Francesco Spallotta

Oxidative Stress and Epigenetic Regulation in Ageing and Age-Related Diseases

Recent statistics indicate that the human population is ageing rapidly. Healthy, but also diseased, elderly people are increasing. This trend is particularly evident in Western countries, where healthier living conditions and better cures are available. To understand the process leading to age-associated alterations is, therefore, of the highest relevance for the development of new treatments for age-associated diseases, such as cancer, diabetes, Alzheimer and cardiovascular accidents. Mechanistically, it is well accepted that the accumulation of intracellular damage determined by reactive oxygen species (ROS) might orchestrate the progressive loss of control over biological homeostasis and the functional impairment typical of aged tissues. Here, we review how epigenetics takes part in the control of stress stimuli and the mechanisms of ageing physiology and physiopathology. Alteration of epigenetic enzyme activity, histone modifications and DNA-methylation is, in fact, typically associated with the ageing process. Specifically, ageing presents peculiar epigenetic markers that, taken altogether, form the still ill-defined “ageing epigenome”. The comprehension of mechanisms and pathways leading to epigenetic modifications associated with ageing may help the development of anti-ageing therapies.

Nitric Oxide Modulates Chromatin Folding in Human Endothelial Cells via Protein Phosphatase 2A Activation and Class II Histone Deacetylases Nuclear Shuttling

Nitric oxide (NO) modulates important endothelial cell (EC) functions and gene expression by a molecular mechanism which is still poorly characterized. Here we show that in human umbilical vein ECs (HUVECs) NO inhibited serum-induced histone acetylation and enhanced histone deacetylase (HDAC) activity. By immunofluorescence and Western blot analyses it was found that NO induced class II HDAC4 and 5 nuclear shuttling and that class II HDACs selective inhibitor MC1568 rescued serum-dependent histone acetylation above control level in NO-treated HUVECs. In contrast, class I HDACs inhibitor MS27–275 had no effect, indicating a specific role for class II HDACs in NO-dependent histone deacetylation. In addition, it was found that NO ability to induce HDAC4 and HDAC5 nuclear shuttling involved the activation of the protein phosphatase 2A (PP2A). In fact, HDAC4 nuclear translocation was impaired in ECs expressing small-t antigen and exposed to NO. Finally, in cells engineered to express a HDAC4-Flag fusion protein, NO induced the formation of a macromolecular complex including HDAC4, HDAC3, HDAC5, and an active PP2A. The present results show that NO-dependent PP2A activation plays a key role in class II HDACs nuclear translocation.

Nε-lysine acetylation determines dissociation from GAP junctions and lateralization of connexin 43 in normal and dystrophic heart

Wanting to explore the epigenetic basis of Duchenne cardiomyopathy, we found that global histone acetylase activity was abnormally elevated and the acetylase P300/CBP-associated factor (PCAF) coimmunoprecipitated with connexin 43 (Cx43), which was Nε-lysine acetylated and lateralized in mdx heart. This observation was paralleled by Cx43 dissociation from N-cadherin and zonula occludens 1, whereas pp60-c-Src association was unaltered. In vivo treatment of mdx with the pan-histone acetylase inhibitor anacardic acid significantly reduced Cx43 Nε-lysine acetylation and restored its association to GAP junctions (GJs) at intercalated discs. Noteworthy, in normal as well as mdx mice, the class IIa histone deacetylases 4 and 5 constitutively colocalized with Cx43 either at GJs or in the lateralized compartments. The class I histone deacetylase 3 was also part of the complex. Treatment of normal controls with the histone deacetylase pan-inhibitor suberoylanilide hydroxamic acid (MC1568) or the class IIa-selective inhibitor 3-{4-[3-(3-fluorophenyl)-3-oxo-1-propen-1-yl]-1-methyl-1H-pyrrol-2-yl}-N-hydroxy-2-propenamide (MC1568) determined Cx43 hyperacetylation, dissociation from GJs, and distribution along the long axis of ventricular cardiomyocytes. Consistently, the histone acetylase activator pentadecylidenemalonate 1b (SPV106) hyperacetylated cardiac proteins, including Cx43, which assumed a lateralized position that partly reproduced the dystrophic phenotype. In the presence of suberoylanilide hydroxamic acid, cell to cell permeability was significantly diminished, which is in agreement with a Cx43 close conformation in the consequence of hyperacetylation. Additional experiments, performed with Cx43 acetylation mutants, revealed, for the acetylated form of the molecule, a significant reduction in plasma membrane localization and a tendency to nuclear accumulation. These results suggest that Cx43 Nε-lysine acetylation may have physiopathological consequences for cell to cell coupling and cardiac function.

Nitric oxide determines mesodermic differentiation of mouse embryonic stem cells by activating class IIa histone deacetylases: potential therapeutic implications in a mouse model of hindlimb ischemia.

In human endothelial cells, nitric oxide (NO) results in class IIa histone deacetylases (HDACs) activation and marked histone deacetylation. It is unknown whether similar epigenetic events occur in embryonic stem cells (ESC) exposed to NO and how this treatment could influence ESC therapeutic potential during tissue regeneration.

The Histone Acetylase Activator Pentadecylidenemalonate 1b Rescues Proliferation and Differentiation in the Human Cardiac Mesenchymal Cells of Type 2 Diabetic Patients

This study investigates the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of H3K9Ac and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5-related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-α general control of amino acid synthesis (GCN5a), determining a relative decrease in total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients.

Histone deacetylase inhibitors: Keeping momentum for neuromuscular and cardiovascular diseases treatment

Histone deacetylases (HDACs) are enzymes with a pleiotropic range of intracellular localizations and actions. They are principally involved in the withdrawal of acetyl-groups from a large number of nuclear and cytoplasmic proteins including nuclear core histones as well as cytoskeletal proteins and metabolically relevant enzymes. Initial findings indicated that HDAC inhibitors (DIs) could be successfully applied in a variety of cancer treatment protocols as a consequence of their anti-proliferative and pro-apoptotic properties. Recent observations, however, enlightened the important therapeutic effects of DIs in experimental animal models for arthritis, neurodegenerative and neuromuscular disorders, heart ischemia, cardiac hypertrophy, heart failure and arrhythmias. A small number of clinical trials are now open or planned for the near future to verify the therapeutic properties of DIs in non-cancer-related diseases. This review summarizes some of the most important observations and concepts aroused by the most recent experimental application of DIs to neuromuscular and cardiac diseases.

Altered SDF-1-mediated differentiation of bone marrow-derived endothelial progenitor cells in diabetes mellitus

In diabetic patients and animal models of diabetes mellitus (DM), circulating endothelial progenitor cell (EPC) number is lower than in normoglycaemic conditions and EPC angiogenic properties are inhibited. Stromal cell derived factor‐1 (SDF‐1) plays a key role in bone marrow (BM) c‐kit+ stem cell mobilization into peripheral blood (PB), recruitment from PB into ischemic tissues and differentiation into endothelial cells. The aim of the present study was to examine the effect of DM in vivo and in vitro, on murine BM‐derived c‐kit+ cells and on their response to SDF‐1. Acute hindlimb ischemia was induced in streptozotocin‐treated DM and control mice; circulating c‐kit+ cells exhibited a rapid increase followed by a return to control levels which was significantly faster in DM than in control mice. CXCR4 expression by BM c‐kit+ cells as well as SDF‐1 protein levels in the plasma and in the skeletal muscle, both before and after the induction of ischemia, were similar between normoglycaemic and DM mice. However, BM‐derived c‐kit+ cells from DM mice exhibited an impaired differentiation towards the endothelial phenotype in response to SDF‐1; this effect was associated with diminished protein kinase phosphorylation. Interestingly, SDF‐1 ability to induce differentiation of c‐kit+ cells from DM mice was restored when cells were cultured under normoglycaemic conditions whereas c‐kit+ cells from normoglycaemic mice failed to differentiate in response to SDF‐1 when they were cultured in hyperglycaemic conditions. These results show that DM diminishes circulating c‐kit+ cell number following hindlimb ischemia and inhibits SDF‐1‐mediated AKT phosphorylation and differentiation towards the endothelial phenotype of BM‐derived c‐kit+ cells.

The histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces cardiac arrhythmias in dystrophic mice

AIMS The effect of histone deacetylase inhibitors on dystrophic heart function is not established. To investigate this aspect, dystrophic mdx mice and wild-type (WT) animals were treated 90 days either with suberoylanilide hydroxamic acid (SAHA, 5 mg/kg/day) or with an equivalent amount of vehicle. METHODS AND RESULTS The following parameters were evaluated: (i) number of ventricular arrhythmias in resting and stress conditions (restraint test) or after aconitine administration; (ii) cardiac excitability, conduction velocity, and refractoriness; (iii) expression and distribution of connexins (Cxs) and Na(v)1.5 sodium channel. Ventricular arrhythmias were negligible in all resting animals. During restraint, however, an increase in the number of arrhythmias was detected in vehicle-treated mdx mice (mdx-V) when compared with SAHA-treated mdx (mdx-SAHA) mice or normal control (WT-V). Interestingly, aconitine, a sodium channel pharmacologic opener, induced ventricular arrhythmias in 83% of WT-V mice, 11% of mdx-V, and in 57% of mdx-SAHA. Epicardial multiple lead recording revealed a prolongation of the QRS complex in mdx-V mice in comparison to WT-V and WT-SAHA mice, paralleled by a significant reduction in impulse propagation velocity. These alterations were efficiently counteracted by SAHA. Molecular analyses revealed that in mdx mice, SAHA determined Cx remodelling of Cx40, Cx37 and Cx32, whereas expression levels of Cx43 and Cx45 were unaltered. Remarkably, Cx43 lateralization observed in mdx control animals was reversed by SAHA treatment which also re-induced Na(v)1.5 expression. CONCLUSION SAHA attenuates arrhythmias in mdx mice by a mechanism in which Cx remodelling and sodium channel re-expression could play an important role.

RNA-seq of the aging brain in the short-lived fish N. furzeri – conserved pathways and novel genes associated with neurogenesis

The brains of teleost fish show extensive adult neurogenesis and neuronal regeneration. The patterns of gene regulation during fish brain aging are unknown. The short‐lived teleost fish Nothobranchius furzeri shows markers of brain aging including reduced learning performances, gliosis, and reduced adult neurogenesis. We used RNA‐seq to quantify genome‐wide transcript regulation and sampled five different time points to characterize whole‐genome transcript regulation during brain aging of N. furzeri. Comparison with human datasets revealed conserved up‐regulation of ribosome, lysosome, and complement activation and conserved down‐regulation of synapse, mitochondrion, proteasome, and spliceosome. Down‐regulated genes differ in their temporal profiles: neurogenesis and extracellular matrix genes showed rapid decay, synaptic and axonal genes a progressive decay. A substantial proportion of differentially expressed genes (~40%) showed inversion of their temporal profiles in the last time point: spliceosome and proteasome showed initial down‐regulation and stress‐response genes initial up‐regulation. Extensive regulation was detected for chromatin remodelers of the DNMT and CBX families as well as members of the polycomb complex and was mirrored by an up‐regulation of the H3K27me3 epigenetic mark. Network analysis showed extensive coregulation of cell cycle/DNA synthesis genes with the uncharacterized zinc‐finger protein ZNF367 as central hub. In situ hybridization showed that ZNF367 is expressed in neuronal stem cell niches of both embryonic zebrafish and adult N. furzeri. Other genes down‐regulated with age, not previously associated with adult neurogenesis and with similar patterns of expression are AGR2, DNMT3A, KRCP, MEX3A, SCML4, and CBX1. CBX7, on the other hand, was up‐regulated with age.

A nitric oxide-dependent cross-talk between class I and III histone deacetylases accelerates skin repair.

Background: Nitric oxide (NO) regulates class I and IIa histone deacetylase (HDAC) function. NO production is regulated by class III HDACs (sirtuins). Results: NO functions as a bridging molecule between class I and sirtuins (SIRTs). Conclusion: The SIRT-NO-class I HDAC axis provides key signals during wound repair. Significance: Modulation of HDAC activity may play an important role in tissue regeneration. In a mouse model of skin repair we found that the class I-IIa histone deacetylase inhibitor trichostatin A accelerated tissue regeneration. Unexpectedly, this effect was suppressed by Sirtinol, a class III histone deacetylase (HDAC) (sirtuin)-selective inhibitor. The role of sirtuins (SIRTs) was then investigated by using resveratrol and a novel SIRT1-2-3 activator, the MC2562 compound we synthesized recently. Both resveratrol and MC2562 were effective in accelerating wound repair. The local administration of natural or synthetic SIRT activators, in fact, significantly accelerated skin regeneration by increasing keratinocyte proliferation. In vitro experiments revealed that the activation of SIRTs stimulated keratinocyte proliferation via endothelial NO synthase phosphorylation and NO production. In this condition, the class I member HDAC2 was found S-nitrosylated on cysteine, a post-transduction modification associated with loss of activity and DNA binding capacity. After deacetylase inhibitor or SIRT activator treatment, ChIP showed, in fact, a significant HDAC2 detachment from the promoter region of insulin growth factor I (IGF-I), fibroblast growth factor 10 (FGF-10), and Epithelial Growth Factor (EGF), which may be the final recipients and effectors of the SIRT-NO-HDAC signaling cascade. Consistently, the effect of SIRT activators was reduced in the presence of NG-nitro-l-arginine methyl ester (l-NAME), a general inhibitor of NO synthesis. In conclusion, the NO-dependent cross-talk among class III and I histone deacetylases suggests an unprecedented signaling pathway important for skin repair.