Wirkt die CaVbeta3-Untereinheit protektiv bei chronisch entzündlichen ZNS-Erkrankungen?
Final Report Abstract
Calcium influx into neurons has been suggested to mediate neurodegeneration under autoimmune inflammatory conditions, such as multiple sclerosis (MS), with the N-type Cavs playing a potentially important role. However, upon comparing the pathology of the pore-forming Cav subunit knockout (Cav2.2 KO) with that of the Cavβ3 KO, differences in the progression of neurodegeneration in EAE were observed. Whereas ablation of Cav2.2 resulted in reduced disease severity, removal of Cavβ3 resulted in a more severe disease course and also increased neurodegeneration. However, upon further investigation, the increased neurodegeneration in Cavβ3 KOs did not appear to be a result of either aberrant neuronal survival or differences in T cell polarisation. We therefore turned our attention to the blood-brain barrier (BBB). Since increased T cell infiltration was observed in the optic nerves, and correlated with increased axonal damage as assessed by beta-amyloid precursor protein accumulation, it is likely that increased T cell infiltration may induce RGC degeneration via secondary degeneration resulting from increased axonal damage in the optic nerves. The BBB of the brain microvasculature is composed of three cellular elements; astrocytic end-feet, endothelial cells and pericytes. To determine if Cavβ3 influences BBB integrity we assessed the permeability. Cavβ3 desensitizes cells to low IP3 concentrations. IP3-dependent Ca release (measured by Fura-2 in the absence of extracellular Ca) evoked by agonists (Bradykinin or LPA) is increased in isolated primary fibroblasts from Cavβ3 KO mice compared to wild-type. Loading fibroblasts isolated from wild-type and β3 KO with caged IP3 and subsequent uncaging by UV flash photolysis with increasing energy revealed that in the absence of Cavβ3 the cells are sensitized to low IP3 concentrations. healthy animals by both Evan’s Blue intravenous injection followed by colorimetric measurement of extravasated Evan’s Blue from the spinal cord, and quantification of infiltrating T cells in healthy animals. These preliminary investigations demonstrated that in Cavβ3 KO healthy mice, there is an enhanced extravasation of Evan’s Blue as well as significantly elevated numbers of both T cells and macrophages/activated, indicating an increased BBB permeability in Cavβ3 KO mice. Thus, it appears that increased disturbances in BBB integrity in Cavβ3 KO mice may explain the increased ability for T cell infiltration, and possibly ensuing neurodegeneration. Calcium is known to be one of the key regulators of BBB permeability but how Cavβ3 may play a role is at present unknown. However, IP3-dependent calcium waves between astrocytes and endothelial cells have been observed and have been suggested to mediate astrocyte-endothelial cell communication at the BBB. Since our data provides evidence that Cavβ3 exerts a regulatory influence on intracellular calcium release from the endoplasmic reticulum via IP3-receptor function, it remains to be determined if this occurs within cells of the BBB, and whether this is essential for maintaining BBB integrity, particularly under autoimmune conditions.
