Application of molecular barcoding and engineered viral vectors for the determination of causative transcriptomic changes in Parkinson’s disease and generation of a fluorescent reporter model for studying cell-cell communication in the brain

Abstract

Parkinson’s disease (PD) is a neurodegenerative disorder with toxic forms of alpha synuclein (α-syn) accumulating in the midbrain dopaminergic (DA) neurons, leading to cell death. In the first part of the study we wanted to investigate the midbrain DA population and their gene expression relative to the intracellular α-syn load. To this end, human wildtype α-syn was packaged inside custom, uniquely barcoded adeno-associated virus vectors (AAVs) and injected into the substantia nigra of the midbrain of TH-Cre mice. This enabled an expression of AAVs specifically in the DA cells. Genes belonging to several pathways were enriched as the α-syn load increased, particularly genes involved in calcium signalling.

Arc protein is essential in synaptic functioning and memory due to its ability to self-assemble into a capsid-like structure and it has been proposed to be able to transfer mRNA across synapses. However, evidence for this is lacking. For the second project, we aimed to knock in a fluorescent protein into the Arc N-terminal domain in mouse using HiTi (CRISPR/Cas9 Homologus Independent Targeted Integration). To this end a bioinformatic pipeline was developed to assess the frequency of correctly formed fusion genes and the translated proteins when different integration sites on Arc were used. A site was found at the 5’-end of Arc, termed 9[+], which successfully produced a fluorescent Arc reporter model, thus enabling the future studies of intercellular transfer of Arc in an intact healthy and diseased brain.

Popular Abstract

What happens in brain cells with a gradual increase in a toxic protein in Parkinson’s disease and where can one attach a glowing tag in a protein that is believed to transfer RNA between brain cells?

Understanding gene changes the dopamine producing cells undergo when alpha synuclein builds up in them
Parkinson’s disease (PD) is one of the most common neurodegenerative diseases characterised by a build-up of a toxic form of a protein alpha synuclein in the dopamine producing cells of the midbrain. Dopamine is important for functions such as movements, which is why PD patients tend to have issues such as rigidity and slowness. As the alpha synuclein builds up in the dopamine cells, it becomes increasingly difficult for the cells to work as they normally would. This eventually leads to the death of these cells. However, we do not yet know exactly why they die. This is because we do not know how the gene expression changes relative to the gradual increase of this protein in the dopamine cells. For the first part of the project, we have designed viruses specific for dopamine cells with alpha synuclein packaged in them. Each alpha synuclein RNA molecule also has a unique barcode attached to them. An RNA molecule can generally be seen as a recipe for a protein and is transcribed from a given gene. We use single cell RNA sequencing methods to capture the RNA inside the cells. On top of this we can also count the number of alpha synclein molecules within a given cell due to them possessing a unique barcode which we can detect with the sequencing methods. Thus, we can detect which genes’ mRNA is found in cells and how this changes based on the number of alpha synclein molecules found in them.

We discovered genes relating to calcium signalling were consistently increased once there were more than 3 alpha synuclein copies in the dopamine producing cells. This may for instance be due to alpha synuclein blocking the ability of the cells to transmit signals between one another. Calcium is crucial in this process and thus the cells may try to compensate for the disruption by increasing calcium levels. Furthermore, internal communication mechanisms of cells termed MAPK and cAMP were also increased when the cell contained between 4-15 copies. These mechanisms are used inside cells to transmit multiple different signals for example when cells kill themselves. The maximum number of copies the cell contained was 93. Furthermore, alpha synuclein also infected a small portion of other than dopamine producing cells. In these cells, the kind of genes relating to potential overstimulation of the cells were detected. This may be in response to poorer functioning of these cells as the alpha synuclein may disturb some of the important processes. However, the results were limited and it was not possible to capture the full breadth of the viral barcodes and thus the alpha synuclein copies. Nevertheless the results provide a preliminary idea of the kind of changes occurring in the cells at the different stages of the alpha synuclein’s build-up.

Finding a site to stick a fluorescent tag in the Arc gene
Arc is a protein involved in cell-cell communication processes such as memory and it has been shown to be affected in neurodegenerative diseases. It is able to self-assemble into viral capsid-like structures and it has been theorised that the protein could this way transport its mRNA between cells. Nevertheless, the evidence for the transport of Arc between cells is lacking. A development of a fluorescent reporter model of Arc would enable its tracking in healthy and diseased brain. Fluorescent refers to a chemically glowing protein which can then be tracked under a microscope. The fluorescent tag can be packaged inside viruses which can subsequently be injected into cells. On top of this, we also inject a CRISPR/Cas9-based system named HITI which enables the cutting of the cell’s DNA at the location we want. Adding these things together enables us to insert the fluorescent tag at a location of interest after the site has been cut. In other words the gene can be seen as a strip of paper containing a recipe for a cookie which we can cut at different sites using “gene scissors” and then glue a new piece of paper, i.e. the tag, in between these strips. However, it can be difficult to know where in the Arc gene we can add the tag without disrupting the gene’s ability to give instructions for the creation of a protein that works as usual. Thus, we tried out cutting the Arc gene at multiple sites using HiTi and then adding the tag into such parts. Afterwards we retrieved the Arc DNA. To analyse this data we developed a bioinformatic pipeline to find the frequency of correctly formed Arc genes with a fluorescent tag in them. The pipeline also assessed how many of them formed a functional protein. Out of different sites where we added the tag, we found only a single site which produced a high frequency of successfully tagged Arc genes and the subsequent functional protein. The protein’s functionality was further confirmed by stimulating a memory mechanism in a certain area of the rat brain. We could see that Arc with a fluorescent tag in it became highly expressed in this area, indicating that the protein has retained its function.

Supervisor: Tomas Björklund
Group: Molecular neuromodulation group, Wallenberg Neurocenter

• author: Räsänen, Atte Oskari
• supervisor: Tomas Björklund
• organization: Degree Projects in Bioinformatics
• course: BINP51 20221
• year: 2023
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9113242
• date added to LUP: 2023-04-05 15:29:58
• date last changed: 2023-04-05 15:29:58