SIRT1 Inhibition Alleviates Gene Silencing in Fragile X Mental Retardation Syndrome

Section on Genomic Structure and Function, Laboratory of Molecular and Cellular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS Genetics (Impact Factor: 7.53). 03/2008; 4(3):e1000017. DOI: 10.1371/journal.pgen.1000017
Source: PubMed


Author Summary
Fragile X syndrome is the leading cause of heritable intellectual disability. The affected gene, FMR1, encodes FMRP, a protein that regulates the synthesis of a number of important neuronal proteins. The causative mutation is an increase in the number of CGG•CCG-repeats found at the beginning of the FMR1 gene. Alleles with >200 repeats are silenced. The silencing process involves DNA methylation as well as modifications to the histone proteins around which the DNA is wrapped in vivo. Treatment with 5-azadeoxycytidine, a DNA methyltransferase inhibitor, reactivates the gene. However, this reagent is toxic and since no DNA demethylase has been found in humans, methylation inhibitors are not useful in cells like neurons that no longer divide. We show here that splitomicin is also able to reactivate the Fragile X allele. It does so by inhibiting a protein deacetylase, SIRT1, thus favoring the action of another enzyme, hMOF that reverses the SIRT1 modification. We also found that 5-azadeoxycytidine acts, at least in part, by reversing the effect of SIRT1. However, since splitomicin reactivation occurred without DNA demethylation, DNA replication is not necessary for its efficacy. Thus, unlike DNA methylation inhibitors, SIRT1 inhibitors may be able to reactivate Fragile X alleles in neurons.

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    • "Recently, the class III HDAC inhibitor nicotinamide (Ghosh and Feany, 2004) has been shown to increase frataxin expression and decrease H3K9me3 and H3K27me3 at the FXN gene in FRDA cells and mouse models and this compound is now in early stage clinical trials (Chan et al., 2013) (Table 2). Furthermore, other HDAC inhibitors such as sirtinol (Ota et al., 2006), splitomicin (Biacsi et al., 2008), LBH589 (Garbes et al., 2009), and oxamflatin (Kim et al., 1999) have shown positive effects in other "
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    • "Second, even when treating for these prolonged periods, the reported increase in FMR1 transcript levels in FXS patient cell lines reached only up to ~20% of healthy control levels. Indeed, 24-hour 5-azacytidine treatment which is more comparable and amendable to screening conditions only resulted in an increase of FMR1 levels to 4% of that of healthy controls, a change likely to small to be detected in a microtiter plate format with statistical significance [16]. Third, the observed maximum treatment effect of up to 20% of normal levels was restricted to some lymphoblast lines from selected patients while the effect was much lower or even absent in other lymphoblast or fibroblast cell lines derived from other FXS patients, a result attributed to differential methylation status of the patients FMR1 promoter regions. "
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    ABSTRACT: Background Hypermethylation of the fragile X mental retardation 1 gene FMR1 results in decreased expression of FMR1 protein FMRP, which is the underlying cause of Fragile X syndrome – an incurable neurological disorder characterized by mental retardation, anxiety, epileptic episodes and autism. Disease-modifying therapies for Fragile X syndrome are thus aimed at treatments that increase the FMRP expression levels in the brain. We describe the development and characterization of two assays for simple and quantitative detection of FMRP protein. Method Antibodies coupled to fluorophores that can be employed for time-resolved Förster’s resonance energy transfer were used for the development of homogeneous, one-step immunodetection. Purified recombinant human FMRP and patient cells were used as control samples for assay development. Results The assays require small sample amounts, display high stability and reproducibility and can be used to quantify endogenous FMRP in human fibroblasts and peripheral blood mononuclear cells. Application of the assays to FXS patient cells showed that the methods can be used both for the characterization of clinical FXS patient samples as well as primary readouts in drug-discovery screens aimed at increasing endogenous FMRP levels in human cells. Conclusion This study provides novel quantitative detection methods for FMRP in FXS patient cells. Importantly, due to the simplicity of the assay protocol, the method is suited to be used in screening applications to identify compounds or genetic interventions that result in increased FMRP levels in human cells.
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