Integrated multi-omics analysis of PBX1 in mouse adult neural stem- and progenitor cells identifies a transcriptional module that functionally links PBX1 to TCF3/4.

Developmental transcription factors act in networks, but how these networks achieve cell- and tissue specificity is still poorly understood. Here, we explored pre-B cell leukemia homeobox 1 (PBX1) in adult neurogenesis combining genomic, transcriptomic, and proteomic approaches. ChIP-seq analysis uncovered PBX1 binding to numerous genomic sites. Integration of PBX1 ChIP-seq with ATAC-seq data predicted interaction partners, which were subsequently validated by mass spectrometry. Whole transcriptome spatial RNA analysis revealed shared expression dynamics of Pbx1 and interacting factors. Among these were class I bHLH proteins TCF3 and TCF4. RNA-seq following Pbx1, Tcf3 or Tcf4 knockdown identified proliferation- and differentiation associated genes as shared targets, while sphere formation assays following knockdown argued for functional cooperativity of PBX1 and TCF3 in progenitor cell proliferation. Notably, while physiological PBX1-TCF interaction has not yet been described, chromosomal translocation resulting in genomic TCF3::PBX1 fusion characterizes a subtype of acute lymphoblastic leukemia. Introducing Pbx1 into Nalm6 cells, a pre-B cell line expressing TCF3 but lacking PBX1, upregulated the leukemogenic genes BLK and NOTCH3, arguing that functional PBX1-TCF cooperativity likely extends to hematopoiesis. Our study hence uncovers a transcriptional module orchestrating the balance between progenitor cell proliferation and differentiation in adult neurogenesis with potential implications for leukemia etiology.

DNA methylation-based classification of central nervous system tumours.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

The endothelial-enriched lncRNA LINC00607 mediates angiogenic function.

Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates, post-atherosclerotic cultured endothelial cells from patients and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1 to ultimately mediate angiogenesis.

Integrative multi-omics reveals two biologically distinct groups of pilocytic astrocytoma.

Pilocytic astrocytoma (PA), the most common pediatric brain tumor, is driven by aberrant mitogen-activated protein kinase signaling most commonly caused by BRAF gene fusions or activating mutations. While 5-year overall survival rates exceed 95%, tumor recurrence or progression constitutes a major clinical challenge in incompletely resected tumors. Here, we used similarity network fusion (SNF) analysis in an integrative multi-omics approach employing RNA transcriptomic and mass spectrometry-based proteomic profiling to molecularly characterize PA tissue samples from 62 patients. Thereby, we uncovered that PAs segregated into two molecularly distinct groups, namely, Group 1 and Group 2, which were validated in three non-overlapping cohorts. Patients with Group 1 tumors were significantly younger and showed worse progression-free survival compared to patients with group 2 tumors. Ingenuity pathways analysis (IPA) and gene set enrichment analysis (GSEA) revealed that Group 1 tumors were enriched for immune response pathways, such as interferon signaling, while Group 2 tumors showed enrichment for action potential and neurotransmitter signaling pathways. Analysis of immune cell-related gene signatures showed an enrichment of infiltrating T Cells in Group 1 versus Group 2 tumors. Taken together, integrative multi-omics of PA identified biologically distinct and prognostically relevant tumor groups that may improve risk stratification of this single pathway driven tumor type.

MR imaging profile and histopathological characteristics of tumour vasculature, cell density and proliferation rate define two distinct growth patterns of human brain metastases from lung cancer.

PURPOSE: Non-invasive prediction of the tumour of origin giving rise to brain metastases (BMs) using MRI measurements obtained in radiological routine and elucidating the biological basis by matched histopathological analysis. METHODS: Preoperative MRI and histological parameters of 95 BM patients (female, 50; mean age 59.6 ± 11.5 years) suffering from different primary tumours were retrospectively analysed. MR features were assessed by region of interest (ROI) measurements of signal intensities on unenhanced T1-, T2-, diffusion-weighted imaging and apparent diffusion coefficient (ADC) normalised to an internal reference ROI. Furthermore, we assessed BM size and oedema as well as cell density, proliferation rate, microvessel density and vessel area as histopathological parameters. RESULTS: Applying recursive partitioning conditional inference trees, only histopathological parameters could stratify the primary tumour entities. We identified two distinct BM growth patterns depending on their proliferative status: Ki67high BMs were larger (p = 0.02), showed less peritumoural oedema (p = 0.02) and showed a trend towards higher cell density (p = 0.05). Furthermore, Ki67high BMs were associated with higher DWI signals (p = 0.03) and reduced ADC values (p = 0.004). Vessel density was strongly reduced in Ki67high BM (p < 0.001). These features differentiated between lung cancer BM entities (p ≤ 0.03 for all features) with SCLCs representing predominantly the Ki67high group, while NSCLCs rather matching with Ki67low features. CONCLUSION: Interpretable and easy to obtain MRI features may not be sufficient to predict directly the primary tumour entity of BM but seem to have the potential to aid differentiating high- and low-proliferative BMs, such as SCLC and NSCLC.

Profiling the neurovascular unit unveils detrimental effects of osteopontin on the blood-brain barrier in acute ischemic stroke.

Blood-brain barrier (BBB) dysfunction, characterized by degradation of BBB junctional proteins and increased permeability, is a crucial pathophysiological feature of acute ischemic stroke. Dysregulation of multiple neurovascular unit (NVU) cell types is involved in BBB breakdown in ischemic stroke that may be further aggravated by reperfusion therapy. Therefore, therapeutic co-targeting of dysregulated NVU cell types in acute ischemic stroke constitutes a promising strategy to preserve BBB function and improve clinical outcome. However, methods for simultaneous isolation of multiple NVU cell types from the same diseased central nervous system (CNS) tissue, crucial for the identification of therapeutic targets in dysregulated NVU cells, are lacking. Here, we present the EPAM-ia method, that facilitates simultaneous isolation and analysis of the major NVU cell types (endothelial cells, pericytes, astrocytes and microglia) for the identification of therapeutic targets in dysregulated NVU cells to improve the BBB function. Applying this method, we obtained a high yield of pure NVU cells from murine ischemic brain tissue, and generated a valuable NVU transcriptome database ( https://bioinformatics.mpi-bn.mpg.de/SGD_Stroke ). Dissection of the NVU transcriptome revealed Spp1, encoding for osteopontin, to be highly upregulated in all NVU cells 24 h after ischemic stroke. Upregulation of osteopontin was confirmed in stroke patients by immunostaining, which was comparable with that in mice. Therapeutic targeting by subcutaneous injection of an anti-osteopontin antibody post-ischemic stroke in mice resulted in neutralization of osteopontin expression in the NVU cell types investigated. Apart from attenuated glial activation, osteopontin neutralization was associated with BBB preservation along with decreased brain edema and reduced risk for hemorrhagic transformation, resulting in improved neurological outcome and survival. This was supported by BBB-impairing effects of osteopontin in vitro. The clinical significance of these findings is that anti-osteopontin antibody therapy might augment current approved reperfusion therapies in acute ischemic stroke by minimizing deleterious effects of ischemia-induced BBB disruption.

New Fully Automated Preparation of High Apparent Molar Activity 68Ga-FAPI-46 on a Trasis AiO Platform.

A large number of applications for fibroblast activation protein inhibitors (FAPI)-based PET agents have been evaluated in conditions ranging from cancer to non-malignant diseases such as myocardial infarction. In particular, 68Ga-FAPI-46 was reported to have a high specificity and affinity for FAP-expressing cells, a fast and high accumulation in tumor lesions/injuries together with a fast body clearance when investigated in vivo. Due to the increasing interest in the use of the agent both preclinically and clinically, we developed an automated synthesis for the production of 68Ga-FAPI-46 on a Trasis AiO platform. The new synthetic procedure, which included the processing of the generator eluate using a strong cation exchange resin and a final purification step through an HLB followed by a QMA cartridge, yielded 68Ga-FAPI-46 with high radiochemical purity (>98%) and apparent molar activity (271.1 ± 105.6 MBq/nmol). Additionally, the in vitro and in vivo properties of the product were assessed on glioblastoma cells and mouse model. Although developed for the preparation of 68Ga-FAPI-46 for preclinical use, our method can be adapted for clinical production as a reliable alternative to the manual (i.e., cold kit) or modular systems preparations already described in the literature.

Role of angiopoietin-2 in venous thrombus resolution and chronic thromboembolic disease.

BACKGROUND: Defective angiogenesis, incomplete thrombus revascularisation and fibrosis are considered critical pathomechanisms of chronic thromboembolic pulmonary hypertension (CTEPH) after pulmonary embolism. Angiopoietin-2 (ANGPT2) has been shown to regulate angiogenesis, but its importance for thrombus resolution and remodelling is unknown. METHODS: ANGPT2 plasma concentrations were measured in patients with CTEPH (n=68) and acute pulmonary embolism (n=84). Tissue removed during pulmonary endarterectomy (PEA) for CTEPH was analysed (immuno)histologically. A mouse model of inferior vena cava ligation was used to study the kinetics of venous thrombus resolution in wild-type mice receiving recombinant ANGPT2 via osmotic pumps, and in transgenic mice overexpressing ANGPT2 in endothelial cells. RESULTS: Circulating ANGPT2 levels were higher in CTEPH patients compared to patients with idiopathic pulmonary arterial hypertension and healthy controls, and decreased after PEA. Plasma ANGPT2 levels were elevated in patients with pulmonary embolism and diagnosis of CTEPH during follow-up. Histological analysis of PEA specimens confirmed increased ANGPT2 expression, and low levels of phosphorylated TIE2 were observed in regions with early-organised pulmonary thrombi, myofibroblasts and fibrosis. Microarray and high-resolution microscopy analysis could localise ANGPT2 overexpression to endothelial cells, and hypoxia and transforming growth factor-ß1 were identified as potential stimuli. Gain-of-function experiments in mice demonstrated that exogenous ANGPT2 administration and transgenic endothelial ANGPT2 overexpression resulted in delayed venous thrombus resolution, and thrombi were characterised by lower TIE2 phosphorylation and fewer microvessels. CONCLUSION: Our findings suggest that ANGPT2 delays venous thrombus resolution and that overexpression of ANGPT2 contributes to thrombofibrosis and may thus support the transition from pulmonary embolism to CTEPH.

Linking epigenetic signature and metabolic phenotype in IDH mutant and IDH wildtype diffuse glioma.

AIMS: Changes in metabolism are known to contribute to tumour phenotypes. If and how metabolic alterations in brain tumours contribute to patient outcome is still poorly understood. Epigenetics impact metabolism and mitochondrial function. The aim of this study is a characterisation of metabolic features in molecular subgroups of isocitrate dehydrogenase mutant (IDHmut) and isocitrate dehydrogenase wildtype (IDHwt) gliomas. METHODS: We employed DNA methylation pattern analyses with a special focus on metabolic genes, large-scale metabolism panel immunohistochemistry (IHC), qPCR-based determination of mitochondrial DNA copy number and immune cell content using IHC and deconvolution of DNA methylation data. We analysed molecularly characterised gliomas (n = 57) for in depth DNA methylation, a cohort of primary and recurrent gliomas (n = 22) for mitochondrial copy number and validated these results in a large glioma cohort (n = 293). Finally, we investigated the potential of metabolic markers in Bevacizumab (Bev)-treated gliomas (n = 29). RESULTS: DNA methylation patterns of metabolic genes successfully distinguished the molecular subtypes of IDHmut and IDHwt gliomas. Promoter methylation of lactate dehydrogenase A negatively correlated with protein expression and was associated with IDHmut gliomas. Mitochondrial DNA copy number was increased in IDHmut tumours and did not change in recurrent tumours. Hierarchical clustering based on metabolism panel IHC revealed distinct subclasses of IDHmut and IDHwt gliomas with an impact on patient outcome. Further quantification of these markers allowed for the prediction of survival under anti-angiogenic therapy. CONCLUSION: A mitochondrial signature was associated with increased survival in all analyses, which could indicate tumour subgroups with specific metabolic vulnerabilities.

HIF-1α is involved in blood-brain barrier dysfunction and paracellular migration of bacteria in pneumococcal meningitis.

Bacterial meningitis is a deadly disease most commonly caused by Streptococcus pneumoniae, leading to severe neurological sequelae including cerebral edema, seizures, stroke, and mortality when untreated. Meningitis is initiated by the transfer of S. pneumoniae from blood to the brain across the blood-cerebrospinal fluid barrier or the blood-brain barrier (BBB). The underlying mechanisms are still poorly understood. Current treatment strategies include adjuvant dexamethasone for inflammation and cerebral edema, followed by antibiotics. The success of dexamethasone is however inconclusive, necessitating new therapies for controlling edema, the primary reason for neurological complications. Since we have previously shown a general activation of hypoxia inducible factor (HIF-1α) in bacterial infections, we hypothesized that HIF-1α, via induction of vascular endothelial growth factor (VEGF) is involved in transmigration of pathogens across the BBB. In human, murine meningitis brain samples, HIF-1α activation was observed by immunohistochemistry. S. pneumoniae infection in brain endothelial cells (EC) resulted in in vitro upregulation of HIF-1α/VEGF (Western blotting/qRT-PCR) associated with increased paracellular permeability (fluorometry, impedance measurements). This was supported by bacterial localization at cell-cell junctions in vitro and in vivo in brain ECs from mouse and humans (confocal, super-resolution, electron microscopy, live-cell imaging). Hematogenously infected mice showed increased permeability, S. pneumoniae deposition in the brain, along with upregulation of genes in the HIF-1α/VEGF pathway (RNA sequencing of brain microvessels). Inhibition of HIF-1α with echinomycin, siRNA in bEnd5 cells or using primary brain ECs from HIF-1α knock-out mice revealed reduced endothelial permeability and transmigration of S. pneumoniae. Therapeutic rescue using the HIF-1α inhibitor echinomycin resulted in increased survival and improvement of BBB function in S. pneumoniae-infected mice. We thus demonstrate paracellular migration of bacteria across BBB and a critical role for HIF-1α/VEGF therein and hence propose targeting this pathway to prevent BBB dysfunction and ensuing brain damage in infections.

Papillary glioneuronal tumor (PGNT) exhibits a characteristic methylation profile and fusions involving PRKCA.

Papillary glioneuronal tumor (PGNT) is a WHO-defined brain tumor entity that poses a major diagnostic challenge. Recently, SLC44A1-PRKCA fusions have been described in PGNT. We subjected 28 brain tumors from different institutions histologically diagnosed as PGNT to molecular and morphological analysis. Array-based methylation analysis revealed that 17/28 tumors exhibited methylation profiles typical for other tumor entities, mostly dysembryoplastic neuroepithelial tumor and hemispheric pilocytic astrocytoma. Conversely, 11/28 tumors exhibited a unique profile, thus constituting a distinct methylation class PGNT. By screening the extended Heidelberg cohort containing over 25,000 CNS tumors, we identified three additional tumors belonging to this methylation cluster but originally histologically diagnosed otherwise. RNA sequencing for the detection of SLC44A1-PRKCA fusions could be performed on 19 of the tumors, 10 of them belonging to the methylation class PGNT. In two additional cases, SLC44A1-PRKCA fusions were confirmed by FISH. We detected fusions involving PRKCA in all cases of this methylation class with material available for analyses: the canonical SLC44A1-PRKCA fusion was observed in 11/12 tumors, while the remaining case exhibited a NOTCH1-PRKCA fusion. Neither of the fusions was found in the tumors belonging to other methylation classes. Our results point towards a high misclassification rate of the morphological diagnosis PGNT and clearly demonstrate the necessity of molecular analyses. PRKCA fusions are highly diagnostic for PGNT, and detection by RNA sequencing enables the identification of rare fusion partners. Methylation analysis recognizes a unique methylation class PGNT irrespective of the nature of the PRKCA fusion.

Long Noncoding RNA MANTIS Facilitates Endothelial Angiogenic Function.

BACKGROUND: The angiogenic function of endothelial cells is regulated by numerous mechanisms, but the impact of long noncoding RNAs (lncRNAs) has hardly been studied. We set out to identify novel and functionally important endothelial lncRNAs. METHODS: Epigenetically controlled lncRNAs in human umbilical vein endothelial cells were searched by exon-array analysis after knockdown of the histone demethylase JARID1B. Molecular mechanisms were investigated by RNA pulldown and immunoprecipitation, mass spectrometry, microarray, several knockdown approaches, CRISPR-Cas9, assay for transposase-accessible chromatin sequencing, and chromatin immunoprecipitation in human umbilical vein endothelial cells. Patient samples from lung and tumors were studied for MANTIS expression. RESULTS: A search for epigenetically controlled endothelial lncRNAs yielded lncRNA n342419, here termed MANTIS, as the most strongly regulated lncRNA. Controlled by the histone demethylase JARID1B, MANTIS was downregulated in patients with idiopathic pulmonary arterial hypertension and in rats treated with monocrotaline, whereas it was upregulated in carotid arteries of Macaca fascicularis subjected to atherosclerosis regression diet, and in endothelial cells isolated from human glioblastoma patients. CRISPR/Cas9-mediated deletion or silencing of MANTIS with small interfering RNAs or GapmeRs inhibited angiogenic sprouting and alignment of endothelial cells in response to shear stress. Mechanistically, the nuclear-localized MANTIS lncRNA interacted with BRG1, the catalytic subunit of the switch/sucrose nonfermentable chromatin-remodeling complex. This interaction was required for nucleosome remodeling by keeping the ATPase function of BRG1 active. Thereby, the transcription of key endothelial genes such as SOX18, SMAD6, and COUP-TFII was regulated by ensuring efficient RNA polymerase II machinery binding. CONCLUSION: MANTIS is a differentially regulated novel lncRNA facilitating endothelial angiogenic function.

Functional morphology of the blood-brain barrier in health and disease.

The adult quiescent blood-brain barrier (BBB), a structure organised by endothelial cells through interactions with pericytes, astrocytes, neurons and microglia in the neurovascular unit, is highly regulated but fragile at the same time. In the past decade, there has been considerable progress in understanding not only the molecular pathways involved in BBB development, but also BBB breakdown in neurological diseases. Specifically, the Wnt/ß-catenin, retinoic acid and sonic hedgehog pathways moved into the focus of BBB research. Moreover, angiopoietin/Tie2 signalling that is linked to angiogenic processes has gained attention in the BBB field. Blood vessels play an essential role in initiation and progression of many diseases, including inflammation outside the central nervous system (CNS). Therefore, the potential influence of CNS blood vessels in neurological diseases associated with BBB alterations or neuroinflammation has become a major focus of current research to understand their contribution to pathogenesis. Moreover, the BBB remains a major obstacle to pharmaceutical intervention in the CNS. The complications may either be expressed by inadequate therapeutic delivery like in brain tumours, or by poor delivery of the drug across the BBB and ineffective bioavailability. In this review, we initially describe the cellular and molecular components that contribute to the steady state of the healthy BBB. We then discuss BBB alterations in ischaemic stroke, primary and metastatic brain tumour, chronic inflammation and Alzheimer's disease. Throughout the review, we highlight common mechanisms of BBB abnormalities among these diseases, in particular the contribution of neuroinflammation to BBB dysfunction and disease progression, and emphasise unique aspects of BBB alteration in certain diseases such as brain tumours. Moreover, this review highlights novel strategies to monitor BBB function by non-invasive imaging techniques focussing on ischaemic stroke, as well as novel ways to modulate BBB permeability and function to promote treatment of brain tumours, inflammation and Alzheimer's disease. In conclusion, a deep understanding of signals that maintain the healthy BBB and promote fluctuations in BBB permeability in disease states will be key to elucidate disease mechanisms and to identify potential targets for diagnostics and therapeutic modulation of the BBB.

Dexamethasone-induced leukocytosis is associated with poor survival in newly diagnosed glioblastoma.

Despite its well-characterized side effects, dexamethasone is widely used in the pre-, peri- and postoperative neurosurgical setting due to its effective relief of tumor-induced symptoms through the reduction of tumor-associated edema. However, some patients show laboratory-defined dexamethasone induced elevation of white blood cell count, and its impact on glioblastoma progression is unknown. We retrospectively analyzed 113 patients with newly diagnosed glioblastoma to describe the incidence, risk factors and clinical features of dexamethasone-induced leukocytosis in primary glioblastoma patients. We further conducted an immunohistochemical analysis of the granulocyte and lymphocyte tumor-infiltration in the available corresponding histological sections. Patient age was identified to be a risk factor for the development of dexamethasone-induced leukocytosis (p < 0.05). The presence of dexamethasone-induced leukocytosis decreased overall survival (HR 2.25 95% CI [1.15-4.38]; p < 0.001) and progression-free survival (HR 2.23 95% CI [1.09-4.59]; p < 0.01). Furthermore, patients with dexamethasone-induced leukocytosis had significantly reduced CD15 + granulocytic- (p < 0.05) and CD3 + lymphocytic tumour infiltration (p < 0.05). We identified a subgroup of glioblastoma patients that are at particularly high risk for poor outcome upon dexamethasone treatment. Therefore, restrictive dosage or other edema reducing substances should be considered in patients with dexamethasone-induced leukocytosis.

ß-Catenin Is Required for Endothelial Cyp1b1 Regulation Influencing Metabolic Barrier Function.

UNLABELLED: The canonical Wnt/ß-catenin signaling pathway is crucial for blood-brain barrier (BBB) formation in brain endothelial cells. Although glucose transporter 1, claudin-3, and plasmalemma vesicular-associated protein have been identified as Wnt/ß-catenin targets in brain endothelial cells, further downstream targets relevant to BBB formation and function are incompletely explored. By Affymetrix expression analysis, we show that the cytochrome P450 enzyme Cyp1b1 was significantly decreased in ß-catenin-deficient mouse endothelial cells, whereas its close homolog Cyp1a1 was upregulated in an aryl hydrocarbon receptor-dependent manner, hence indicating that ß-catenin is indispensable for Cyp1b1 but not for Cyp1a1 expression. Functionally, Cyp1b1 could generate retinoic acid from retinol leading to cell-autonomous induction of the barrier-related ATP-binding cassette transporter P-glycoprotein. Cyp1b1 could also generate 20-hydroxyeicosatetraenoic acid from arachidonic acid, decreasing endothelial barrier function in vitro In mice in vivo pharmacological inhibition of Cyp1b1 increased BBB permeability for small molecular tracers, and Cyp1b1 was downregulated in glioma vessels in which BBB function is lost. Hence, we propose Cyp1b1 as a target of ß-catenin indirectly influencing BBB properties via its metabolic activity, and as a potential target for modulating barrier function in endothelial cells. SIGNIFICANCE STATEMENT: Wnt/ß-catenin signaling is crucial for blood-brain barrier (BBB) development and maintenance; however, its role in regulating metabolic characteristics of endothelial cells is unclear. We provide evidence that ß-catenin influences endothelial metabolism by transcriptionally regulating the cytochrome P450 enzyme Cyp1b1 Furthermore, expression of its close homolog Cyp1a1 was inhibited by ß-catenin. Functionally, Cyp1b1 generated retinoic acid as well as 20-hydroxyeicosatetraenoic acid that regulated P-glycoprotein and junction proteins, respectively, thereby modulating BBB properties. Inhibition of Cyp1b1 in vivo increased BBB permeability being in line with its downregulation in glioma endothelia, potentially implicating Cyp1b1 in other brain pathologies. In conclusion, Wnt/ß-catenin signaling regulates endothelial metabolic barrier function through Cyp1b1 transcription.

Extracellular vesicle-mediated transfer of genetic information between the hematopoietic system and the brain in response to inflammation.

Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.

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.

ß-Catenin-Gli1 interaction regulates proliferation and tumor growth in medulloblastoma.

BACKGROUND: The Wnt/beta-catenin and the Hedgehog (Hh) pathway interact in various cell types while eliciting opposing or synergistic cellular effects. Both pathways are known as exclusive drivers of two distinct molecular subtypes of medulloblastoma (MB). In sonic hedgehog (Shh)-driven MB, activation of Wnt signaling has been shown to suppress tumor growth by either beta-catenin-dependent or -independent inhibition of Shh signaling. However, mechanistic insight in how beta-catenin inhibits the Hh pathway is not known. FINDINGS: Here we show that beta-catenin stabilization by the glycogen synthase kinase 3 inhibitor lithium chloride (LiCl) reduced growth of primary hedgehog-driven MB tumor spheres from patched heterozygous mice (Ptch(+/-)) in vitro. LiCl treatment of MB spheres down-regulated the Hh target Gli1, whereas the repressive Gli3 protein (Gli3R) was increased. Mechanistically, we show by co-immunoprecipitation and proximity ligation assay that stabilized beta-catenin physically interacts with Gli1, leading to Gli1 sequestration and inhibition of its transcriptional activity. Reduction of Hh signaling upon LiCl stimulation resulted in reduced proliferation, sphere self renewal, a G2/M arrest and induction of a senescent-like state, indicated by p21 upregulation and by increased staining of senescence-associated beta-galactosidase (SA-betaGal). Moreover, LiCl treatment of subcutaneously transplanted MB cells significantly reduced tumor initiation defined as "tumor take". Although tumor progression was similar, LiCl-treated tumors showed decreased mitotic figures and phospho-histone H3 staining. CONCLUSION: We propose that beta-catenin stabilization increases its physical interaction with Gli1, leading to Gli1 degradation and inhibition of Hh signaling, thereby promoting tumor cell senescence and suppression of "tumor take" in mice.

Tumour necrosis factor receptor superfamily member 9 (TNFRSF9) is up-regulated in reactive astrocytes in human gliomas.

AIMS: The prognosis of patients with malignant gliomas is still dismal despite maximum treatment. Novel therapeutic alternatives targeting tumorigenic pathways are, therefore, demanded. In murine glioma models, targeting of tumour necrosis factor receptor superfamily (TNFRSF) 9 led to complete tumour eradication. Thus, TNFRSF9 might also constitute a promising target in human diffuse gliomas. As there is a lack of data, we aimed to define the expression pattern and cellular source of TNFRSF9 in human gliomas. METHODS: We investigated TNFRSF9 expression in normal human central nervous system (CNS) tissue and glioma specimens using immunohistochemistry, immunofluorescence and Western blotting techniques. RESULTS: Our results show that TNFRSF9 is considerably up-regulated in human gliomas when compared with normal brain tissue. In addition, our data provides evidence for an immune cell-independent de novo expression pattern of TNFRSF9 in mainly non-neoplastic reactive astrocytes and excludes classic immunological cell types, namely lymphocytes and microglia as the source of TNFRSF9. Moreover, TNFRSF9 is predominantly expressed in a perivascular and peritumoural distribution with significantly higher expression in IDH-1 mutant gliomas. CONCLUSIONS: Our findings provide a novel, TNFRSF9-positive, reactive astrocytic phenotype and challenge the therapeutic suitability of TNFRSF9 as a promising target for human gliomas.

Paired box gene 8 (PAX8) expression is associated with sonic hedgehog (SHH)/wingless int (WNT) subtypes, desmoplastic histology and patient survival in human medulloblastomas.

AIMS: The paired box gene 8 (PAX8) plays crucial roles in organ patterning and cellular differentiation during development and tumorigenesis. Although its function is partly understood in vertebrate development, there is poor data concerning human central nervous system (CNS) development and brain tumours. METHODS: We investigated developing human (n = 19) and mouse (n = 3) brains as well as medulloblastomas (MBs) (n = 113) for PAX8 expression by immunohistochemistry. Human MB cell lines were assessed for PAX8 expression using polymerase chain reaction and immunoblotting and analysed for growth and migration following PAX8 knock-down by small interfering ribonucleic acid (siRNA). RESULTS: PAX8 protein expression was associated with germinal layers in human and murine forebrain and hindbrain development. PAX8 expression significantly decreased over time in the external granule cell layer but increased in the internal granule cell layer. In MB subtypes, we observed an association of PAX8 expression with sonic hedgehog (SHH) and wingless int subtypes but not with group 3 and 4 MBs. Beyond that, we detected high PAX8 levels in desmoplastic MB subtypes. Univariate analyses revealed high PAX8 levels as a prognostic factor associated with a significantly better patient prognosis in human MB (overall survival: Log-Rank P = 0.0404, Wilcoxon P = 0.0280; progression-free survival: Log-Rank P = 0.0225; Wilcoxon P = 0.0136). In vitro assays revealed increased proliferation and migration of MB cell lines after PAX8 siRNA knock-down. CONCLUSION: In summary, high PAX8 expression is linked to better prognosis in MBs potentially by suppressing both proliferative and migratory properties of MB cells. The distinct spatio-temporal expression pattern of PAX8 during brain development might contribute to the understanding of distinct MB subtype histogenesis.