An invitro model for the study of perivascular clearance of amyloid. Relevance to Alzheimer's disease.
Lead Research Organisation:
University of Southampton
Department Name: Clinical and Experimental Sciences
Abstract
One of the major features of the ageing brain is the deposition of proteins like amyloid-beta (Abeta) in the walls of capillaries and arteries. Abeta is deposited preferentially along fused endothelial-astroglial capillary basement membrane and the basement membrane surrounding vascular smooth muscle cells in larger vessels, surprisingly it not found in arterial endothelial or astroglial basement membranes. As these matricies have different composition this suggests a component specific deposition. The brain lacks traditional lymphatic vessels for the elimination of proteins and solutes. Interstitial fluid of the brain is cleared along the basement membranes of capillaries and arteries, effectively the lymphatic drainage
pathways of the brain and this process fails with ageing and with possession of Apolipoprotein E4 (ApoE4) genotype.
In this project we are aiming to firstly create in vitro a model of the intramural perivascular drainage pathways, consisting of the basement membranes of endothelial, vascular smooth muscle, pericyte and astrocyte cells. Secondly, we aim to flow soluble amyloidbeta over the cell types, in an effort to create the first in vitro model of perivascular drainage of Abeta, relevant to the pathogenesis of Alzheimer's disease.
pathways of the brain and this process fails with ageing and with possession of Apolipoprotein E4 (ApoE4) genotype.
In this project we are aiming to firstly create in vitro a model of the intramural perivascular drainage pathways, consisting of the basement membranes of endothelial, vascular smooth muscle, pericyte and astrocyte cells. Secondly, we aim to flow soluble amyloidbeta over the cell types, in an effort to create the first in vitro model of perivascular drainage of Abeta, relevant to the pathogenesis of Alzheimer's disease.
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M014932/1 | 01/10/2015 | 30/09/2019 | |||
| 1673877 | Studentship | BB/M014932/1 | 01/10/2015 | 30/09/2019 | Abby Keable |
| Description | Alzheimer's is a disease that causes dementia and is responsible for approximately 2/3 of all dementia cases. In Alzheimer's disease the brain is no longer able to efficiently remove waste and instead it begins to accumulate in the tiny drainage channels along the walls of cerebral blood vessels. Age is a major risk factor for development of Alzheimer's disease and people with a certain genetic background (ApoE4) are also at higher risk. The tiny channels for the drainage of waste, known as basement membranes, are too small to be seen on scans such as MRI or PET and cannot be easily accessed for experiments. My project involves using the cells that make up a cerebral blood vessel and assembling them in vitro to create a model of a blood vessel and its associated basement membrane that is easy to handle, easy to image and easy to manipulate experimentally. There are many stages involved in assembling this complex multi-cell model from characterising properties of individual cell lines to finding the optimum number of cells of each type to grow together to calibrating and optimising the application of flow. This award is still active therefore this system is still a work in progress however each stage has yielded significant findings. Key findings are as follows: Each cell type produces a specific set of components that makes up the channels for drainage (basement membranes) and these are unique for each cell type in the brain. This may help to explain the route that waste travels along as it exits the brain. The number of cells, their shape and the basement membranes are altered with an ApoE4 genotype providing a possible explanation for the earlier and more sever accumulation of waste in individuals with and ApoE4 genetic background. The millifluidics system provided by Kirkstall can be used to apply a flow of fluid to cells that is of sufficient speed and pressure to mimic the drainage of waste in the brain. Cells expressing ApoE4 are not able to remove waste as efficiently as ApoE2 or ApoE3 cells when flow was applied with the millifluidics system supporting the earlier findings that differences in cell shape and basement membrane composition affect the ability to efficiently remove waste. |
| Exploitation Route | This project is still active and as such not all of the research objectives are complete. Once the model is complete people will be able to use it as a tool to test whether drainage along the walls of cerebral blood vessels can be improved to prevent the accumulation of waste. This is an area of interest for pharmaceutical companies and is a potential target for therapeutics. There are many research groups that use the cells that I use for my model for various purposes and the information obtained about the composition of the basement membranes may prove useful for these groups and help to further their research. Also the data obtained showing the differences between cells of different ApoE genotypes may help us understand why ApoE4 increases the risk of Alzheimer's disease. |
| Sectors | Education,Pharmaceuticals and Medical Biotechnology |
| Title | in vitro model of intramural drainage of amyloid beta using millifluidics |
| Description | Using the Quasi Vivo system developed by Kirkstall Ltd and human brain vascular smooth muscle cells purchased from Sciencell I was able to produce a flow of different species of amyloid beta to a layer of smooth muscle cells and assess the impact on the cells and their basement membranes. The interconnected cell culture flow system was set up according to manufacturer instructions and calibrated for a four chamber set-up. A coverslip containing a layer of smooth muscle cells was loaded into each chamber and the system was filled with culture media that contained either 100nM amyloid beta 1-40, 100nM amyloid beta 1-42 or 100nM dextran as a vehicle control. The peristaltic pump was set to an outlet speed of 10ul/min to mimic the flow of interstitial fluid and the whole system was placed inside an incubator for 48 hours. Cells were fixed with formaldehyde and immunostained to assess the profile of the basement membrane proteins and examined with confocal microscopy. |
| Type Of Material | Model of mechanisms or symptoms - in vitro |
| Year Produced | 2019 |
| Provided To Others? | No |
| Impact | My results indicate that it is amyloid beta 1-42 that had the most significant impact on the basement membrane of smooth muscle cells causing a significant decrease in collagen IV and laminin. This likely causes a dynamic remodelling of the basement membrane and disrupts the glycoprotein:proteoglycan ratio which is though to impede intramural periarterial drainage and result in cerebral amyloid angiopathy. Amyloid beta 1-42 is typically found in plaques but was found to be deposited in the cerebral vessels of patients receiving amyloid beta 42 immunotherapy so my results provide a plausible explanation as to the failure of the trials and the increased CAA seen in the patients post-mortem. |
| Description | Oral presentation at the Advances in Cell and Tissue Culture conference (22nd-24th May 2017) |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Presented an overview of the aims and objectives of my project to an audience with an interest in cell and tissue culture and animal replacement. Also presented preliminary findings using the Quasi Vivo millifluidics system provided by my industrial partners (Kirkstall) to the other users of the system who were in attendance at the conference. Also took part in a Quasi Vivo user group meeting, which took place after the conference, where all the users of the various Quasi Vivo systems have the opportunity to informally talk about their use of the system and any problems associated with it, which helps with troubleshooting and product development. |
| Year(s) Of Engagement Activity | 2017 |
| Description | Oral presentation at the British Neuropathological Society annual meeting |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | As a result there is potential for a collaboration with Sheffield as my results complement their own findings |
| Year(s) Of Engagement Activity | 2018 |
| Description | Poster presentation at the 120th meeting of the British Neuropathological Society |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Presenting an academic poster with latest research findings to the British Neuropathological Society. This gave me the opportunity to discuss my work with other people in similar research fields and also see what other work is being done in my field of research. |
| Year(s) Of Engagement Activity | 2019 |