A transcriptomic study of Williams-Beuren syndrome associated genes in mouse embryonic stem cells.

Williams-Beuren syndrome (WBS) is a relatively rare disease caused by the deletion of 1.5 to 1.8 Mb on chromosome 7 which contains approximately 28 genes. This multisystem disorder is mainly characterized by supravalvular aortic stenosis, mental retardation, and distinctive facial features. We generated mouse embryonic stem (ES) cells clones expressing each of the 4 human WBS genes (WBSCR1, GTF2I, GTF2IRD1 and GTF2IRD2) found in the specific delated region 7q11.23 causative of the WBS. We generated at least three stable clones for each gene with stable integration in the ROSA26 locus of a tetracycline-inducible upstream of the coding sequence of the genet tagged with a 3xFLAG epitope. Three clones for each gene were transcriptionally profiled in inducing versus non-inducing conditions for a total of 24 profiles. This small collection of human WBS-ES cell clones represents a resource to facilitate the study of the function of these genes during differentiation.

Uroplakin traffic through the Golgi apparatus induces its fragmentation: new insights from novel in vitro models.

Uroplakins (UPs) play an essential role in maintaining an effective urothelial permeability barrier at the level of superficial urothelial cell (UC) layer. Although the organization of UPs in the apical plasma membrane (PM) of UCs is well known, their transport in UCs is only partially understood. Here, we dissected trafficking of UPs and its differentiation-dependent impact on Golgi apparatus (GA) architecture. We demonstrated that individual subunits UPIb and UPIIIa are capable of trafficking from the endoplasmic reticulum to the GA in UCs. Moreover, UPIb, UPIIIa or UPIb/UPIIIa expressing UCs revealed fragmentation and peripheral redistribution of Golgi-units. Notably, expression of UPIb or UPIb/UPIIIa triggered similar GA fragmentation in MDCK and HeLa cells that do not express UPs endogenously. The colocalization analysis of UPIb/UPIIIa-EGFP and COPI, COPII or clathrin suggested that UPs follow constitutively the post-Golgi route to the apical PM. Depolymerisation of microtubules leads to complete blockade of the UPIb/UPIIIa-EGFP post-Golgi transport, while disassembly of actin filaments shows significantly reduced delivery of UPIb/UPIIIa-EGFP to the PM. Our findings show the significant effect of the UPs expression on the GA fragmentation, which enables secretory Golgi-outpost to be distributed as close as possible to the sites of cargo delivery at the PM.

A systems biology approach reveals new endoplasmic reticulum-associated targets for the correction of the ATP7B mutant causing Wilson disease.

Copper (Cu) is an important trace element required for the activity of essential enzymes. However, excess Cu compromises the redox balance in cells and tissues causing serious toxicity. The process of disposal of excess Cu from organisms relies on the activity of Cu-transporting ATPase ATP7B. ATP7B is mainly expressed in liver hepatocytes where it sequesters the potentially toxic metal and mediates its excretion into the bile. Mutations in the ATP7B gene cause Wilson disease (WD), which is characterized by the accumulation of toxic Cu in the liver due to the scarce expression of ATP7B as well as the failure of ATP7B mutants to pump Cu and/or traffic to the Cu-excretion sites. The most frequent ATP7B mutant, H1069Q, still presents a significant Cu-transporting activity, but undergoes retention within the endoplasmic reticulum (ER) where the mutant is rapidly degraded. Expression of this ATP7B mutant has been recently reported to activate the p38 and JNK stress kinase pathways, which, in turn, trigger quality control mechanisms leading to the arrest of ATP7B-H1069Q in the ER and to the acceleration of its degradation. However, the main molecular players operating in these p38/JNK-dependent ER quality control pathways remain to be discovered. By using a combination of RNAseq, bioinformatics and RNAi approaches, we found a cluster of ER quality control genes whose expression is controlled by p38 and JNK and is required for the efficient retention of the ATP7B-H1069Q mutant in the ER. Silencing these genes reduced the accumulation of the ATP7B mutant in the ER and facilitated the mutant sorting and export to the Golgi and post-Golgi copper excretion sites. In sum, our findings reveal the ER-associated genes that could be utilized for the correction of ATP7B mutants and, hence, for the normalization of Cu homeostasis in Wilson disease.

Identification of p38 MAPK and JNK as new targets for correction of Wilson disease-causing ATP7B mutants.

UNLABELLED: Wilson disease (WD) is an autosomal recessive disorder that is caused by the toxic accumulation of copper (Cu) in the liver. The ATP7B gene, which is mutated in WD, encodes a multitransmembrane domain adenosine triphosphatase that traffics from the trans-Golgi network to the canalicular area of hepatocytes, where it facilitates excretion of excess Cu into the bile. Several ATP7B mutations, including H1069Q and R778L that are two of the most frequent variants, result in protein products, which, although still functional, remain in the endoplasmic reticulum. Thus, they fail to reach Cu excretion sites, resulting in the toxic buildup of Cu in the liver of WD patients. Therefore, correcting the location of these mutants by leading them to the appropriate functional sites in the cell should restore Cu excretion and would be beneficial to help large cohorts of WD patients. However, molecular targets for correction of endoplasmic reticulum-retained ATP7B mutants remain elusive. Here, we show that expression of the most frequent ATP7B mutant, H1069Q, activates p38 and c-Jun N-terminal kinase signaling pathways, which favor the rapid degradation of the mutant. Suppression of these pathways with RNA interference or specific chemical inhibitors results in the substantial rescue of ATP7B(H1069Q) (as well as that of several other WD-causing mutants) from the endoplasmic reticulum to the trans-Golgi network compartment, in recovery of its Cu-dependent trafficking, and in reduction of intracellular Cu levels. CONCLUSION: Our findings indicate p38 and c-Jun N-terminal kinase as intriguing targets for correction of WD-causing mutants and, hence, as potential candidates, which could be evaluated for the development of novel therapeutic strategies to combat WD. (Hepatology 2016;63:1842-1859).

Wilson disease protein ATP7B utilizes lysosomal exocytosis to maintain copper homeostasis.

Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease.

Cytosolic phospholipase A2ε drives recycling through the clathrin-independent endocytic route.

Previous studies have demonstrated that membrane tubule-mediated transport events in biosynthetic and endocytic routes require phospholipase A2 (PLA2) activity. Here, we show that cytosolic phospholipase A2ε (cPLA2ε, also known as PLA2G4E) is targeted to the membrane compartments of the clathrin-independent endocytic route through a C-terminal stretch of positively charged amino acids, which allows the enzyme to interact with phosphoinositide lipids [especially PI(4,5)P2] that are enriched in clathrin-independent endosomes. Ablation of cPLA2ε suppressed the formation of tubular elements that carry internalized clathrin-independent cargoes, such as MHC-I, CD147 and CD55, back to the cell surface and, therefore, caused their intracellular retention. The ability of cPLA2ε to support recycling through tubule formation relies on the catalytic activity of the enzyme, because the inactive cPLA2ε(S420A) mutant was not able to recover either tubule growth or transport from clathrin-independent endosomes. Taken together, our findings indicate that cPLA2ε is a new important regulator of trafficking processes within the clathrin-independent endocytic and recycling route. The affinity of cPLA2ε for this pathway supports a new hypothesis that different PLA2 enzymes use selective targeting mechanisms to regulate tubule formation locally during specific trafficking steps in the secretory and/or endocytic systems.

Gene transfer of master autophagy regulator TFEB results in clearance of toxic protein and correction of hepatic disease in alpha-1-anti-trypsin deficiency.

Alpha-1-anti-trypsin deficiency is the most common genetic cause of liver disease in children and liver transplantation is currently the only available treatment. Enhancement of liver autophagy increases degradation of mutant, hepatotoxic alpha-1-anti-trypsin (ATZ). We investigated the therapeutic potential of liver-directed gene transfer of transcription factor EB (TFEB), a master gene that regulates lysosomal function and autophagy, in PiZ transgenic mice, recapitulating the human hepatic disease. Hepatocyte TFEB gene transfer resulted in dramatic reduction of hepatic ATZ, liver apoptosis and fibrosis, which are key features of alpha-1-anti-trypsin deficiency. Correction of the liver phenotype resulted from increased ATZ polymer degradation mediated by enhancement of autophagy flux and reduced ATZ monomer by decreased hepatic NFκB activation and IL-6 that drives ATZ gene expression. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease of alpha-1-anti-trypsin deficiency. This study may pave the way towards applications of TFEB gene transfer for treatment of a wide spectrum of human disorders due to intracellular accumulation of toxic proteins.

Reverse engineering a mouse embryonic stem cell-specific transcriptional network reveals a new modulator of neuronal differentiation.

Gene expression profiles can be used to infer previously unknown transcriptional regulatory interaction among thousands of genes, via systems biology 'reverse engineering' approaches. We 'reverse engineered' an embryonic stem (ES)-specific transcriptional network from 171 gene expression profiles, measured in ES cells, to identify master regulators of gene expression ('hubs'). We discovered that E130012A19Rik (E13), highly expressed in mouse ES cells as compared with differentiated cells, was a central 'hub' of the network. We demonstrated that E13 is a protein-coding gene implicated in regulating the commitment towards the different neuronal subtypes and glia cells. The overexpression and knock-down of E13 in ES cell lines, undergoing differentiation into neurons and glia cells, caused a strong up-regulation of the glutamatergic neurons marker Vglut2 and a strong down-regulation of the GABAergic neurons marker GAD65 and of the radial glia marker Blbp. We confirmed E13 expression in the cerebral cortex of adult mice and during development. By immuno-based affinity purification, we characterized protein partners of E13, involved in the Polycomb complex. Our results suggest a role of E13 in regulating the division between glutamatergic projection neurons and GABAergic interneurons and glia cells possibly by epigenetic-mediated transcriptional regulation.

A mouse embryonic stem cell bank for inducible overexpression of human chromosome 21 genes.

BACKGROUND: Dosage imbalance is responsible for several genetic diseases, among which Down syndrome is caused by the trisomy of human chromosome 21. RESULTS: To elucidate the extent to which the dosage imbalance of specific human chromosome 21 genes perturb distinct molecular pathways, we developed the first mouse embryonic stem (ES) cell bank of human chromosome 21 genes. The human chromosome 21-mouse ES cell bank includes, in triplicate clones, 32 human chromosome 21 genes, which can be overexpressed in an inducible manner. Each clone was transcriptionally profiled in inducing versus non-inducing conditions. Analysis of the transcriptional response yielded results that were consistent with the perturbed gene's known function. Comparison between mouse ES cells containing the whole human chromosome 21 (trisomic mouse ES cells) and mouse ES cells overexpressing single human chromosome 21 genes allowed us to evaluate the contribution of single genes to the trisomic mouse ES cell transcriptome. In addition, for the clones overexpressing the Runx1 gene, we compared the transcriptome changes with the corresponding protein changes by mass spectroscopy analysis. CONCLUSIONS: We determined that only a subset of genes produces a strong transcriptional response when overexpressed in mouse ES cells and that this effect can be predicted taking into account the basal gene expression level and the protein secondary structure. We showed that the human chromosome 21-mouse ES cell bank is an important resource, which may be instrumental towards a better understanding of Down syndrome and other human aneuploidy disorders.