protein levels of microglial markers Iba1 and MHCII were measured using Western blot. qPCR was a method used to measure the mRNA expression of Ciita to see if IFN-γ stimulated the gene in BV2 cells. mRNA levels of Cd74, a component of MHCII formation and trafficking, was also measured using qPCR, together with Tnfα and Iba1. Immunocytochemistry was used to see if BV2 cells were activated by IFN-γ. The results show no clear activation of BV2 cells using IFN-γ. The conclusion is to set up a clear protocol for BV2 activation using IFN-γ. In that way, the project will form the basis for studying HMC3 human microglia cells and primary rat microglia together with silencing Ciita with shRNA. The effect of Ciita levels on α-syn uptake, processing and spread, will then be better understood. (Less)
- Popular Abstract
- CIITA: A key player in Parkinson's diseased brains?
The ability to move voluntarily is not often thought about until that ability is lost. Parkinson's disease (PD) is a neurodegenerative disease in the brain that affects your ability to move. A fundamental cause of PD is a protein called alpha-synuclein (α-syn) which, when aggregated, leads to insoluble masses called Lewi bodies. These Lewi bodies cause neurons involved in the voluntary motor movement to die. The immune system recognizes α-syn as a foreign molecule, also known as an antigen. This activity launches an immune response in the brain. Microglia, the antigen-presenting cells in the brain, surveys the environment, uptakes the antigens, and communicates the information to... (More) - CIITA: A key player in Parkinson's diseased brains?
The ability to move voluntarily is not often thought about until that ability is lost. Parkinson's disease (PD) is a neurodegenerative disease in the brain that affects your ability to move. A fundamental cause of PD is a protein called alpha-synuclein (α-syn) which, when aggregated, leads to insoluble masses called Lewi bodies. These Lewi bodies cause neurons involved in the voluntary motor movement to die. The immune system recognizes α-syn as a foreign molecule, also known as an antigen. This activity launches an immune response in the brain. Microglia, the antigen-presenting cells in the brain, surveys the environment, uptakes the antigens, and communicates the information to immune cells. These microglia have a protein called major histocompatibility complex II (MHCII), which is involved in antigen presentation. CIITA is a gene that regulates the expression of MHCII. Variation in the gene alters the severity of neurodegeneration in PD rat models. Another study has linked Ciita variability to microglial α-syn uptake, processing, and spread. The project aimed to study this effect in microglial cells.
The project's primary method was using mouse microglial cells, called BV2 cells. A protein called IFN-γ was used to stimulate the cells in three different concentrations and durations. Western blot, a method to detect proteins, was used to detect the upregulation of MHCII, among other proteins. Similarly, qPCR, another technique used to quantify mRNA expression, measured the mRNA expression of the genes involved in BV2 and Ciita activation.
However, the results show limited activation of BV2 cells using IFN-γ. These results were because of the high variability in the data. The conclusion is to set up a clear protocol for BV2 cell activation using IFN-γ. In that way, the project will form the basis for future research studying the effect of Ciita levels on α-syn uptake, processing, and spread. When the role of Ciita on α-syn is established, medical therapeutics can be designed to help cure PD.
Master’s Degree Project in Molecular Biology 60 credits 2022
Department of Biology, Lund University
Advisor: Maria Swanberg
Translational Neurogenetics, A10, Department of Experimental Medical Science (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9103402
- author
- Zolfeghari, Sam
- supervisor
- organization
- course
- MOBN03 20212
- year
- 2022
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 9103402
- date added to LUP
- 2022-11-21 14:46:45
- date last changed
- 2022-11-21 14:46:45
@misc{9103402,
abstract = {{Parkinson's disease (PD) is a neurodegenerative disease that is characterized by alpha-synuclein (α-syn) aggregations and loss of dopaminergic neurons. The cause for PD is still being researched but neuroinflammation has gained increasing awareness. Polymorphism in class II, major histocompatibility complex, transactivator (Ciita), which regulates major histocompatibility complex II (MHCII) expression, has shown to affect the aggregation and spread of α-syn in vivo. However, it is unknown how different levels of MHCII affects α-syn uptake, processing and spread. This project aimed to optimize an in vitro setup to study the relation between MHCII levels to α-syn uptake, processing and spread. Interferon gamma (IFN-γ) was used to stimulate BV2 mouse microglia cells, a cell line often used in in vitro models. The protein levels of microglial markers Iba1 and MHCII were measured using Western blot. qPCR was a method used to measure the mRNA expression of Ciita to see if IFN-γ stimulated the gene in BV2 cells. mRNA levels of Cd74, a component of MHCII formation and trafficking, was also measured using qPCR, together with Tnfα and Iba1. Immunocytochemistry was used to see if BV2 cells were activated by IFN-γ. The results show no clear activation of BV2 cells using IFN-γ. The conclusion is to set up a clear protocol for BV2 activation using IFN-γ. In that way, the project will form the basis for studying HMC3 human microglia cells and primary rat microglia together with silencing Ciita with shRNA. The effect of Ciita levels on α-syn uptake, processing and spread, will then be better understood.}},
author = {{Zolfeghari, Sam}},
language = {{eng}},
note = {{Student Paper}},
title = {{Optimization of an in vitro model to elucidate the role of Ciita levels on alpha-synuclein uptake, processing and spread}},
year = {{2022}},
}