Poly(ADP-ribose) polymerase gene disruption conferred mice resistant to streptozotocin-induced diabetes

Streptozotocin (STZ), a glucose analogue known to induce diabetes in experimental animals, causes DNA strand breaks and subsequent activation of poly(ADPribose) polymerase (Parp). Because Parp uses NAD as a substrate, extensive DNA damage will result in reduction of cellular NAD level. In fact, STZ induces NAD depletion and cell death in isolated pancreatic islets in vitro. Activation of Parp therefore is thought to play an important role in STZ-induced diabetes. In the present study, we established Parp-deficient (Parp−/−) mice by disrupting Parp exon 1 by using the homologous recombination technique. These mice were used to examine the possible involvement of Parp in STZ-induced β-cell damage in vivo. The wild-type (Parp+/+) mice showed significant increases in blood glucose concentration from 129 mg/dl to 218, 370, 477, and 452 mg/dl on experimental days 1, 7, 21, and 60, respectively, after a single injection of 180 mg STZ/kg body weight. In contrast, the concentration of blood glucose in Parp−/− mice remained normal up to day 7, slightly increased on day 21, but returned to normal levels on day 60. STZ injection caused extensive necrosis in the islets of Parp+/+ mice on day 1, with subsequent progressive islet atrophy and loss of functional β cells from day 7. In contrast, the extent of islet β-cell death and dysfunction was markedly less in Parp−/− mice. Our findings clearly implicate Parp activation in islet β-cell damage and glucose intolerance induced by STZ in vivo.

Increased anxiety of mice lacking the serotonin1A receptor

Brain serotonin (5-HT) has been implicated in a number of physiological processes and pathological conditions. These effects are mediated by at least 14 different 5-HT receptors. We have inactivated the gene encoding the 5-HT1A receptor in mice and found that receptor-deficient animals have an increased tendency to avoid a novel and fearful environment and to escape a stressful situation, behaviors consistent with an increased anxiety and stress response. Based on the role of the 5-HT1A receptor in the feedback regulation of the 5-HT system, we hypothesize that an increased serotonergic neurotransmission is responsible for the anxiety-like behavior of receptor-deficient animals. This view is consistent with earlier studies showing that pharmacological activation of the 5-HT system is anxiogenic in animal models and also in humans.

Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II

The classically recognized functions of the renin–angiotensin system are mediated by type 1 (AT1) angiotensin receptors. Whereas man possesses a single AT1 receptor, there are two AT1 receptor isoforms in rodents (AT1A and AT1B) that are products of separate genes (Agtr1a and Agtr1b). We have generated mice lacking AT1B (Agtr1b −/−) and both AT1A and AT1B receptors (Agtr1a −/−Agtr1b −/−). Agtr1b −/− mice are healthy, without an abnormal phenotype. In contrast, Agtr1a −/−Agtr1b −/− mice have diminished growth, vascular thickening within the kidney, and atrophy of the inner renal medulla. This phenotype is virtually identical to that seen in angiotensinogen-deficient (Agt−/−) and angiotensin-converting enzyme-deficient (Ace −/−) mice that are unable to synthesize angiotensin II. Agtr1a −/−Agtr1b −/− mice have no systemic pressor response to infusions of angiotensin II, but they respond normally to another vasoconstrictor, epinephrine. Blood pressure is reduced substantially in the Agtr1a −/− Agtr1b −/− mice and following administration of an angiotensin converting enzyme inhibitor, their blood pressure increases paradoxically. We suggest that this is a result of interruption of AT2-receptor signaling. In summary, our studies suggest that both AT1 receptors promote somatic growth and maintenance of normal kidney structure. The absence of either of the AT1 receptor isoforms alone can be compensated in varying degrees by the other isoform. These studies reaffirm and extend the importance of AT1 receptors to mediate physiological functions of the renin–angiotensin system.

Long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin

The ob/ob mouse is genetically deficient in leptin and exhibits a phenotype that includes obesity and non-insulin-dependent diabetes melitus. This phenotype closely resembles the morbid obesity seen in humans. In this study, we demonstrate that a single intramuscular injection of a recombinant adeno-associated virus (AAV) vector encoding mouse leptin (rAAV-leptin) in ob/ob mice leads to prevention of obesity and diabetes. The treated animals show normalization of metabolic abnormalities including hyperglycemia, insulin resistance, impaired glucose tolerance, and lethargy. The effects of a single injection have lasted through the 6-month course of the study. At all time points measured the circulating levels of leptin in the serum were similar to age-matched control C57 mice. These results demonstrate that maintenance of normal levels of leptin (2–5 ng/ml) in the circulation can prevent both the onset of obesity and associated non-insulin-dependent diabetes. Thus a single injection of a rAAV vector expressing a therapeutic gene can lead to complete and long-term correction of a genetic disorder. Our study demonstrates the long-term correction of a disease caused by a genetic defect and proves the feasibility of using rAAV-based vectors for the treatment of chronic disorders like obesity.

The role of the D2 dopamine receptor (D2R) in A2A adenosine receptor (A2AR)-mediated behavioral and cellular responses as revealed by A2A and D2 receptor knockout mice

The A2AR is largely coexpressed with D2Rs and enkephalin mRNA in the striatum where it modulates dopaminergic activity. Activation of the A2AR antagonizes D2R-mediated behavioral and neurochemical effects in the basal ganglia through a mechanism that may involve direct A2AR–D2R interaction. However, whether the D2R is required for the A2AR to exert its neural function is an open question. In this study, we examined the role of D2Rs in A2AR-induced behavioral and cellular responses, by using genetic knockout (KO) models (mice deficient in A2ARs or D2Rs or both). Behavioral analysis shows that the A2AR agonist 2–4-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine reduced spontaneous as well as amphetamine-induced locomotion in both D2 KO and wild-type mice. Conversely, the nonselective adenosine antagonist caffeine and the A2AR antagonist 8-(3-chlorostyryl)caffeine produced motor stimulation in mice lacking the D2R, although the stimulation was significantly attentuated. At the cellular level, A2AR inactivation counteracted the increase in enkephalin expression in striatopallidal neurons caused by D2R deficiency. Consistent with the D2 KO phenotype, A2AR inactivation partially reversed both acute D2R antagonist (haloperidol)-induced catalepsy and chronic haloperidol-induced enkephalin mRNA expression. Together, these results demonstrate that A2ARs elicit behavioral and cellular responses despite either the genetic deficiency or pharmacological blockade of D2Rs. Thus, A2AR-mediated neural functions are partially independent of D2Rs. Moreover, endogenous adenosine acting at striatal A2ARs may be most accurately viewed as a facilitative modulator of striatal neuronal activity rather than simply as an inhibitory modulator of D2R neurotransmission.

Defective localization of the NADPH phagocyte oxidase to Salmonella-containing phagosomes in tumor necrosis factor p55 receptor-deficient macrophages

Tumor necrosis factor receptor (TNFR) p55-knockout (KO) mice are susceptible profoundly to Salmonella infection. One day after peritoneal inoculation, TNFR-KO mice harbor 1,000-fold more bacteria in liver and spleen than wild-type mice despite the formation of well organized granulomas. Macrophages from TNFR-KO mice produce abundant quantities of reactive oxygen and nitrogen species in response to Salmonella but nevertheless exhibit poor bactericidal activity. Treatment with IFN-γ enhances killing by wild-type macrophages but does not restore the killing defect of TNFR-KO cells. Bactericidal activity of macrophages can be abrogated by a deletion in the gene encoding TNFα but not by saturating concentrations of TNF-soluble receptor, suggesting that intracellular TNFα can regulate killing of Salmonella by macrophages. Peritoneal macrophages from TNFR-KO mice fail to localize NADPH oxidase-containing vesicles to Salmonella-containing vacuoles. A TNFR-KO mutation substantially restores virulence to an attenuated mutant bacterial strain lacking the type III secretory system encoded by Salmonella pathogenicity island 2 (SPI2), suggesting that TNFα and SPI2 have opposing actions on a common pathway of vesicular trafficking. TNFα–TNFRp55 signaling plays a critical role in the immediate innate immune response to an intracellular pathogen by optimizing the delivery of toxic reactive oxygen species to the phagosome.

Adeno-associated viral vector-mediated gene transfer results in long-term enzymatic and functional correction in multiple organs of Fabry mice

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A). This enzyme deficiency leads to impaired catabolism of α-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop painful neuropathy and vascular occlusions that progressively lead to cardiovascular, cerebrovascular, and renal dysfunction and early death. Although enzyme replacement therapy and bone marrow transplantation have shown promise in the murine analog of Fabry disease, gene therapy holds a strong potential for treating this disease in humans. Delivery of the normal α-gal A gene (cDNA) into a depot organ such as liver may be sufficient to elicit corrective circulating levels of the deficient enzyme. To investigate this possibility, a recombinant adeno-associated viral vector encoding human α-gal A (rAAV-AGA) was constructed and injected into the hepatic portal vein of Fabry mice. Two weeks postinjection, α-gal A activity in the livers of rAAV-AGA-injected Fabry mice was 20–35% of that of the normal mice. The transduced animals continued to show higher α-gal A levels in liver and other tissues compared with the untouched Fabry controls as long as 6 months after treatment. In parallel to the elevated enzyme levels, we see significant reductions in Gb3 levels to near normal at 2 and 5 weeks posttreatment. The lower Gb3 levels continued in liver, spleen, and heart, up to 25 weeks with no significant immune response to the virus or α-gal A. Also, no signs of liver toxicity occurred after the rAAV-AGA administration. These findings suggest that an AAV-mediated gene transfer may be useful for the treatment of Fabry disease and possibly other metabolic disorders.

Male fertility defects in mice lacking the serine protease inhibitor protease nexin-1

Understanding infertility and sterility requires knowledge of the molecular mechanisms underlying sexual reproduction. We have found that male mice deficient for the gene encoding the protease inhibitor protease nexin-1 (PN-1) show a marked impairment in fertility from the onset of sexual maturity. Absence of PN-1 results in altered semen protein composition, which leads to inadequate semen coagulation and deficient vaginal plug formation upon copulation. Progressive morphological changes of the seminal vesicles also are observed. Consistent with these findings, abnormal PN-1 expression was found in the semen of men displaying seminal dysfunction. The data demonstrate that the level of extracellular proteolytic activity is a critical element in controlling male fertility.

Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation

Liver-specific and nonliver-specific methionine adenosyltransferases (MATs) are products of two genes, MAT1A and MAT2A, respectively, that catalyze the formation of S-adenosylmethionine (AdoMet), the principal biological methyl donor. Mature liver expresses MAT1A, whereas MAT2A is expressed in extrahepatic tissues and is induced during liver growth and dedifferentiation. To examine the influence of MAT1A on hepatic growth, we studied the effects of a targeted disruption of the murine MAT1A gene. MAT1A mRNA and protein levels were absent in homozygous knockout mice. At 3 months, plasma methionine level increased 776% in knockouts. Hepatic AdoMet and glutathione levels were reduced by 74 and 40%, respectively, whereas S-adenosylhomocysteine, methylthioadenosine, and global DNA methylation were unchanged. The body weight of 3-month-old knockout mice was unchanged from wild-type littermates, but the liver weight was increased 40%. The Affymetrix genechip system and Northern and Western blot analyses were used to analyze differential expression of genes. The expression of many acute phase-response and inflammatory markers, including orosomucoid, amyloid, metallothionein, Fas antigen, and growth-related genes, including early growth response 1 and proliferating cell nuclear antigen, is increased in the knockout animal. At 3 months, knockout mice are more susceptible to choline-deficient diet-induced fatty liver. At 8 months, knockout mice developed spontaneous macrovesicular steatosis and predominantly periportal mononuclear cell infiltration. Thus, absence of MAT1A resulted in a liver that is more susceptible to injury, expresses markers of an acute phase response, and displays increased proliferation.

Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy.

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin. Significant nNOS immunoreactivity and enzyme activity was absent in skeletal muscle tissues from patients with Duchenne muscular dystrophy. Similarly, in dystrophin-deficient skeletal muscles from mdx mice both soluble and particulate nNOS was greatly reduced compared with C57 control mice. nNOS mRNA was also reduced in mdx muscle in contrast to mRNA levels for a dystrophin binding protein, alpha 1-syntrophin. nNOS levels increased dramatically from 2 to 52 weeks of age in C57 skeletal muscle, which may indicate a physiological role for NO in aging-related processes. Biochemical purification readily dissociates nNOS from the dystrophin-glycoprotein complex. Thus, nNOS is not an integral component of the dystrophin-glycoprotein complex and is not simply another dystrophin-associated protein since the expression of both nNOS mRNA and protein is affected by dystrophin expression.

Deficits in memory and hippocampal long-term potentiation in mice with reduced calbindin D28K expression.

The influx of calcium into the postsynaptic neuron is likely to be an important event in memory formation. Among the mechanisms that nerve cells may use to alter the time course or size of a spike of intracellular calcium are cytosolic calcium binding or "buffering" proteins. To consider the role in memory formation of one of these proteins, calbindin D28K, which is abundant in many neurons, including the CA1 pyramidal cells of the hippocampus, transgenic mice deficient in calbindin D28K have been created. These mice show selective impairments in spatial learning paradigms and fail to maintain long-term potentiation. These results suggest a role for calbindin D28K protein in temporally extending a neuronal calcium signal, allowing the activation of calcium-dependent intracellular signaling pathways underlying memory function.

Granzyme A is critical for recovery of mice from infection with the natural cytopathic viral pathogen, ectromelia.

Cytolytic lymphocytes are of cardinal importance in the recovery from primary viral infections. Both natural killer cells and cytolytic T cells mediate at least part of their effector function by target cell lysis and DNA fragmentation. Two proteins, perforin and granzyme B, contained within the cytoplasmic granules of these cytolytic effector cells have been shown to be directly involved in these processes. A third protein contained within these granules, granzyme A, has so far not been attributed with any biological relevance. Using mice deficient for granzyme A, we show here that granzyme A plays a crucial role in recovery from the natural mouse pathogen, ectromelia, by mechanisms other than cytolytic activity.

Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice.

Macrophages secrete a variety of proteinases that are thought to participate in remodeling of the extracellular matrix associated with inflammatory processes. We have eliminated expression of the macrophage metalloelastase (MME) gene by targeted disruption to assess the role of this protein in macrophage-mediated proteolysis. We found that the macrophages of MME-deficient (MME-/-) mice have a markedly diminished capacity to degrade extracellular matrix components. In addition, MME-/- macrophages are essentially unable to penetrate reconstituted basement membranes in vitro and in vivo. MME is therefore required for macrophage-mediated extracellular matrix proteolysis and tissue invasion.

Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor.

The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF.

Cytochrome P450 1A1 promoter as a genetic switch for the regulatable and physiological expression of a plasma protein in transgenic mice.

Transgenic and gene knockout techniques allow for in vivo study of the consequences of adding or subtracting specific genes. However, in some instances, such as the study of lethal mutations or of the physiological consequences of changing gene expression, turning on and off an introduced gene at will would be advantageous. We have used cytochrome p450 1A1 promoter to drive expression of the human apolipoprotein E (apoE) gene in transgenic mice. In six independent lines, robust expression of the transgene depended upon injection of the inducer beta-naphthoflavone, whereas the seventh line had high basal expression that was augmented further by the inducer. The low level of basal expression in an inducer-dependent line was confirmed upon breeding the transgene onto the hypercholesterolemic apoE-deficient background. In the basal state transgene expression was physiologically insignificant, as these mice were as hypercholesterolemic as their nontransgenic apoE-deficient littermates. When injected with the inducer, plasma cholesterol levels of the transgenic mice decreased dramatically as apoE expression was induced to yield greater than physiological levels in plasma. The inducer could pass transplacentally from an injected mother to her fetuses with concomitant induction of fetal transgene mRNA. Inducer could also pass via breast milk from an injected mother to her suckling neonatal pups, giving rise to the induction of human apoE in neonate plasma. These finding suggest a strategy to temporarily ameliorate genetic deficiencies that would otherwise lead to fetal or neonatal lethality.