The blood-brain barrier (BBB) prevents the entrance of circulating molecules and immune cells into the central nervous system. The barrier is formed by specialized brain endothelial cells that are interconnected by tight junctions (TJ). A defective function of the BBB has been described for a variety of neuroinflammatory diseases, indicating that proper regulation is essential for maintaining brain homeostasis. Under pathological conditions, reactive oxygen species (ROS) significantly contribute to BBB dysfunction and inflammation in the brain by enhancing cellular migration. However, a detailed study about the molecular mechanism by which ROS alter BBB integrity has been lacking. Here we demonstrate that ROS alter BBB integrity, which is paralleled by cytoskeleton rearrangements and redistribution and disappearance of TJ proteins claudin-5 and occludin. Specific signaling pathways, including RhoA and PI3 kinase, mediated observed processes and specific inhibitors of these pathways prevented ROS-induced monocyte migration across an in vitro model of the BBB. Interestingly, these processes were also mediated by protein kinase B (PKB/Akt), a previously unknown player in cytoskeleton and TJ dynamics that acted downstream of RhoA and PI3 kinase. Our study reveals new insights into molecular mechanisms underlying BBB regulation and provides novel opportunities for the treatment of neuroinflammatory diseases.
Background In brain tissues from multiple sclerosis (MS) patients, clusters of activated HLA-DR-expressing microglia, also referred to as preactive lesions, are located throughout the normal-appearing white matter. The aim of this study was to gain more insight into the frequency, distribution and cellular architecture of preactive lesions using a large cohort of well-characterized MS brain samples. Methods Here, we document the frequency of preactive lesions and their association with distinct white matter lesions in a cohort of 21 MS patients. Immunohistochemistry was used to gain further insight into the cellular and molecular composition of preactive lesions. Results Preactive lesions were observed in a majority of MS patients (67%) irrespective of disease duration, gender or subtype of disease. Microglial clusters were predominantly observed in the vicinity of active demyelinating lesions and are not associated with T cell infiltrates, axonal alterations, activated astrocytes or blood–brain barrier disruption. Microglia in preactive lesions consistently express interleukin-10 and TNF-α, but not interleukin-4, whereas matrix metalloproteases-2 and −9 are virtually absent in microglial nodules. Interestingly, key subunits of the free-radical-generating enzyme NADPH oxidase-2 were abundantly expressed in microglial clusters. Conclusions The high frequency of preactive lesions suggests that it is unlikely that most of them will progress into full-blown demyelinating lesions. Preactive lesions are not associated with blood–brain barrier disruption, suggesting that an intrinsic trigger of innate immune activation, rather than extrinsic factors crossing a damaged blood–brain barrier, induces the formation of clusters of activated microglia.
HERV-W was expressed in demyelinated lesions from MS brains, which were all positive for this endogenous pathogenic protein. Pronounced HERV-W immunoreactivity in active MS lesions was intimately associated with areas of active demyelination throughout the successive stages of lesion evolution in MS brains. Based on its pathogenic potential, this HERV-W (MSRV) endogenous toxin thus appears to be a novel therapeutic target in MS. It also has a unique positioning as an early and lifelong expressed pathogenic agonist, acting upstream the pathways in which dysregulated physiological effectors are usually targeted by present therapeutic strategies for MS.
This article is available online at http://www.jlr.org stigmasterol (ethyl group at C24, ⌬ 22) ( 1, 2 ). In contrast to cholesterol, these sterols are exclusively derived from the diet and cannot be synthesized endogenously in mammals. A high plant sterol intake (2-2.5 g/day) leads to reduced total and LDL-cholesterol ( ف 12%) in the circulation ( 3, 4 ). Therefore, plant sterols are frequently applied as functional, nonprescription food additives to prevent atherosclerosis and cardiovascular diseases ( 5 ). However, administration of high-dose plant sterols results in increased serum plant sterol concentrations ( 6 ). Hard end-point studies showing an effect on the number of cardiovascular events or on mortality after long-term intake of high-dose plant sterols are lacking ( 7 ), as are insights into the underlying molecular mechanisms ( 8 ). Because adverse events upon plant sterol administration in animal studies are increasingly being reported to cause adverse events ( 9 ), it is a current topic of debate ( 10 ).In contrast to peripheral tissues, all cholesterol within the central nervous system is synthesized in situ because circulating cholesterol is not able to cross the blood-brain barrier (BBB) ( 11,12 ). Recently, we reported that circulating plant sterols, in contrast to cholesterol, can enter the brains of ATP binding cassette g5 (Abcg5)-defi cient mice, a model for phytosterolemia ( 13 ). ABCG5 and ABCG8 act as functional heterodimer transporters at the apical membranes of enterocytes and hepatocytes, where they excrete plant sterols into the intestinal lumen and bile, Abstract Plant sterols such as sitosterol and campesterol are frequently administered as cholesterol-lowering supplements in food. Recently, it has been shown in mice that, in contrast to the structurally related cholesterol, circulating plant sterols can enter the brain. We questioned whether the accumulation of plant sterols in murine brain is reversible. After being fed a plant sterol ester-enriched diet for 6 weeks, C57BL/6NCrl mice displayed signifi cantly increased concentrations of plant sterols in serum, liver, and brain by 2-to 3-fold. Blocking intestinal sterol uptake for the next 6 months while feeding the mice with a plant stanol ester -enriched diet resulted in strongly decreased plant sterol levels in serum and liver, without affecting brain plant sterol levels. Relative to plasma concentrations, brain levels of campesterol were higher than sitosterol, suggesting that campesterol traverses the blood-brain barrier more effi ciently. In vitro experiments with brain endothelial cell cultures showed that campesterol crossed the blood-brain barrier more effi ciently than sitosterol. We conclude that, over a 6-month period, plant sterol accumulation in murine brain is virtually irreversible. Plant sterols differ structurally from cholesterol by an additional methyl or ethyl group at C24 and/or a double bond at C22 ( ⌬ 22). The most prevalent plant sterols are campesterol (methyl group at C24), sitosterol (ethyl group at C24)...
The choroid plexus (CP) is strategically located between the peripheral blood and the cerebrospinal fluid, and is involved in the regulation of central nervous system (CNS) homeostasis. In multiple sclerosis (MS), demyelination and inflammation occur in the CNS. While experimental animal models of MS pointed to the CP as a key route for immune cell invasion of the CNS, little is known about the distribution of immune cells in the human CP during progressive phases of MS. Here, we use immunohistochemistry and confocal microscopy to explore the main immune cell populations in the CP of progressive MS patients and non-neuroinflammatory controls, in terms of abundance and location within the distinct CP compartments. We show for the first time that the CP stromal density of granulocytes and CD8+ T cells is higher in progressive MS patients compared to controls. In line with previous studies, the CP of both controls and progressive MS patients contains relatively high numbers of macrophages and dendritic cells. Moreover, we found virtually no B cells or plasma cells in the CP. MHCII+ antigenpresenting cells were often found in close proximity to T cells, suggesting constitutive CNS immune monitoring functions of the CP. Together, our data highlights the role of the CP in immune homeostasis and indicates the occurrence of mild inflammatory processes in the CP of progressive MS patients. However, our findings suggest that the CP is only marginally involved in immune cell migration into the CNS in chronic MS.
Recent evidence suggests that reactive oxygen species (ROS) produced by inflammatory cells drive axonal degeneration in active multiple sclerosis (MS) lesions by inducing mitochondrial dysfunction. Mitochondria are endowed with a variety of antioxidant enzymes, including peroxiredoxin-3 and thioredoxin-2, which are involved in limiting ROS-induced damage. In this study, we explored the distribution and role of the mitochondrial antioxidants peroxiredoxin-3 and thioredoxin-2 as well as their regulator peroxisome proliferator-activated receptor gamma coactivator1-alpha (PGC-1α) in MS pathogenesis.Immunohistochemical analysis of a large cohort of MS patients revealed a striking upregulation of PGC-1α and downstream mitochondrial antioxidants in active demyelinating MS lesions. Enhanced expression was predominantly observed in reactive astrocytes. To elucidate the functional role of astrocytic PGC-1α in MS pathology, we generated human primary astrocytes that genetically overexpressed PGC-1α. Upon an oxidative insult, these cells were shown to produce less ROS and were found to be more resistant to ROS-induced cell death compared to control cells. Intriguingly, also neuronal cells co-cultured with PGC-1α-overexpressing astrocytes were protected against an exogenous oxidative attack compared to neuronal cells co-cultured with control astrocytes. Finally, enhanced astrocytic PGC-1α levels markedly reduced the production and secretion of the pro-inflammatory mediators interleukin-6 and chemokine (C-C motif) ligand 2. Our findings suggest that increased astrocytic PGC-1α in active MS lesions might initially function as an endogenous protective mechanism to dampen oxidative damage and inflammation thereby reducing neurodegeneration. Activation of PGC-1α therefore represents a promising therapeutic strategy to improve mitochondrial function and repress inflammation.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-014-0170-2) contains supplementary material, which is available to authorized users.
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