Valerie Dubost

Identification of Dlk1-Dio3 imprinted gene cluster noncoding RNAs as novel candidate biomarkers for liver tumor promotion.

The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital (PB) mediated liver tumor promotion in vivo. Molecular profiling (mRNA, microRNA [miRNA], DNA methylation, and proteins) of mouse liver during 13 weeks of PB treatment revealed progressive increases in hepatic expression of long noncoding RNAs and miRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. PB induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was localized to glutamine synthetase-positive hypertrophic perivenous hepatocytes, suggesting a role for β-catenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo genetic dependence on constitutive androstane receptor and β-catenin pathways. Our data identify Dlk1-Dio3 ncRNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds.

Heart Structure-Specific Transcriptomic Atlas Reveals Conserved microRNA-mRNA Interactions

MicroRNAs are short non-coding RNAs that regulate gene expression at the post-transcriptional level and play key roles in heart development and cardiovascular diseases. Here, we have characterized the expression and distribution of microRNAs across eight cardiac structures (left and right ventricles, apex, papillary muscle, septum, left and right atrium and valves) in rat, Beagle dog and cynomolgus monkey using microRNA sequencing. Conserved microRNA signatures enriched in specific heart structures across these species were identified for cardiac valve (miR-let-7c, miR-125b, miR-127, miR-199a-3p, miR-204, miR-320, miR-99b, miR-328 and miR-744) and myocardium (miR-1, miR-133b, miR-133a, miR-208b, miR-30e, miR-499-5p, miR-30e*). The relative abundance of myocardium-enriched (miR-1) and valve-enriched (miR-125b-5p and miR-204) microRNAs was confirmed using in situ hybridization. MicroRNA-mRNA interactions potentially relevant for cardiac functions were explored using anti-correlation expression analysis and microRNA target prediction algorithms. Interactions between miR-1/Timp3, miR-125b/Rbm24, miR-204/Tgfbr2 and miR-208b/Csnk2a2 were identified and experimentally investigated in human pulmonary smooth muscle cells and luciferase reporter assays. In conclusion, we have generated a high-resolution heart structure-specific mRNA/microRNA expression atlas for three mammalian species that provides a novel resource for investigating novel microRNA regulatory circuits involved in cardiac molecular physiopathology.

Phenobarbital Induces Cell Cycle Transcriptional Responses in Mouse Liver Humanized for Constitutive Androstane and Pregnane X Receptors

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CAR(KO)-PXR(KO)), double humanized CAR and PXR (CAR(h)-PXR(h)), and wild-type C57BL/6 mice. Wild-type and CAR(h)-PXR(h) mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CAR(KO)-PXR(KO) mouse livers and largely reversible in wild-type and CAR(h)-PXR(h) mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CAR(h)-PXR(h) mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.

Functional roles of Lgr4 and Lgr5 in embryonic gut, kidney and skin development in mice ☆

Lgr4 and Lgr5 are known markers of adult and embryonic tissue stem cells in various organs. However, whether Lgr4 and Lgr5 are important for embryonic development remains unclear. To study their functions during intestinal crypt, skin and kidney development we now generated mice lacking either Lgr4 (Lgr4KO), Lgr5 (Lgr5KO) or both receptors (Lgr4/5dKO). E16.5 Lgr4KO mice displayed complete loss of Lgr5+/Olfm4+intestinal stem cells, compromised Wnt signaling and impaired proliferation and differentiation of gut epithelium. Similarly, E16.5 Lgr4KO mice showed reduced basal cell proliferation and hair follicle numbers in the developing skin, as well as dilated kidney tubules and ectatic Bowman׳s spaces. Although Lgr4KO and Lgr5KO mice both died perinatally, Lgr5 deletion did not compromise embryonic development of gut, kidney or skin. Concomitant deletion of Lgr4 and Lgr5 did not prevent perinatal lethality, in contrast to a previous report that suggested rescue of Lgr5 KO perinatal lethality by a hypomorphic Lgr4 mutant. While the double deletion did not further promote the phenotypes observed in Lgr4KO intestines, impaired kidney cell proliferation, reduced epidermal thickness, loss of Lgr5+follicular epithelium and impaired hair follicle development were only observed in Lgr4/5dKO mice. This supports complementary functions of both receptors. Our findings clearly establish the importance of Lgr4 and Lgr5 during embryonic gut, skin and kidney development, with a dominant role of Lgr4.

Vascular Origin of Vildagliptin-induced Skin Effects in Cynomolgus Monkeys Pathomechanistic Role of Peripheral Sympathetic System and Neuropeptide Y

The purpose of this article is to characterize skin lesions in cynomolgus monkeys following vildagliptin (dipeptidyl peptidase-4 inhibitor) treatment. Oral vildagliptin administration caused dose-dependent and reversible blister formation, peeling and flaking skin, erosions, ulcerations, scabs, and sores involving the extremities at ≥5 mg/kg/day and necrosis of the tail and the pinnae at ≥80 mg/kg/day after 3 weeks of treatment. At the affected sites, the media and the endothelium of dermal arterioles showed hypertrophy/hyperplasia. Skin lesion formation was prevented by elevating ambient temperature. Vildagliptin treatment also produced an increase in blood pressure and heart rate likely via increased sympathetic tone. Following treatment with vildagliptin at 80 mg/kg/day, the recovery time after lowering the temperature in the feet of monkeys and inducing cold stress was prolonged. Ex vivo investigations showed that small digital arteries from skin biopsies of vildagliptin-treated monkeys exhibited an increase in neuropeptide Y–induced vasoconstriction. This finding correlated with a specific increase in NPY and in NPY1 receptors observed in the skin of vildagliptin-treated monkeys. Present data provide evidence that skin effects in monkeys are of vascular origin and that the effects on the NPY system in combination with increased peripheral sympathetic tone play an important pathomechanistic role in the pathogenesis of cutaneous toxicity.

Editor’s Highlight: Comparative Renal Safety Assessment of the Hepatitis B Drugs, Adefovir, Tenofovir, Telbivudine and Entecavir in Rats

The aim of this study was to determine the relative safety of 4 antiviral drugs (telbivudine, tenofovir, adefovir, and entecavir) against hepatitis B virus with respect to kidney function and toxicity in male Sprague Dawley rats. The antiviral drugs were administered once daily for 4 weeks by oral gavage at ∼10 and 25–40 times the human equivalent dose. Main assessments included markers of renal toxicity in urine, magnetic resonance imaging (MRI) of kidney function, histopathology, and electron microscopic examination. Administration of adefovir at 11 and 28 mg/kg for 4 weeks caused functional and morphological kidney alterations in a time- and dose-dependent manner, affecting mainly the proximal tubules and suggesting a mechanism of toxicity related to mitochondrial degeneration/depletion. Of note, the observed adefovir-induced reduction of kidney function was not detected by the standard method of glomerular filtration rate (GFR) measurements (clearance rate of the endogenous marker, creatinine), thereby emphasizing the superiority of MRI in terms of sensitive detection of GFR in rats. For the low dose of 300 mg/kg of tenofovir, minor kidney effects such as nuclear enlargement in the tubular epithelium, and hyaline droplets accumulation were detected, which was also observed for the low dose (11 mg/kg) of adefovir. No assessments could be done at the higher dose of 600/1000 mg/kg tenofovir due to gastrointestinal tract toxicity which prevented treatment of the animals for longer than 1 week. Entecavir at 1 and 3 mg/kg and telbivudine at 600 and 1600 mg/kg caused no toxicologically relevant effects on the kidney.

Fingolimod inhibits brain atrophy and promotes brain-derived neurotrophic factor in an animal model of multiple sclerosis

Longitudinal brain atrophy quantification is a critical efficacy measurement in multiple sclerosis (MS) clinical trials and the determination of No Evidence of Disease Activity (NEDA). Utilising fingolimod as a clinically validated therapy we evaluated the use of repeated brain tissue volume measures during chronic experimental autoimmune encephalomyelitis (EAE) as a new preclinical efficacy measure. Brain volume changes were quantified using magnetic resonance imaging (MRI) at 7 Tesla and correlated to treatment-induced brain derived neurotrophic factor (BDNF) measured in blood, cerebrospinal fluid, spinal cord and brain. Serial brain MRI measurements revealed slow progressive brain volume loss in vehicle treated EAE mice despite a stable clinical score. Fingolimod (1 mg/kg) significantly ameliorated brain tissue atrophy in the cerebellum and striatum when administered from established EAE disease onwards. Fingolimod-dependent tissue preservation was associated with induction of BDNF specifically within the brain and co-localized with neuronal soma. In contrast, therapeutic teriflunomide (3 mg/kg) treatment failed to inhibit CNS autoimmune mediated brain degeneration. Finally, weekly anti-IL-17A antibody (15 mg/kg) treatment was highly efficacious and preserved whole brain, cerebellum and striatum volume. Fingolimod-mediated BDNF increases within the CNS may contribute to limiting progressive tissue loss during chronic neuroinflammation.

Xenobiotic CAR Activators Induce Dlk1-Dio3 Locus Noncoding RNA Expression in Mouse Liver

Derisking xenobiotic-induced nongenotoxic carcinogenesis (NGC) represents a significant challenge during the safety assessment of chemicals and therapeutic drugs. The identification of robust mechanism-based NGC biomarkers has the potential to enhance cancer hazard identification. We previously demonstrated Constitutive Androstane Receptor (CAR) and WNT signaling-dependent up-regulation of the pluripotency associated Dlk1-Dio3 imprinted gene cluster noncoding RNAs (ncRNAs) in the liver of mice treated with tumor-promoting doses of phenobarbital (PB). Here, we have compared phenotypic, transcriptional ,and proteomic data from wild-type, CAR/PXR double knock-out and CAR/PXR double humanized mice treated with either PB or chlordane, and show that hepatic Dlk1-Dio3 locus long ncRNAs are upregulated in a CAR/PXR-dependent manner by two structurally distinct CAR activators. We further explored the specificity of Dlk1-Dio3 locus ncRNAs as hepatic NGC biomarkers in mice treated with additional compounds working through distinct NGC modes of action. We propose that up-regulation of Dlk1-Dio3 cluster ncRNAs can serve as an early biomarker for CAR activator-induced nongenotoxic hepatocarcinogenesis and thus may contribute to mechanism-based assessments of carcinogenicity risk for chemicals and novel therapeutics.

Differential expression of dipeptidyl peptidase IV in human versus cynomolgus monkey skin eccrine sweat glands

Dipeptidyl peptidase IV (DPP4) is a peptidase whose inhibition is beneficial in Type II diabetes treatment. Several evidences suggest potential implication of DPP4 in skin disorders such as psoriasis, keloids and fibrotic skin diseases where its inhibition could also be beneficial. DPP4 expression in human skin was described mainly in dermal fibroblasts and a subset of keratinocytes in the basal layer. Of importance in the perspective of preclinical experimentation, DPP4 distribution in skin of non-human primate species has not been documented. This report evidences unexpected differences between a set of human and cynomolgus monkey skin samples revealing a major expression of DPP4 in eccrine sweat glands of cynomolgus monkeys but not in humans. This represents a unique distinctive feature compared to the conserved expression of dipeptidyl peptidases 8 and 9 and potential relevant DPP4 substrates such as neuropeptide Y (NPY) and receptors (NPY-receptor 1 and Neurokinin receptor). Finally the observation that cathepsin D, an unrelated protease, shows the opposite expression compared to DPP4 (present in human but not in cynomolgus monkey eccrine sweat glands) could indicate that human eccrine sweat glands evolved a divergent protease repertoire compared to non-human primates. These unexpected differences in the eccrine sweat glands protease repertoire will need to be confirmed extending the analysis to a major number of donors but could imply possible biochemical divergences, reflecting the functional evolution of the glands and the control of their activity. Our findings also demonstrate that non-human primates studies aiming at understanding DPP4 function in skin biology are not readily translatable to human.

The Bace-1 inhibitor CNP520 for prevention trials in Alzheimer's disease

The beta‐site amyloid precursor protein cleaving enzyme‐1 (BACE‐1) initiates the generation of amyloid‐β (Aβ), and the amyloid cascade leading to amyloid plaque deposition, neurodegeneration, and dementia in Alzheimers disease (AD). Clinical failures of anti‐Aβ therapies in dementia stages suggest that treatment has to start in the early, asymptomatic disease states. The BACE‐1 inhibitor CNP520 has a selectivity, pharmacodynamics, and distribution profile suitable for AD prevention studies. CNP520 reduced brain and cerebrospinal fluid (CSF) Aβ in rats and dogs, and Aβ plaque deposition in APP‐transgenic mice. Animal toxicology studies of CNP520 demonstrated sufficient safety margins, with no signs of hair depigmentation, retina degeneration, liver toxicity, or cardiovascular effects. In healthy adults ≥ 60 years old, treatment with CNP520 was safe and well tolerated and resulted in robust and dose‐dependent Aβ reduction in the cerebrospinal fluid. Thus, long‐term, pivotal studies with CNP520 have been initiated in the Generation Program.