Faculty Bibliography

Introduction: Noncoding small RNAs in the retina regulate gene expression by targeting and repressing mRNA. Small RNAs are secreted in extracellular vesicles (EVs). Analysis of EVs released from developing retinal tissue is an essential step in elucidating the role of EV molecular cargo and signalling during retinogenesis. A number of canonical genes are associated with retinal cell fate determination during development, but EV-mediated gene regulation in the retinal microenvironment remains undefined. In this study, we characterize the microRNA, tRNA, and piRNA composition of EVs secreted from human induced pluripotent stem cell (hiPSCs) - derived 3D retinas at three developmental time points that correlate with hallmarks of retinal cell differentiation and lamination in vivo.
Method(s): Retinal organoids were generated from hiPSCs. We selected three developmental time points (day 42, 63 and 90) that represent distinctive stages during normal retinal cell fate specification and lamination. We analysed the release rate, concentration, morphology and content (miRNA, tRNA and pi- RNA) of EVs released from human hiPSCs-derived 3D retinas.
Result(s): The genetic signalling, developmental time course and morphogenesis of these retinal organoids were comparable to those of developing human retinas in vivo. According to Gene Ontology analysis, miRNA targets at the earliest stage of development were more relevant to early differentiation and cell morphogenesis, whereas miRNA targets at the later stages were more relevant to cell proliferation, cell differentiation, and cell migration. Summary/Conclusion: For the first time, this work demonstrates the rate of release and concentration of EVs from developing hiPSC-derived 3D retinal tissue. We report a large variety of small RNAs in EVs from hiPSC-derived 3D retinas, including miRNAs, tRNAs and piRNAs. The full range of small RNAs detected in our EVs may act as regulatory elements to modulate gene activity and may serve as biomarkers of normal development. This work represents the first sequencing analysis of small RNA species contained in hiPSCderived 3D retinas and their released EVs

Uroplakin (UP) tetraspanins and their associated proteins are major mammalian urothelial differentiation products that form unique 2D-crystals of 16-nm particles ("urothelial plaques") covering the apical urothelial surface. Although uroplakins are highly expressed only in mouse urothelium and are often referred to as being urothelium-specific, they are also expressed in several nonurothelial cell types in stomach, kidney, prostate, epididymis, testis/sperms and ovary/oocytes. In oocytes, uroplakins co-localize with CD9 on cell surface and multivesicular body-derived exosomes, and the cytoplasmic tail of UPIIIa undergoes a conserved fertilization-dependent, Fyn-mediated tyrosine-phosphorylation that also occurs in Xenopus laevis eggs. Uroplakin knockout and antibody blocking reduce mouse eggs' fertilization rate in in vitro fertilization assays, and UPII/IIIa double-knockout mice have a smaller litter size. Phylogenetic analyses showed that uroplakin sequences underwent significant mammal-specific changes. These results suggest that, by mediating signal transduction and modulating membrane stability that do not require 2D-crystal formation, uroplakins can perform conserved and more ancestral fertilization functions in mouse and frog eggs. Uroplakins acquired the ability to form 2D- crystalline plaques during mammalian divergence enabling them to perform additional functions, including umbrella cell enlargement and the formation of permeability and mechanical barriers, in order to protect/modify the apical surface of the modern-day mammalian urothelium.

Germ granules are non-membranous ribonucleoprotein granules deemed the hubs for post-transcriptional gene regulation and functionally linked to germ cell fate across species. Little is known about the physical properties of germ granules and how these relate to germ cell function. Here we study two types of germ granules in the Drosophila embryo: cytoplasmic germ granules that instruct primordial germ cells (PGCs) formation and nuclear germ granules within early PGCs with unknown function. We show that cytoplasmic and nuclear germ granules are phase transitioned condensates nucleated by Oskar protein that display liquid as well as hydrogel-like properties. Focusing on nuclear granules, we find that Oskar drives their formation in heterologous cell systems. Multiple, independent Oskar protein domains synergize to promote granule phase separation. Deletion of Oskar's nuclear localization sequence specifically ablates nuclear granules in cell systems. In the embryo, nuclear germ granules promote germ cell divisions thereby increasing PGC number for the next generation.

Aim of the study/UNASSIGNED:Interferon (IFN)-α is now established as a treatment modality in various human cancers. The IFN-α-inducible human "myxovirus resistance protein A" (MxA) is a cytoplasmic dynamin-family large GTPase primarily characterized for its broad-spectrum antiviral activity and, more recently, for its anti-tumor and anti-metastasis effects. We characterized the association of IFN-α-induced MxA with cytoplasmic structures in human Huh7 cancer cells and in primary endothelial cells. Material and methods/UNASSIGNED:We re-evaluated the long-standing inference that MxA associated with the smooth ER using double-label immunofluorescence techniques and the ER structural protein RTN4 as a marker for smooth ER in IFN-α-treated cells. We also evaluated the relationship of exogenously expressed HA-MxA and GFP-MxA with mitochondria, and characterized cytoplasmic GFP-MxA structures using correlated light and electron microscopy (CLEM). Results and conclusions/UNASSIGNED:We discovered that IFN-α-induced endogenous MxA associated with variably-sized endosome-like and reticular cytoplasmic structures which were distinct from the ER. Thin-section EM studies of GFP-MxA expressing Huh7 cells showed that GFP-MxA formed variably-sized clusters of vesiculotubular elements to form endosome-like "MxA bodies". Many of these clusters stretched out alongside cytoskeletal elements to give the appearance of a cytoplasmic "MxA reticulum". This MxA meshwork was distinct from but adjacent to mitochondria. GFP-MxA expressing Huh7 cells showed reduced MitoTracker uptake and swollen mitochondria by thin-section EM. The new data identify cytoplasmic MxA structures as novel organelles, and suggest cross-talk between MxA structures and mitochondria that might account for the increased anti-tumoral efficacy of IFN-α combined with ligands that activate other pattern-sensing receptor pathways.

Plakophilin-2 (PKP2) is a component of the desmosome and known for its role in cell-cell adhesion. Mutations in human PKP2 associate with a life-threatening arrhythmogenic cardiomyopathy, often of right ventricular predominance. Here, we use a range of state-of-the-art methods and a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mouse to demonstrate that in addition to its role in cell adhesion, PKP2 is necessary to maintain transcription of genes that control intracellular calcium cycling. Lack of PKP2 reduces expression of Ryr2 (coding for Ryanodine Receptor 2), Ank2 (coding for Ankyrin-B), Cacna1c (coding for CaV1.2) and Trdn (coding for triadin), and protein levels of calsequestrin-2 (Casq2). These factors combined lead to disruption of intracellular calcium homeostasis and isoproterenol-induced arrhythmias that are prevented by flecainide treatment. We propose a previously unrecognized arrhythmogenic mechanism related to PKP2 expression and suggest that mutations in PKP2 in humans may cause life-threatening arrhythmias even in the absence of structural disease.It is believed that mutations in desmosomal adhesion complex protein plakophilin 2 (PKP2) cause arrhythmia due to loss of cell-cell communication. Here the authors show that PKP2 controls the expression of proteins involved in calcium cycling in adult mouse hearts, and that lack of PKP2 can cause arrhythmia in a structurally normal heart.

Recapitulation of lung development from human pluripotent stem cells (hPSCs) in three dimensions (3D) would allow deeper insight into human development, as well as the development of innovative strategies for disease modelling, drug discovery and regenerative medicine. We report here the generation from hPSCs of lung bud organoids (LBOs) that contain mesoderm and pulmonary endoderm and develop into branching airway and early alveolar structures after xenotransplantation and in Matrigel 3D culture. Expression analysis and structural features indicated that the branching structures reached the second trimester of human gestation. Infection in vitro with respiratory syncytial virus, which causes small airway obstruction and bronchiolitis in infants, led to swelling, detachment and shedding of infected cells into the organoid lumens, similar to what has been observed in human lungs. Introduction of mutation in HPS1, which causes an early-onset form of intractable pulmonary fibrosis, led to accumulation of extracellular matrix and mesenchymal cells, suggesting the potential use of this model to recapitulate fibrotic lung disease in vitro. LBOs therefore recapitulate lung development and may provide a useful tool to model lung disease.

Reticulon-4 (RTN4) (also called Nogo-A/B) and the atlastin (ATL) GTPases have critical roles in the formation of tubules and sheets of the standard peripheral endoplasmic reticulum (ER) in mammalian cells. The interferon-inducible protein MxA (myxovirus resistance protein A) is a dynamin-family, atlastin-like GTPase with membrane-binding and tubulation activity. While MxA has been investigated extensively for its antiviral effects, less is known about its role in cellular physiology in the uninfected cell. Over the last 15 years MxA has been represented to localize to "subcompartments of the smooth ER." However these prior studies did not include ER structural proteins as markers. In contrast, using RTN4 as an ER marker, we discovered that MxA expressed in human cell lines associated with large tubuloreticular structures that were distinct from the standard RTN4/ATL3-based ER. Our methods included immunofluorescence studies, thin-section EM, and single and double-label immunoEM. MxA generated large variably sized tubuloreticular structures that were RTN-4 negative. In contrast to HAtagged ATL3 which colocalized with RTN4 and increased ER sheets at three-way junctions, HA-tagged MxA tubules were independent of RTN4 and MxA did not affect the standard RTN4-based ER. Thus, remarkably, one and the same cell contained two distinct tubuloreticular systems - one based on RTN4/ATL3 and the other on MxA. The MxA-reticulum is a novel organelle which is positive for EEA1, clathrin light chain, Rab5, Rab11 and GRP78/BiP, but not for LAMP2, Rab7 and syntaxin 17. The present studies advance the novel paradigm that different atlastin-like GTPases can generate distinct ER-like tubuloreticular systems within the same cell. The new data require reinterpretion of prior studies in the MxA field over the last decade

BACKGROUND: Gaucher disease (GD) is a genetic disease caused by mutations in the GBA1 gene which result in reduced enzymatic activity of beta-glucocerebrosidase (GCase). This study identified the progranulin (PGRN) gene (GRN) as another gene associated with GD. METHODS: Serum levels of PGRN were measured from 115 GD patients and 99 healthy controls, whole GRN gene from 40 GD patients was sequenced, and the genotyping of 4 SNPs identified in GD patients was performed in 161 GD and 142 healthy control samples. Development of GD in PGRN-deficient mice was characterized, and the therapeutic effect of rPGRN on GD analyzed. FINDINGS: Serum PGRN levels were significantly lower in GD patients (96.65+/-53.45ng/ml) than those in healthy controls of the general population (164.99+/-43.16ng/ml, p<0.0001) and of Ashkenazi Jews (150.64+/-33.99ng/ml, p<0.0001). Four GRN gene SNPs, including rs4792937, rs78403836, rs850713, and rs5848, and three point mutations, were identified in a full-length GRN gene sequencing in 40 GD patients. Large scale SNP genotyping in 161 GD and 142 healthy controls was conducted and the four SNP sites have significantly higher frequency in GD patients. In addition, "aged" and challenged adult PGRN null mice develop GD-like phenotypes, including typical Gaucher-like cells in lung, spleen, and bone marrow. Moreover, lysosomes in PGRN KO mice exhibit a tubular-like appearance. PGRN is required for the lysosomal appearance of GCase and its deficiency leads to GCase accumulation in the cytoplasm. More importantly, recombinant PGRN is therapeutic in various animal models of GD and human fibroblasts from GD patients. INTERPRETATION: Our data demonstrates an unknown association between PGRN and GD and identifies PGRN as an essential factor for GCase's lysosomal localization. These findings not only provide new insight into the pathogenesis of GD, but may also have implications for diagnosis and alternative targeted therapies for GD.

[This corrects the article DOI: 10.1371/journal.pgen.1005625.].