MAGT1 Deficiency Dysregulates Platelet Cation Homeostasis and Accelerates Arterial Thrombosis and Ischemic Stroke in Mice.

BACKGROUND: MAGT1 (magnesium transporter 1) is a subunit of the oligosaccharide protein complex with thiol-disulfide oxidoreductase activity, supporting the process of N-glycosylation. MAGT1 deficiency was detected in human patients with X-linked immunodeficiency with magnesium defect syndrome and congenital disorders of glycosylation, resulting in decreased cation responses in lymphocytes, thereby inhibiting the immune response against viral infections. Curative hematopoietic stem cell transplantation of patients with X-linked immunodeficiency with magnesium defect causes fatal bleeding and thrombotic complications. METHODS: We studied the role of MAGT1 deficiency in platelet function in relation to arterial thrombosis and hemostasis using several in vitro experimental settings and in vivo models of arterial thrombosis and transient middle cerebral artery occlusion model of ischemic stroke. RESULTS: MAGT1-deficient mice (Magt1-/y) displayed accelerated occlusive arterial thrombus formation in vivo, a shortened bleeding time, and profound brain damage upon focal cerebral ischemia. These defects resulted in increased calcium influx and enhanced second wave mediator release, which further reinforced platelet reactivity and aggregation responses. Supplementation of MgCl2 or pharmacological blockade of TRPC6 (transient receptor potential cation channel, subfamily C, member 6) channel, but not inhibition of store-operated calcium entry, normalized the aggregation responses of Magt1-/y platelets to the control level. GP (glycoprotein) VI activation of Magt1-/y platelets resulted in hyperphosphorylation of Syk (spleen tyrosine kinase), LAT (linker for activation of T cells), and PLC (phospholipase C) γ2, whereas the inhibitory loop regulated by PKC (protein kinase C) was impaired. A hyperaggregation response to the GPVI agonist was confirmed in human platelets isolated from a MAGT1-deficient (X-linked immunodeficiency with magnesium defect) patient. Haploinsufficiency of TRPC6 in Magt1-/y mice could normalize GPVI signaling, platelet aggregation, and thrombus formation in vivo. CONCLUSIONS: These results suggest that MAGT1 and TRPC6 are functionally linked. Therefore, deficiency or impaired functionality of MAGT1 could be a potential risk factor for arterial thrombosis and stroke.

Electrical Stimulation of the Mesencephalic Locomotor Region Has No Impact on Blood-Brain Barrier Alterations after Cerebral Photothrombosis in Rats.

Blood-brain barrier (BBB) disruption is a critical event after ischemic stroke, which results in edema formation and hemorrhagic transformation of infarcted tissue. BBB dysfunction following stroke is partly mediated by proinflammatory agents. We recently have shown that high frequency stimulation of the mesencephalic locomotor region (MLR-HFS) exerts an antiapoptotic and anti-inflammatory effect in the border zone of cerebral photothrombotic stroke in rats. Whether MLR-HFS also has an impact on BBB dysfunction in the early stage of stroke is unknown. In this study, rats were subjected to photothrombotic stroke of the sensorimotor cortex and implantation of a stimulating microelectrode into the ipsilesional MLR. Thereafter, either HFS or sham stimulation of the MLR was applied for 24 h. After scarifying the rats, BBB disruption was assessed by determining albumin extravasation and tight junction integrity (claudin 3, claudin 5, and occludin) using Western blot analyses and immunohistochemistry. In addition, by applying zymography, expression of pro-metalloproteinase-9 (pro-MMP-9) was analyzed. No differences were found regarding infarct size and BBB dysfunction between stimulated and unstimulated animals 24 h after induction of stroke. Our results indicate that MLR-HFS neither improves nor worsens the damaged BBB after stroke. Attenuating cytokines/chemokines in the perilesional area, as mediated by MLR-HFS, tend to play a less significant role in preventing the BBB integrity.

Pretreatment with rivaroxaban attenuates stroke severity in rats by a dual antithrombotic and anti-inflammatory mechanism.

Stroke outcome is more favourable in patients receiving oral anticoagulants compared with non-anticoagulated patients. The reasons for this "stroke-attenuating" property of oral anticoagulants are largely unknown. This study examined whether prestroke anticoagulation with rivaroxaban, a novel direct factor Xa inhibitor, influences stroke severity, thrombin-mediated intracerebral thrombus formation and pro-inflammatory processes in a rat model of brain ischaemia/reperfusion injury. Male Wistar rats were anticoagulated with rivaroxaban and subjected to 90 minutes of transient middle cerebral artery occlusion. Infarct size, functional outcome and the occurrence of intracranial haemorrhage (ICH) were assessed until day 7. Thrombin generation was determined by measuring the amount of thrombin/antithrombin complex. Intracerebral thrombus formation was evaluated by histology and Western blot. CD68-immunoreactivity and the expression of cytokines and adhesion molecules were investigated to assess postischaemic inflammation. The integrity of the blood-brain barrier was analysed using fluorescein isothiocyanate-dextran. Rats pretreated with rivaroxaban developed significantly smaller strokes and less severe functional deficits compared with controls. Although rivaroxaban strongly reduced thrombin-mediated thrombus formation, this was not accompanied by an increased risk of ICH. In addition, rivaroxaban dampened the inflammatory response in the ischaemic brain by downregulating ICAM-1 expression and the activation of CD68+-immune cells. In contrast, rivaroxaban had no effect on the integrity of the blood-brain barrier after stroke. Here, we identified reduced thrombo-inflammation as a major determinant of the stroke-protective property of rivaroxaban in rats. Further studies are needed to assess the therapeutic potential of novel oral anticoagulants in the acute phase after a stroke.

Fingolimod (FTY720-P) Does Not Stabilize the Blood-Brain Barrier under Inflammatory Conditions in an in Vitro Model.

Breakdown of the blood-brain barrier (BBB) is an early hallmark of multiple sclerosis (MS), a progressive inflammatory disease of the central nervous system. Cell adhesion in the BBB is modulated by sphingosine-1-phosphate (S1P), a signaling protein, via S1P receptors (S1P1). Fingolimod phosphate (FTY720-P) a functional S1P1 antagonist has been shown to improve the relapse rate in relapsing-remitting MS by preventing the egress of lymphocytes from lymph nodes. However, its role in modulating BBB permeability-in particular, on the tight junction proteins occludin, claudin 5 and ZO-1-has not been well elucidated to date. In the present study, FTY720-P did not change the transendothelial electrical resistance in a rat brain microvascular endothelial cell (RBMEC) culture exposed to inflammatory conditions and thus did not decrease endothelial barrier permeability. In contrast, occludin was reduced in RBMEC culture after adding FTY720-P. Additionally, FTY720-P did not alter the amount of endothelial matrix metalloproteinase (MMP)-9 and MMP-2 in RBMEC cultures. Taken together, our observations support the assumption that S1P1 plays a dual role in vascular permeability, depending on its ligand. Thus, S1P1 provides a mechanistic basis for FTY720-P-associated disruption of endothelial barriers-such as the blood-retinal barrier-which might result in macular edema.

Endothelial TWIK-related potassium channel-1 (TREK1) regulates immune-cell trafficking into the CNS.

The blood-brain barrier (BBB) is an integral part of the neurovascular unit (NVU). The NVU is comprised of endothelial cells that are interconnected by tight junctions resting on a parenchymal basement membrane ensheathed by pericytes, smooth muscle cells and a layer of astrocyte end feet. Circulating blood cells, such as leukocytes, complete the NVU. BBB disruption is common in several neurological diseases, but the molecular mechanisms involved remain largely unknown. We analyzed the role of TWIK-related potassium channel-1 (TREK1, encoded by KCNK2) in human and mouse endothelial cells and the BBB. TREK1 was downregulated in endothelial cells by treatment with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Blocking TREK1 increased leukocyte transmigration, whereas TREK1 activation had the opposite effect. We identified altered mitogen-activated protein (MAP) kinase signaling, actin remodeling and upregulation of cellular adhesion molecules as potential mechanisms of increased migration in TREK1-deficient (Kcnk2(-/-)) cells. In Kcnk2(-/-) mice, brain endothelial cells showed an upregulation of the cellular adhesion molecules ICAM1, VCAM1 and PECAM1 and facilitated leukocyte trafficking into the CNS. Following the induction of experimental autoimmune encephalomyelitis (EAE) by immunization with a myelin oligodendrocyte protein (MOG)35-55 peptide, Kcnk2(-/-) mice showed higher EAE severity scores that were accompanied by increased cellular infiltrates in the central nervous system (CNS). The severity of EAE was attenuated in mice given the amyotrophic lateral sclerosis drug riluzole or fed a diet enriched with linseed oil (which contains the TREK-1 activating omega-3 fatty acid α-linolenic acid). These beneficial effects were reduced in Kcnk2(-/-) mice, suggesting TREK-1 activating compounds may be used therapeutically to treat diseases related to BBB dysfunction.

Glatiramer acetate attenuates pro-inflammatory T cell responses but does not directly protect neurons from inflammatory cell death.

Glatiramer acetate (GA) is a synthetic, random, basic copolymer capable of modulating adaptive T cell responses. In animal models of various inflammatory and degenerative central nervous system disorders, GA-induced T cells cross the blood-brain barrier, secrete high levels of anti-inflammatory cytokines and neurotrophins, and thus both reduce neuronal damage and promote neurogenesis. Recently, it has been suggested that GA itself may permeate the (impaired) blood-brain-barrier and directly protect neurons under conditions of inflammation-mediated neurodegeneration. To test this hypothesis, we examined the direct effects of GA on neuronal functionality and T cell-mediated neuronal apoptosis in culture, acute brain slices, and focal experimental autoimmune encephalomyelitis. GA caused a depolarization of the resting membrane potential and led to an immediate impairment of action potential generation in neurons. Moreover, GA-incubated neurons underwent dose-dependent apoptosis. Apoptosis of ovalbumin peptide-loaded major histocompatibility complex class I-expressing neurons induced by ovalbumin-specific effector T cells could be reduced by pre-incubation of T cells, but not neurons with GA. Similar results could be found using acute brain slices. In focal experimental autoimmune encephalomyelitis, lesion size and neuronal apoptosis could be limited by pretreating rats with GA, whereas intracerebral GA application into the inflammatory lesion had no effect on neuronal survival. Our data suggest that GA attenuates adaptive pro-inflammatory T cell responses, but does not exert direct neuroprotective effects.