CNS angiogenesis and barriergenesis occur simultaneously.

The blood-brain barrier (BBB) plays a vital role in the central nervous system (CNS). A comprehensive understanding of BBB development has been hampered by difficulties in observing the differentiation of brain endothelial cells (BECs) in real-time. Here, we generated two transgenic zebrafish line, Tg(glut1b:mCherry) and Tg(plvap:EGFP), to serve as in vivo reporters of BBB development. We showed that barriergenesis (i.e. the induction of BEC differentiation) occurs immediately as endothelial tips cells migrate into the brain parenchyma. Using the Tg(glut1b:mCherry) transgenic line, we performed a genetic screen and identified a zebrafish mutant with a nonsense mutation in gpr124, a gene known to play a role in CNS angiogenesis and BBB development. We also showed that our transgenic plvap:EGFP line, a reporter of immature brain endothelium, is initially expressed in newly formed brain endothelial cells, but subsides during BBB maturation. Our results demonstrate the ability to visualize the in vivo differentiation of brain endothelial cells into the BBB phenotype and establish that CNS angiogenesis and barriergenesis occur simultaneously.

Leveraging Zebrafish To Study Bona Fide Astrocytes.

In a recent study, Chen and colleagues demonstrated that zebrafish spinal cord radial glia differentiate into cells that are similar to mammalian astrocytes. This study highlights the validity of the zebrafish model for discovering molecular mechanisms governing astrocyte function.

Fishing for Contact: Modeling Perivascular Glioma Invasion in the Zebrafish Brain.

Glioblastoma multiforme (GBM) is a highly invasive, central nervous system (CNS) cancer for which there is no cure. Invading tumor cells evade treatment, limiting the efficacy of the current standard of care regimen. Understanding the underlying invasive behaviors that support tumor growth may allow for generation of novel GBM therapies. Zebrafish (Danio rerio) are attractive for genetics and live imaging and have, in recent years, emerged as a model system suitable for cancer biology research. While other groups have studied CNS tumors using zebrafish, few have concentrated on the invasive behaviors supporting the development of these diseases. Previous studies demonstrated that one of the main mechanisms of GBM invasion is perivascular invasion, i.e., single tumor cell migration along blood vessels. Here, we characterize phenotypes, methodology, and potential therapeutic avenues for utilizing zebrafish to model perivascular GBM invasion. Using patient-derived xenolines or an adherent cell line, we demonstrate tumor expansion within the zebrafish brain. Within 24-h postintracranial injection, D54-MG-tdTomato glioma cells produce fingerlike projections along the zebrafish brain vasculature. As few as 25 GBM cells were sufficient to promote single cell vessel co-option. Of note, these tumor-vessel interactions are CNS specific and do not occur on pre-existing blood vessels when injected into the animal's peripheral tissue. Tumor-vessel interactions increase over time and can be pharmacologically disrupted through inhibition of Wnt signaling. Therefore, zebrafish serve as a favorable model system to study perivascular glioma invasion, one of the deadly characteristics that make GBM so difficult to treat.

Using Zebrafish to Elucidate Glial-Vascular Interactions During CNS Development.

An emerging area of interest in Neuroscience is the cellular relationship between glia and blood vessels, as many of the presumptive support roles of glia require an association with the vasculature. These interactions are best studied in vivo and great strides have been made using mice to longitudinally image glial-vascular interactions. However, these methods are cumbersome for developmental studies, which could benefit from a more accessible system. Zebrafish (Danio rerio) are genetically tractable vertebrates, and given their translucency, are readily amenable for daily live imaging studies. We set out to examine whether zebrafish glia have conserved traits with mammalian glia regarding their ability to interact with and maintain the developing brain vasculature. We utilized transgenic zebrafish strains in which oligodendrocyte transcription factor 2 (olig2) and glial fibrillary acidic protein (gfap) identify different glial populations in the zebrafish brain and document their corresponding relationship with brain blood vessels. Our results demonstrate that olig2+ and gfap+ zebrafish glia have distinct lineages and each interact with brain vessels as previously observed in mouse brain. Additionally, we manipulated these relationships through pharmacological and genetic approaches to distinguish the roles of these cell types during blood vessel development. olig2+ glia use blood vessels as a pathway during their migration and Wnt signaling inhibition decreases their single-cell vessel co-option. By contrast, the ablation of gfap+ glia at the beginning of CNS angiogenesis impairs vessel development through a reduction in Vascular endothelial growth factor (Vegf), supporting a role for gfap+ glia during new brain vessel formation in zebrafish. This data suggests that zebrafish glia, akin to mammalian glia, have different lineages that show diverse interactions with blood vessels, and are a suitable model for elucidating glial-vascular relationships during vertebrate brain development.

Transcriptional Regulation of Amino Acid Transport in Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is a deadly brain tumor with a large unmet therapeutic need. Here, we tested the hypothesis that wild-type p53 is a negative transcriptional regulator of SLC7A11, the gene encoding the System xc- (SXC) catalytic subunit, xCT, in GBM. We demonstrate that xCT expression is inversely correlated with p53 expression in patient tissue. Using representative patient derived (PDX) tumor xenolines with wild-type, null, and mutant p53 we show that p53 expression negatively correlates with xCT expression. Using chromatin immunoprecipitation studies, we present a molecular interaction whereby p53 binds to the SLC7A11 promoter, suppressing gene expression in PDX GBM cells. Accordingly, genetic knockdown of p53 increases SLC7A11 transcript levels; conversely, over-expressing p53 in p53-null GBM cells downregulates xCT expression and glutamate release. Proof of principal studies in mice with flank gliomas demonstrate that daily treatment with the mutant p53 reactivator, PRIMA-1Met, results in reduced tumor growth associated with reduced xCT expression. These findings suggest that p53 is a molecular switch for GBM glutamate biology, with potential therapeutic utility.

The predictive capability of immunohistochemistry and DNA sequencing for determining TP53 functional mutation status: a comparative study of 41 glioblastoma patients.

Tumor protein 53 (p53) regulates fundamental pathways of cellular growth and differentiation. Aberrant p53 expression in glioblastoma multiforme, a terminal brain cancer, has been associated with worse patient outcomes and decreased chemosensitivity. Therefore, correctly identifying p53 status in glioblastoma is of great clinical significance. p53 immunohistochemistry is used to detect pathological presence of the TP53 gene product. Here, we examined the relationship between p53 immunoreactivity and TP53 mutation status by DNA Sanger sequencing in adult glioblastoma. Of 41 histologically confirmed samples, 27 (66%) were immunopositive for a p53 mutation via immunohistochemistry. Utilizing gene sequencing, we identified only eight samples (20%) with TP53 functional mutations and one sample with a silent mutation. Therefore, a ≥10% p53 immunohistochemistry threshold for predicting TP53 functional mutation status in glioma is insufficient. Implementing this ≥10% threshold, we demonstrated a remarkably low positive-predictive value (30%). Furthermore, the sensitivity and specificity with ≥10% p53 immunohistochemistry to predict TP53 functional mutation status were 100% and 42%, respectively. Our data suggests that unless reliable sequencing methodology is available for confirming TP53 status, raising the immunoreactivity threshold would increase positive and negative predictive values as well as the specificity without changing the sensitivity of the immunohistochemistry assay.

Combating malignant astrocytes: Strategies mitigating tumor invasion.

Malignant gliomas are glial-derived, primary brain tumors that carry poor prognosis. Existing therapeutics are largely ineffective and dramatically affect quality of life. The standard of care details a taxing combination of surgical resection, radiation of the resection cavity, and temozolomide (TMZ) chemotherapy, with treatment extending life by only an average of months (Maher et al., 2001; Stupp et al., 2005). Despite scientific and technological advancement, surgery remains the most important treatment modality. Therapeutic obstacles include xenobiotic protection conveyed by the blood-brain barrier (Zhang et al., 2015), invasiveness and therapeutic resistance of tumor cell populations (Bao et al., 2006), and distinctive attributes of secondary glioma occurrence (Ohgaki and Kleihues, 2013). While these brain malignancies can be classified by grade or grouped by molecular subclass, each tumor presents itself as its own complication. Based on all of these obstacles, new therapeutic approaches are urgently needed. These will likely emerge from numerous exciting studies of glioma biology that are ongoing and reviewed here. These show unexpected roles for ion channels, amino-acid transporters, and connexin gap junctions in supporting the invasive growth of gliomas. These studies have identified a number of proteins that may be targeted for therapy in the future.

CDK2 inhibitors as candidate therapeutics for cisplatin- and noise-induced hearing loss.

Hearing loss caused by aging, noise, cisplatin toxicity, or other insults affects 360 million people worldwide, but there are no Food and Drug Administration-approved drugs to prevent or treat it. We screened 4,385 small molecules in a cochlear cell line and identified 10 compounds that protected against cisplatin toxicity in mouse cochlear explants. Among them, kenpaullone, an inhibitor of multiple kinases, including cyclin-dependent kinase 2 (CDK2), protected zebrafish lateral-line neuromasts from cisplatin toxicity and, when delivered locally, protected adult mice and rats against cisplatin- and noise-induced hearing loss. CDK2-deficient mice displayed enhanced resistance to cisplatin toxicity in cochlear explants and to cisplatin- and noise-induced hearing loss in vivo. Mechanistically, we showed that kenpaullone directly inhibits CDK2 kinase activity and reduces cisplatin-induced mitochondrial production of reactive oxygen species, thereby enhancing cell survival. Our experiments have revealed the proapoptotic function of CDK2 in postmitotic cochlear cells and have identified promising therapeutics for preventing hearing loss.

Carboxylesterases: General detoxifying enzymes.

Carboxylesterases (CE) are members of the esterase family of enzymes, and as their name suggests, they are responsible for the hydrolysis of carboxylesters into the corresponding alcohol and carboxylic acid. To date, no endogenous CE substrates have been identified and as such, these proteins are thought to act as a mechanism to detoxify ester-containing xenobiotics. As a consequence, they are expressed in tissues that might be exposed to such agents (lung and gut epithelia, liver, kidney, etc.). CEs demonstrate very broad substrate specificities and can hydrolyze compounds as diverse as cocaine, oseltamivir (Tamiflu), permethrin and irinotecan. In addition, these enzymes are irreversibly inhibited by organophosphates such as Sarin and Tabun. In this overview, we will compare and contrast the two human enzymes that have been characterized, and evaluate the biology of the interaction of these proteins with organophosphates (principally nerve agents).