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Ex vivo treatment with a novel synthetic aminoglycoside NB54 in primary fibroblasts from Rett syndrome patients suppresses MECP2 nonsense mutations.

Vecsler M, Ben Zeev B, Nudelman I, Anikster Y, Simon AJ, Amariglio N, Rechavi G, Baasov T, Gak E - PLoS ONE (2011)

Bottom Line: Recently developed synthetic NB aminoglycosides have demonstrated significantly improved effects compared to gentamicin evident in substantially higher suppression and reduced acute toxicity in vitro.In addition, the recovered MeCP2 was translocated to the cell nucleus and moreover led to parallel increase in one of the most important MeCP2 downstream effectors, the brain derived neurotrophic factor (BDNF).Our findings suggest that NB54 may induce restoration of the potentially functional MeCP2 in primary RTT fibroblasts and encourage further studies of NB54 and other rationally designed aminoglycoside derivatives as potential therapeutic agents for nonsense MECP2 mutations in RTT.

View Article: PubMed Central - PubMed

Affiliation: Sagol Neuroscience Center, Sheba Medical Center, Tel Hashomer, Israel.

ABSTRACT

Background: Nonsense mutations in the X-linked methyl CpG-binding protein 2 (MECP2) comprise a significant proportion of causative MECP2 mutations in Rett syndrome (RTT). Naturally occurring aminoglycosides, such as gentamicin, have been shown to enable partial suppression of nonsense mutations related to several human genetic disorders, however, their clinical applicability has been compromised by parallel findings of severe toxic effects. Recently developed synthetic NB aminoglycosides have demonstrated significantly improved effects compared to gentamicin evident in substantially higher suppression and reduced acute toxicity in vitro.

Results: We performed comparative study of suppression effects of the novel NB54 and gentamicin on three MECP2 nonsense mutations (R294X, R270X and R168X) common in RTT, using ex vivo treatment of primary fibroblasts from RTT patients harboring these mutations and testing for the C-terminal containing full-length MeCP2. We observed that NB54 induces dose-dependent suppression of MECP2 nonsense mutations more efficiently than gentamicin, which was evident at concentrations as low as 50 µg/ml. NB54 read-through activity was mutation specific, with maximal full-length MeCP2 recovery in R168X (38%), R270X (27%) and R294X (18%). In addition, the recovered MeCP2 was translocated to the cell nucleus and moreover led to parallel increase in one of the most important MeCP2 downstream effectors, the brain derived neurotrophic factor (BDNF).

Conclusion: Our findings suggest that NB54 may induce restoration of the potentially functional MeCP2 in primary RTT fibroblasts and encourage further studies of NB54 and other rationally designed aminoglycoside derivatives as potential therapeutic agents for nonsense MECP2 mutations in RTT.

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MECP2 mRNA expression levels following treatment with NB54.Total mRNA was purified from NB54-treated (100 µg/ml for 5days) and untreated R294X fibroblasts. Expression levels of both MECP2_e1 and _e2 isoforms were determined by real-time qPCR using GAPDH levels as an internal reference. Experiments were performed in triplicates.
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pone-0020733-g003: MECP2 mRNA expression levels following treatment with NB54.Total mRNA was purified from NB54-treated (100 µg/ml for 5days) and untreated R294X fibroblasts. Expression levels of both MECP2_e1 and _e2 isoforms were determined by real-time qPCR using GAPDH levels as an internal reference. Experiments were performed in triplicates.

Mentions: Mutations specific effects of NB54 were further reproduced treating R294X, R270X and R168X fibroblasts with 400 µg/ml NB54 for 5 days (Fig. 2A and B) that was previously found optimal in R294X (Fig. 1A). This experiment independently demonstrated that NB54 was more effective in R168X (UGA G), with increased 38% read-through compared to 18% and 27% in R294X (UGA U) and R270X (UGA A), respectively. The difference between this and previous findings (Fig. 1) of NB54 effect at the concentration of 400 µg/ml may be an outcome of XCI variability. Studying expression MECP2 RNA levels in R294X fibroblasts treated with 100 µg/ml NB54 for 5 days, we observed insignificant differences in MECP2_e1 and _e2 expression before and after NB54 treatment (less than 25%, Student's t test p>0.05) and could be attributed to XCI variability (Fig. 3), thereby supporting the notion that NB54 has no effect on the mRNA level.


Ex vivo treatment with a novel synthetic aminoglycoside NB54 in primary fibroblasts from Rett syndrome patients suppresses MECP2 nonsense mutations.

Vecsler M, Ben Zeev B, Nudelman I, Anikster Y, Simon AJ, Amariglio N, Rechavi G, Baasov T, Gak E - PLoS ONE (2011)

MECP2 mRNA expression levels following treatment with NB54.Total mRNA was purified from NB54-treated (100 µg/ml for 5days) and untreated R294X fibroblasts. Expression levels of both MECP2_e1 and _e2 isoforms were determined by real-time qPCR using GAPDH levels as an internal reference. Experiments were performed in triplicates.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113846&req=5

pone-0020733-g003: MECP2 mRNA expression levels following treatment with NB54.Total mRNA was purified from NB54-treated (100 µg/ml for 5days) and untreated R294X fibroblasts. Expression levels of both MECP2_e1 and _e2 isoforms were determined by real-time qPCR using GAPDH levels as an internal reference. Experiments were performed in triplicates.
Mentions: Mutations specific effects of NB54 were further reproduced treating R294X, R270X and R168X fibroblasts with 400 µg/ml NB54 for 5 days (Fig. 2A and B) that was previously found optimal in R294X (Fig. 1A). This experiment independently demonstrated that NB54 was more effective in R168X (UGA G), with increased 38% read-through compared to 18% and 27% in R294X (UGA U) and R270X (UGA A), respectively. The difference between this and previous findings (Fig. 1) of NB54 effect at the concentration of 400 µg/ml may be an outcome of XCI variability. Studying expression MECP2 RNA levels in R294X fibroblasts treated with 100 µg/ml NB54 for 5 days, we observed insignificant differences in MECP2_e1 and _e2 expression before and after NB54 treatment (less than 25%, Student's t test p>0.05) and could be attributed to XCI variability (Fig. 3), thereby supporting the notion that NB54 has no effect on the mRNA level.

Bottom Line: Recently developed synthetic NB aminoglycosides have demonstrated significantly improved effects compared to gentamicin evident in substantially higher suppression and reduced acute toxicity in vitro.In addition, the recovered MeCP2 was translocated to the cell nucleus and moreover led to parallel increase in one of the most important MeCP2 downstream effectors, the brain derived neurotrophic factor (BDNF).Our findings suggest that NB54 may induce restoration of the potentially functional MeCP2 in primary RTT fibroblasts and encourage further studies of NB54 and other rationally designed aminoglycoside derivatives as potential therapeutic agents for nonsense MECP2 mutations in RTT.

View Article: PubMed Central - PubMed

Affiliation: Sagol Neuroscience Center, Sheba Medical Center, Tel Hashomer, Israel.

ABSTRACT

Background: Nonsense mutations in the X-linked methyl CpG-binding protein 2 (MECP2) comprise a significant proportion of causative MECP2 mutations in Rett syndrome (RTT). Naturally occurring aminoglycosides, such as gentamicin, have been shown to enable partial suppression of nonsense mutations related to several human genetic disorders, however, their clinical applicability has been compromised by parallel findings of severe toxic effects. Recently developed synthetic NB aminoglycosides have demonstrated significantly improved effects compared to gentamicin evident in substantially higher suppression and reduced acute toxicity in vitro.

Results: We performed comparative study of suppression effects of the novel NB54 and gentamicin on three MECP2 nonsense mutations (R294X, R270X and R168X) common in RTT, using ex vivo treatment of primary fibroblasts from RTT patients harboring these mutations and testing for the C-terminal containing full-length MeCP2. We observed that NB54 induces dose-dependent suppression of MECP2 nonsense mutations more efficiently than gentamicin, which was evident at concentrations as low as 50 µg/ml. NB54 read-through activity was mutation specific, with maximal full-length MeCP2 recovery in R168X (38%), R270X (27%) and R294X (18%). In addition, the recovered MeCP2 was translocated to the cell nucleus and moreover led to parallel increase in one of the most important MeCP2 downstream effectors, the brain derived neurotrophic factor (BDNF).

Conclusion: Our findings suggest that NB54 may induce restoration of the potentially functional MeCP2 in primary RTT fibroblasts and encourage further studies of NB54 and other rationally designed aminoglycoside derivatives as potential therapeutic agents for nonsense MECP2 mutations in RTT.

Show MeSH
Related in: MedlinePlus