A multilevel screening pipeline in zebrafish identifies therapeutic drugs for GAN.

Giant axonal neuropathy (GAN) is a fatal neurodegenerative disorder for which there is currently no treatment. Affecting the nervous system, GAN starts in infancy with motor deficits that rapidly evolve toward total loss of ambulation. Using the gan zebrafish model that reproduces the loss of motility as seen in patients, we conducted the first pharmacological screening for the GAN pathology. Here, we established a multilevel pipeline to identify small molecules restoring both the physiological and the cellular deficits in GAN. We combined behavioral, in silico, and high-content imaging analyses to refine our Hits to five drugs restoring locomotion, axonal outgrowth, and stabilizing neuromuscular junctions in the gan zebrafish. The postsynaptic nature of the drug's cellular targets provides direct evidence for the pivotal role the neuromuscular junction holds in the restoration of motility. Our results identify the first drug candidates that can now be integrated in a repositioning approach to fasten therapy for the GAN disease. Moreover, we anticipate both our methodological development and the identified hits to be of benefit to other neuromuscular diseases.

HT-smFISH: a cost-effective and flexible workflow for high-throughput single-molecule RNA imaging.

The ability to visualize RNA in its native subcellular environment by using single-molecule fluorescence in situ hybridization (smFISH) has reshaped our understanding of gene expression and cellular functions. A major hindrance of smFISH is the difficulty to perform systematic experiments in medium- or high-throughput formats, principally because of the high cost of generating the individual fluorescent probe sets. Here, we present high-throughput smFISH (HT-smFISH), a simple and cost-efficient method for imaging hundreds to thousands of single endogenous RNA molecules in 96-well plates. HT-smFISH uses RNA probes transcribed in vitro from a large pool of unlabeled oligonucleotides. This allows the generation of individual probes for many RNA species, replacing commercial DNA probe sets. HT-smFISH thus reduces costs per targeted RNA compared with many smFISH methods and is easily scalable and flexible in design. We provide a protocol that combines oligo pool design, probe set generation, optimized hybridization conditions and guidelines for image acquisition and analysis. The pipeline requires knowledge of standard molecular biology tools, cell culture and fluorescence microscopy. It is achievable in ~20 d. In brief, HT-smFISH is tailored for medium- to high-throughput screens that image RNAs at single-molecule sensitivity.

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy.

Tumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

Ki-67 regulates global gene expression and promotes sequential stages of carcinogenesis.

Ki-67 is a nuclear protein that is expressed in all proliferating vertebrate cells. Here, we demonstrate that, although Ki-67 is not required for cell proliferation, its genetic ablation inhibits each step of tumor initiation, growth, and metastasis. Mice lacking Ki-67 are resistant to chemical or genetic induction of intestinal tumorigenesis. In established cancer cells, Ki-67 knockout causes global transcriptome remodeling that alters the epithelial-mesenchymal balance and suppresses stem cell characteristics. When grafted into mice, tumor growth is slowed, and metastasis is abrogated, despite normal cell proliferation rates. Yet, Ki-67 loss also down-regulates major histocompatibility complex class I antigen presentation and, in the 4T1 syngeneic model of mammary carcinoma, leads to an immune-suppressive environment that prevents the early phase of tumor regression. Finally, genes involved in xenobiotic metabolism are down-regulated, and cells are sensitized to various drug classes. Our results suggest that Ki-67 enables transcriptional programs required for cellular adaptation to the environment. This facilitates multiple steps of carcinogenesis and drug resistance, yet may render cancer cells more susceptible to antitumor immune responses.

Measuring the subcellular compartmentalization of viral infections by protein complementation assay.

The recent emergence and reemergence of viruses in the human population has highlighted the need to develop broader panels of therapeutic molecules. High-throughput screening assays opening access to untargeted steps of the viral replication cycle will provide powerful leverage to identify innovative antiviral molecules. We report here the development of an innovative protein complementation assay, termed αCentauri, to measure viral translocation between subcellular compartments. As a proof of concept, the Centauri fragment was either tethered to the nuclear pore complex or sequestered in the nucleus, while the complementary α fragment (<16 amino acids) was attached to the integrase proteins of infectious HIV-1. The translocation of viral ribonucleoproteins from the cytoplasm to the nuclear envelope or to the nucleoplasm efficiently reconstituted superfolder green fluorescent protein or NanoLuc αCentauri reporters. These fluorescence- or bioluminescence-based assays offer a robust readout of specific steps of viral infection in a multiwell format that is compatible for high-throughput screening and is validated by a short hairpin RNA-based prototype screen.

Modulation of innate immune signaling by a Coxiella burnetii eukaryotic-like effector protein.

The Q fever agent Coxiella burnetii uses a defect in organelle trafficking/intracellular multiplication (Dot/Icm) type 4b secretion system (T4SS) to silence the host innate immune response during infection. By investigating C. burnetii effector proteins containing eukaryotic-like domains, here we identify NopA (nucleolar protein A), which displays four regulator of chromosome condensation (RCC) repeats, homologous to those found in the eukaryotic Ras-related nuclear protein (Ran) guanine nucleotide exchange factor (GEF) RCC1. Accordingly, NopA is found associated with the chromatin nuclear fraction of cells and uses the RCC-like domain to interact with Ran. Interestingly, NopA triggers an accumulation of Ran-GTP, which accumulates at nucleoli of transfected or infected cells, thus perturbing the nuclear import of transcription factors of the innate immune signaling pathway. Accordingly, qRT-PCR analysis on a panel of cytokines shows that cells exposed to the C. burnetii nopA::Tn or a Dot/Icm-defective dotA::Tn mutant strain present a functional innate immune response, as opposed to cells exposed to wild-type C. burnetii or the corresponding nopA complemented strain. Thus, NopA is an important regulator of the innate immune response allowing Coxiella to behave as a stealth pathogen.

Combined strategy of siRNA and osteoclast actin cytoskeleton automated imaging to identify novel regulators of bone resorption shows a non-mitotic function for anillin.

Osteoclasts are the main cells responsible for the resorption of mineralized extracellular matrices. They are the major targets for anti-resorptive therapies to manage osteoporosis, a major public health problem. Osteoclasts are giant multinucleated cells that can organize their a unique adhesion structure based on a belt of podosomes, which is the keystone of the bone resorption apparatus. We combined differential transcriptomics and siRNA screening approaches to get a broader view of cytoskeletal regulators that participate in the control of osteoclast cytoskeleton and identify novel regulators of bone resorption by osteoclasts. We identified 20 new candidate regulators of osteoclasts cytoskeleton including Fkbp15, Spire1, Tacc2 and RalA, for which we confirmed they are necessary for proper organization of the podosome belt. We also showed that Anillin, well known for its role during cytokinesis, is essential in osteoclasts for correct podosome patterning and efficient bone resorption. In particular, Anillin controls the levels of the GTPase RhoA, a known regulator of osteoclast cytoskeleton and resorption activity. Finally, we set up and validated an automated imaging strategy based on open-source software for automatic and objective measurement of actin cytoskeleton organization in osteoclasts. We provide these pipelines that are useful to automatically assess the effect of collections of siRNAs or chemical compounds on osteoclast cytoskeleton or differentiation.