A novel, ataxic mouse model of ataxia telangiectasia caused by a clinically relevant nonsense mutation.

Ataxia Telangiectasia (A-T) and Ataxia with Ocular Apraxia Type 1 (AOA1) are devastating neurological disorders caused by null mutations in the genome stability genes, A-T mutated (ATM) and Aprataxin (APTX), respectively. Our mechanistic understanding and therapeutic repertoire for treating these disorders are severely lacking, in large part due to the failure of prior animal models with similar null mutations to recapitulate the characteristic loss of motor coordination (i.e., ataxia) and associated cerebellar defects. By increasing genotoxic stress through the insertion of null mutations in both the Atm (nonsense) and Aptx (knockout) genes in the same animal, we have generated a novel mouse model that for the first time develops a progressively severe ataxic phenotype associated with atrophy of the cerebellar molecular layer. We find biophysical properties of cerebellar Purkinje neurons (PNs) are significantly perturbed (e.g., reduced membrane capacitance, lower action potential [AP] thresholds, etc.), while properties of synaptic inputs remain largely unchanged. These perturbations significantly alter PN neural activity, including a progressive reduction in spontaneous AP firing frequency that correlates with both cerebellar atrophy and ataxia over the animal's first year of life. Double mutant mice also exhibit a high predisposition to developing cancer (thymomas) and immune abnormalities (impaired early thymocyte development and T-cell maturation), symptoms characteristic of A-T. Finally, by inserting a clinically relevant nonsense-type null mutation in Atm, we demonstrate that Small Molecule Read-Through (SMRT) compounds can restore ATM production, indicating their potential as a future A-T therapeutic.

Mechanisms by which S-albuterol induces human bronchial smooth muscle cell proliferation.

BACKGROUND: Racemic albuterol is a 50:50 mixture of the R-isomer, levalbuterol, and the S-isomer, S-albuterol. S-Albuterol increases airway hyperresponsiveness to spasmogens, exacerbates asthmatic conditions and stimulates cell growth, whereas levalbuterol attenuates cell growth in culture. The mechanisms of S-albuterol-induced cell proliferation are not well understood. We studied the role of albuterol isomers and intracellular cell cycle regulators on proliferation of human bronchial smooth muscle cells. METHODS: Serum-starved cells (72 h) were fed test agents for 24 h and cell proliferation was measured. The expression of nuclear factor-kappaB inhibitory protein IkappaBalpha, nuclear factor-kappaB, cyclin-dependent kinases 2 and 4, interleukin (IL)-6, and retinoblastoma and platelet-activating factor (PAF) receptor protein were measured by Western blotting. RESULTS: S-Albuterol, PAF and platelet-derived growth factor stimulated cell proliferation, but levalbuterol and the racemic mixture inhibited cell proliferation compared with the effect of 5% fetal bovine serum alone. The proliferative effect of platelet-derived growth factor on S-albuterol was not additive, suggesting that the 2 mediators act by different mechanisms. S-Albuterol induced greater expression of all the measured proteins than either levalbuterol, the racemic mixture or 5% fetal bovine serum. S-Albuterol stimulated IL-6 secretion and abolished the ability of levalbuterol to inhibit IL-6 secretion. CONCLUSION: Our data show that S-albuterol stimulates cell proliferation by activating expression and phosphorylation of several intracellular mitogenic proteins and may exacerbate asthma by stimulating the release of IL-6. Induction of PAF receptor protein expression by S-albuterol strongly suggests that S-albuterol may exert its adverse effects by binding to a G protein-coupled receptor such as the PAF receptor.

Role of platelet-activating factor in pulmonary vascular remodeling associated with chronic high altitude hypoxia in ovine fetal lambs.

Platelet-activating factor (PAF) is implicated in pathogenesis of chronic hypoxia-induced pulmonary hypertension in some animal models and in neonates. Effects of chronic hypoxia on PAF receptor (PAF-R) system in fetal pulmonary vasculature are unknown. We investigated the effect of chronic high altitude hypoxia (HAH) in fetal lambs [pregnant ewes were kept at 3,801 m (12,470 ft) altitude from approximately 35 to 145 days gestation] on PAF-R-mediated effects in the pulmonary vasculature. Age-matched controls were kept at sea level. Intrapulmonary arteries were isolated, and smooth muscle cells (SMC-PA) were cultured from HAH and control fetuses. To determine presence of pulmonary vascular remodeling, lung tissue sections were subjected to morphometric analysis. Percentage medial wall thickness was significantly increased (P < 0.05) in arteries at all levels in the HAH lambs. PAF-R protein expression studied by immunocytochemistry and Western blot analysis on lung tissue SMC-PA demonstrated greater PAF-R expression in HAH lambs. PAF-R binding (femtomoles per 10(6) cells) in HAH SMC-PA was 90.3 +/- 4.08 and 66% greater than 54.3 +/- 4.9 in control SMC-PA. Pulmonary arteries from HAH fetuses synthesized >3-fold PAF than vessels from controls. Compared with controls SMC-PA of HAH lambs demonstrated 139% and 40% greater proliferation in 10% FBS alone and with 10 nM PAF, respectively. Our data demonstrate that exposure of ovine fetuses to HAH will result in significant upregulation of PAF synthesis, PAF-R expression, and PAF-R-mediated effects in pulmonary arteries. These findings suggest that increased PAF-R protein expression and increased PAF binding contribute to pulmonary vascular remodeling in these animals and may predispose them to persistent pulmonary hypertension after birth.