Anne Marie Gillespie

Primary afferent tachykinins are required to experience moderate to intense pain

The excitatory neurotransmitter glutamate coexists with the peptide known as substance P in primary afferents that respond to painful stimulation. Because blockers of glutamate receptors reliably reduce pain behaviour, it is assumed that ‘pain’ messages are mediated by glutamate action on dorsal horn neurons. The contribution of substance P, however, is still unclear. We have now disrupted the mouse preprotachykinin A gene (PPT-A), which encodes substance P and a related tachykinin, neurokinin A (ref. 5). We find that although the behavioural response to mildly painful stimuli is intact in these mice, the response to moderate to intense pain is significantly reduced. Neurogenic inflammation, which results from peripheral release of substance P and neurokinin A (ref. 6), is almost absent in the mutant mice. We conclude that the release of tachykinins from primary afferent pain-sensing receptors (nociceptors) is required to produce moderate to intense pain.

The Use of Transgenic and Mutant Mice to Study Oxygen Free Radical Metabolism

ABSTRACT: To distinguish the role of Mn superoxide dismutase (MnSOD) from that of cytoplasmic CuZn superoxide dismutase (CuZnSOD), the mouse MnSOD gene (Sod2) was inactivated by homologous recombination. Sod2−/− mice on a CD1 (outbred) genetic background die within the first 10 days of life (mean, 5.4 days) with a complex phenotype that includes dilated cardiomyopathy, accumulation of lipid in liver and skeletal muscle, metabolic acidosis and ketosis, and a severe reduction in succinate dehydrogenase (complex II) and aconitase (a TCA cycle enzyme) activities in the heart and, to a lesser extent, in other organs. These findings indicate that MnSOD is required to maintain the integrity of mitochondrial enzymes susceptible to direct inactivation by superoxide. On the other hand, Lebovitz et al. reported an independently derived MnSod null mouse (Sod2tmlLeb) on a mixed C57BL/6 and 129Sv background with a different phenotype. Because a difference in genetic background is the most likely explanation for the phenotypic differences, the two mutant lines were crossed into different genetic backgrounds for further analyses. To study the phenotype of Sod2tmlLeb mice CD1 background, the Sod2tmlLeb mice were crossed to CD1 for two generations before the −/+ mice were intercrossed to generate −/− mice. The life span distribution of CD1〈Sod2−/−〉Leb was shifted to the left, indicating a shortened life span on the CD1 background. Furthermore, the CD1〈Sod2−/−〉Leb mice develop metabolic acidosis at an early stage as was observed with CD1〈Sod2−/−〉Cje. When Sod2tmlCje was placed on C57BL/6J (B6) background, the −/− mice were found to die either during midgestation or within the first 4 days after birth. However, when the B6〈Sod2−/+〉Cje were crossed with DBA/2J (D2) for the generation of B6D2F2〈Sod2−/−〉Cje mice, an entirely different phenotype, similar to that described by Lebovitz et al., was observed. The F2 Sod−/− mice were able to survive up to 18 days, and the animals that lived for more than 15 days displayed neurological abnormalities including ataxia and seizures. Their hearts were not as severely affected as were those of the CD1 mice, and neurological degeneration rather than heart defect appears to be the cause of death.

Identification and characterization of a new Down syndrome model, Ts[Rb(12.1716)]2Cje, resulting from a spontaneous Robertsonian fusion between T(1716)65Dn and mouseChromosome 12

The segmental trisomy model, Ts65Dn, has been a valuable resource for the study of the molecular and developmental processes associated with the pathogenesis of Down syndrome. However, male infertility and poor transmission of the small marker chromosome, T(1716)65Dn, carrying the distal end of mouse Chromosome 16 (MMU16) are limiting factors in the efficient production of these animals for experimental purposes. We describe here the identification and preliminary characterization of mice, designated Ts[Rb(12.1716)]2Cje, carrying a chromosomal rearrangement of the Ts65Dn genome whereby the marker chromosome has been translocated to Chromosome 12 (MMU12) forming a Robertsonian chromosome. This stable rearrangement confers fertility in males and increases the frequency of transmitted segmental trisomy through the female germline. We confirm retention of a dosage imbalance of human Chromosome 21 (HSA21)-homologous genes from App to the telomere and expression levels similar to Ts65Dn within the triplicated region. In addition, we characterized the dendritic morphology of granule cells in the fascia dentata in Ts[Rb(12.1716)]2Cje and 2N control mice. Quantitative confocal microscopy revealed decreased spine density on the dendrites of dentate granule cells and significantly enlarged dendritic spines affecting the entire population in Ts[Rb(12.1716)]2Cje as compared to 2N controls. These findings document that the structural dendritic spine abnormalities are similar to those previously observed in Ts65Dn mice. We conclude that this new model of Down syndrome offers reproductive advantages without sacrificing the integrity of the Ts65Dn model.

Genetic modification of the dilated cardiomyopathy and neonatal lethality phenotype of mice lacking manganese superoxide dismutase

Oxygen radical-mediated tissue damage has been implicated in a large number of pathological conditions including ischemia reperfusion injury (Chart 1994, Euler 1995, Kinouchi et al. 1991, Samaja et al. 1994), neurodegenerative diseases (Fahn and Cohen 1992, Jenner 1994, Lafon-Kazal et al. 1993), neonatal hyperoxic lung injury (Davis et al. 1993), atherosclerosis (Halliwell 1993), and the aging process (Ku et al. 1993, Orr and Sohal 1994). Therefore, many studies have been focused on enzymes and molecules that can scavenge oxygen radicals for their potential in the prevention and/ or therapy of these disorders. Under normal circumstances, the majority of oxygen radicals are generated in the mitochondria as a byproduct of electron transport and oxidative phosphorylation required for the production of ATP. Cells are protected against this metabolically-induced oxidative stress by several oxygen radical scavengers, including the superoxide dismutases, catalase, glutathione peroxidase, and reduced glutathione. Among these, one form of superoxide dismutase, manganese superoxide dismutase (MnSOD), has been the subject of particular interest because it is located within mitochondria, can be induced by several cytokines and superoxide itself, and appears to be involved in other processes, including tumor suppression and cellular differentiation (Akashi et al. 1995, Church et al. 1993, Harris et al. 1991, Sato et al. 1995, St. Clair et al. 1994). To investigate the role of MnSOD and to distinguish its actions from those of cytoplasmic CuZnSOD, the mouse MnSOD gene (Sod2) was inactivated by homologous recombination (Li et al. 1995) to delete exon 3. Phenotypic analysis of the mutant animals placed on the CD1 tml ~f (an outbred strain) genetic background (CD1Sod2<tm 1Cje>, formerly designated asSod2)indicated that whereas there is no detectable atypical phenotype in the heterozygous mutants (Sod2-~+) up to 9 months of age, homozygous mutant mice (Sod2-~-) die within the first 10 days or life. The major phenotype abnormalities observed with Sod2-~-mice include: 1 ) dilated cardiomyopathy with a thin left ventricular wall and enlarged left ventricular cavity; 2) accumulation of lipid in liver and skeletal muscle; 3) metabolic acidosis with increased ketones and decreased bicarbonate in the plasma; 4) excretion of 3-hydroxy-3-methylglutaric and 3-hydroxy3-methylglutaconic acids, 5) respiratory alkalosis, as an attempt to compensate for the metabolic acidosis; and 6) a severe reduction in succinate dehydrogenase (complex II) and aconitase (a TCA cycle enzyme) activities in the heart and, to a lesser extent, in other organs. These findings indicate that MnSOD is required for the normal biological function of tissues by maintaining the integrity of mitochondrial enzymes susceptible to direct inactivation by superoxide. MnSOD mutant mice [designated Sod2 (r~lBcM] have also been generated by Lebovitz et al. (1996) by gene targeting to produce a deletion of exons 1 and 2. Homozygous S o d 2 ~IBcM mutant mice on a mixed genetic background survived for as long as 18 days. These mice are anemic and exhibit progressive motor abnormalities characterized by weakness, rapid fatigue and ataxia. In addition, lack of myelination was observed in the spinal cord, and dystrophic neurons were seen scattered throughout the CNS. Possible explanations for the discrepancy of the phenotypes observed in the two different strains of MnSOD deficient mice include differences between the targeting vectors used to generate the mutations, between the embryonic stem cells employed, and/or between the genetic backgrounds on which the mutant mice were bred. A major influence of genetic background on the mutant phenotype of several strains of knockout mice has been reported (Threadgill et al. 1995; Rozmahel et al. 1996), and these phenotypic differences have become quite important for revealing the existence of interesting genetic modifiers of the ef fects of the primary mutations. Therefore, Sod2<tmlCje> -/mice were generated on two backgrounds in addition to the CD1 background originally used: 8 to 10 generations of backcrosses to C57BL/6J (designated B6) which made them 99.6 to 99.9% congenic on B6, and B6D2 F2 [the C57BL/6J heterozygotes were crossed to DBA/2 (designated D2) to generate B6D2 F1 animals, and then these Fls were intercrossed to generate B6D2 F2 animals for phenotypic analysis. In contrast to the 10 and 18 day survival times of CD1 Sod2<tmlCje> and SOD2 ~ScM, respectively, the liveborn -/mice on C57BL/6J background (B6<Sod2-/->) survived for only up to 4 days, with an average life span of 1.5 days. In addition, about half of the B6<Sod2-/-> mice died about day 15 of gestation, and the -/fetuses appeared very pale. On the other hand, the liveborn B6D2 F2<Sod2-/-> mice survived for up to 18 days with an average life span of 11 days. The long-lived B6D2 F2Sod2-/-> mice (> 15 days) displayed neurological abnormalities which included ataxia and seizures. How-

Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins

Secretory diarrheas caused by bacterial enterotoxins, including cholera and travelers diarrhea, remain a major global health problem. Inappropriate activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel occurs in these diarrheas. We previously reported that the benzopyrimido‐pyrrolooxazinedione (R)‐BPO‐27 inhibits CFTR chloride conductance with low‐nanomolar potency. Here, we demonstrate using experimental mouse models and human enterocyte cultures the potential utility of (R)‐BPO‐27 for treatment of secretory diarrheas caused by cholera and Escherichia coli enterotoxins. (R)‐BPO‐27 fully blocked CFTR chloride conductance in epithelial cell cultures and intestine after cAMP agonists, cholera toxin, or heat‐stable enterotoxin of E. coli (STa toxin), with IC50 down to ~5 nM. (R)‐BPO‐27 prevented cholera toxin and STa toxin‐induced fluid accumulation in small intestinal loops, with IC50 down to 0.1 μg/kg. (R)‐BPO‐27 did not impair intestinal fluid absorption or inhibit other major intestinal transporters. Pharmacokinetics in mice showed >90% oral bioavailability with sustained therapeutic serum levels for >4 h without the significant toxicity seen with 7‐d administration at 5 μg/kg/d. As evidence to support efficacy in human diarrheas, (R)‐BPO‐27 blocked fluid secretion in primary cultures of enteroids from human small intestine and anion current in enteroid monolayers. These studies support the potential utility of (R)‐BPO‐27 for therapy of CFTR‐mediated secretory diarrheas.—Cil, O., Phuan, P.‐W., Gillespie, A.M., Lee, S., Tradtrantip, L., Yin, J., Tse, M., Zachos, N.C., Lin, R., Donowitz, M., Verkman, A.S. Benzopyrimido‐pyrrolo‐oxazine‐dione CFTR inhibitor (R)‐BPO‐27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins. FASEB J. 31, 751–760 (2017). http://www.fasebj.org