A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations.

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

Comparative transcriptomics coupled to developmental grading via transgenic zebrafish reporter strains identifies conserved features in neutrophil maturation.

Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defense. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here we show, by generating and analysing transgenic zebrafish strains representing distinct neutrophil differentiation stages, a high-resolution transcriptional profile of neutrophil maturation. We link gene expression at each stage to characteristic transcription factors, including C/ebp-ß, which is important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human samples confirms high molecular similarity of immature stages and discriminates zebrafish-specific from pan-species gene signatures. Applying the pan-species neutrophil maturation signature to RNA-sequencing data from human neuroblastoma patients reveals association between metastatic tumor cell infiltration in the bone marrow and an overall increase in mature neutrophils. Our detailed neutrophil maturation atlas thus provides a valuable resource for studying neutrophil function at different stages across species in health and disease.

High-content drug screening in zebrafish xenografts reveals high efficacy of dual MCL-1/BCL-XL inhibition against Ewing sarcoma.

Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.

YB-1 regulates mesothelioma cell migration via snail but not EGFR, MMP1, EPHA5 or PARK2.

Pleural mesothelioma (PM) is characterized by rapid growth, local invasion, and limited therapeutic options. The multifunctional oncoprotein Y-box-binding protein-1 (YB-1) is frequently overexpressed in cancer and its inhibition reduces aggressive behavior in multiple tumor types. Here, we investigated the effects of YB-1 on target gene regulation and PM cell behavior. Whereas siRNA-mediated YB-1 knockdown reduced cell motility, YB-1 overexpression resulted in scattering, increased migration, and intravasation in vitro. Furthermore, YB-1 stimulated PM cell spreading in zebrafish. Combined knockdown and inducible overexpression of YB-1 allowed bidirectional control and rescue of cell migration, the pattern of which was closely followed by the mRNA and protein levels of EGFR and the protein level of snail, whereas the mRNA levels of MMP1, EPHA5, and PARK2 showed partial regulation by YB-1. Finally, we identified snail as a critical regulator of YB-1-mediated cell motility in PM. This study provides insights into the mechanism underlying the aggressive nature of PM and highlights the important role of YB-1 in this cancer. In this context, we found that YB-1 closely regulates EGFR and snail, and, moreover, that YB-1-induced cell migration depends on snail.

Fibroblast growth factor receptor 4 promotes glioblastoma progression: a central role of integrin-mediated cell invasiveness.

Glioblastoma (GBM) is characterized by a particularly invasive phenotype, supported by oncogenic signals from the fibroblast growth factor (FGF)/ FGF receptor (FGFR) network. However, a possible role of FGFR4 remained elusive so far. Several transcriptomic glioma datasets were analyzed. An extended panel of primary surgical specimen-derived and immortalized GBM (stem)cell models and original tumor tissues were screened for FGFR4 expression. GBM models engineered for wild-type and dominant-negative FGFR4 overexpression were investigated regarding aggressiveness and xenograft formation. Gene set enrichment analyses of FGFR4-modulated GBM models were compared to patient-derived datasets. Despite widely absent in adult brain, FGFR4 mRNA was distinctly expressed in embryonic neural stem cells and significantly upregulated in glioblastoma. Pronounced FGFR4 overexpression defined a distinct GBM patient subgroup with dismal prognosis. Expression levels of FGFR4 and its specific ligands FGF19/FGF23 correlated both in vitro and in vivo and were progressively upregulated in the vast majority of recurrent tumors. Based on overexpression/blockade experiments in respective GBM models, a central pro-oncogenic function of FGFR4 concerning viability, adhesion, migration, and clonogenicity was identified. Expression of dominant-negative FGFR4 resulted in diminished (subcutaneous) or blocked (orthotopic) GBM xenograft formation in the mouse and reduced invasiveness in zebrafish xenotransplantation models. In vitro and in vivo data consistently revealed distinct FGFR4 and integrin/extracellular matrix interactions. Accordingly, FGFR4 blockade profoundly sensitized FGFR4-overexpressing GBM models towards integrin/focal adhesion kinase inhibitors. Collectively, FGFR4 overexpression contributes to the malignant phenotype of a highly aggressive GBM subgroup and is associated with integrin-related therapeutic vulnerabilities.

Preclinical testing of CAR T cells in zebrafish xenografts.

Engineered chimeric antigen receptor T cells (CAR T cells) have emerged as a promising immunotherapy for cancer and have proven to be effective for B cell malignancies. Currently, great efforts are undertaken to expand the application of CAR T cells to other cancer entities, to increase the efficacy of CAR T cell-mediated killing of cancer cells and to reduce possible side effects of CAR T cell therapy. This creates a need for preclinical models to test the many emerging novel CAR designs. Traditionally, mouse xenograft models are applied to investigate the efficacy of CAR T cells in vivo. Here, we describe a complementing xenograft protocol for testing CAR T cells against human leukemia cells in zebrafish embryos. The embryonic zebrafish xenograft promises to be a fast and cost-efficient model and particularly offers live imaging opportunities of CAR T cell distribution and killing of cancer cells in vivo.

A Preclinical Embryonic Zebrafish Xenograft Model to Investigate CAR T Cells In Vivo.

Chimeric antigen receptor (CAR) T cells have proven to be a powerful cellular therapy for B cell malignancies. Massive efforts are now being undertaken to reproduce the high efficacy of CAR T cells in the treatment of other malignancies. Here, predictive preclinical model systems are important, and the current gold standard for preclinical evaluation of CAR T cells are mouse xenografts. However, mouse xenograft assays are expensive and slow. Therefore, an additional vertebrate in vivo assay would be beneficial to bridge the gap from in vitro to mouse xenografts. Here, we present a novel assay based on embryonic zebrafish xenografts to investigate CAR T cell-mediated killing of human cancer cells. Using a CD19-specific CAR and Nalm-6 leukemia cells, we show that live observation of killing of Nalm-6 cells by CAR T cells is possible in zebrafish embryos. Furthermore, we applied Fiji macros enabling automated quantification of Nalm-6 cells and CAR T cells over time. In conclusion, we provide a proof-of-principle study that embryonic zebrafish xenografts can be used to investigate CAR T cell-mediated killing of tumor cells. This assay is cost-effective, fast, and offers live imaging possibilities to directly investigate CAR T cell migration, engagement, and killing of effector cells.

A Putative Mechanism for Magnetoreception by Electromagnetic Induction in the Pigeon Inner Ear.

A diverse array of vertebrate species employs the Earth's magnetic field to assist navigation. Despite compelling behavioral evidence that a magnetic sense exists, the location of the primary sensory cells and the underlying molecular mechanisms remain unknown [1]. To date, most research has focused on a light-dependent radical-pair-based concept and a system that is proposed to rely on biogenic magnetite (Fe3O4) [2, 3]. Here, we explore an overlooked hypothesis that predicts that animals detect magnetic fields by electromagnetic induction within the semicircular canals of the inner ear [4]. Employing an assay that relies on the neuronal activity marker C-FOS, we confirm that magnetic exposure results in activation of the caudal vestibular nuclei in pigeons that is independent of light [5]. We show experimentally and by physical calculations that magnetic stimulation can induce electric fields in the pigeon semicircular canals that are within the physiological range of known electroreceptive systems. Drawing on this finding, we report the presence of a splice isoform of a voltage-gated calcium channel (CaV1.3) in the pigeon inner ear that has been shown to mediate electroreception in skates and sharks [6]. We propose that pigeons detect magnetic fields by electromagnetic induction within the semicircular canals that is dependent on the presence of apically located voltage-gated cation channels in a population of electrosensory hair cells.

The Parkinson's disease-linked Leucine-rich repeat kinase 2 (LRRK2) is required for insulin-stimulated translocation of GLUT4.

Mutations within Leucine-rich repeat kinase 2 (LRRK2) are associated with late-onset Parkinson's disease. The physiological function of LRRK2 and molecular mechanism underlying the pathogenic role of LRRK2 mutations remain uncertain. Here, we investigated the role of LRRK2 in intracellular signal transduction. We find that deficiency of Lrrk2 in rodents affects insulin-dependent translocation of glucose transporter type 4 (GLUT4). This deficit is restored during aging by prolonged insulin-dependent activation of protein kinase B (PKB, Akt) and Akt substrate of 160 kDa (AS160), and is compensated by elevated basal expression of GLUT4 on the cell surface. Furthermore, we find a crucial role of Rab10 phosphorylation by LRRK2 for efficient insulin signal transduction. Translating our findings into human cell lines, we find comparable molecular alterations in fibroblasts from Parkinson's patients with the known pathogenic G2019S LRRK2 mutation. Our results highlight the role of LRRK2 in insulin-dependent signalling with potential therapeutic implications.

Mutations in MAST1 Cause Mega-Corpus-Callosum Syndrome with Cerebellar Hypoplasia and Cortical Malformations.

Corpus callosum malformations are associated with a broad range of neurodevelopmental diseases. We report that de novo mutations in MAST1 cause mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCC-CH-CM) in the absence of megalencephaly. We show that MAST1 is a microtubule-associated protein that is predominantly expressed in post-mitotic neurons and is present in both dendritic and axonal compartments. We further show that Mast1 null animals are phenotypically normal, whereas the deletion of a single amino acid (L278del) recapitulates the distinct neurological phenotype observed in patients. In animals harboring Mast1 microdeletions, we find that the PI3K/AKT3/mTOR pathway is unperturbed, whereas Mast2 and Mast3 levels are diminished, indicative of a dominant-negative mode of action. Finally, we report that de novo MAST1 substitutions are present in patients with autism and microcephaly, raising the prospect that mutations in this gene give rise to a spectrum of neurodevelopmental diseases.

Subcellular analysis of pigeon hair cells implicates vesicular trafficking in cuticulosome formation and maintenance.

Hair cells are specialized sensors located in the inner ear that enable the transduction of sound, motion, and gravity into neuronal impulses. In birds some hair cells contain an iron-rich organelle, the cuticulosome, that has been implicated in the magnetic sense. Here, we exploit histological, transcriptomic, and tomographic methods to investigate the development of cuticulosomes, as well as the molecular and subcellular architecture of cuticulosome positive hair cells. We show that this organelle forms rapidly after hatching in a process that involves vesicle fusion and nucleation of ferritin nanoparticles. We further report that transcripts involved in endocytosis, extracellular exosomes, and metal ion binding are differentially expressed in cuticulosome positive hair cells. These data suggest that the cuticulosome and the associated molecular machinery regulate the concentration of iron within the labyrinth of the inner ear, which might indirectly tune a magnetic sensor that relies on electromagnetic induction.

Glycine-alanine dipeptide repeat protein contributes to toxicity in a zebrafish model of C9orf72 associated neurodegeneration.

BACKGROUND: The most frequent genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) is the expansion of a GGGGCC hexanucleotide repeat in a non-coding region of the chromosome 9 open reading frame 72 (C9orf72) locus. The pathological hallmarks observed in C9orf72 repeat expansion carriers are the formation of RNA foci and deposition of dipeptide repeat (DPR) proteins derived from repeat associated non-ATG (RAN) translation. Currently, it is unclear whether formation of RNA foci, DPR translation products, or partial loss of C9orf72 predominantly drive neurotoxicity in vivo. By using a transgenic approach in zebrafish we address if the most frequently found DPR in human ALS/FTLD brain, the poly-Gly-Ala (poly-GA) protein, is toxic in vivo. METHOD: We generated several transgenic UAS responder lines that express either 80 repeats of GGGGCC alone, or together with a translation initiation ATG codon forcing the translation of GA80-GFP protein upon crossing to a Gal4 driver. The GGGGCC repeat and GA80 were fused to green fluorescent protein (GFP) lacking a start codon to monitor protein translation by GFP fluorescence. RESULTS: Zebrafish transgenic for the GGGGCC repeat lacking an ATG codon showed very mild toxicity in the absence of poly-GA. However, strong toxicity was induced upon ATG initiated expression of poly-GA, which was rescued by injection of an antisense morpholino interfering with start codon dependent poly-GA translation. This morpholino only interferes with GA80-GFP translation without affecting repeat transcription, indicating that the toxicity is derived from GA80-GFP. CONCLUSION: These novel transgenic C9orf72 associated repeat zebrafish models demonstrate poly-GA toxicity in zebrafish. Reduction of poly-GA protein rescues toxicity validating this therapeutic approach to treat C9orf72 repeat expansion carriers. These novel animal models provide a valuable tool for drug discovery to reduce DPR associated toxicity in ALS/FTLD patients with C9orf72 repeat expansions.

η-Secretase processing of APP inhibits neuronal activity in the hippocampus.

Alzheimer disease (AD) is characterized by the accumulation of amyloid plaques, which are predominantly composed of amyloid-ß peptide. Two principal physiological pathways either prevent or promote amyloid-ß generation from its precursor, ß-amyloid precursor protein (APP), in a competitive manner. Although APP processing has been studied in great detail, unknown proteolytic events seem to hinder stoichiometric analyses of APP metabolism in vivo. Here we describe a new physiological APP processing pathway, which generates proteolytic fragments capable of inhibiting neuronal activity within the hippocampus. We identify higher molecular mass carboxy-terminal fragments (CTFs) of APP, termed CTF-η, in addition to the long-known CTF-α and CTF-ß fragments generated by the α- and ß-secretases ADAM10 (a disintegrin and metalloproteinase 10) and BACE1 (ß-site APP cleaving enzyme 1), respectively. CTF-η generation is mediated in part by membrane-bound matrix metalloproteinases such as MT5-MMP, referred to as η-secretase activity. η-Secretase cleavage occurs primarily at amino acids 504-505 of APP695, releasing a truncated ectodomain. After shedding of this ectodomain, CTF-η is further processed by ADAM10 and BACE1 to release long and short Aη peptides (termed Aη-α and Aη-ß). CTFs produced by η-secretase are enriched in dystrophic neurites in an AD mouse model and in human AD brains. Genetic and pharmacological inhibition of BACE1 activity results in robust accumulation of CTF-η and Aη-α. In mice treated with a potent BACE1 inhibitor, hippocampal long-term potentiation was reduced. Notably, when recombinant or synthetic Aη-α was applied on hippocampal slices ex vivo, long-term potentiation was lowered. Furthermore, in vivo single-cell two-photon calcium imaging showed that hippocampal neuronal activity was attenuated by Aη-α. These findings not only demonstrate a major functionally relevant APP processing pathway, but may also indicate potential translational relevance for therapeutic strategies targeting APP processing.

TREM2 mutations implicated in neurodegeneration impair cell surface transport and phagocytosis.

Genetic variants in the triggering receptor expressed on myeloid cells 2 (TREM2) have been linked to Nasu-Hakola disease, Alzheimer's disease (AD), Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia (FTD), and FTD-like syndrome without bone involvement. TREM2 is an innate immune receptor preferentially expressed by microglia and is involved in inflammation and phagocytosis. Whether and how TREM2 missense mutations affect TREM2 function is unclear. We report that missense mutations associated with FTD and FTD-like syndrome reduce TREM2 maturation, abolish shedding by ADAM proteases, and impair the phagocytic activity of TREM2-expressing cells. As a consequence of reduced shedding, TREM2 is virtually absent in the cerebrospinal fluid (CSF) and plasma of a patient with FTD-like syndrome. A decrease in soluble TREM2 was also observed in the CSF of patients with AD and FTD, further suggesting that reduced TREM2 function may contribute to increased risk for two neurodegenerative disorders.