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Analysis of alpha-synuclein expression in a novel preclinical rodent model of Parkinson’s disease
Sweden (IFMSA-Sweden) - Lunds Universitet, Lund
Department of experimental medical science
Type of Research Project
- Basic science
What is the background of the project?
The goal of the current project is to develop a novel preclinical rodent model for parkinson’s disease which displays stable behavioural deficits for the purpose of assessing the functionality of stem cell based therapies. Current preclincial rodent models of Parkinson’s disease are based on neurotoxins and do neither fully recapitulate the progressive nature of the disease nor the accumulation of proteins throughout the brain. In the current project we utilise Adeno Associated Viral vectors to overexpress the human alpha-synucelin protein in the midbrain of rats. This overexpression leads to the development of relevant pathology in the nigro-striatal dopamine system in forms of neuronal degeneration, formation of lewy-body like inclusions, denervation of dopamibergic fibres in the striatum as well as axonal dystropy and impaired dopamine release adn reuptake kinetics. Previous studies have shown that the degeneration of the nigrostriatal dopamine system is highly variable using this approach and only 25% of rats do develop a behavioural phenotype. In the current study we aim to aggravate the pathology by co-injection of preformed fibrils which will serve as a template for the misfolding of the alphasynuclein. We will assess wild-type rats of the Sprague-Dawley strian on a series of behavioural tests that has been shown to be sensitive to dopaminergic manipulations. We will then either inject the AAV-vector and Preformed Fibrils simultaneously or sequentially (4 week later) into the midbrain. Behavioural assessments will be conducted in 4 week intervals until the brains are harvested 12 weeks post surgery.
What is the aim of the project?
The aim of the project is the development of a novel preclincial rodent model of Parkinson’s disease. The model shoudl display a stable behavioural phenotype for the assessment of cell based therapies. The tasks of the students in this project involves the post-mortem analysis of the brains to 1.) investigate the pathology and 2.) correlate the pathological changes with the behavioural impairments.
What techniques and methods are used?
The project involves molecular cloning of viral plasmids, production of AAV vectors, behavioural assessment of rodents, stereotaxic surgery, transcardial perfusion, dissection of brain tissue. After generation of the AAV-vector and production of the PFFs we will inject these into the animals which have been stratified into behaviourally matching groups (see above). We will havest the brains after all behavioural data has been collated and use the tissue for post-processing as described below: The methods that will be used are: Sectioning brains on a microtome into 35um thick 1:12 coronal series for the immunohistochemical analysis. Performing Immunohistochemical stainings using antibodies against TH, VMAT, aSyn, pSer129, GFP, NeuN, as well as Iba1/Ox42 (cd11b). These markers will give an indication of dopaminergic degeneration/denervation, development of pathology and immune responses. Analysis of brains through imaging methods. The stained sections will be immaged using standard ligh based microscopy as well as confocal microscopy to assess and quantify the degeneration/denervation of the dopaminergic system as well as the co-expression of proteins inside the dopaminergic system.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
The student is expected to section brains into coronal slices on a freezing sledge microtome. This will be followed by immunohistochemical stainings sin antibodies, for several proteins (see techniques) that are relevant. The stained sections will then be mounted on glass coverslips for quantifications. The student will then quantify the density of the dopaminergic fibre denervation and co-expression of alpha-synuclein related pathology in the dopaminergic cells of the midbrain.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Theoretical teaching will be done when the student arrives. There will be background literature. We have semiars, journal clubs, and labmeetings where the student will participate.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- No specific outcome is expected
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Pre-Medical students from the American-British system - Students in biomedical fields
- Progressive neurodegenerative and behavioural changes induced by AAV-mediated overexpression of α-synuclein in midbrain dopamine neurons. Neurobiology of Disease;Volume 45; Issue 3; March 2012; Pages 939-953 (https://doi.org/10.1016/j.nbd.2011.12.013)
- Chapter 5 - Viral vector-mediated overexpression of α-synuclein as a progressive model of Parkinson’s disease. Progress in Brain Research;Volume 184; 2010; Pages 89-111 (https://doi.org/10.1016/S0079-6123(10)84005-1)
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