Florence Bernex

Corrective GUSB Transfer to the Canine Mucopolysaccharidosis VII Brain

Severe deficiency in lysosomal β-glucuronidase (β-glu) enzymatic activity results in mucopolysaccharidosis (MPS) VII, an orphan disease with symptoms often appearing in early childhood. Symptoms are variable, but many patients have multiple organ disorders including neurological defects. At the cellular level, deficiency in β-glu activity leads to abnormal accumulation of glycosaminoglycans (GAGs), and secondary accumulation of GM2 and GM3 gangliosides, which have been linked to neuroinflammation. There have been encouraging gene transfer studies in the MPS VII mouse brain, but this is the first study attempting the correction of the >200-fold larger and challenging canine MPS VII brain. Here, the efficacy of a helper-dependent (HD) canine adenovirus (CAV-2) vector harboring a human GUSB expression cassette (HD-RIGIE) in the MPS VII dog brain was tested. Vector genomes, β-glu activity, GAG content, lysosome morphology and neuropathology were analyzed and quantified. Our data demonstrated that CAV-2 vectors preferentially transduced neurons and axonal retrograde transport from the injection site to efferent regions was efficient. HD-RIGIE injections, associated with mild and transient immunosuppression, corrected neuropathology in injected and noninjected structures throughout the cerebrum. These data support the clinical evaluation of HD CAV-2 vectors to treat the neurological defects associated with MPS VII and possibly other neuropathic lysosomal storage diseases.

High expression of the RNA-binding protein RBPMS2 in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract and are often associated with KIT or PDGFRA gene mutations. GIST cells might arise from the interstitial cells of Cajal (ICCs) or from a mesenchymal precursor that is common to ICCs and smooth muscle cells (SMCs). Here, we analyzed the mRNA and protein expression of RNA-Binding Protein with Multiple Splicing-2 (RBPMS2), an early marker of gastrointestinal SMC precursors, in human GISTs (n=23) by in situ hybridization, quantitative RT-PCR analysis and immunohistochemistry. The mean RBPMS2 mRNA level in GISTs was 42-fold higher than in control gastrointestinal samples (p<0.001). RBPMS2 expression was not correlated with KIT and PDGFRA expression levels, but was higher in GISTs harboring KIT mutations than in tumors with wild type KIT and PDGFRA or in GISTs with PDGFRA mutations that were characterized by the lowest RBPMS2 levels. Moreover, RBPMS2 levels were 64-fold higher in GIST samples with high risk of aggressive behavior than in adult control gastrointestinal samples and 6.2-fold higher in high risk than in low risk GIST specimens. RBPMS2 protein level was high in 87% of the studied GISTs independently of their histological classification. Finally, by inhibiting the KIT signaling pathway in GIST882 cells, we show that RBPMS2 expression is independent of KIT activation. In conclusion, RBPMS2 is up-regulated in GISTs compared to normal adult gastrointestinal tissues, indicating that RBPMS2 might represent a new diagnostic marker for GISTs and a potential target for cancer therapy.

Coxsackievirus Adenovirus Receptor Loss Impairs Adult Neurogenesis, Synapse Content, and Hippocampus Plasticity.

Although we are beginning to understand the late stage of neurodegenerative diseases, the molecular defects associated with the initiation of impaired cognition are poorly characterized. Here, we demonstrate that in the adult brain, the coxsackievirus and adenovirus receptor (CAR) is located on neuron projections, at the presynapse in mature neurons, and on the soma of immature neurons in the hippocampus. In a proinflammatory or diseased environment, CAR is lost from immature neurons in the hippocampus. Strikingly, in hippocampi of patients at early stages of late-onset Alzheimers disease (AD), CAR levels are significantly reduced. Similarly, in triple-transgenic AD mice, CAR levels in hippocampi are low and further reduced after systemic inflammation. Genetic deletion of CAR from the mouse brain triggers deficits in adult neurogenesis and synapse homeostasis that lead to impaired hippocampal plasticity and cognitive deficits. We propose that post-translational CAR loss of function contributes to cognitive defects in healthy and diseased-primed brains. SIGNIFICANCE STATEMENT This study addressed the role of the coxsackievirus and adenovirus receptor (CAR), a single-pass cell adhesion molecule, in the adult brain. Our results demonstrate that CAR is expressed by mature neurons throughout the brain. In addition, we propose divergent roles for CAR in immature neurons, during neurogenesis, and at the mature synapse. Notably, CAR loss of function also affects hippocampal plasticity.

Corrective GUSB transfer to the canine mucopolysaccharidosis VII cornea using a helper-dependent canine adenovirus vector.

Corneal transparency is maintained, in part, by specialized fibroblasts called keratocytes, which reside in the fibrous lamellae of the stroma. Corneal clouding, a condition that impairs visual acuity, is associated with numerous diseases, including mucopolysaccharidosis (MPS) type VII. MPS VII is due to deficiency in β-glucuronidase (β-glu) enzymatic activity, which leads to accumulation of glycosaminoglycans (GAGs), and secondary accumulation of gangliosides. Here, we tested the efficacy of canine adenovirus type 2 (CAV-2) vectors to transduce keratocyte in vivo in mice and nonhuman primates, and ex vivo in dog and human corneal explants. Following efficacy studies, we asked if we could treat corneal clouding by the injection a helper-dependent (HD) CAV-2 vector (HD-RIGIE) harboring the human cDNA coding for β-glu (GUSB) in the canine MPS VII cornea. β-Glu activity, GAG content, and lysosome morphology and physiopathology were analyzed. We found that HD-RIGIE injections efficiently transduced coxsackievirus adenovirus receptor-expressing keratocytes in the four species and, compared to mock-injected controls, improved the pathology in the canine MPS VII cornea. The key criterion to corrective therapy was the steady controlled release of β-glu and its diffusion throughout the collagen-dense stroma. These data support the continued evaluation of HD CAV-2 vectors to treat diseases affecting corneal keratocytes.

Combination of nutritional polyphenols supplementation with exercise training counteracts insulin resistance and improves endurance in high-fat diet-induced obese rats

Separately, polyphenols and exercise are known to prevent insulin resistance (IR) but their combined curative effects on established obesity and IR require further investigation. Therefore, we compared the metabolic effects of a combination of exercise and grape polyphenols supplementation in obese IR rats with high-fat diet (EXOPP) to the effect of high-fat diet alone (HF) or with a nutritional supplementation of grape polyphenols (PP) or with endurance exercise (EXO) during 8 wks. We observed an improvement of systemic and skeletal muscle insulin sensitivity in EXO and EXOPP rats. EXOPP rats compared to HF rats presented a lower insulinemia and HOMA-IR with higher liver and muscle glycogen contents. Interestingly, EXOPP rats had a 68% enhanced endurance capacity compared to EXO rats with also a higher activation of AMPK compared to sedentary and EXO rats with increased lipid oxidation. Together, our results suggest that grape polyphenols supplementation combined with exercise has a synergistic effect by increasing muscle lipid oxidation and sparing glycogen utilization which thus enhances endurance capacity. Our data highlight that in cases of established obesity and IR, the combination of nutritional grape polyphenols supplementation and exercise heighten and intensify their individual metabolic effects.

A histopathological classification system of Tyr::NRASQ61K murine melanocytic lesions: A reproducible simplified classification

Genetically engineered mouse models offer essential opportunities to investigate the mechanisms of initiation and progression in melanoma. Here, we report a new simplified histopathology classification of mouse melanocytic lesions in Tyr::NRASQ61K derived models, using an interactive decision tree that produces homogeneous categories. Reproducibility for this classification system was evaluated on a panel of representative cases of murine melanocytic lesions by pathologists and basic scientists. Reproducibility, measured as inter‐rater agreement between evaluators using a modified Fleiss’ kappa statistic, revealed a very good agreement between observers. Should this new simplified classification be adopted, it would create a robust system of communication between researchers in the field of mouse melanoma models.

Checkpoint kinase 1 inhibition sensitises transformed cells to dihydroorotate dehydrogenase inhibition

Reduction in nucleotide pools through the inhibition of mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) has been demonstrated to effectively reduce cancer cell proliferation and tumour growth. The current study sought to investigate whether this antiproliferative effect could be enhanced by combining Chk1 kinase inhibition. The pharmacological activity of DHODH inhibitor teriflunomide was more selective towards transformed mouse embryonic fibroblasts than their primary or immortalised counterparts, and this effect was amplified when cells were subsequently exposed to PF477736 Chk1 inhibitor. Flow cytometry analyses revealed substantial accumulations of cells in S and G2/M phases, followed by increased cytotoxicity which was characterised by caspase 3-dependent induction of cell death. Associating PF477736 with teriflunomide also significantly sensitised SUM159 and HCC1937 human triple negative breast cancer cell lines to dihydroorotate dehydrogenase inhibition. The main characteristic of this effect was the sustained accumulation of teriflunomide-induced DNA damage as cells displayed increased phospho serine 139 H2AX (γH2AX) levels and concentration-dependent phosphorylation of Chk1 on serine 345 upon exposure to the combination as compared with either inhibitor alone. Importantly a similar significant increase in cell death was observed upon dual siRNA mediated depletion of Chk1 and DHODH in both murine and human cancer cell models. Altogether these results suggest that combining DHODH and Chk1 inhibitions may be a strategy worth considering as a potential alternative to conventional chemotherapies.Reduction in nucleotide pools through the inhibition of mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) has been demonstrated to effectively reduce cancer cell proliferation and tumour growth. The current study sought to investigate whether this antiproliferative effect could be enhanced by combining Chk1 kinase inhibition. The pharmacological activity of DHODH inhibitor teriflunomide was more selective towards transformed mouse embryonic fibroblasts than their primary or immortalised counterparts, and this effect was amplified when cells were subsequently exposed to PF477736 Chk1 inhibitor. Flow cytometry analyses revealed substantial accumulations of cells in S and G2/M phases, followed by increased cytotoxicity which was characterised by caspase 3-dependent induction of cell death. Associating PF477736 with teriflunomide also significantly sensitised SUM159 and HCC1937 human triple negative breast cancer cell lines to dihydroorotate dehydrogenase inhibition. The main characteristic of this effect was the sustained accumulation of teriflunomide-induced DNA damage as cells displayed increased phospho serine 139 H2AX (γH2AX) levels and concentration-dependent phosphorylation of Chk1 on serine 345 upon exposure to the combination as compared with either inhibitor alone. Importantly a similar significant increase in cell death was observed upon dual siRNA mediated depletion of Chk1 and DHODH in both murine and human cancer cell models. Altogether these results suggest that combining DHODH and Chk1 inhibitions may be a strategy worth considering as a potential alternative to conventional chemotherapies.