Extracellular vesicles derived from the choroid plexus trigger the differentiation of neural stem cells.

The choroid plexus secrets cerebrospinal fluid (CSF) composed of electrolytes, cytokines, growth factors, metabolites and extracellular vesicles (EVs) that flow through the interconnected brain ventricles. On their course, CSF components can act as signals that affect, for example, neural stem cells (NSCs) residing in niches of the ventricular wall. We studied EV-born CSF signals in an in vitro culture system. We purified EVs from the secretome of a choroid plexus cell line (Z310 cells), and from primary choroid plexus cultures and co-cultured those EVs with NSCs isolated from the niche of the lateral and the third ventricle. EVsZ310 and EVsCHP were purified by differential centrifugation. This yielded fractions of EVs of 50-150-nm diameter that induced a complex multicellular network formation and NSC differentiation. Both types of EV converted the round NSCs to cells that extended long processes that contacted nearby, alike-shaped cells. Mass spectrometry showed that the differentiation-inducing EVZ310 were enriched for membrane and membrane-associated proteins involved in cell differentiation, membrane trafficking, and membrane organization. We hypothesize that this type of EV Z310 cargo causes changes of stem cell morphology that leads to multicellular networks in the niches. This cell-shape transition may represent an initial step in NSC differentiation.

Cilia-driven flows in the brain third ventricle.

The brain ventricles are interconnected, elaborate cavities that traverse the brain. They are filled with cerebrospinal fluid (CSF) that is, to a large part, produced by the choroid plexus, a secretory epithelium that reaches into the ventricles. CSF is rich in cytokines, growth factors and extracellular vesicles that glide along the walls of ventricles, powered by bundles of motile cilia that coat the ventricular wall. We review the cellular and biochemical properties of the ventral part of the third ventricle that is surrounded by the hypothalamus. In particular, we consider the recently discovered intricate network of cilia-driven flows that characterize this ventricle and discuss the potential physiological significance of this flow for the directional transport of CSF signals to cellular targets located either within the third ventricle or in the adjacent hypothalamic brain parenchyma. Cilia-driven streams of signalling molecules offer an exciting perspective on how fluid-borne signals are dynamically transmitted in the brain. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

Monoclonal antibody G250 targeting CA â ¨: Binding specificity, internalization and therapeutic effects in a non-renal cancer model.

G250 (Girentuximab) is a chimeric IgG1 monoclonal antibody (MAb) currently being evaluated as an immunotherapy for kidney cancer. It targets carbonic anhydrase protein (CA Ⅸ), a transmembrane carbonic anhydrase (CA) isoform, which is regulated by VHL/HIF pathway and hence expressed in the majority of renal cell carcinomas (RCCs) as well as in hypoxic non­RCC tumours. CA Ⅸ functions in pH regulation and cell migration/invasion, and supports tumour cell survival in hypoxia and/or acidosis. It contains a highly active extracellular catalytic domain (CA) extended N-terminally with a proteoglycan-like region and C-terminally with short transmembrane and intracellular regions. Here we characterize the binding and internalization properties of G250, as well as its therapeutic effects in animal model, and discuss the impact of G250­mediated immunotherapy in non­RCC tumours. We demonstrated that G250 MAb recognizes a conformational epitope in the CA domain, detects the soluble CA Ⅸ ectodomain (ECD), but not the splicing variant, and does not cross-react with CA Ⅰ, Ⅱ, and Ⅻ isoforms. We showed that G250 internalizes via clathrin-coated vesicles, escapes degradation in lysosomes and enters the recycling pathway via the perinuclear compartment. This results in long intracellular persistence and enables consecutive internalization cycles. Moreover, the recycled antibody maintains an intact Fc portion potentially capable of continuous induction of antibody-dependent cell-mediated cytotoxicity (ADCC) response, thus explaining its therapeutic efficacy. Finally, we showed that G250 treatment is effective against HT-29 colorectal carcinoma xenografts that differ from RCC by more heterogeneous, hypoxia-related expression of CA Ⅸ. These results suggest potential therapeutic usefulness of the G250 MAb in non-RCC tumours.

Carnosine inhibits carbonic anhydrase IX-mediated extracellular acidosis and suppresses growth of HeLa tumor xenografts.

BACKGROUND: Carbonic anhydrase IX (CA IX) is a transmembrane enzyme that is present in many types of solid tumors. Expression of CA IX is driven predominantly by the hypoxia-inducible factor (HIF) pathway and helps to maintain intracellular pH homeostasis under hypoxic conditions, resulting in acidification of the tumor microenvironment. Carnosine (ß-alanyl-L-histidine) is an anti-tumorigenic agent that inhibits the proliferation of cancer cells. In this study, we investigated the role of CA IX in carnosine-mediated antitumor activity and whether the underlying mechanism involves transcriptional and translational modulation of HIF-1α and CA IX and/or altered CA IX function. METHODS: The effect of carnosine was studied using two-dimensional cell monolayers of several cell lines with endogenous CA IX expression as well as Madin Darby canine kidney transfectants, three-dimensional HeLa spheroids, and an in vivo model of HeLa xenografts in nude mice. mRNA and protein expression and protein localization were analyzed by real-time PCR, western blot analysis, and immunofluorescence staining, respectively. Cell viability was measured by a flow cytometric assay. Expression of HIF-1α and CA IX in tumors was assessed by immunohistochemical staining. Real-time measurement of pH was performed using a sensor dish reader. Binding of CA IX to specific antibodies and metabolon partners was investigated by competitive ELISA and proximity ligation assays, respectively. RESULTS: Carnosine increased the expression levels of HIF-1α and HIF targets and increased the extracellular pH, suggesting an inhibitory effect on CA IX-mediated acidosis. Moreover, carnosine significantly inhibited the growth of three-dimensional spheroids and tumor xenografts compared with untreated controls. Competitive ELISA showed that carnosine disrupted binding between CA IX and antibodies specific for its catalytic domain. This finding was supported by reduced formation of the functional metabolon of CA IX and anion exchanger 2 in the presence of carnosine. CONCLUSIONS: Our results indicate that interaction of carnosine with CA IX leads to conformational changes of CA IX and impaired formation of its metabolon, which in turn disrupts CA IX function. These findings suggest that carnosine could be a promising anticancer drug through its ability to attenuate the activity of CA IX.