VIB Ugent Center for Inflammation Research Roos Vandenbroucke Lab
Abstract bachelor project FBT 2020-21: The anti-inflammatory effect of mesenchymal stem cell-derived extracellular vesicles on Niemann-Pick disease type C1 pathology
Niemann-Pick disease type C1 (NPC1) is a lysosomal storage disorder caused by loss-of-function mutations of the Npc1-gene. The disease has many clinical features, including inflammation. Despite many years of research, a cure for this disease is not available. This bachelor thesis aims to investigate the anti-inflammatory effect of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) on NPC1 pathology. The ultimate purpose of this research is to understand the disease better and to take the first steps into finding a possible cure. For this, two separate groups of Npc1-/- mice are treated with MSC-EVs fraction 16.3 or MSC-EVs fraction 41.5, delivered by an external lab (from the University Hospital of Essen). Furthermore, Npc1+/+ as well as Npc1-/- mice are given four intravenous injections with either a buffer or platelet-derived extracellular vesicles (EVs) as a control.
The mice are weighed before each injection. The weight gain of mice treated with MSC-EVs 41.5 is remarkable higher than the other groups. Approximately two weeks after the first injection, the mice are sacrificed. Bio-Plex is performed on both cerebrospinal fluid (CSF) and serum to determine the concentration of specific chemo- and cytokines. The results show a difference in cytokine levels between Npc1+/+ and Npc1-/- mice. Furthermore, it can be derived that treatment with MSC-EV 41.5 can revert the phenotype of Npc1-/- mice to the phenotype of Npc1+/+ mice. In serum, the concentration of interleukin (IL)- 12 is decreased to normal levels in mice treated with MSC-EVs 41.5. On the other hand, no differences in the concentration of keratinocyte-derived chemokine (KC) are seen between the groups. However, the concentration of KC in CSF of mice treated with MSC-EV 41.5 is decreased compared to treatment with MSC-EV 16.3.
Also, qPCR is performed on the liver, lung, spleen and different brain regions. In general, Npc1+/+ mice show lower expression levels of chemo- and cytokines compared to Npc1-/- mice. Additionally, the relative expression of tumour necrosis factor (Tnf), Il-1β, C-C motif chemokine ligand (Ccl) 2, Ccl3, Ccl5 and C-X-C motif chemokine ligand (Cxcl) 10 of
Npc1-/- mice treated with MSC-EV 41.5 is decreased to levels of Npc1+/+ mice. In some cases, T-test shows a significant difference between Npc1+/+ control mice and Npc1-/- mice treated with MSC-EV 16.3, but also between Npc1-/- control mice and Npc1-/- mice treated with MSC-EV 41.5. Inducible nitric oxide synthase (Inos) and ‘nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alfa’ (Iκbα) don’t show any pattern in any of the investigated tissues. Il-4 and Il-6 show this pattern in the prefrontal cortex and the prefrontal cortex and olfactory bulb respectively.
Finally, immunohistochemistry (IHC) is performed. The stainings show mostly a difference between Npc1+/+- and Npc1-/- mice, but not between treatment with MSC-EV 16.3 or 41.5. Only ‘glial fibrillary acidic protein’ (GFAP)-staining (except for the cortex-region) and ‘ionized calcium-binding adapter molecule 1’ (Iba1)-staining (only in the cortex-region) do show a difference between Npc1+/+- and Npc1-/- mice.
Ultimately, it can be concluded that of the two MSC-EV fractions that have been tested, only fraction 41.5 has an anti-inflammatory effect on NPC1 pathology. Possible further experiments could be a repeat of the experiment, but the sampling could take place a few weeks later so that possible changes in immune cells and cellular processes have time to develop. Also, IHC could be performed on the peripheral organs to see whether MSC-EV 41.5 can normalize the morphology.
Abstract bachelor project 2 FBT2019-20: 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 1 FBT 2019-20: 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.
Lien Van Hoecke