Cristina Fillat

Diabetes Risk Gene and Wnt Effector Tcf7l2/TCF4 Controls Hepatic Response to Perinatal and Adult Metabolic Demand

Most studies on TCF7L2 SNP variants in the pathogenesis of type 2 diabetes (T2D) focus on a role of the encoded transcription factor TCF4 in β cells. Here, a mouse genetics approach shows that removal of TCF4 from β cells does not affect their function, whereas manipulating TCF4 levels in the liver has major effects on metabolism. In Tcf7l2(-/-) mice, the immediate postnatal surge in liver metabolism does not occur. Consequently, pups die due to hypoglycemia. By combining chromatin immunoprecipitation with gene expression profiling, we identify a TCF4-controlled metabolic gene program that isxa0acutely activated in the postnatal liver. In concordance, adult liver-specific Tcf7l2 knockout micexa0show reduced hepatic glucose production during fasting and display improved glucose homeostasis when maintained on high-fat diet. Furthermore, liver-specific TCF4 overexpression increases hepatic glucose production. These observations imply that TCF4 directly activates metabolic genes and that inhibition of Wnt signaling may be beneficial in metabolic disease.

Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice

Huntingtons disease (HD) is a dominantly inherited neurodegenerative disorder caused by expanded CAG repeats in the huntingtin (HTT) gene. Although several palliative treatments are available, there is currently no cure and patients generally die 10–15 y after diagnosis. Several promising approaches for HD therapy are currently in development, including RNAi and antisense analogs. We developed a complementary strategy to test repression of mutant HTT with zinc finger proteins (ZFPs) in an HD model. We tested a “molecular tape measure” approach, using long artificial ZFP chains, designed to bind longer CAG repeats more strongly than shorter repeats. After optimization, stable ZFP expression in a model HD cell line reduced chromosomal expression of the mutant gene at both the protein and mRNA levels (95% and 78% reduction, respectively). This was achieved chromosomally in the context of endogenous mouse HTT genes, with variable CAG-repeat lengths. Shorter wild-type alleles, other genomic CAG-repeat genes, and neighboring genes were unaffected. In vivo, striatal adeno-associated virus viral delivery in R6/2 mice was efficient and revealed dose-dependent repression of mutant HTT in the brain (up to 60%). Furthermore, zinc finger repression was tested at several levels, resulting in protein aggregate reduction, reduced decline in rotarod performance, and alleviation of clasping in R6/2 mice, establishing a proof-of-principle for synthetic transcription factor repressors in the brain.

Regulated expression of human insulin in the liver of transgenic mice corrects diabetic alterations.

Transgenic mice expressing the P‐enolpyruvate carboxykinase (PEPCK)/human insulin chimeric gene have been obtained as a model to study the feasibility of gene therapy for diabetes. These transgenic animals were healthy and normoglycemic and expressed human insulin in a physiologically regulated manner, mainly in the liver. Streptozotocin‐treated transgenic mice had high levels of human insulin immunoreactivity in serum and showed a significant decrease (up to 40%) in glycemia compared with streptozotocin‐treated control mice. The expression of genes involved in liver glucose metabolism, such as glucokinase, pyruvate kinase, and PEPCK, which is markedly altered by diabetes, was significantly recovered in transgenic mice treated with streptozotocin. In addition, the activity of both glucokinase and glycogen synthase, and the content of glucose 6‐phosphate and glycogen, were normal in the liver, even when transgenic animals were treated with diabetogenic doses of streptozotocin. These results constitute an indication in vivo that diabetes gene therapy is possible, by means of the production of insulin in extrapancreatic tissues.—Valera, A., Fillat, C., Costa, C., Sabater, J., Visa, J., Pujol, A., Bosch, F. Regulated expression of human insulin in the liver of transgenic mice corrects diabetic alterations. FASEB J. 8: 440‐447; 1994.

Murine models for Down syndrome.

The availability of the recently published DNA sequence of human chromosome 21 (HSA21) is a landmark contribution that will have an immediate impact on the study of the role of specific genes to Down syndrome (DS). Trisomy 21 or DS is the only autosomal aneuploidy that is not lethal in the fetal or early postnatal period. DS phenotypes show variable penetrance, affecting many different organs, including brain (mental retardation, early onset of Alzheimers disease, AD), muscle (hypotonia), skeleton, and blood. DS phenotypes may stem directly from the cumulative effect of overexpression of specific HSA21 gene products or indirectly through the interaction of these gene products with the whole genome, transcriptome, or proteome. Mouse genetic models have played an important role in the elucidation of the contribution of specific genes to the DS phenotype. To date, the strategies used for modeling DS in mice have been three: (1) to assess single-gene contributions to DS phenotype, using transgenic techniques to create models overexpressing single or combinations of genes, (2) to assess the effects of overexpressing large foreign DNA pieces, introduced on yeast artificial chromosomes (YACs) or bacterial artificial chromosomes (BACs) into transgenic mice, and (3) mouse trisomies that carry all or part of MMU16, which has regions of conserved homology with HSA21. Here we review the existing murine models and the relevance of their contribution to DS research.

Motor phenotypic alterations in TgDyrk1a transgenic mice implicate DYRK1A in Down syndrome motor dysfunction.

Motor deficits are among the most frequent impairments in Down syndrome (DS), but their neuropathological and molecular bases remain elusive. Here we investigate the motor profile of transgenic mice overexpressing Dyrk1a, Tg(Dyrk1a)1Cff (hereafter TgDyrk1a), a candidate gene hypothesized to cause some of the neurological defects associated with DS. We have previously shown DYRK1A expression in the cerebellum and functionally related structures, most brainstem motor nuclei and spinal cord, supporting a role for Dyrk1a in controlling motor function. Here we demonstrate that TgDyrk1a mice present DYRK1A overexpression in these areas along with specific motor dysfunction. The main finding that emerged was impairment of motor learning and alteration of the organization of locomotor behavior, which agrees with reported clinical observations in subjects with DS. These results confirm and extend previous data and provide further insight to the functional domains that might be altered in TgDyrk1a mice and underlying molecular mechanisms of DS motor dysfunction.

Irreversible electroporation shows efficacy against pancreatic carcinoma without systemic toxicity in mouse models

Pancreatic ductal adenocarcinoma (PDAC) therapies show limited success. Irreversible electroporation (IRE) is an innovative loco-regional therapy in which high-voltage pulses are applied to induce plasma membrane defects leading to cellular death. In the present study we evaluated the feasibility of IRE against PDAC. IRE treatment exhibited significant antitumor effects and prolonged survival in mice with orthotopic xenografts. Extensive tumor necrosis, reduced tumor cell proliferation and disruption of microvessels were observed at different days post-IRE. Animals had transient increases in transaminases, amylase and lipase enzymes that normalized at 24h post-IRE. These results suggest that IRE could be an effective treatment for locally advanced pancreatic tumors.

Targeting Dyrk1A with AAVshRNA Attenuates Motor Alterations in TgDyrk1A, a Mouse Model of Down Syndrome

Genetic-dissection studies carried out with Down syndrome (DS) murine models point to the critical contribution of Dyrk1A overexpression to the motor abnormalities and cognitive deficits displayed in DS individuals. In the present study we have used a murine model overexpressing Dyrk1A (TgDyrk1A mice) to evaluate whether functional CNS defects could be corrected with an inhibitory RNA against Dyrk1A, delivered by bilateral intrastriatal injections of adeno-associated virus type 2 (AAVshDyrk1A). We report that AAVshDyrk1A efficiently transduced HEK293 cells and primary neuronal cultures, triggering the specific inhibition of Dyrk1A expression. Injecting the vector into the striata of TgDyrk1A mice resulted in a restricted, long-term transduction of the striatum. This gene therapy was found to be devoid of toxicity and succeeded in normalizing Dyrk1A protein levels in TgDyrk1A mice. Importantly, the behavioral studies of the adult TgDyrk1A mice treated showed a reversal of corticostriatal-dependent phenotypes, as revealed by the attenuation of their hyperactive behavior, the restoration of motor-coordination defects, and an improvement in sensorimotor gating. Taken together, the data demonstrate that normalizing Dyrk1A gene expression in the striatum of adult TgDyrk1A mice, by means of AAVshRNA, clearly reverses motor impairment. Furthermore, these results identify Dyrk1A as a potential target for therapy in DS.

Enhanced pancreatic tumor regression by a combination of adenovirus and retrovirus-mediated delivery of the herpes simplex virus thymidine kinase gene

We have evaluated the effectiveness of combining the different characteristics of retrovirus and adenovirus to apply the herpes simplex virus thymidine kinase gene (HSVtk) and ganciclovir (GCV) treatment for gene therapy of pancreatic cancer. Transduction of NP-18 human pancreatic cells in culture by either the adenoviral vector (ADV/tk) or the retroviral vector (Rv/tk) followed by GCV treatment resulted in a GCV dose-dependent cytotoxic effect. A bystander effect was determined, both in NP-18 cultures and in xenogeneic cell mixtures of NP-18 and PA317 cells. Studies in vivo indicated that the effectiveness of tumor regression after HSVtk gene transfer and GCV treatment was dependent first on the tumor size at the time of viral injection and secondly, in large tumors, on the type of virus administered. The administration of the viral combination (ADV/tk + vector producer cells VPC-Rv/tk) was the best approach tested and resulted in a dramatic reduction in tumor mass after 4 days of GCV treatment which was maintained for the treatment period. Remarkably, two animals presented a complete eradication of the tumor. Thus, the HSVtk/GCV system when administered using a viral combination (ADV/tk + VPC-Rv/tk), may be a promising suicide gene therapy for pancreatic carcinomas.

Regulated Segregation of Kinase Dyrk1A during Asymmetric Neural Stem Cell Division Is Critical for EGFR-Mediated Biased Signaling

Stem cell division can result in two sibling cells exhibiting differential mitogenic and self-renewing potential. Here, we present evidence that the dual-specificity kinase Dyrk1A is part of a molecular pathway involved in the regulation of biased epidermal growth factor receptor (EGFR) signaling in the progeny of dividing neural stem cells (NSC) of the adult subependymal zone (SEZ). We show that EGFR asymmetry requires regulated sorting and that a normal Dyrk1a dosage is required to sustain EGFR in the two daughters of a symmetrically dividing progenitor. Dyrk1A is symmetrically or asymmetrically distributed during mitosis, and biochemical analyses indicate that it prevents endocytosis-mediated degradation of EGFR by a mechanism that requires phosphorylation of the EGFR signaling modulator Sprouty2. Finally, Dyrk1a heterozygous NSCs exhibit defects in self-renewal, EGF-dependent cell-fate decisions, and long-term persistence in vivo, suggesting that symmetrical divisions play a role in the maintenance of the SEZ reservoir.

Transgenic mice overexpressing the full-length neurotrophin receptor TrkC exhibit increased catecholaminergic neuron density in specific brain areas and increased anxiety-like behavior and panic reaction

Accumulating evidence has suggested that neurotrophins participate in the pathophysiology of mood disorders. We have developed transgenic mice overexpressing the full-length neurotrophin-3 receptor TrkC (TgNTRK3) in the central nervous system. TgNTRK3 mice show increased anxiety-like behavior and enhancement of panic reaction in the mouse defense test battery, along with an increase in the number and density of catecholaminergic (tyrosine hydroxylase positive) neurons in locus coeruleus and substantia nigra. Furthermore, treatment of TgNTRK3 mice with diazepam significantly attenuated the anxiety-like behaviors in the plus maze. These results provide evidence for the involvement of TrkC in the development of noradrenergic neurons in the central nervous system with consequences on anxiety-like behavior and panic reaction. Thus, changes in TrkC expression levels could contribute to the phenotypic expression of panic disorder through a trophic effect on noradrenergic neurons in the locus coeruleus. Our results demonstrate that the elevated NT3-TrkC tone via overexpression of TrkC in the brain may constitute a molecular mechanism for the expression of anxiety and anxiety.