ABHD11, a new diacylglycerol lipase involved in weight gain regulation.

Obesity epidemic continues to spread and obesity rates are increasing in the world. In addition to public health effort to reduce obesity, there is a need to better understand the underlying biology to enable more effective treatment and the discovery of new pharmacological agents. Abhydrolase domain-containing protein 11 (ABHD11) is a serine hydrolase enzyme, localized in mitochondria, that can synthesize the endocannabinoid 2-arachidonoyl glycerol (2AG) in vitro. In vivo preclinical studies demonstrated that knock-out ABHD11 mice have a similar 2AG level as WT mice and exhibit a lean metabolic phenotype. Such mice resist to weight gain in Diet Induced Obesity studies (DIO) and display normal biochemical plasma parameters. Metabolic and transcriptomic analyses on serum and tissues of ABHD11 KO mice from DIO studies show a modulation in bile salts associated with reduced fat intestinal absorption. These data suggest that modulating ABHD11 signaling pathway could be of therapeutic value for the treatment of metabolic disorders.

A 3D Human Liver Model of Nonalcoholic Steatohepatitis.

Background and Aims: To better understand nonalcoholic steatohepatitis (NASH) disease progression and to evaluate drug targets and compound activity, we undertook the development of an in vitro 3D model to mimic liver architecture and the NASH environment. Methods: We have developed an in vitro preclinical 3D NASH model by coculturing primary human hepatocytes, human stellate cells, liver endothelial cells and Kupffer cells embedded in a hydrogel of rat collagen on a 96-well plate. A NASH-like environment was induced by addition of medium containing free fatty acids and tumor necrosis factor-α. This model was then characterized by biochemical, imaging and transcriptomics analyses. Results: We succeeded in defining suitable culture conditions to maintain the 3D coculture for up to 10 days in vitro, with the lowest level of steatosis and reproducible low level of inflammation and fibrosis. NASH disease was induced with a custom medium mimicking NASH features. The cell model exhibited the key NASH disease phenotypes of hepatocyte injury, steatosis, inflammation, and fibrosis. Hepatocyte injury was highlighted by a decrease of CYP3A4 expression and activity, without loss of viability up to day 10. Moreover, the model was able to stimulate a stable inflammatory and early fibrotic environment, with expression and secretion of several cytokines. A global gene expression analysis confirmed the NASH induction. Conclusions: This is a new in vitro model of NASH disease consisting of four human primary cell-types that exhibits most features of the disease. The 10-day cell viability and cost effectiveness of the model make it suitable for medium throughput drug screening and provide attractive avenues to better understand disease physiology and to identify and characterize new drug targets.

A Human Stem Cell Model of Fabry Disease Implicates LIMP-2 Accumulation in Cardiomyocyte Pathology.

Here, we have used patient-derived induced pluripotent stem cell (iPSC) and gene-editing technology to study the cardiac-related molecular and functional consequences of mutations in GLA causing the lysosomal storage disorder Fabry disease (FD), for which heart dysfunction is a major cause of mortality. Our in vitro model recapitulated clinical data with FD cardiomyocytes accumulating GL-3 and displaying an increased excitability, with altered electrophysiology and calcium handling. Quantitative proteomics enabled the identification of >5,500 proteins in the cardiomyocyte proteome and secretome, and revealed accumulation of the lysosomal protein LIMP-2 and secretion of cathepsin F and HSPA2/HSP70-2 in FD. Genetic correction reversed these changes. Overexpression of LIMP-2 directly induced the secretion of cathepsin F and HSPA2/HSP70-2, implying causative relationship, and led to massive vacuole accumulation. In summary, our study has revealed potential new cardiac biomarkers for FD, and provides valuable mechanistic insight into the earliest pathological events in FD cardiomyocytes.

Type 2/Th2-driven inflammation impairs olfactory sensory neurogenesis in mouse chronic rhinosinusitis model.

BACKGROUND: Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is a chronic inflammatory disease often accompanied by impairment of sense of smell. This symptom has been somewhat overlooked, and its relationship to inflammatory cytokines, tissue compression, neuronal loss, and neurogenesis is still unclear. METHODS: In order to elucidate potential mechanisms leading to CRS in humans, we have established a type 2/T helper type 2 cell (Th2)-mediated allergic CRS mouse model, based on house dust mite (HDM) and Staphylococcus aureus enterotoxin B (SEB) sensitization. The inflammatory status of the olfactory epithelium (OE) was assessed using histology, biochemistry, and transcriptomics. The sense of smell was evaluated by studying olfactory behavior and recording electro-olfactograms (EOGs). RESULTS: After 22 weeks, a typical type 2/Th2-mediated inflammatory profile was obtained, as demonstrated by increased interleukin (IL)-4, IL-5, and IL-13 in the OE. The number of mast cells and eosinophils was increased, and infiltration of these cells into the olfactory mucosa was also observed. In parallel, transcriptomic and histology analyses indicated a decreased number of immature olfactory neurons, possibly due to decreased renewal. However, the number of mature sensory neurons was not affected and neither the EOG nor olfactory behavior was impaired. CONCLUSION: Our mouse model of CRS displayed an allergic response to HDM + SEB administration, including the type 2/Th2 inflammatory profile characteristic of human eosinophilic CRSwNP. Although the sense of smell did not appear to be altered in these conditions, the data reveal the influence of chronic inflammation on olfactory neurogenesis, suggesting that factors unique to humans may be involved in CRSwNP-associated anosmia.

Prostaglandin EP2 receptor signaling protects human trabecular meshwork cells from apoptosis induced by ER stress through down-regulation of p53.

E-prostanoid receptor subtype 2 (EP2) agonists are currently under clinical development as hypotensive agents for the treatment of ocular hypertension. However, the effects of EP2 receptor agonists on trabecular meshwork (TM) alterations leading to primary open-angle glaucoma (POAG) are still unknown. Here, we evaluated whether EP2 receptor activation exhibits protective functions on TM cell death induced by endoplasmic reticulum (ER) stress. We show that the EP2 receptor agonist butaprost protects TM cell death mediated by the ER stress inducer tunicamycin through a cyclic AMP (cAMP)-dependent mechanism, but independent of the classical cAMP sensors, protein kinase A and exchange proteins activated by cAMP. The ER stress-induced intrinsic apoptosis inhibited by the EP2 receptor agonist was correlated with a decreased accumulation of the cellular stress sensor p53. In addition, p53 down-regulation was associated with inhibition of its transcriptional activity, which led to decreased expression of the pro-apoptotic p53-upregulated modulator of apoptosis (PUMA). The stabilization of p53 by nutlin-3a abolished butaprost-mediated cell death protection. In conclusion, we showed that EP2 receptor activation protects against ER stress-dependent mitochondrial apoptosis through down-regulation of p53. The specific inhibition of this pathway could reduce TM alterations observed in POAG patients.

Effective clearance of GL-3 in a human iPSC-derived cardiomyocyte model of Fabry disease.

Fabry disease, a rare X-linked α-galactosidase A deficiency, causes progressive lysosomal accumulation of globotriaosylceramide (GL-3) in a variety of cell types. As the disease progresses, renal failure, left ventricular hypertrophy, and strokes may occur. Enzyme replacement therapy (ERT), with recombinant α-galactosidase A, is currently available for use to reduce GL-3 deposits. However, although it improves cardiac function and decreases left ventricular mass, GL-3 clearance upon ERT has been demonstrated in cardiac capillary endothelium but not in cardiomyocytes of patients. Relevant models are needed to understand the pathogenesis of cardiac disease and explore new therapeutic approaches. We generated induced pluripotent stem cells (iPSC) from Fabry patients and differentiated them into cardiomyocytes. In these cells, GL-3 accumulates in the lysosomes over time, resulting in phenotypic changes similar to those found in cardiac tissue from Fabry patients. Using this human in vitro model, we demonstrated that substrate reduction therapy via glucosylceramide synthase inhibition was able to prevent accumulation and to clear lysosomal GL-3 in cardiomyocytes. This new in vitro model recapitulates essential features of cardiomyocytes from patients with Fabry disease and therefore provides a useful and relevant tool for further investigations of new therapy.

Generation of a drug-inducible reporter system to study cell reprogramming in human cells.

BACKGROUND: Strategies on the basis of doxycycline-inducible lentiviruses in mouse cells allowed the examination of mechanisms governing somatic cell reprogramming. RESULTS: Using a doxycycline-inducible human reprogramming system, we identified unreported miRs enhancing reprogramming efficiency. CONCLUSION: We generated a drug-inducible human reprogramming reporter system as an invaluable tool for genetic or chemical screenings. SIGNIFICANCE: These cellular systems provide a tool to enable the advancement of reprogramming technologies in human cells. Reprogramming of somatic cells into induced pluripotent stem cells is achieved by the expression of defined transcription factors. In the last few years, reprogramming strategies on the basis of doxycycline-inducible lentiviruses in mouse cells became highly powerful for screening purposes when the expression of a GFP gene, driven by the reactivation of endogenous stem cell specific promoters, was used as a reprogramming reporter signal. However, similar reporter systems in human cells have not been generated. Here, we describe the derivation of drug-inducible human fibroblast-like cell lines that express different subsets of reprogramming factors containing a GFP gene under the expression of the endogenous OCT4 promoter. These cell lines can be used to screen functional substitutes for reprogramming factors or modifiers of reprogramming efficiency. As a proof of principle of this system, we performed a screening of a library of pluripotent-enriched microRNAs and identified hsa-miR-519a as a novel inducer of reprogramming efficiency.

Comparison of promoter region constructs for in vivo intramuscular expression.

BACKGROUND: High transgene expression is generally expected after gene transfer. However, different level, kinetics and localization of expression might be needed for relevant therapeutic applications. Former studies have compared various promoter regions driving gene expression leading to conflicting results. In the present work, two promoter families have been compared using the efficient in vivo intramuscular electrotransfer technique. METHODS: Three promoter regions were constructed by associating the strong ubiquitous cytomegalovirus (CMV) enhancer-promoter to its homologous intron A or to a heterologous intron, or to a hybrid intron. Promoter regions derived from the muscle creatine kinase (MCK) promoter were also studied. The expression of the same transgene (SeAP or neurotrophin-3) under control of these different promoters was compared after plasmid electrotransfer in mouse tibialis-cranialis skeletal muscle. RESULTS: Heterologous intron association to the CMV promoter did not modify gene expression kinetics nor increase gene expression level. Usefulness of intron A or hybrid intron association to the CMV promoter depended on the gene. The various MCK promoters drove efficient gene expression but lower than that obtained with the CMV promoter. Furthermore, peak value was reached earlier with MCK promoter regions (14 days). CONCLUSION: For applications of gene transfer restricted to skeletal muscle, the MCK promoter or a MCK promoter variant would be a promising alternative to the CMV promoter. Indeed, it has been demonstrated that the use of MCK promoter limits humoral and cell-mediated immune responses. Furthermore, the MCK promoter decreases the initial expression peak that may be detrimental, drives a sustained gene expression, and improves gene transfer safety.

Positive in vivo heterologous gene regulation by electric pulses delivery with metallothionein I gene promoter.

BACKGROUND: In vivo electrotransfer is a physical method of gene delivery in various tissues and organs. It is a promising strategy for the systemic secretion of therapeutic proteins and for DNA vaccination. Nevertheless, for the success of gene therapy in clinics, it is essential to develop gene regulation systems. The existing systems described in the literature all rely on the creation of an artificial transcription factor and/or an inducer drug. New strategies based on endogenous regulatable elements are being developed. We have previously identified the murine metallothionein promoter as an endogenous promoter inducible by controlled electric stimuli applied for electrotransfer experiments. We report here a regulation strategy based on this murine metallothionein promoter in a plasmid context using electric pulses delivery as an inducer. METHODS: Plasmids containing different constructions of the murine metallothionein I (mMT-I) promoter were transfected in mice tibialis-cranalis muscles using the simple skeletal muscle electrotransfer method. The regulation system was studied with the murine secreted alkaline phosphatase (MUSEAP) reporter gene. RESULTS: The mMT-I promoter can be transiently induced in vivo by application of electric fields. Its inducibility was analyzed in a plasmid context. We demonstrated that the mechanism of this transcriptional induction is not mediated by the cellular entry of metal ions. The ARE (antioxidant-responsive element) sequence was identified as the element responsive to the electric field stimulation. CONCLUSIONS: This time-control of the expression of a therapeutic gene by physical stimuli could be of value in the context of gene regulation for gene therapy.

Heterogeneous nuclear ribonucleoprotein A1 is a novel internal ribosome entry site trans-acting factor that modulates alternative initiation of translation of the fibroblast growth factor 2 mRNA.

Alternative initiation of translation of the human fibroblast growth factor 2 (FGF-2) mRNA at five in-frame CUG or AUG translation initiation codons requires various RNA cis-acting elements, including an internal ribosome entry site (IRES). Here we describe the purification of a trans-acting factor controlling FGF-2 mRNA translation achieved by several biochemical purification approaches. We have identified the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as a factor that binds to the FGF-2 5'-leader RNA and that also complements defective FGF-2 translation in vitro in rabbit reticulocyte lysate. Recombinant hnRNP A1 stimulates in vitro translation at the four IRES-dependent initiation codons but has no effect on the cap-dependent initiation codon. Consistent with a role of hnRNP A1 in the control of alternative initiation of translation, short interfering RNA-mediated knock down of hnRNP A1 specifically inhibits translation at the four IRES-dependent initiation codons. Furthermore, hnRNP A1 binds to the FGF-2 IRES, implicating this interaction in the control of alternative initiation of translation.

Transcriptional activation of the metallothionein I gene by electric pulses in vivo: basis for the development of a new gene switch system.

BACKGROUND: In vivo gene transfer to skeletal muscle is a promising strategy for the treatment of muscular disorders and for the systemic delivery of therapeutic proteins. Nevertheless, for a safe and effective protein production, the spatial and temporal control of gene expression is critical. The existing regulating systems rely on the use of an exogenously regulatory protein and/or an inducer drug whose pharmacological properties are of major concerns for therapeutic applications in humans. Therefore, new strategies based on endogenous regulatable elements have been explored. METHODS: Gene expression profiles of skeletal muscle submitted or not to electrical pulses and harvested at different times were compared using the Affymetrix GeneChip technology. The endogenous metallothionein promoter was studied by Northern blot and semiquantitative and quantitative RT-PCR. The inducibility of the metallothionein I promoter placed in a plasmid exogenous context was studied using the murine SEAP reporter gene. RESULTS: The expression of metallothionein I mRNA is significantly increased 6 h after electric pulses delivery. This induction is transient. Identical MT-I expression level is observed after several sequential series of pulses delivery. We demonstrated as well that the MT-II promoter was sensitive to electric pulses delivery. Moreover, the metallothionein I promoter, placed in a plasmid context in front of a reporter gene, was also activated by the application of transient electric field. CONCLUSIONS: We identified a promoter highly inducible by the controlled electric stimuli applied for electrotransfer experiments. The use of the metallothionein promoter is promising for the time-control by physical stimuli of the expression of a therapeutic gene.