Proteomics on human cerebral cavernous malformations reveals novel biomarkers in neurovascular dysfunction for the disease pathology.

BACKGROUND: Cerebral cavernous malformation (CCM) is a disease associated with an elevated risk of focal neurological deficits, seizures, and hemorrhagic stroke. The disease has an inflammatory profile and improved knowledge of CCM pathology mechanisms and exploration of candidate biomarkers will enable new non-invasive treatments. METHODS: We analyzed protein signatures in human CCM tissue samples by using a highly specific and sensitive multiplexing technique, proximity extension assay. FINDINGS: Data analysis revealed CCM specific proteins involved in endothelial dysfunction/inflammation/activation, leukocyte infiltration/chemotaxis, hemostasis, extracellular matrix dysfunction, astrocyte and microglial cell activation. Biomarker expression profiles matched bleeding status, especially with higher levels of inflammatory markers and activated astrocytes in ruptured than non-ruptured samples, some of these biomarkers are secreted into blood or urine. Furthermore, analysis was also done in a spatially resolving manner by separating the lesion area from the surrounding brain tissue. Our spatial studies revealed that although appearing histologically normal, the CCM border areas were pathological when compared to control brain tissues. Moreover, the functional relevance of CD93, ICAM-1 and MMP9, markers related to endothelial cell activation and extracellular matrix was validated by a murine pre-clinical CCM model. INTERPRETATION: Here we present a novel strategy for proteomics analysis on human CCMs, offering a possibility for high-throughput protein screening acquiring data on the local environment in the brain. Our data presented here describe CCM relevant brain proteins and specifically those which are secreted can serve the need of circulating CCM biomarkers to predict cavernoma's risk of bleeding.

Altered cytoskeletal status in the transition from proneural to mesenchymal glioblastoma subtypes.

Glioblastoma is a highly aggressive brain tumor with poor patient prognosis. Treatment outcomes remain limited, partly due to intratumoral heterogeneity and the invasive nature of the tumors. Glioblastoma cells invade and spread into the surrounding brain tissue, and even between hemispheres, thus hampering complete surgical resection. This invasive motility can arise through altered properties of the cytoskeleton. We hypothesize that cytoskeletal organization and dynamics can provide important clues to the different malignant states of glioblastoma. In this study, we investigated cytoskeletal organization in glioblastoma cells with different subtype expression profiles, and cytoskeletal dynamics upon subtype transitions. Analysis of the morphological, migratory, and invasive properties of glioblastoma cells identified cytoskeletal components as phenotypic markers that can serve as diagnostic or prognostic tools. We also show that the cytoskeletal function and malignant properties of glioblastoma cells shift during subtype transitions induced by altered expression of the neurodevelopmental transcription factor SOX2. The potential of SOX2 re-expression to reverse the mesenchymal subtype into a more proneural subtype might open up strategies for novel glioblastoma treatments.

ELTD1 deletion reduces vascular abnormality and improves T-cell recruitment after PD-1 blockade in glioma.

BACKGROUND: Tumor vessels in glioma are molecularly and functionally abnormal, contributing to treatment resistance. Proteins differentially expressed in glioma vessels can change vessel phenotype and be targeted for therapy. ELTD1 (Adgrl4) is an orphan member of the adhesion G-protein-coupled receptor family upregulated in glioma vessels and has been suggested as a potential therapeutic target. However, the role of ELTD1 in regulating vessel function in glioblastoma is poorly understood. METHODS: ELTD1 expression in human gliomas and its association with patient survival was determined using tissue microarrays and public databases. The role of ELTD1 in regulating tumor vessel phenotype was analyzed using orthotopic glioma models and ELTD1-/- mice. Endothelial cells isolated from murine gliomas were transcriptionally profiled to determine differentially expressed genes and pathways. The consequence of ELTD1 deletion on glioma immunity was determined by treating tumor-bearing mice with PD-1-blocking antibodies. RESULTS: ELTD1 levels were upregulated in human glioma vessels, increased with tumor malignancy, and were associated with poor patient survival. Progression of orthotopic gliomas was not affected by ELTD1 deletion, however, tumor vascular function was improved in ELTD1-/- mice. Bioinformatic analysis of differentially expressed genes indicated increased inflammatory response and decreased proliferation in tumor endothelium in ELTD1-/- mice. Consistent with an enhanced inflammatory response, ELTD1 deletion improved T-cell infiltration in GL261-bearing mice after PD-1 checkpoint blockade. CONCLUSION: Our data demonstrate that ELTD1 participates in inducing vascular dysfunction in glioma, and suggest that targeting of ELTD1 may normalize the vessels and improve the response to immunotherapy.

Propranolol Reduces the Development of Lesions and Rescues Barrier Function in Cerebral Cavernous Malformations: A Preclinical Study.

[Figure: see text].

Mapping endothelial-cell diversity in cerebral cavernous malformations at single-cell resolution.

Cerebral cavernous malformation (CCM) is a rare neurovascular disease that is characterized by enlarged and irregular blood vessels that often lead to cerebral hemorrhage. Loss-of-function mutations to any of three genes results in CCM lesion formation; namely, KRIT1, CCM2, and PDCD10 (CCM3). Here, we report for the first time in-depth single-cell RNA sequencing, combined with spatial transcriptomics and immunohistochemistry, to comprehensively characterize subclasses of brain endothelial cells (ECs) under both normal conditions and after deletion of Pdcd10 (Ccm3) in a mouse model of CCM. Integrated single-cell analysis identifies arterial ECs as refractory to CCM transformation. Conversely, a subset of angiogenic venous capillary ECs and respective resident endothelial progenitors appear to be at the origin of CCM lesions. These data are relevant for the understanding of the plasticity of the brain vascular system and provide novel insights into the molecular basis of CCM disease at the single cell level.

Fine-Tuning of Sox17 and Canonical Wnt Coordinates the Permeability Properties of the Blood-Brain Barrier.

RATIONALE: The microvasculature of the central nervous system includes the blood-brain barrier (BBB), which regulates the permeability to nutrients and restricts the passage of toxic agents and inflammatory cells. Canonical Wnt/ß-catenin signaling is responsible for the early phases of brain vascularization and BBB differentiation. However, this signal declines after birth, and other signaling pathways able to maintain barrier integrity at postnatal stage are still unknown. OBJECTIVE: Sox17 (SRY [sex-determining region Y]-box 17) constitutes a major downstream target of Wnt/ß-catenin in endothelial cells and regulates arterial differentiation. In the present article, we asked whether Sox17 may act downstream of Wnt/ß-catenin in inducing BBB differentiation and maintenance. METHODS AND RESULTS: Using reporter mice and nuclear staining of Sox17 and ß-catenin, we report that although ß-catenin signaling declines after birth, Sox17 activation increases and remains high in the adult. Endothelial-specific inactivation of Sox17 leads to increase of permeability of the brain microcirculation. The severity of this effect depends on the degree of BBB maturation: it is strong in the embryo and progressively declines after birth. In search of Sox17 mechanism of action, RNA sequencing analysis of gene expression of brain endothelial cells has identified members of the Wnt/ß-catenin signaling pathway as downstream targets of Sox17. Consistently, we found that Sox17 is a positive inducer of Wnt/ß-catenin signaling, and it acts in concert with this pathway to induce and maintain BBB properties. In vivo, inhibition of the ß-catenin destruction complex or expression of a degradation-resistant ß-catenin mutant, prevent the increase in permeability and retina vascular malformations observed in the absence of Sox17. CONCLUSIONS: Our data highlight a novel role for Sox17 in the induction and maintenance of the BBB, and they underline the strict reciprocal tuning of this transcription factor and Wnt/ß-catenin pathway. Modulation of Sox17 activity may be relevant to control BBB permeability in pathological conditions.

Paternal easiRNAs regulate parental genome dosage in Arabidopsis.

The regulation of parental genome dosage is of fundamental importance in animals and plants, as exemplified by X-chromosome inactivation and dosage compensation. The 'triploid block' is a classic example of dosage regulation in plants that establishes a reproductive barrier between species differing in chromosome number1,2. This barrier acts in the embryo-nourishing endosperm tissue and induces the abortion of hybrid seeds through a yet unknown mechanism 3 . Here we show that depletion of paternal epigenetically activated small interfering RNAs (easiRNAs) bypasses the triploid block in response to increased paternal ploidy in Arabidopsis thaliana. Paternal loss of the plant-specific RNA polymerase IV suppressed easiRNA formation and rescued triploid seeds by restoring small-RNA-directed DNA methylation at transposable elements (TEs), correlating with reduced expression of paternally expressed imprinted genes (PEGs). Our data suggest that easiRNAs form a quantitative signal for paternal chromosome number and that their balanced dosage is required for post-fertilization genome stability and seed viability.

The meiotic regulator JASON utilizes alternative translation initiation sites to produce differentially localized forms.

The JASON (JAS) protein plays an important role in maintaining an organelle band across the equator of male meiotic cells during the second division, with its loss leading to unreduced pollen in Arabidopsis. In roots cells, JAS localizes to the Golgi, tonoplast and plasma membrane. Here we explore the mechanism underlying the localization of JAS. Overall, our data show that leaky ribosom scanning and alternative translation initiation sites (TISs) likely leads to the formation of two forms of JAS: a long version with an N-terminal Golgi localization signal and a short version with a different N-terminal signal targeting the protein to the plasma membrane. The ratio of the long and short forms of JAS is developmentally regulated, with both being produced in roots but the short form being predominant and functional during meiosis. This regulation of TISs in meiocytes ensures that the short version of JAS is formed during meiosis to ensure separation of chromosome groups and the production of reduced pollen. We hypothesize that increased occurrence of unreduced pollen under stress conditions may be a consequence of altered usage of JAS TISs during stress.

Transcriptome profiling of tobacco (Nicotiana tabacum) pollen and pollen tubes.

BACKGROUND: Pollen tube growth is essential for plant reproduction and represents a widely employed model to investigate polarized cell expansion, a process important for plant morphogenesis and development. Cellular and regulatory mechanisms underlying pollen tube elongation are under intense investigation, which stands to greatly benefit from a comprehensive understanding of global gene expression profiles in pollen and pollen tubes. Here, RNA sequencing technology was applied to de novo assemble a Nicotiana tabacum male gametophytic transcriptome and to compare transcriptome profiles at two different stages of gametophyte development: mature pollen grains (MPG) and pollen tubes grown for six hours in vitro (PT6). RESULTS: De novo assembly of data obtained by 454 sequencing of a normalized cDNA library representing tobacco pollen and pollen tube mRNA (pooled mRNA isolated from mature pollen grains [MPG] and from pollen tubes grown in vitro for 3 [PT3] or 6 [PT6] hours) resulted in the identification of 78,364 unigenes. Among these unigenes, which mapped to 24,933 entries in the Sol Genomics Network (SGN) N. tabacum unigene database, 24,672 were predicted to represent full length cDNAs. In addition, quantitative analyses of data obtained by Illumina sequencing of two separate non-normalized MPG and PT6 cDNA libraries showed that 8979 unigenes were differentially expressed (differentially expressed unigenes: DEGs) between these two developmental stages at a FDR q-value of <0.0001. Interestingly, whereas most of these DEGs were downregulated in PT6, the minor fraction of DEGs upregulated in PT6 was enriched for GO (gene ontology) functions in pollen tube growth or fertilization. CONCLUSIONS: A major output of our study is the development of two different high-quality databases representing the tobacco male gametophytic transcriptome and containing encompassing information about global changes in gene expression after pollen germination. Quantitative analyses of these databases 1) indicated that roughly 30% of all tobacco genes are expressed in the male gametophyte, and 2) support previous observations suggesting a global reduction of transcription after pollen germination. Interestingly, a small number of genes, many of which predicted to function in pollen tube growth or fertilization, were found to be upregulated in elongating pollen tubes despite globally reduced transcription.

Recent Sex Chromosome Divergence despite Ancient Dioecy in the Willow Salix viminalis.

Sex chromosomes can evolve when recombination is halted between a pair of chromosomes, and this can lead to degeneration of the sex-limited chromosome. In the early stages of differentiation sex chromosomes are homomorphic, and even though homomorphic sex chromosomes are very common throughout animals and plants, we know little about the evolutionary forces shaping these types of sex chromosomes. We used DNA- and RNA-Seq data from females and males to explore the sex chromosomes in the female heterogametic willow, Salix viminalis, a species with ancient dioecy but with homomorphic sex chromosomes. We detected no major sex differences in read coverage in the sex determination (SD) region, indicating that the W region has not significantly degenerated. However, single nucleotide polymorphism densities in the SD region are higher in females compared with males, indicating very recent recombination suppression, followed by the accumulation of sex-specific single nucleotide polymorphisms. Interestingly, we identified two female-specific scaffolds that likely represent W-chromosome-specific sequence. We show that genes located in the SD region display a mild excess of male-biased expression in sex-specific tissue, and we use allele-specific gene expression analysis to show that this is the result of masculinization of expression on the Z chromosome rather than degeneration of female-expression on the W chromosome. Together, our results demonstrate that insertion of small DNA fragments and accumulation of sex-biased gene expression can occur before the detectable decay of the sex-limited chromosome.