ABCMdb

PMID: 19136563

Du M, Keeling KM, Fan L, Liu X, Bedwell DM
Poly-L-aspartic acid enhances and prolongs gentamicin-mediated suppression of the CFTR-G542X mutation in a cystic fibrosis mouse model.
J Biol Chem. 2009 Mar 13;284(11):6885-92. Epub 2009 Jan 9., 2009-03-13 [PubMed]

Sentences

No. Mutations Sentence Comment

35 ABCC7 p.Gly542* Printed in the U.S.A. MARCH 13, 2009•VOLUME 284•NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 6885 an increased and prolonged level of suppression of the CFTR-G542X nonsense mutation in a CF mouse model. Login to comment 55 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* Mice and Treatment Protocols-The CFTR-G542X mice used in this study contained the Cftrtm1Cam knock-out (20) and expressed a human CFTR transgene with the G542X premature stop mutation (3, 4, 21) (referred to as Cftr-/- hCFTR-G542X mice). Login to comment 57 ABCC7 p.Gly542* Transcription of the hCFTR-G542X transgene was driven by the rat intestinal fatty acid-binding protein promoter. Login to comment 114 ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* PAA Enhances Gentamicin-induced Readthrough in Cftr-/- hCFTR-G542X Mice-We previously reported that once daily subcutaneous injections of 5 mg/kg gentamicin or 15 mg/kg amikacin resulted in suppression of the hCFTR-G542X mutation and a partial restoration of CFTR protein and function in Cftr-/- hCFTR-G542X mice (4). Login to comment 117 ABCC7 p.Gly542* Because our in vitro results indicated that the co-administration of gentamicin plus PAA enhanced suppression, we next investigated whether PAA could enhance the readthrough of a nonsense mutation by a low dose of gentamicin in Cftr-/- hCFTR-G542X mice. Login to comment 121 ABCC7 p.Gly542* ABCC7 p.Gly542* To systematically examine the effect of PAA on readthrough of the hCFTR-G542X nonsense mutation, Cftr-/- hCFTR-G542X mice were included in six different treatment groups as shown in Fig. 4A. Treatments consisted of subcutaneous injections of 5 mg/kg gentamicin alone or 5 mg/kg gentamicin plus 70 mg/kg PAA delivered once daily in a hind limb. Login to comment 122 ABCC7 p.Gly542* ABCC7 p.Gly542* Groups 1-3 contained Cftr-/- hCFTR-G542X mice that were treated as indicated for 14 days. Group 1 contained control mice that were left untreated for 14 days. Group 2 contained mice treated with gentamicin alone for 14 days, whereas group 3 contained Cftr-/- hCFTR-G542X mice that were administered gentamicin plus PAA for 14 days. Login to comment 124 ABCC7 p.Gly542* Groups 4-6 contained Cftr-/- hCFTR-G542X mice that were again treated for 14 days as described above, followed by a 4-day chase period before assays were performed. Login to comment 134 ABCC7 p.Gly542* ABCC7 p.Gly542* PAA Enhances CFTR Nonsense Codon Readthrough 6888 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 11•MARCH As controls, both wild type mice (Cftrϩ/ϩ , group 8) and Cftr knock-out mice without the hCFTR-G542X transgene (Cftr-/- , group 10) were treated with gentamicin plus PAA for 14 days with continuing administration of PAA for another 4 days. Login to comment 136 ABCC7 p.Gly542* The results of short circuit current measurements from the 10 different treatment groups of Cftr-/- hCFTR-G542X, wild type mice, and Cftr-/- mice are shown in Fig. 4B. A summary and statistical analysis of short circuit currents from the 10 different treatment groups are shown in Table 2. Login to comment 137 ABCC7 p.Gly542* The results from group 1 (untreated Cftr-/- hCFTR-G542X mice) revealed that 11% of samples (3/28) exhibited cAMP-stimulated short circuit currents, resulting in an average current of only 0.2 ␮A/cm2 . Login to comment 138 ABCC7 p.Gly542* ABCC7 p.Gly542* The infrequent response observed in untreated Cftr-/- hCFTR-G542X mice may be attributable to a low baseline level of endogenous readthrough of the hCFTR-G542X transgene, because these currents are not observed in Cftr-/- mice that do not carry the transgene, as shown below and discussed in a previous report (21). Login to comment 151 ABCC7 p.Gly542* Effect of gentamicin treatment (؎PAA) on cAMP-activated transepithelialchloridecurrentsinintestinaltissuesfromCftr-/- hCFTR-G542X. Login to comment 152 ABCC7 p.Gly542* A, diagram of different treatment groups of Cftr-/- hCFTR-G542X mice, as well as wild type (Cftrϩ/ϩ ) and Cftr-/- controls. Login to comment 157 ABCC7 p.Gly542* Cftr-/- hCFTR-G542X Cftr؉/؉ Cftr-/- Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Group 10 Treatment (14 days) None Gent Gent PAA Gent Gent PAA Gent PAA None Gent PAA None Gent PAA Chase (4 days) No No No Yes Yes Yes ϩPAA No Yes ϩPAA No Yes ϩPAA Positives/total 3/28 9/26 8/18 1/12 6/17 9/18 14/15 13/14 0/8 0/8 Positives (%) 11 35 44 8 35 50 93 93 0 0 Mean current (␮A/cm2 ) 0.2 0.7 1.1 0.13 1.2 2.1 5.0 5.9 0 0 p value (relative to group 1)a Ͻ0.05 Ͻ0.05 0.35 Ͻ0.05 Ͻ0.01 p value (relative to group 4)b Ͻ0.05 Ͻ0.05 Wild type current (%) 4 14 22 3 24 42 100 118 0 0 a p values of mean currents measured in groups 2-6 are relative to the mean current measured in group 1. b p values of mean currents measured in groups 5 and 6 are relative to the mean current measured in group 4. Login to comment 161 ABCC7 p.Gly542* When considered together, these results indicate that the co-administration of PAA significantly increases the average cAMP-stimulated short circuit current induced by gentamicin in hCFTR-G542X mice. Login to comment 168 ABCC7 p.Gly542* Together, these results confirmed that the CFTR activity that appears following gentamicin (or gentamicin plus PAA) treatment is dependent upon the presence of the hCFTR-G542X transgene. Login to comment 187 ABCC7 p.Gly542* Samples from the duodenum of Cftr-/- hCFTR-G542X mice were incubated with either preimmuneserumorCFTR-NBD1serum.Afterincubationofthesamplewithafluorescentsecondaryantibody, the samples were visualized by fluorescence microscopy. Login to comment 197 ABCC7 p.Gly542* ABCC7 p.Gly542* Our results with Cftr-/- hCFTR-G542X mice suggest that the higher level of accumulated intracellular gentamicin also remains accessible for ribosome binding in vivo, because the co-administration of gentamicin with PAA had a stimulatory effect on readthrough of the hCFTR-G542X mutation. Login to comment 200 ABCC7 p.Gly542* These results are consistent with our in vitro readthrough results and strongly suggest that PAA co-administration increases the intracellular aminoglycoside concentration accessible to ribosomes in the intestinal tissues of Cftr-/- hCFTR-G542X mice. Login to comment 202 ABCC7 p.Gly542* When Cftr-/- hCFTR-G542X mice were administered gentamicin for 14 days and then left for 4 days without treatment, we found that the CFTR protein and activity was reduced to a negligible level (3% of the mean wild type cAMP-activated short circuit current). Login to comment 204 ABCC7 p.Gly542* Because readthrough of the CFTR-G542X mutation probably ceases shortly after the gentamicin treatment is terminated, this loss of CFTR activity probably reflects the turnover of the hCFTR protein synthesized during the treatment period. Login to comment 208 ABCC7 p.Gly542* ABCC7 p.Gly542* These results demonstrate that the co-administration of gentamicin with PAA not only enhances the suppression of the hCFTR-G542X nonsense mutation in Cftr-/- hCFTR-G542X mice but also extends the time period during which hCFTR activity and protein can be detected following the termination of gentamicin treatment. Login to comment