VIB Ugent Center for Inflammation Research Roos Vandenbroucke Lab
Abstract bachelor project 2019-20 project 2: Unraveling the choroid plexus heterogeneity in health and disease
The choroid plexus is a highly vascularized tree-like structure, attached to the ventricle wall via a stalk region in all four ventricles of the brain. Preliminary studies show that the choroid plexus has a role in communication between body and brain, especially under inflammatory conditions.
In this project, we aimed to unravel the spatial and cellular heterogeneity of the choroid plexus in health and systemic inflammation.
Information about the different cell populations and their transcriptome of the choroid plexus in health and systemic inflammation are determined by single cell RNA-sequencing. This dataset will be validated by using a wide variety of technologies such as mRNA stainings using hybridization chain reaction and protein stainings using immunohistochemistry on specific choroid plexus tissue sections and whole mount samples. To validate the number and variety of immune cells in the choroid plexus and cerebrospinal fluid, flow cytometry is performed. For the spatial determination of immune cells, multiplex immune cell stainings by Akoya CODEX are performed.
In the single cell RNA sequencing dataset, two fibroblast subtypes in the choroid plexus were revealed. Spatial validation of the two subtypes was performed with mRNA stainings which gave multiple conclusions. Type I fibroblasts (Dpep1+) are always located in the stromal site while type II fibroblasts (Igfbp6+) are always located in the stalk regions. There is never overlap between the two fibroblasts types. The next step is studying their function in health and disease. A ConA blood vessel staining was successful to visualize the blood vessels in the choroid plexus. Akoya CODEX validated the presence of macrophage subtypes and absence of T and B cells in the choroid plexus during systemic inflammation. However, other immune cells were not identified. Other technologies like immunohistochemistry and flow cytometry can be used to study these cells. Flow cytometry validated the presence and quantification of macrophages, dendritic cells, monocytes and neutrophils and absence of T and B cells. The quantification results are in line with the single cell RNA-sequencing dataset.
To further unravel the choroid plexus heterogeneity and understanding its response on systemic inflammation, stainings targeting other cell types will be performed, in combination with the optimized ConA protocol. This way, the spatial location of the immune cells can be studied.
Abstract bachelor project 2019-20 project 1: Characterization of a gut-originating mouse model for Parkinson’s disease
Background: Parkinson’s disease (PD) is the second most common age-related neurodegenerative disorder after Alzheimer’s disease, affecting the motoric functions of patients. The dopaminergic neurons of PD patients are degenerated by the formation of misfolded, aggregated alpha-synuclein (α-syn) proteins. PD is characterized by its motoric symptoms, such as bradykinesia and tremor at rest. However, non-motor symptoms, such as constipation, precede the onset of motoric symptoms by at least two decades. Some studies have claimed that the α-syn pathology manifests in the gut and then traverses to the brain. The vagus nerve, a bundle of fibres that innervates major organs (e.g. the gut), seems to be the entry route to the brain.
Aim: The goal of this study is to prove the progression of the α-syn pathology from the gut to the brain by using a gut-wall mouse model. Additionally, a comparison in the α-syn species (human and mouse) is conducted, based on the quality controls established to validate the successful aggregation of the α-syn.
Methods: The spreading of misfolded α-syn from the gut to the brain is performed by injecting mouse models into two locations of the pyloric stomach and upper duodenum. After some time, the gut tissues as well as the brain are extracted. The inflammatory gene expression in the gut tissues is detected as well as through western blotting. An additional colorimetric staining of the brain is performed. The human and mouse α-syn proteins are compared by quality controls, such as the turbidity (visual) test, the sedimentation assay, the thioflavin T fluorescence assay, the transmission electron microscopy visualisation and the blue native PAGE.
Results: The quality controls of both human and mouse α-syn showed a reliable generation of pre-formed fibrils. The mouse α-syn showed faster aggregation of monomeric α-syn in the thioflavin T fluorescence assay. The aggregated α-syn of the mouse is much longer than that of the human. But the shortened fibrils of the mouse α-syn are much smaller than the human α-syn, despite same conditions used. The inflammatory gene expression results were dismissible, due to the fact that the results could not be statistically reliably interpreted. The western blot did not detect any α-syn protein. The colorimetric staining of the brain showed a significant activation of microglia at the dorsal motor nucleus of the vagus nerve level.
Conclusion: For the inflammatory gene expression analysis a bigger experimental mouse model needs to be used. To verify the results of the western blot, a colorimetric staining of the same tissues is suggested. Current quality control test show that the mouse α-syn could potentially induce better aggregation on endogenous α-syn. For verification in vivo test to compare the effect of the proteins are suggested.