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T Cell Infiltration in Central Nervous System and Their Association with Brain Calcification in Slc20a2-deficient Mice

ABSTRACT: Primary familial brain calcification (PFBC) is a rare neurodegenerative and neuropsychiatric disorder characterized by bilateral and symmetric brain calcification along the microvessels or inside neuronal cells in the basal ganglia, thalamus, and cerebellum. Neurological symptoms typically include movement disorders, cognitive impairment, and neuropsychiatric signs. Slc20a2 homozygous (HO) knockout mice can simulate the phenotype of brain calcification observed in humans. However, the cellular and molecular mechanisms associated with brain calcification, particularly at an extremely early stage (before calcification emerges), remain largely unknown. Herein, we quantified central nervous system (CNS)-infiltrating immune cells in different age groups and found that CD45+CD3+ T cells were significantly increased in the brain parenchyma even in the pre-calcification stage of 1-month-old Slc20a2-HO mice. Further immunophenotyping demonstrated that CD3+CD4-CD8- and CD3+CD4+ T cells were the two main subtypes increased in the brain. The accumulation of total T cells appears to be associated with the severity of brain calcification. Paracellular and transcellular permeability of the blood-brain barrier increased in endothelial cells of brain microvessels in Slc20a2-HO mice. The expression of endothelial cell adhesion molecules was increased, while that of tight and adherens junction proteins was decreased, reflecting the molecular basis of T cell recruitment to endothelial cells and paracellular migration into the brain. IgG and albumin assays revealed an increased leakage of endothelial cells and the process of T cell transcellular migration was captured by immunoelectron microscopy, suggesting the enhancement of transcytosis or endocytosis in brain endothelial cells of Slc20a2-HO mice. The sphingosine 1-phosphate receptor modulator, FTY720, could significantly inhibit brain calcification, probably by reducing the CNS infiltration of T cells, subsequently modulating neuroinflammation, and enhancing the phagocytosis of CNS resident immune cells. This study demonstrated that CNS-infiltrating T cells were associated with the progression of brain calcification and suggested that the impairment of blood-brain barrier permeability, which was closely related to T cell invasion into the CNS, could be explained by an increase in the paracellular and transcellular pathways of brain endothelial cells in Slc20a2-HO mice. Moreover, FTY720 was a potential drug that could be used to treat patients with PFBC in the future

ORGANISM(S): Mus musculus

PROVIDER: GSE218335 | GEO | 2022/11/23

REPOSITORIES: GEO

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Project description:The blood-brain barrier (BBB) dynamically controls and maintains a precisely balanced brain microenvironment necessary for reliable functioning. It is made up of highly specialized endothelial cells (ECs) in the lining of the vascular wall. These ECs are surrounded by the basal lamina, astrocytic perivascular endfeet, pericytes, microglia and neuronal processes that have been shown to contribute to barrier function1. In essence, the brain endothelium limits both transcellular and paracellular passage of cells and molecules into the central nervous system (CNS). Transcellular passage of hydrophilic molecules is limited due to a low rate of transcytotic vesicles, an extremely low pinocytotic activity, expression of active efflux transporters, and high metabolic activity. Paracellular diffusion of hydrophilic molecules and trafficking of immune cells is restricted by a network of tight junctions (TJs)2-5. Due to these characteristics, the BBB is able to protect the CNS from sudden changes in blood composition and uncontrolled influx of immune cells. In a large number of neuro-inflammatory diseases, an impaired function and an opening of the BBB are observed. In diseases of the CNS like multiple sclerosis or stroke or after brain trauma, the BBB becomes inflamed (mediated by for example reactive oxygen species (ROS) and hypoxia) and its function severely impaired which gives rise to enhanced cellular infiltration, thus contributing to tissue damage and neurological deficits. Due to the specialized nature of brain ECs, transmigration of leukocytes through cerebrovascular endothelium is likely to differ from that in other vascular beds. Generally, the transmigration process is closely controlled by the interaction of leukocytes with the endothelial surface followed by low velocity rolling, arrest, firm adhesion, and finally transmigration. For the diapedesis of immune cells across the cerebral vasculature a re-arrangement of the cytoskeleton and opening of the TJ complexes is required to allow cells to pass into the sub endothelial space. In the initial step of the adhesive cascade leukocytes adhere to the endothelium with low affinity. This adhesion is mediated by several members of the selectin family and their corresponding ligands. Despite the low affinity of these interactions, resulting contact of leukocytes with the endothelium leads to further activation of both cell types and finally transmigration of the leukocytes through the BBB6,7. The glycosylation of endothelial cells of different vascular bed origins To date it is unknown which specific carbohydrate structures on the BBB endothelium mediate leukocyte capture and rolling, and whether these structures are differentially expressed onto inflamed brain EC compared to normal brain ECs and vascular beds of other organs. Initial studies using qPCR and FACS analysis within our group reveal that brain endothelial cells have a different profile in their glycosylation-related genes compared to microvascular ECs upon inflammation, which may result in a different glycosylation profile of adhesive structures and may underlie rolling, adhesion and diapedesis of leukocytes. In this project we wish to identify specific, glycosylated structures on brain endothelial cells that mediate capture, rolling and diapedesis of leukocytes in the brain. To investigate the expression of glycosyltransferases in dendritic cells and the changes in expression associated with maturation. RNA preparations from stimulated and non-stimulated hcMEC/D3 (human brain endothelial cells line) and FMVEC (human promary microvascular endothelial cells isolated from foreskins) were sent to both Microarray Core (E) and Core(C). The RNA was put on an RNeasy Column, amplified, labeled, and hybridized to the Glycov3 microarrays. Data was sent to Dr. van Kooyk's lab for analysis.

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T Cell Infiltration in Central Nervous System and Their Association with Brain Calcification in Slc20a2-deficient Mice

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