John N. Snouwaert

An Animal Model for Cystic Fibrosis Made by Gene Targeting

Cystic fibrosis results from defects in the gene encoding a cyclic adenosine monophosphate-dependent chloride ion channel known as the cystic fibrosis transmembrane conductance regulator (CFTR). To create an animal model for cystic fibrosis, mice were generated from embryonic stem cells in which the CFTR gene was disrupted by gene targeting. Mice homozygous for the disrupted gene display many features common to young human cystic fibrosis patients, including failure to thrive, meconium ileus, alteration of mucous and serous glands, and obstruction of glandlike structures with inspissated eosinophilic material. Death resulting from intestinal obstruction usually occurs before 40 days of age.

The prostaglandin receptor EP4 triggers remodelling of the cardiovascular system at birth

Survival of newborn placental mammals depends on closure of the ductus arteriosus (DA), an arterial connection in the fetus which directs blood away from the pulmonary circulation and towards the placenta where oxygenation occurs. Here we show that morphological changes resulting in closure of the DA in mice are virtually identical to those observed in larger mammals, including humans, and that maintenance of the DA in the open, or patent, state in fetal mice is dependent on prostaglandin synthesis. This requirement is absent in mice lacking the prostaglandin E2 EP4 receptor (EP4(−/−) mice). In EP 4(−/−) mice of the 129 strain, remodelling of the DA fails to occur after birth, resulting in a left-to-right shunt of blood and subsequently in death. This suggests that the neonatal drop in prostaglandin E2 (refs 3,4,5,6,7) that triggers ductal closure is sensed through the EP4 receptor. In contrast, 5% of EP4(−/−) mice of mixed genetic background survive, and selective breeding of these mice leads to a 21% survival rate, suggesting that alleles at other loci can provide an alternative mechanism for ductal closure.

BRCA1 deficient embryonic stem cells display a decreased homologous recombination frequency and an increased frequency of non-homologous recombination that is corrected by expression of a Brca1 transgene

BRCA1 is a nuclear phosphoprotein that has been classified as a tumor suppressor based on the fact that women carrying a mutated copy of the BRCA1 gene are at increased risk of developing breast and ovarian cancer. The association of BRCA1 with RAD51 has led to the hypothesis that BRCA1 is involved in DNA repair. We describe here the generation and analysis of murine embryonic stem (ES) cell lines in which both copies of the murine homologue of the human BRCA1 gene have been disrupted by gene targeting. We show that exogenous DNA introduced into these BRCA1 deficient cells by electroporation is randomly integrated into the genome at a significantly higher rate than in wild type ES cells. In contrast, integration of exogenous DNA by homologous recombination occurs in BRCA1 deficient cells at a significantly lower rate than in wild type controls. When BRCA1 expression is re-established at 5 – 10% of normal levels by introduction of a Brca1 transgene into BRCA1 deficient ES cells, the frequency of random integration is reduced to wild type levels, although the frequency of homologous recombination is not significantly improved. These results suggest that BRCA1 plays a role in determining the response of cells to double stranded DNA breaks.

Deficits in sensorimotor gating and tests of social behavior in a genetic model of reduced NMDA receptor function

Reduced NMDA receptor function is hypothesized to contribute to the pathophysiology of schizophrenia. In order to model chronic and developmental NMDA receptor hypofunction, a mouse line was developed that expresses low levels of the NMDA R1 subunit (NR1) of the NMDA receptor. The present study tested the hypothesis that these NR1 hypomorphic mice would exhibit deficits in sensorimotor and conspecific interactions, analogous to deficits observed in schizophrenic patients. F1 hybrid mice homozygous for the NR1 hypomorphic mutation (NR1 -/-) were generated by crossing heterozygous mice (NR1 +/-) from C57BL/6 and 129 Sv/Ev backgrounds. To assess sensorimotor gating, mice were tested in the paradigm of prepulse inhibition of acoustic startle. The NR1 hypomorphic mice exhibited increased acoustic startle responses and also showed deficits in prepulse inhibition. Startle responses were differentially altered by predator odor exposure in the male NR1 -/- mice, in comparison to control mice. In a test of social affiliation, the wild type mice spent significantly more time investigating a novel mouse in comparison to the NR1 -/- mice. In a resident-intruder test, marked deficits were found in sex-specific aggressive behavior between the wild type and mutant mice. These data support the contention that the NR1 hypomorphic mice exhibit alterations in sensorimotor gating and typical conspecific interactions, reminiscent of behavioral disturbances associated with schizophrenia. The NR1 hypomorphic mice could represent a model system to explore novel treatment and preventative strategies for certain symptoms of schizophrenia.

AT1A Angiotensin Receptors in the Renal Proximal Tubule Regulate Blood Pressure

Hypertension affects more than 1.5 billion people worldwide but the precise cause of elevated blood pressure (BP) cannot be determined in most affected individuals. Nonetheless, blockade of the renin-angiotensin system (RAS) lowers BP in the majority of patients with hypertension. Despite its apparent role in hypertension pathogenesis, the key cellular targets of the RAS that control BP have not been clearly identified. Here we demonstrate that RAS actions in the epithelium of the proximal tubule have a critical and nonredundant role in determining the level of BP. Abrogation of AT(1) angiotensin receptor signaling in the proximal tubule alone is sufficient to lower BP, despite intact vascular responses. Elimination of this pathway reduces proximal fluid reabsorption and alters expression of key sodium transporters, modifying pressure-natriuresis and providing substantial protection against hypertension. Thus, effectively targeting epithelial functions of the proximal tubule of the kidney should be a useful therapeutic strategy in hypertension.

NLRP1 dependent pyroptosis leads to acute lung injury and morbidity in mice

Acute inflammation in response to both exogenous and endogenous danger signals can lead to the assembly of cytoplasmic inflammasomes that stimulate the activation of caspase-1. Subsequently, caspase-1 facilitates the maturation and release of cytokines and also, under some circumstances, the induction of cell death by pyroptosis. Using a mouse line lacking expression of NLRP1, we show that assembly of this inflammasome in cells is triggered by a toxin from anthrax and that it initiates caspase-1 activation and release of IL-1β. Furthermore, NLRP1 inflammasome activation also leads to cell death, which escalates over 3 d following exposure to the toxin and culminates in acute lung injury and death of the mice. We show that these events are not dependent on production of IL-1β by the inflammasome but are dependent on caspase-1 expression. In contrast, muramyl dipeptide-mediated inflammasome formation is not dependent on NLRP1 but NLRP3. Taken together, our findings show that assembly of the NLRP1 inflammasome is sufficient to initiate pyroptosis, which subsequently leads to a self-amplifying cascade of cell injury within the lung from which the lung cannot recover, eventually resulting in catastrophic consequences for the organism.

Microsomal Prostaglandin E Synthase-2 Is Not Essential For In Vivo Prostaglandin E2 Biosynthesis

Prostaglandin E(2) (PGE(2)) plays an important role in the normal physiology of many organ systems. Increased levels of this lipid mediator are associated with many disease states, and it potently regulates inflammatory responses. Three enzymes capable of in vitro synthesis of PGE(2) from the cyclooxygenase metabolite PGH(2) have been described. Here, we examine the contribution of one of these enzymes to PGE(2) production, mPges-2, which encodes microsomal prostaglandin synthase-2 (mPGES-2), by generating mice homozygous for the null allele of this gene. Loss of mPges-2 expression did not result in a measurable decrease in PGE(2) levels in any tissue or cell type examined from healthy mice. Taken together, analysis of the mPGES-2 deficient mouse lines does not substantiate the contention that mPGES-2 is a PGE(2) synthase.

Alterations in regional brain metabolism in genetic and pharmacological models of reduced NMDA receptor function

A mouse line has been developed that expresses low levels of the NMDA R1 (NR1) subunit of the NMDA receptor [Cell 98 (1999) 427]. These NR1 hypomorphic mice represent an experimental model of reduced NMDA receptor function that may be relevant to the pathophysiology of schizophrenia. To further characterize the neurobiological phenotype resulting from developmental NMDA receptor hypofunction, regional brain metabolic activity was assessed by autoradiographic analysis of 14C-2-deoxyglucose (2-DG) uptake. In addition, ligand binding to NMDA, AMPA, and kainate receptors was measured by quantitative autoradiography. MK-801 binding to NMDA receptors was reduced markedly throughout the brain of the NR1 hypomorphic mice. However, no alteration in 3H-AMPA or 3H-kainate binding was apparent in any region examined. Neuroanatomically specific alterations in regional 2-DG uptake were observed in the NR1 hypomorphic animals. Reduced relative 2-DG uptake was observed in the medial prefrontal and anterior cingulate cortices. Altered patterns of 2-DG uptake were also found in neocortical regions, with selective reductions of uptake in layer 6 in frontal regions of somatosensory and motor cortices. These data indicate alterations in cortical circuitry in the NR1 hypomorphic animals and are consistent with functional imaging studies in chronic schizophrenia patients which typically show reduced frontal cortical metabolic activity. Reduced relative 2-DG uptake was also found in the caudate, accumbens, hippocampus, and select thalamic regions in the NR1-deficient mice. However, in many other brain regions no alteration in 2-DG uptake was observed. The alterations in 2-DG uptake in the NR1 hypomorphic mice were distinctly different compared to those observed after acute challenge with the selective NMDA antagonist MK-801 in wild-type mice. The altered patterns of brain 2-DG uptake in the NR1 hypomorphic mice found in the present work, together with the altered behavioral phenotypes previously described, suggest that the mice may provide a valuable model to study novel therapeutic strategies to counteract the neurobiological consequences of chronic developmental NMDA receptor hypofunction.

Amphetamine-induced Fos is reduced in limbic cortical regions but not in the caudate or accumbens in a genetic model of NMDA receptor hypofunction.

A mouse strain has been developed that expresses low levels of the NR1 subunit of the NMDA receptor. These mice are a model of chronic developmental NMDA receptor hypofunction and may therefore have relevance to the hypothesized NMDA receptor hypofunction in schizophrenia. Many schizophrenia patients show exaggerated behavioral and neuronal responses to amphetamine compared to healthy subjects. Studies were designed to determine if the NR1-deficient mice would exhibit enhanced sensitivity to amphetamine. Effects of amphetamine on behavioral activation and Fos induction were compared between the NR1-deficient mice and wild-type controls. The NR1 hypomorphic mice and controls exhibited similar locomotor activation after administration of amphetamine at 2 mg/kg. The mutant mice showed slightly reduced peak locomotor activity and slightly increased stereotypy after 4 mg/kg amphetamine. There were no differences in Fos induction in response to amphetamine in the caudate putamen, nucleus accumbens, medial or central amygdala nuclei, or bed nucleus of the stria terminalis. However, amphetamine-induced Fos was substantially attenuated in the medial frontal (infralimbic) and cingulate cortices, basolateral amygdala, and in the lateral septum of the mutant mice. The results suggest a neuroanatomically selective activation deficit to amphetamine challenge in the NR1-deficient mice.

Thromboxane Receptors in Smooth Muscle Promote Hypertension, Vascular Remodeling, and Sudden Death

The prostanoid thromboxane A2 has been implicated to contribute to the pathogenesis of many cardiovascular diseases, including hypertension. To study the role of vascular thromboxane-prostanoid (TP) receptors in blood pressure regulation, we generated mice with cell-specific deletion of TP receptors in smooth muscle using Cre/Loxp technology. We crossed the KISM22&agr;-Cre transgenic mouse line expressing Cre recombinase in smooth muscle cells with a mouse line bearing a conditional allele of the Tbxa2r gene (Tp flox ). In KISM22&agr;-Cre + Tp flox/flox (TP-SMKO) mice, TP receptors were efficiently deleted from vascular smooth muscle cells. In TP-SMKOs, acute vasoconstrictor responses to the TP agonist U46619 were attenuated to a similar extent in both the peripheral and renal circulations. Yet, acute vascular responses to angiotensin II were unaffected at baseline and after chronic angiotensin II administration. Infusion of high-dose U46619 caused circulatory collapse and death in a majority of control mice but had negligible hemodynamic effects in TP-SMKOs, which were completely protected from U46619-induced sudden death. Baseline blood pressures were normal in TP-SMKOs. However, the absence of TP receptors in vascular smooth muscle cells was associated with significant attenuation of angiotensin II–induced hypertension and diminished vascular remodeling. This was also associated with reduced urinary thromboxane production after chronic angiotensin II. Thus, TP receptors in vascular smooth muscle cells play a major role in mediating the actions of thromboxane A2 in TP agonist-induced shock, hypertension, and vascular remodeling of the aorta.