Elisabetta Soragni

The HDAC inhibitor 4b ameliorates the disease phenotype and transcriptional abnormalities in Huntington's disease transgenic mice

Transcriptional dysregulation has emerged as a core pathologic feature of Huntingtons disease (HD), one of several triplet-repeat disorders characterized by movement deficits and cognitive dysfunction. Although the mechanisms contributing to the gene expression deficits remain unknown, therapeutic strategies have aimed to improve transcriptional output via modulation of chromatin structure. Recent studies have demonstrated therapeutic effects of commercially available histone deacetylase (HDAC) inhibitors in several HD models; however, the therapeutic value of these compounds is limited by their toxic effects. Here, beneficial effects of a novel pimelic diphenylamide HDAC inhibitor, HDACi 4b, in an HD mouse model are reported. Chronic oral administration of HDACi 4b, beginning after the onset of motor deficits, significantly improved motor performance, overall appearance, and body weight of symptomatic R6/2300Q transgenic mice. These effects were associated with significant attenuation of gross brain-size decline and striatal atrophy. Microarray studies revealed that HDACi 4b treatment ameliorated, in part, alterations in gene expression caused by the presence of mutant huntingtin protein in the striatum, cortex, and cerebellum of R6/2300Q transgenic mice. For selected genes, HDACi 4b treatment reversed histone H3 hypoacetylation observed in the presence of mutant huntingtin, in association with correction of mRNA expression levels. These findings suggest that HDACi 4b, and possibly related HDAC inhibitors, may offer clinical benefit for HD patients and provide a novel set of potential biomarkers for clinical assessment.

HDAC inhibitors correct frataxin deficiency in a Friedreich ataxia mouse model.

Background Friedreich ataxia, an autosomal recessive neurodegenerative and cardiac disease, is caused by abnormally low levels of frataxin, an essential mitochondrial protein. All Friedreich ataxia patients carry a GAA⋅TTC repeat expansion in the first intron of the frataxin gene, either in the homozygous state or in compound heterozygosity with other loss-of-function mutations. The GAA expansion inhibits frataxin expression through a heterochromatin-mediated repression mechanism. Histone modifications that are characteristic of silenced genes in heterochromatic regions occur at expanded alleles in cells from Friedreich ataxia patients, including increased trimethylation of histone H3 at lysine 9 and hypoacetylation of histones H3 and H4. Methodology/Principal Findings By chromatin immunoprecipitation, we detected the same heterochromatin marks in homozygous mice carrying a (GAA)230 repeat in the first intron of the mouse frataxin gene (KIKI mice). These animals have decreased frataxin levels and, by microarray analysis, show significant gene expression changes in several tissues. We treated KIKI mice with a novel histone deacetylase inhibitor, compound 106, which substantially increases frataxin mRNA levels in cells from Friedreich ataxia individuals. Treatment increased histone H3 and H4 acetylation in chromatin near the GAA repeat and restored wild-type frataxin levels in the nervous system and heart, as determined by quantitative RT-PCR and semiquantitative western blot analysis. No toxicity was observed. Furthermore, most of the differentially expressed genes in KIKI mice reverted towards wild-type levels. Conclusions/Significance Lack of acute toxicity, normalization of frataxin levels and of the transcription profile changes resulting from frataxin deficiency provide strong support to a possible efficacy of this or related compounds in reverting the pathological process in Friedreich ataxia, a so far incurable neurodegenerative disease.

Chemical Probes Identify a Role for Histone Deacetylase 3 in Friedreich's Ataxia Gene Silencing

We recently identified a class of pimelic diphenylamide histone deacetylase (HDAC) inhibitors that show promise as therapeutics in the neurodegenerative diseases Friedreichs ataxia (FRDA) and Huntingtons disease. Here, we describe chemical approaches to identify the HDAC enzyme target of these inhibitors. Incubation of a trifunctional activity-based probe with a panel of class I and class II recombinant HDAC enzymes, followed by click chemistry addition of a fluorescent dye and gel electrophoresis, identifies HDAC3 as a unique high-affinity target of the probe. Photoaffinity labeling in a nuclear extract prepared from human lymphoblasts with the trifunctional probe, followed by biotin addition through click chemistry, streptavidin enrichment, and Western blotting also identifies HDAC3 as the preferred cellular target of the inhibitor. Additional inhibitors with different HDAC specificity profiles were synthesized, and results from transcription experiments in FRDA cells point to a unique role for HDAC3 in gene silencing in Friedreichs ataxia.

A nutrient‐regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii

Important morphogenetic transitions in fungi are triggered by starvation‐induced changes in the expression of structural surface proteins. Here, we report that nutrient deprivation causes a strong and reversible up‐regulation of TbSP1, a surface‐associated, Ca2+‐dependent phospholipase from the mycorrhizal fungus Tuber borchii. TbSP1 is the first phospholipase A2 to be described in fungi and identifies a novel class of phospholipid‐hydrolyzing enzymes. The TbSP1 phospholipase, which is synthesized initially as a pre‐protein, is processed efficiently and secreted during the mycelial phase. The mature protein, however, also localizes to the inner cell wall layer, close to the plasma membrane, in both free‐living and symbiosis‐engaged hyphae. It thus appears that a dual localization phospholipase A2 is involved in the adaptation of a symbiotic fungus to conditions of persistent nutritional limitation. Moreover, the fact that TbSP1‐related sequences are present in Streptomyces and Neurospora, and not in wholly sequenced non‐filamentous microorganisms, points to a general role for TbSP1 phospholipases A2 in the organization of multicellular filamentous structures in bacteria and fungi.

Role of Mismatch Repair Enzymes in GAA·TTC Triplet-repeat Expansion in Friedreich Ataxia Induced Pluripotent Stem Cells

Background: Friedreich ataxia is caused by a GAA·TTC triplet-repeat expansion in the first intron of the FXN gene. Results: Expansion of the repeats is observed in induced pluripotent stem cells (iPSCs) and can be blocked with either shRNAs to mismatch repair enzymes or small molecules targeting the repeats. Conclusion: MutSα and MutSβ are involved in repeat expansion. Significance: iPSCs provide a model system for studying triplet-repeat expansion. The genetic mutation in Friedreich ataxia (FRDA) is a hyperexpansion of the triplet-repeat sequence GAA·TTC within the first intron of the FXN gene. Although yeast and reporter construct models for GAA·TTC triplet-repeat expansion have been reported, studies on FRDA pathogenesis and therapeutic development are limited by the availability of an appropriate cell model in which to study the mechanism of instability of the GAA·TTC triplet repeats in the human genome. Herein, induced pluripotent stem cells (iPSCs) were generated from FRDA patient fibroblasts after transduction with the four transcription factors Oct4, Sox2, Klf4, and c-Myc. These cells were differentiated into neurospheres and neuronal precursors in vitro, providing a valuable cell model for FRDA. During propagation of the iPSCs, GAA·TTC triplet repeats expanded at a rate of about two GAA·TTC triplet repeats/replication. However, GAA·TTC triplet repeats were stable in FRDA fibroblasts and neuronal stem cells. The mismatch repair enzymes MSH2, MSH3, and MSH6, implicated in repeat instability in other triplet-repeat diseases, were highly expressed in pluripotent stem cells compared with fibroblasts and neuronal stem cells and occupied FXN intron 1. In addition, shRNA silencing of MSH2 and MSH6 impeded GAA·TTC triplet-repeat expansion. A specific pyrrole-imidazole polyamide targeting GAA·TTC triplet-repeat DNA partially blocked repeat expansion by displacing MSH2 from FXN intron 1 in FRDA iPSCs. These studies suggest that in FRDA, GAA·TTC triplet-repeat instability occurs in embryonic cells and involves the highly active mismatch repair system.

Epigenetic therapy for Friedreich ataxia.

To investigate whether a histone deacetylase inhibitor (HDACi) would be effective in an in vitro model for the neurodegenerative disease Friedreich ataxia (FRDA) and to evaluate safety and surrogate markers of efficacy in a phase I clinical trial in patients.

Long intronic GAA•TTC repeats induce epigenetic changes and reporter gene silencing in a molecular model of Friedreich ataxia

Friedreich ataxia (FRDA) is caused by hyperexpansion of GAA•TTC repeats located in the first intron of the FXN gene, which inhibits transcription leading to the deficiency of frataxin. The FXN gene is an excellent target for therapeutic intervention since (i) 98% of patients carry the same type of mutation, (ii) the mutation is intronic, thus leaving the FXN coding sequence unaffected and (iii) heterozygous GAA•TTC expansion carriers with ∼50% decrease of the frataxin are asymptomatic. The discovery of therapeutic strategies for FRDA is hampered by a lack of appropriate molecular models of the disease. Herein, we present the development of a new cell line as a molecular model of FRDA by inserting 560 GAA•TTC repeats into an intron of a GFP reporter minigene. The GFP_(GAA•TTC)560 minigene recapitulates the molecular hallmarks of the mutated FXN gene, i.e. inhibition of transcription of the reporter gene, decreased levels of the reporter protein and hypoacetylation and hypermethylation of histones in the vicinity of the repeats. Additionally, selected histone deacetylase inhibitors, known to stimulate the FXN gene expression, increase the expression of the GFP_(GAA•TTC)560 reporter. This FRDA model can be adapted to high-throughput analyses in a search for new therapeutics for the disease.

A high-affinity ammonium transporter from the mycorrhizal ascomycete Tuber borchii☆

An ammonium transporter cDNA, named TbAMT1, was isolated from the ectomycorrhizal ascomycetous truffle Tuber borchii. The polypeptide encoded by TbAMT1 (52 kDa) functionally complements ammonium uptake-defective yeast mutants and shares sequence similarity with previously characterized ammonium transporters from Saccharomyces (Mep) and Arabidopsis (AtAMT1). Structural characteristics common to the Mep/Amt family and peculiar features of the Tuber transporter have been evidenced by a detailed topological model of the TbAMT1 protein, which predicts 11 transmembrane helices with an N terminus(OUT)/C terminus(IN) orientation. As revealed by uptake/competition experiments conducted in yeast, TbAMT1 is a high-affinity transporter with an apparent K(m) for ammonium of 2 microM. The TbAMT1 mRNA was very slowly, yet specifically upregulated in nitrogen-deprived T. borchii mycelia. Instead, a much faster return to basal expression levels was observed upon resupplementation of either ammonium or nitrate, which thus appear to be utilized as equally effective nitrogen sources by Tuber mycelia.

RNA Toxicity and Missplicing in the Common Eye Disease Fuchs Endothelial Corneal Dystrophy

Background: Expansion of intronic (CTG·CAG)n repeats in TCF4 is found in most Fuchs endothelial corneal dystrophy (FECD) patients. Results: RNA foci co-localizing with the splicing factor MBNL1 are found in FECD cells, and changes in mRNA splicing occur. Conclusion: Trinucleotide repeat expansion in FECD is associated with RNA focus formation and missplicing. Significance: RNA toxicity occurs in a disease affecting millions of patients. Fuchs endothelial corneal dystrophy (FECD) is an inherited degenerative disease that affects the internal endothelial cell monolayer of the cornea and can result in corneal edema and vision loss in severe cases. FECD affects ∼5% of middle-aged Caucasians in the United States and accounts for >14,000 corneal transplantations annually. Among the several genes and loci associated with FECD, the strongest association is with an intronic (CTG·CAG)n trinucleotide repeat expansion in the TCF4 gene, which is found in the majority of affected patients. Corneal endothelial cells from FECD patients harbor a poly(CUG)n RNA that can be visualized as RNA foci containing this condensed RNA and associated proteins. Similar to myotonic dystrophy type 1, the poly(CUG)n RNA co-localizes with and sequesters the mRNA-splicing factor MBNL1, leading to missplicing of essential MBNL1-regulated mRNAs. Such foci and missplicing are not observed in similar cells from FECD patients who lack the repeat expansion. RNA-Seq splicing data from the corneal endothelia of FECD patients and controls reveal hundreds of differential alternative splicing events. These include events previously characterized in the context of myotonic dystrophy type 1 and epithelial-to-mesenchymal transition, as well as splicing changes in genes related to proposed mechanisms of FECD pathogenesis. We report the first instance of RNA toxicity and missplicing in a common non-neurological/neuromuscular disease associated with a repeat expansion. The FECD patient population with this (CTG·CAG)n trinucleotide repeat expansion exceeds that of the combined number of patients in all other microsatellite expansion disorders.

Rationale for the development of 2-aminobenzamide histone deacetylase inhibitors as therapeutics for Friedreich ataxia.

Numerous studies have pointed to histone deacetylase inhibitors as potential therapeutics for various neurodegenerative diseases, and clinical trials with several histone deacetylase inhibitors have been performed or are under way. However, histone deacetylase inhibitors tested to date either are highly cytotoxic or have very low specificities for different histone deacetylase enzymes. The authors’ laboratories have identified a novel class of histone deacetylase inhibitors (2-aminobenzamides) that reverses heterochromatin-mediated silencing of the frataxin (FXN) gene in Friedreich ataxia. The authors have identified the histone deacetylase enzyme isotype target of these compounds and present evidence that compounds that target this enzyme selectively increase FXN expression from pathogenic alleles. Studies with model compounds show that these histone deacetylase inhibitors increase FXN messenger RNA levels in the brain in mouse models for Friedreich ataxia and relieve neurological symptoms observed in mouse models and support the notion that this class of molecules may serve as therapeutics for the human disease.