Julie Mazzolini

Adenosine 3':5' monophosphate (cAMP) is a ubiquitous signalling molecule generated by the adenylyl cyclase family of proteins which is encoded by ten genes. The biological significance of this diversity is not well understood. In mammals, transmembrane adenylyl cyclase 9 (AC9) is resistant to regulation by heterotrimeric G proteins. A major facet of this resistance is auto-inhibition - in the presence of activated Gsα, AC9 is inhibited by its C-terminal domain. Here, we examined the natural evolution of this paradoxical control mechanism. At the primary sequence level, the hallmarks of auto-inhibition are apparent in all vertebrates, none are found in invertebrates. Teleost-specific genome duplication (TGD) resulted in two AC9 ohnologues one of which lacks the hallmarks of auto-inhibition. We cloned the cDNAs of the AC9 ohnologues of zebrafish and demonstrated the predicted difference in autoinhibition. The tissue distributions of the adcy9 genes of teleost species also point their subfunctionalization. Above all, auto-inhibited adcy9 is largely restricted to the brain indicating a fundamental role in brain development or function. Our findings document a quantum leap of the regulation of the enzymatic activity of AC9 in vertebrates and the potency of TGD to meet an adaptational challenge through functionally diversified ohnologues.

Background: Extracellular vesicles (EVs) are critical mediators of intercellular communication in development, physiology, and disease. In vivo models such as Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio (zebrafish) now provide powerful platforms to visualize EV dynamics in real time. However, the full potential of these models remains underutilized due to the lack of reliable, cell-specific EV labelling tools and robust EV isolation protocols. Here, we present an optimized workflow for the isolation of EVs from zebrafish larvae and the in vivo labelling of EVs in a cell-type-specific manner. Methods: To isolate EVs from larval zebrafish, we used size exclusion chromatography (SEC). By comparing different tissue digestion methods and performing step-by-step optimisation of sample preparation prior to SEC, we established a novel protocol that enables EV isolation without compromising cell viability. EV size and concentration were assessed by nanoparticle tracking analysis (NTA), with subsequent characterization by transmission electron microscopy (TEM) and Western blotting. To evaluate the sensitivity of our protocol, we treated zebrafish larvae with GW4869, a known inhibitor of EV biogenesis, and assessed the dose-dependent effects on EV release. To specifically label EVs from distinct cell types, we have generated a UAS: CD63-GFP construct which can be expressed under control of the Gal4 transcriptional activator. Results: Through a systematic comparison of tissue dissociation techniques, we identify Bacillus licheniformis protease as a superior alternative to conventional collagenase treatment, which compromises cell integrity. Treatment with GW4869 confirmed that EV biogenesis and release can be inhibited in a dose-dependent manner and demonstrated that our protocol is sensitive enough to detect and quantify changes in EV levels. To enable cell-specific EV tracking in vivo, we combined the UAS: CD63-GFP construct with a radial glia-specific Gal4 driver line, providing a proof-of-concept for targeted EV imaging in intact tissues. Conclusions: These advances provide a versatile toolkit for mechanistic studies of EV function in vivo. The broad availability of cell-type-specific Gal4 driver lines in zebrafish and Drosophila will now allow researchers to trace EV dynamics from virtually any cell type, while our isolation protocol enables rigorous, quantitative EV analyses across developmental and pathological contexts.

Microglia actively promotes the growth of high‐grade gliomas. Within the glioma microenvironment an amoeboid microglial morphology has been observed, however the underlying causes and the related impact on microglia functions and their tumor promoting activities is unclear. Using the advantages of the larval zebrafish model, we identified the underlying mechanism and show that microglial morphology and functions are already impaired during glioma initiation stages. The presence of pre‐neoplastic HRasV12 expressing cells induces an amoeboid morphology of microglia, increases microglial numbers and decreases their motility and phagocytic activity. RNA sequencing analysis revealed lower expression levels of the actin nucleation promoting factor wasla in microglia. Importantly, a microglia specific rescue of wasla expression restores microglial morphology and functions. This results in increased phagocytosis of pre‐neoplastic cells and slows down tumor progression. In conclusion, we identified a mechanism that de‐activates core microglial functions within the emerging glioma microenvironment. Restoration of this mechanism might provide a way to impair glioma growth.

Summary: Microglia actively promote the growth of high-grade gliomas. Within the glioma microenvironment an activated (amoeboid) microglial morphology has been observed, however the underlying causes and the related impact on microglia functions and their tumour promoting activities is unclear. Using the advantages of the larval zebrafish model, we demonstrate that pre-neoplastic glioma cells have an immediate impact on microglial morphology and functions. Overexpression of human HRasV12 in proliferating domains of the larval brain induces an amoeboid morphology of microglia, increases microglial numbers and decreases their motility and phagocytic activity. RNA sequencing analysis revealed lower expression levels of the actin nucleation promoting factor wasla in microglia. Importantly, a microglia specific rescue of wasla expression restores microglial morphology and functions. This results in increased phagocytosis of pre-neoplastic cells and slows down tumour progression. In conclusion, we identified a mechanism that de-activates core microglial functions within the emerging glioma microenvironment.

Iron is typically the dominant metal in the ultrafine fraction of airborne particulate matter. Various studies have investigated the toxicity of inhaled nano-sized iron oxide particles (FeO_xNPs) but their results have been contradictory, with some indicating no or minor effects and others finding effects including oxidative stress and inflammation. Most studies, however, did not use materials reflecting the characteristics of FeO_xNPs present in the environment. We, therefore, analysed the potential toxicity of FeO_xNPs of different forms (Fe₃O₄, α-Fe₂O₃ and γ-Fe₂O₃) reflecting the characteristics of high iron content nano-sized particles sampled from the environment, both individually and in a mixture (FeO_x-mix). A preliminary in vitro study indicated Fe₃O₄ and FeO_x-mix were more cytotoxic than either form of Fe₂O₃ in human bronchial epithelial cells (BEAS-2B). Follow-up in vitro (0.003, 0.03, 0.3 µg/mL, 24 h) and in vivo (Sprague–Dawley rats, nose-only exposure, 50 µg/m³ and 500 µg/m³, 3 h/d × 3 d) studies therefore focused on these materials. Experiments in vitro explored responses at the molecular level via multi-omics analyses at concentrations below those at which significant cytotoxicity was evident to avoid detection of responses secondary to toxicity. Inhalation experiments used aerosol concentrations chosen to produce similar levels of particle deposition on the airway surface as were delivered in vitro. These were markedly higher than environmental concentrations. No clinical signs of toxicity were seen nor effects on BALF cell counts or LDH levels. There were also no significant changes in transcriptomic or metabolomic responses in lung or BEAS-2B cells to suggest adverse effects.

Microglia are the resident macrophages of the brain. Over the past decade, our understanding of the function of these cells has significantly improved. Microglia do not only play important roles in the healthy brain but are involved in almost every brain pathology. Gene expression profiling allowed to distinguish microglia from other macrophages and revealed that the full microglia signature can only be observed in vivo. Thus, animal models are irreplaceable to understand the function of these cells. One of the popular models to study microglia is the zebrafish larva. Due to their optical transparency and genetic accessibility, zebrafish larvae have been employed to understand a variety of microglia functions in the living brain. Here, we performed RNA sequencing of larval zebrafish microglia at different developmental time points: 3, 5, and 7 days post fertilization (dpf). Our analysis reveals that larval zebrafish microglia rapidly acquire the core microglia signature and many typical microglia genes are expressed from 3 dpf onwards. The majority of changes in gene expression happened between 3 and 5 dpf, suggesting that differentiation mainly takes place during these days. Furthermore, we compared the larval microglia transcriptome to published data sets of adult zebrafish microglia, mouse microglia, and human microglia. Larval microglia shared a significant number of expressed genes with their adult counterparts in zebrafish as well as with mouse and human microglia. In conclusion, our results show that larval zebrafish microglia mature rapidly and express the core microglia gene signature that seems to be conserved across species.

Previously we described direct cellular interactions between microglia and AKT1+ brain tumour cells in zebrafish (Chia et al., 2018). However, it was unclear how these interactions were initiated: it was also not clear if they had an impact on the growth of tumour cells. Here, we show that neoplastic cells hijack mechanisms that are usually employed to direct microglial processes towards highly active neurons and injuries in the brain. We show that AKT1+ cells possess dynamically regulated high intracellular Ca²⁺ levels. Using a combination of live imaging, genetic and pharmacological tools, we show that these Ca²⁺ transients stimulate ATP-mediated interactions with microglia. Interfering with Ca²⁺ levels, inhibiting ATP release and CRISPR-mediated mutation of the p2ry12 locus abolishes these interactions. Finally, we show that reducing the number of microglial interactions significantly impairs the proliferation of neoplastic AKT1 cells. In conclusion, neoplastic cells repurpose the endogenous neuron to microglia signalling mechanism via P2ry12 activation to promote their own proliferation.

Complementary methods to observe frustrated phagocytosis and phagosome closure using total internal reflection fluorescence microscopy (TIRFM) are described here. Frustrated phagocytosis occurs when phagocytic cells are exposed to an opsonized surface and spread as if trying to engulf it, allowing for the observation of phagocytic spreading and the biochemical events that directly precede it. Phagosome formation and closure is an inherently three-dimensional process though, and cannot be studied in the “frustrated” situation. Here we describe a method to visualize with unprecedented high-resolution phagosome formation and closure in three dimensions. It allows for observation of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody. Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 °C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 °C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) > 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

It is now clear that microglia and macrophages are present in brain tumors, but whether or how they affect initiation and development of tumors is not known. Exploiting the advantages of the zebrafish (Danio rerio) model, we showed that macrophages and microglia respond immediately upon oncogene activation in the brain. Overexpression of human AKT1 within neural cells of larval zebrafish led to a significant increase in the macrophage and microglia populations. By using a combination of transgenic and mutant zebrafish lines, we showed that this increase was caused by the infiltration of peripheral macrophages into the brain mediated via Sdf1b-Cxcr4b signaling. Intriguingly, confocal live imaging reveals highly dynamic interactions between macrophages/microglia and pre-neoplastic cells, which do not result in phagocytosis of pre-neoplastic cells. Finally, depletion of macrophages and microglia resulted in a significant reduction of oncogenic cell proliferation. Thus, macrophages and microglia show tumor promoting functions already during the earliest stages of the developing tumor microenvironment.