Atypical sphingosine-1-phosphate metabolites-biological implications of alkyl chain length.

Sphingosine-1-phosphate (S1P) is a bioactive lipid signaling molecule with pleiotropic implications by both auto- and paracrine signaling. Signaling occurs by engaging five G protein-coupled receptors (S1P1-5) or intracellular pathways. While the extensively studied S1P with a chain length of 18 carbon atoms (d18:1 S1P) affects lymphocyte trafficking, immune cell survival and inflammatory responses, the biological implication of atypical S1Ps such as d16:1 or d20:1 remains elusive. As S1P lipids have far-reaching implications in health and disease states in mammalian organisms, the previous contrasting results may be attributed to differences in S1P's alkyl chain length. Current research is beginning to appreciate these less abundant atypical S1P moieties. This review provides an up-to-date foundation of recent findings on the biological implications of atypical S1P chain lengths and offers a perspective on future research endeavors on S1P alkyl chain length-influenced signaling and its implications for drug discovery.

Sepia pharaonis Ink Mitigates Dehydroepiandrosterone-Induced Insulin Resistance in Mouse Model of Polycystic Ovarian Syndrome.

The present study aims to evaluate the effect of Sepia pharaonis ink on insulin resistance in PCOS-induced mice. Treatment with sepia ink in dehydroepiandrosterone (DHEA)-induced PCOS mice at various doses of 50 mg/kg, 100 mg/kg, and 200 mg/kg body weight mitigated the insulin resistance in the study groups with decreased concentration of testosterone and increased concentrations of estrogen and progesterone compared to the PCOS group tested by ELISA. The histopathological analysis and restoration of glucose analysis showed a significant reduction in treatment groups. Reduced expression of insulin resistance genes like androgen receptor (AR), insulin receptor substrate 1 (IRS-1), and insulin-like growth factor1 (IGF-1) by qRT-PCR indicate a positive impact of sepia ink in alleviating the symptoms associated with PCOS. Taken together, the results of this study indicate sepia ink as a promising therapeutic intervention and a possible drug target for insulin resistance in diabetes and gynecological disorders like PCOS.

BI-3231, an enzymatic inhibitor of HSD17B13, reduces lipotoxic effects induced by palmitic acid in murine and human hepatocytes.

17-ß-hydroxysteroid dehydrogenase 13 (HSD17B13), a lipid droplet-associated enzyme, is primarily expressed in the liver and plays an important role in lipid metabolism. Targeted inhibition of enzymatic function is a potential therapeutic strategy for treating steatotic liver disease (SLD). The present study is aimed at investigating the effects of the first selective HSD17B13 inhibitor, BI-3231, in a model of hepatocellular lipotoxicity using human cell lines and primary mouse hepatocytes in vitro. Lipotoxicity was induced with palmitic acid in HepG2 cells and freshly isolated mouse hepatocytes and the cells were coincubated with BI-3231 to assess the protective effects. Under lipotoxic stress, triglyceride (TG) accumulation was significantly decreased in the BI-3231-treated cells compared with that of the control untreated human and mouse hepatocytes. In addition, treatment with BI-3231 led to considerable improvement in hepatocyte proliferation, cell differentiation, and lipid homeostasis. Mechanistically, BI-3231 increased the mitochondrial respiratory function without affecting ß-oxidation. BI-3231 inhibited the lipotoxic effects of palmitic acid in hepatocytes, highlighting the potential of targeting HSD17B13 as a specific therapeutic approach in steatotic liver disease.NEW & NOTEWORTHY 17-ß-Hydroxysteroid dehydrogenase 13 (HSD17B13) is a lipid droplet protein primarily expressed in the liver hepatocytes. HSD17B13 is associated with the clinical outcome of chronic liver diseases and is therefore a target for the development of drugs. Here, we demonstrate the promising therapeutic effect of BI-3231 as a potent inhibitor of HSD17B13 based on its ability to inhibit triglyceride accumulation in lipid droplets (LDs), restore lipid metabolism and homeostasis, and increase mitochondrial activity in vitro.

Expression of PKM2 in wound keratinocytes is coupled to angiogenesis during skin repair in vivo and in HaCaT keratinocytes in vitro.

An injured skin is rapidly restored in a manner of wound healing. We have previously shown that intact insulin signaling and glucose uptake are fundamental to proper wound closure. Consequently, under exacerbated inflammation, compromised insulin action and glucose uptake lead to impaired healing. However, in spite of the increased attention to cell metabolism during tissue regeneration, metabolic mediators that govern cellular and physiological processes throughout skin repair remained largely elusive. Through assessment of mRNA using real-time PCR and protein blot analysis, we report that healing of cutaneous wounds comprise a boosted expression of genes involved in glycolysis, oxidative phosphorylation, pentose phosphate shunt, and glutamine anaplerosis. We further focused on the functional role of pyruvate kinase M (PKM) isoenzymes that catalyze the final and rate-limiting step of glycolysis. Whereas the expression of the metabolic constitutively active Pkm1 isozyme remained almost unchanged, Pkm2 is augmented during the inflammatory phase of healing. The immunohistochemistry and RNA in situ hybridization analysis showed a confined Pkm2 expression to keratinocytes of the hyperproliferative epithelium and, to a lesser extent, infiltrating neutrophils and monocytes as well as later on in macrophages. Notably, the expression of Pkm2 in keratinocytes facing the wound bed side colocalized with VEGF expression. The in vitro knockdown of PKM2 in HaCaT keratinocytes using small interfering (si) RNA confirmed an acute role for PKM2 in facilitating the complete induction of VEGF mRNA and protein expression in keratinocytes; this function is mainly HIF-1α independent. KEY MESSAGES: • Wound healing involves activation of glycolysis, oxidative phosphorylation, pentos-phosphate shunt, and replenishment of tri-carboxylic acid (TCA) cycle through glutamine anaplerosis. • The pyruvate kinase M2 (PKM2) isoform is upregulated during the inflammatory phase of cutaneous healing, mainly in keratinocytes of hyperproliferative epithelia. • In vivo, the expression of VEGF in wound keratinocytes is colocalized with PKM2. • PKM2 is required for full induction of VEGF in HaCaT keratinocytes in vitro.

Reciprocal abrogation of PKM isoforms: contradictory outcomes and differing impact of splicing signal on CRISPR/Cas9 mediates gene editing in keratinocytes.

The healing of wounded skin is a highly organized process involving a massive cell in- and outflux, proliferation and tissue remodelling. It is well accepted that metabolic constraints such as diabetes mellitus, overweight or anorexia impairs wound healing. Indeed, wound inflammation involves a boost of overall metabolic changes. As wound healing converges inflammatory processes that are also common to transformation, we investigate the functional role of the pro-neoplastic factor pyruvate kinase (PK) M2 and its metabolic active splice variant PKM1 in keratinocytes. Particularly, we challenge the impact of reciprocal ablation of PKM1 or two expression. Here, CRISPR/Cas9 genome editing of the PKM gene in HaCaT reveals an unexpected mutational bias at the 3'SS of exon 9, whereas no preference for any particular kind of mutation at exon 10 3' splice, despite the close vicinity (400 nucleotides apart) and sequence similarity between the two sites. Furthermore, as opposed to transient silencing of PKM2, exclusion splicing of PKM2 via genome editing mutually increases PKM1 mRNA and protein expression and compensates for the absence of PKM2, whereas the reciprocal elimination of PKM1 splicing reduces PKM2 expression and impedes cell proliferation, thus unveiling an essential role for PKM1 in growth and metabolic balance of HaCaT keratinocytes.

Behind the Wall-Compartment-Specific Neovascularisation during Post-Stroke Recovery in Mice.

Ischemic stroke is a highly prevalent vascular disease leading to oxygen- and glucose deprivation in the brain. In response, ischemia-induced neovascularization occurs, which is supported by circulating CD34+ endothelial progenitor cells. Here, we used the transient middle cerebral artery occlusion (tMCAO) mouse model to characterize the spatio-temporal alterations within the ischemic core from the acute to the chronic phase using multiple-epitope-ligand cartography (MELC) for sequential immunohistochemistry. We found that around 14 days post-stroke, significant angiogenesis occurs in the ischemic core, as determined by the presence of CD31+/CD34+ double-positive endothelial cells. This neovascularization was accompanied by the recruitment of CD4+ T-cells and dendritic cells as well as IBA1+ and IBA1- microglia. Neighborhood analysis identified, besides pericytes only for T-cells and dendritic cells, a statistically significant distribution as direct neighbors of CD31+/CD34+ endothelial cells, suggesting a role for these cells in aiding angiogenesis. This process was distinct from neovascularization of the peri-infarct area as it was separated by a broad astroglial scar. At day 28 post-stroke, the scar had emerged towards the cortical periphery, which seems to give rise to a neuronal regeneration within the peri-infarct area. Meanwhile, the ischemic core has condensed to a highly vascularized subpial region adjacent to the leptomeningeal compartment. In conclusion, in the course of chronic post-stroke regeneration, the astroglial scar serves as a seal between two immunologically active compartments-the peri-infarct area and the ischemic core-which exhibit distinct processes of neovascularization as a central feature of post-stroke tissue remodeling. Based on our findings, we propose that neovascularization of the ischemic core comprises arteriogenesis as well as angiogenesis originating from the leptomenigeal vasculature.

The atypical sphingosine 1-phosphate variant, d16:1 S1P, mediates CTGF induction via S1P2 activation in renal cell carcinoma.

Sphingosine 1-phosphate (S1P) is a lipid mediator with numerous biological functions. The term 'S1P' mainly refers to the sphingolipid molecule with a long-chain sphingoid base of 18 carbon atoms, d18:1 S1P. The enzyme serine palmitoyltransferase catalyses the first step of the sphingolipid de novo synthesis using palmitoyl-CoA as the main substrate. After further reaction steps, d18:1 S1P is generated. However, also stearyl-CoA or myristoyl-CoA can be utilised by the serine palmitoyltransferase, which at the end of the S1P synthesis pathway, results in the production of d20:1 S1P and d16:1 S1P respectively. We measured these S1P homologues in mice and renal tissue of patients suffering from renal cell carcinoma (RCC). Our experiments highlight the relevance of d16:1 S1P for the induction of connective tissue growth factor (CTGF) in the human renal clear cell carcinoma cell line A498 and human RCC tissue. We show that d16:1 S1P versus d18:1 and d20:1 S1P leads to the highest CTGF induction in A498 cells via S1P2 signalling and that both d16:1 S1P and CTGF levels are elevated in RCC compared to adjacent healthy tissue. Our data indicate that d16:1 S1P modulates conventional S1P signalling by acting as a more potent agonist at the S1P2 receptor than d18:1 S1P. We suggest that elevated plasma levels of d16:1 S1P might play a pro-carcinogenic role in the development of RCC via CTGF induction.

Endothelial Sphingosine-1-Phosphate Receptor 4 Regulates Blood-Brain Barrier Permeability and Promotes a Homeostatic Endothelial Phenotype.

The precise regulation of blood-brain barrier (BBB) permeability for immune cells and blood-borne substances is essential to maintain brain homeostasis. Sphingosine-1-phosphate (S1P), a lipid signaling molecule enriched in plasma, is known to affect BBB permeability. Previous studies focused on endothelial S1P receptors 1 and 2, reporting a barrier-protective effect of S1P1 and a barrier-disruptive effect of S1P2. Here, we present novel data characterizing the expression, localization, and function of the S1P receptor 4 (S1P4) on primary brain microvascular endothelial cells (BMECs). Hitherto, the receptor was deemed to be exclusively immune cell associated. We detected a robust expression of S1P4 in homeostatic murine BMECs (MBMECs), bovine BMECs (BBMECs), and porcine BMECs (PBMECs) and pinpointed its localization to abluminal endothelial membranes via immunoblotting of fractionated brain endothelial membrane fragments. Apical S1P treatment of BMECs tightened the endothelial barrier in vitro, whereas basolateral S1P treatment led to an increased permeability that correlated with S1P4 downregulation. Likewise, downregulation of S1P4 was observed in mouse brain microvessels (MBMVs) after stroke, a neurologic disease associated with BBB impairment. RNA sequencing and qPCR analysis of BMECs suggested the involvement of S1P4 in endothelial homeostasis and barrier function. Using S1P4 knock-out (KO) mice and S1P4 siRNA as well as pharmacological agonists and antagonists of S1P4 both in vitro and in vivo, we demonstrate an overall barrier-protective function of S1P4. We therefore suggest S1P4 as a novel target regulating BBB permeability and propose its therapeutic potential in CNS diseases associated with BBB dysfunction.SIGNIFICANCE STATEMENT Many neurologic diseases including multiple sclerosis and stroke are associated with blood-brain barrier (BBB) impairment and disturbed brain homeostasis. Sphingosine-1-phosphate receptors (S1PRs) are potent regulators of endothelial permeability and pharmacological S1PR modulators are already in clinical use. However, the precise role of S1P for BBB permeability regulation and the function of receptors other than S1P1 and S1P2 therein are still unclear. Our study shows both barrier-disruptive and barrier-protective effects of S1P at the BBB that depend on receptor polarization. We demonstrate the expression and novel barrier-protective function of S1P4 in brain endothelial cells and pinpoint its localization to abluminal membranes. Our work may contribute to the development of novel specific S1PR modulators for the treatment of neurologic diseases associated with BBB impairment.

A Sphingosine 1-Phosphate Gradient Is Linked to the Cerebral Recruitment of T Helper and Regulatory T Helper Cells during Acute Ischemic Stroke.

Emerging evidence suggests a complex relationship between sphingosine 1-phosphate (S1P) signaling and stroke. Here, we show the kinetics of S1P in the acute phase of ischemic stroke and highlight accompanying changes in immune cells and S1P receptors (S1PR). Using a C57BL/6 mouse model of middle cerebral artery occlusion (MCAO), we assessed S1P concentrations in the brain, plasma, and spleen. We found a steep S1P gradient from the spleen towards the brain. Results obtained by qPCR suggested that cells expressing the S1PR type 1 (S1P1+) were the predominant population deserting the spleen. Here, we report the cerebral recruitment of T helper (TH) and regulatory T (TREG) cells to the ipsilateral hemisphere, which was associated with differential regulation of cerebral S1PR expression patterns in the brain after MCAO. This study provides insight that the S1P-S1PR axis facilitates splenic T cell egress and is linked to the cerebral recruitment of S1PR+ TH and TREG cells. Further insights by which means the S1P-S1PR-axis orchestrates neuronal positioning may offer new therapeutic perspectives after ischemic stroke.

Gene Expression Dynamics at the Neurovascular Unit During Early Regeneration After Cerebral Ischemia/Reperfusion Injury in Mice.

With increasing distribution of endovascular stroke therapies, transient middle cerebral artery occlusion (tMCAO) in mice now more than ever depicts a relevant patient population with recanalized M1 occlusion. In this case, the desired therapeutic effect of blood flow restauration is accompanied by breakdown of the blood-brain barrier (BBB) and secondary reperfusion injury. The aim of this study was to elucidate short and intermediate-term transcriptional patterns and the involved pathways covering the different cellular players at the neurovascular unit after transient large vessel occlusion. To achieve this, male C57Bl/6J mice were treated according to an intensive post-stroke care protocol after 60 min occlusion of the middle cerebral artery or sham surgery to allow a high survival rate. After 24 h or 7 days, RNA from microvessel fragments from the ipsilateral and the contralateral hemispheres was isolated and used for mRNA sequencing. Bioinformatic analyses allowed us to depict gene expression changes at two timepoints of neurovascular post-stroke injury and regeneration. We validated our dataset by quantitative real time PCR of BBB-associated targets with well-characterized post-stroke dynamics. Hence, this study provides a well-controlled transcriptome dataset of a translationally relevant mouse model 24 h and 7 days after stroke which might help to discover future therapeutic targets in cerebral ischemia/reperfusion injury.

S1P d20:1, an endogenous modulator of S1P d18:1/S1P2 -dependent signaling.

Sphingosine 1-phosphate (S1P) signaling influences numerous cell biological mechanisms such as differentiation, proliferation, survival, migration, and angiogenesis. Intriguingly, our current knowledge is based solely on the role of S1P with an 18-carbon long-chain base length, S1P d18:1. Depending on the composition of the first and rate-limiting enzyme of the sphingolipid de novo metabolism, the serine palmitoyltransferase, other chain lengths have been described in vivo. While cells are also able to produce S1P d20:1, its abundance and function remains elusive so far. Our experiments are highlighting the role of S1P d20:1 in the mouse central nervous system (CNS) and human glioblastoma. We show here that S1P d20:1 and its precursors are detectable in both healthy mouse CNS-tissue and human glioblastoma. On the functional level, we focused our work on one particular, well-characterized pathway, the induction of cyclooxygenase (COX)-2 expression via the S1P receptor 2 (S1P2 ). Intriguingly, S1P d20:1 only fairly induces COX-2 expression and can block the S1P d18:1-induced COX-2 expression mediated via S1P2 activation in the human glioblastoma cell line LN229. This data indicates that S1P d20:1 might act as an endogenous modulator of S1P signaling via a partial agonism at the S1P2 receptor. While our findings might stimulate further research on the relevance of long-chain base lengths in sphingolipid signaling, the metabolism of S1P d20:1 has to be considered as an integral part of S1P signaling pathways in vivo.

Beta adrenoceptor blockade ameliorates impaired glucose tolerance and alterations of the cerebral ceramide metabolism in an experimental model of ischemic stroke.

BACKGROUND: Sphingolipids are versatile signaling molecules derived from membrane lipids of eukaryotic cells. Ceramides regulate cellular processes such as proliferation, differentiation and apoptosis and are involved in cellular stress responses. Experimental evidence suggests a pivotal role of sphingolipids in the pathogenesis of cardiovascular diseases, including ischemic stroke. A neuroprotective effect has been shown for beta-adrenergic antagonists in rodent stroke models and supported by observational clinical data. However, the exact underlying pathophysiological mechanisms are still under investigation. We aimed to examine the influence of propranolol on the ceramide metabolism in the stroke-affected brain. METHODS: Mice were subjected to 60 or 180 min transient middle cerebral artery occlusion (tMCAO) and infarct size, functional neurological deficits, glucose tolerance, and brain ceramide levels were assessed after 12, 24, and 72 h to evaluate whether the latter two processes occur in a similar time frame. Next, we assessed the effects of propranolol (10 mg/kg bw) at 0, 4 and 8 h after tMCAO and FTY720 (fingolimod; 1 mg/kg) on infarct size, functional outcome, immune cell counts and brain ceramide levels at 24 h after 60 min tMCAO. RESULTS: We found a temporal coincidence between stroke-associated impaired glucose tolerance and brain ceramide accumulation. Whereas propranolol reduced ischemic lesion size, improved functional outcome and reduced brain ceramide accumulation without an effect on circulating immune cells, FTY720 showed the known neuroprotective effect and strong reduction of circulating immune cells without affecting brain ceramide accumulation. CONCLUSIONS: Propranolol ameliorates both stroke-associated impairment of glucose tolerance and brain ceramide accumulation which are temporally linked, strengthening the evidence for a role of the sympathetic nervous system in regulating post-stroke glucose metabolism and its metabolic consequences in the brain.

Sphingosine Kinase 2 Modulates Retinal Neovascularization in the Mouse Model of Oxygen-Induced Retinopathy.

Purpose: Neovascularization is a major cause of blindness in various ocular diseases. Bioactive sphingosine 1-phosphate (S1P), synthesized by two sphingosine kinases (Sphk1, Sphk2), emerged as a key player in a multitude of cellular processes, including cell survival, proliferation, inflammation, migration, and angiogenesis. We investigated the role of Sphk2, S1P, and S1P receptors (S1PR) during retinal neovascularization using the oxygen-induced retinopathy mouse model (OIR). Methods: Sphk2 overexpressing (tgSphk2) and Sphk2 knockout (Sphk2-/-) mice were used in the OIR model, exposed to 75% O2 over 5 days from postnatal day (P)7 to 12 to initiate vessel regression. After returning to room air, these mice developed a marked neovascularization. Retinae recovered from untreated and treated eyes at P7, P12, P14, and P17 were used for lectin-stained retinal whole mounts, mass spectrometry, and quantitative real-time PCR. Results: tgSphk2 mice showed higher retinal S1P concentrations, accelerated retinal angiogenesis, and increased neovascularization. Expression of S1PR, vascular endothelial growth factor α (VEGFα), and angiopoietin 1 and 2 was differentially regulated during the course of OIR in the different genotypes. Sphk2-/- displayed a markedly reduced retinal angiogenesis and neovascularization as well as decreased VEGFα and angiopoietin expression. Conclusions: Using genetic models of Sphk2 overexpression or deletion we demonstrate a strong impact of Sphk2/S1P on retinal vasculopathy and expression of vascular growth factors like VEGF and angiopoietin in the retina. Consequently, Sphk2, S1P, and S1PR may offer attractive novel therapeutic targets for ischemic retinopathies.

Alteration of sphingolipid metabolism as a putative mechanism underlying LPS-induced BBB disruption.

Septic encephalopathy with confusion and agitation occurs early during sepsis and contributes to the severity of the disease. A decrease in the sphingosine-1-phosphate (S1P) blood levels has been shown in patients and in animal models of sepsis. The lipid mediator S1P is known to be involved in endothelial barrier function in a context-dependent manner. We utilized lipopolysaccharide (LPS)-injected mice as a model for septic encephalopathy and first performed tracer permeability assays to assess the blood-brain barrier (BBB) breakdown in vivo. At time points corresponding to the BBB breakdown post LPS injection, we aimed to characterize the regulation of the sphingolipid signaling pathway at the BBB during sepsis. We measured sphingolipid concentrations in blood, in mouse brain microvessels (MBMVs), and brain tissue. We also analyzed the expression of S1P receptors, transporters, and metabolizing enzymes in MBMVs and brain tissue. Primary mouse brain microvascular endothelial cells (MBMECs) were isolated to evaluate the effects of LPS on transendothelial electrical resistance (TEER) as a measure of permeability in vitro. We observed a relevant decrease in S1P levels after LPS injection in all three compartments (blood, MBMVs, brain tissue) that was accompanied by an increased expression of the S1P receptor type 1 and of sphingosine kinase 1 on one hand and of the S1P degrading enzymes lipid phosphate phosphatase 1 (LPP1) and S1P phosphatase 1 on the other hand, as well as a down-regulation of sphingosine kinase 2. Application of LPS to a monolayer of primary MBMECs did not alter TEER, but serum from LPS-treated mice lead to a breakdown of the barrier compared to serum from vehicle-treated mice. We observed profound alterations of the sphingolipid metabolism at the BBB after LPS injection that point toward a therapeutic potential of drugs interfering with this pathway as novel approach for the detrimental overwhelming immune response in sepsis. Read the Editorial Highlight for this article on page 115. Cover Image for this Issue: doi. 10.1111/jnc.14161.

Reliability of infarct volumetry: Its relevance and the improvement by a software-assisted approach.

Despite the efficacy of neuroprotective approaches in animal models of stroke, their translation has so far failed from bench to bedside. One reason is presumed to be a low quality of preclinical study design, leading to bias and a low a priori power. In this study, we propose that the key read-out of experimental stroke studies, the volume of the ischemic damage as commonly measured by free-handed planimetry of TTC-stained brain sections, is subject to an unrecognized low inter-rater and test-retest reliability with strong implications for statistical power and bias. As an alternative approach, we suggest a simple, open-source, software-assisted method, taking advantage of automatic-thresholding techniques. The validity and the improvement of reliability by an automated method to tMCAO infarct volumetry are demonstrated. In addition, we show the probable consequences of increased reliability for precision, p-values, effect inflation, and power calculation, exemplified by a systematic analysis of experimental stroke studies published in the year 2015. Our study reveals an underappreciated quality problem in translational stroke research and suggests that software-assisted infarct volumetry might help to improve reproducibility and therefore the robustness of bench to bedside translation.

Angiopoietin-2-induced blood-brain barrier compromise and increased stroke size are rescued by VE-PTP-dependent restoration of Tie2 signaling.

The homeostasis of the central nervous system is maintained by the blood-brain barrier (BBB). Angiopoietins (Ang-1/Ang-2) act as antagonizing molecules to regulate angiogenesis, vascular stability, vascular permeability and lymphatic integrity. However, the precise role of angiopoietin/Tie2 signaling at the BBB remains unclear. We investigated the influence of Ang-2 on BBB permeability in wild-type and gain-of-function (GOF) mice and demonstrated an increase in permeability by Ang-2, both in vitro and in vivo. Expression analysis of brain endothelial cells from Ang-2 GOF mice showed a downregulation of tight/adherens junction molecules and increased caveolin-1, a vesicular permeability-related molecule. Immunohistochemistry revealed reduced pericyte coverage in Ang-2 GOF mice that was supported by electron microscopy analyses, which demonstrated defective intra-endothelial junctions with increased vesicles and decreased/disrupted glycocalyx. These results demonstrate that Ang-2 mediates permeability via paracellular and transcellular routes. In patients suffering from stroke, a cerebrovascular disorder associated with BBB disruption, Ang-2 levels were upregulated. In mice, Ang-2 GOF resulted in increased infarct sizes and vessel permeability upon experimental stroke, implicating a role of Ang-2 in stroke pathophysiology. Increased permeability and stroke size were rescued by activation of Tie2 signaling using a vascular endothelial protein tyrosine phosphatase inhibitor and were independent of VE-cadherin phosphorylation. We thus identified Ang-2 as an endothelial cell-derived regulator of BBB permeability. We postulate that novel therapeutics targeting Tie2 signaling could be of potential use for opening the BBB for increased CNS drug delivery or tighten it in neurological disorders associated with cerebrovascular leakage and brain edema.

Fingolimod for the treatment of neurological diseases-state of play and future perspectives.

Sphingolipids are a fascinating class of signaling molecules derived from the membrane lipid sphingomyelin. They show abundant expression in the brain. Complex sphingolipids such as glycosphingolipids (gangliosides and cerebrosides) regulate vesicular transport and lysosomal degradation and their dysregulation can lead to storage diseases with a neurological phenotype. More recently, simple sphingolipids such ceramide, sphingosine and sphingosine 1-phosphate (S1P) were discovered to signal in response to many extracellular stimuli. Forming an intricate signaling network, the balance of these readily interchangeable mediators is decisive for cell fate under stressful conditions. The immunomodulator fingolimod is the prodrug of an S1P receptor agonist. Following receptor activation, the drug leads to downregulation of the S1P1 receptor inducing functional antagonism. As the first drug to modulate the sphingolipid signaling pathway, it was marketed in 2010 for the treatment of multiple sclerosis (MS). At that time, immunomodulation was widely accepted as the key mechanism of fingolimod's efficacy in MS. But given the excellent passage of this lipophilic compound into the brain and its massive brain accumulation as well as the abundant expression of S1P receptors on brain cells, it is conceivable that fingolimod also affects brain cells directly. Indeed, a seminal study showed that the protective effect of fingolimod in experimental autoimmune encephalitis (EAE), a murine MS model, is lost in mice lacking the S1P1 receptor on astrocytes, arguing for a specific role of astrocytic S1P signaling in MS. In this review, we discuss the role of sphingolipid mediators and their metabolizing enzymes in neurologic diseases and putative therapeutic strategies arising thereof.