Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex.

BACKGROUND: REST (Repressor-Element 1 [RE1]-silencing transcription factor) inhibits Na+/Ca2+exchanger-1 (Ncx1) transcription in neurons through the binding of RE1 site on brain promoter (Br) after stroke. We identified a new putative RE1 site in Ncx1 heart promoter (Ht) sequence (Ht-RE1) that participates in neuronal Ncx1 transcription. Because REST recruits DNA-methyltransferase-1 (DNMT1) and MeCP2 (methyl-CpG binding protein 2) on different neuronal genes, we investigated the role of this complex in Ncx1 transcriptional regulation after stroke. METHODS AND RESULTS: Luciferase experiments performed in SH-SY5Y cells demonstrated that Br activity was selectively decreased by REST, whereas Ht activity was reduced by DNMT1, MeCP2, and REST. Notably, site-direct mutagenesis of Ht-RE1 prevented REST-dependent downregulation of Ncx1. Furthermore, in temporoparietal cortex of 8-week-old male wild-type mice (C57BL/6) subjected to transient middle cerebral artery occlusion, DNMT1, MeCP2, and REST binding to Ht promoter was increased, with a consequent DNA promoter hypermethylation. Intracerebroventricular injection of siREST prevented DNMT1/MeCP2 binding to Ht and Ncx1 downregulation, thus causing a reduction in stroke-induced damage. Consistently, in cortical neurons subjected to oxygen and glucose deprivation plus reoxygenation Ncx1 knockdown counteracted neuronal protection induced by the demethylating agent 5-azacytidine. For comparisons between 2 experimental groups, Student's t test was used, whereas for more than 2 experimental groups, 1-way ANOVA was used, followed by Tukey or Newman Keuls. Statistical significance was set at P<0.05. CONCLUSIONS: If the results of this study are confirmed in humans, it could be asserted that DNMT1/MeCP2/REST complex disruption could be a new pharmacological strategy to reduce DNA methylation of Ht in the brain, ameliorating stroke damage.

Stroke by inducing HDAC9-dependent deacetylation of HIF-1 and Sp1, promotes TfR1 transcription and GPX4 reduction, thus determining ferroptotic neuronal death.

Background: The inhibition of histone deacetylase 9 (HDAC9) represents a promising druggable target for stroke intervention. Indeed, HDAC9 is overexpressed in neurons after brain ischemia where exerts a neurodetrimental role. However, mechanisms of HDAC9-dependent neuronal cell death are not yet well established. Methods: Brain ischemia was obtained in vitro by primary cortical neurons exposed to glucose deprivation plus reoxygenation (OGD/Rx) and in vivo by transient middle cerebral artery occlusion. Western blot and quantitative real-time polymerase chain reaction were used to evaluate transcript and protein levels. Chromatin immunoprecipitation was used to evaluate the binding of transcription factors to the promoter of target genes. Cell viability was measured by MTT and LDH assays. Ferroptosis was evaluated by iron overload and 4-hydroxynonenal (4-HNE) release. Results: Our results showed that HDAC9 binds to hypoxia-inducible factor 1 (HIF-1) and specificity protein 1 (Sp1), two transcription activators of transferrin 1 receptor (TfR1) and glutathione peroxidase 4 (GPX4) genes, respectively, in neuronal cells exposed to OGD/Rx. Consequently, HDAC9 induced: (1) an increase in protein level of HIF-1 by deacetylation and deubiquitination, thus promoting the transcription of the pro-ferroptotic TfR1 gene; and (2) a reduction in Sp1 protein levels by deacetylation and ubiquitination, thus resulting in a down-regulation of the anti-ferroptotic GPX4 gene. Supporting these results, the silencing of HDAC9 partially prevented either HIF-1 increase and Sp1 reduction after OGD/Rx. Interestingly, silencing of the neurodetrimental factors, HDAC9, HIF-1, or TfR1 or the overexpression of the prosurvival factors Sp1 or GPX4 significantly reduced a well-known marker of ferroptosis 4-HNE after OGD/Rx. More important, in vivo, intracerebroventricular injection of siHDAC9 reduced 4-HNE levels after stroke by preventing: (1) HIF-1 and TfR1 increase and thus the augmented intracellular iron overload; and (2) a reduction of Sp1 and its target gene GPX4. Conclusions: Collectively, results obtained suggest that HDAC9 mediates post-traslational modifications of HIF-1 and Sp1 that, in turn, increases TfR1 and decreases GPX4 expression, thus promoting neuronal ferroptosis in in vitro and in vivo models of stroke.

The Transcriptional Complex Sp1/KMT2A by Up-Regulating Restrictive Element 1 Silencing Transcription Factor Accelerates Methylmercury-Induced Cell Death in Motor Neuron-Like NSC34 Cells Overexpressing SOD1-G93A.

Methylmercury (MeHg) exposure has been related to amyotrophic lateral sclerosis (ALS) pathogenesis and molecular mechanisms of its neurotoxicity has been associated to an overexpression of the Restrictive Element 1 Silencing Transcription factor (REST). Herein, we evaluated the possibility that MeHg could accelerate neuronal death of the motor neuron-like NSC34 cells transiently overexpressing the human Cu2+/Zn2+superoxide dismutase 1 (SOD1) gene mutated at glycine 93 (SOD1-G93A). Indeed, SOD1-G93A cells exposed to 100 nM MeHg for 24 h showed a reduction in cell viability, as compared to cells transfected with empty vector or with unmutated SOD1 construct. Interestingly, cell survival reduction in SOD1-G93A cells was associated with an increase of REST mRNA and protein levels. Furthermore, MeHg increased the expression of the transcriptional factor Sp1 and promoted its binding to REST gene promoter sequence. Notably, Sp1 knockdown reverted MeHg-induced REST increase. Co-immunoprecipitation experiments demonstrated that Sp1 physically interacted with the epigenetic writer Lysine-Methyltransferase-2A (KMT2A). Moreover, knocking-down of KMT2A reduced MeHg-induced REST mRNA and protein increase in SOD1-G93A cells. Finally, we found that MeHg-induced REST up-regulation triggered necropoptotic cell death, monitored by RIPK1 increased protein expression. Interestingly, REST knockdown or treatment with the necroptosis inhibitor Necrostatin-1 (Nec) decelerated MeH-induced cell death in SOD1-G93A cells. Collectively, this study demonstrated that MeHg hastens necroptotic cell death in SOD1-G93A cells via Sp1/KMT2A complex, that by epigenetic mechanisms increases REST gene expression.

GATA3 (GATA-Binding Protein 3)/KMT2A (Lysine-Methyltransferase-2A) Complex by Increasing H3K4-3me (Trimethylated Lysine-4 of Histone-3) Upregulates NCX3 (Na+-Ca2+ Exchanger 3) Transcription and Contributes to Ischemic Preconditioning Neuroprotection.

Background and Purpose: NCX3 (Na+-Ca2+ exchanger 3) plays a relevant role in stroke; indeed its pharmacological blockade or its genetic ablation exacerbates brain ischemic damage, whereas its upregulation takes part in the neuroprotection elicited by ischemic preconditioning. To identify an effective strategy to induce an overexpression of NCX3, we examined transcription factors and epigenetic mechanisms potentially involved in NCX3 gene regulation. Methods: Brain ischemia and ischemic preconditioning were induced in vitro by exposure of cortical neurons to oxygen and glucose deprivation plus reoxygenation (OGD/Reoxy) and in vivo by transient middle cerebral artery occlusion. Western blot and quantitative real-time polymerase chain reaction were used to evaluate transcripts and proteins of GATA3 (GATA-binding protein 3), KMT2A (lysine-methyltransferase-2A), and NCX3. GATA3 and KMT2A binding on NCX3 gene was evaluated by chromatin immunoprecipitation and Rechromatin immunoprecipitation experiments. Results: Among the putative transcription factors sharing a consensus sequence on the ncx3 brain promoter region, GATA3 was the only able to up-regulate ncx3. Interestingly, GATA3 physically interacted with KMT2A, and their overexpression or knocking-down increased or downregulated NCX3 mRNA and protein, respectively. Notably, site-direct mutagenesis of GATA site on ncx3 brain promoter region counteracted GATA3 and KMT2A binding on NCX3 gene. More importantly, we found that in the perischemic cortical regions of preconditioned rats GATA3 recruited KMT2A and the complex H3K4-3me (trimethylated lysine-4 of histone-3) on ncx3 brain promoter region, thus reducing transient middle cerebral artery occlusion­induced damage. Consistently, in vivo silencing of either GATA3 or KMT2A prevented NCX3 upregulation and consequently the neuroprotective effect of preconditioning stimulus. The involvement of GATA3/KMT2A complex in neuroprotection elicited by ischemic preconditioning was further confirmed by in vitro experiments in which the knocking-down of GATA3 and KMT2A reverted the neuroprotection induced by NCX3 overexpression in cortical neurons exposed to anoxic preconditioning followed by oxygen and glucose deprivation plus reoxygenation. Conclusions: Collectively, our results revealed that GATA3/KMT2A complex epigenetically activates NCX3 gene transcription during ischemic preconditioning.

CD4+ T Cell Defects in a Mulibrey Patient With Specific TRIM37 Mutations.

Mulibrey (muscle-liver-brain-eye) syndrome (MUL) is an autosomal recessive disorder caused by mutations in the TRIpartite motif (TRIM)37 gene, encoding for TRIM37 a member of the TRIM E3 ubiquitin ligase protein family. MUL patients are characterized by growth retardation, dysmorphic features, and a wide range of abnormalities affecting different organs. However, T-cell abnormalities have not been observed in MUL subjects, to date. Here we described the immunological features of a MUL child carrying recently identified TRIM37 mutations, a 17q22 deletion of maternal origin combined with a TRIM37 variant of paternal origin. Here we found quantitative and functional defects in CD4+ T cells from this MUL case. Low levels of TRIM37 protein were specifically detected in CD4+ T cells of MUL patient and associated with their altered proliferation and cytokine production. Of note, both CD4+ and CD8+ T lymphocytes of MUL child displayed an effector memory phenotype compared with healthy children. This clinical case research highlighted the possible role of TRIM37 in the control of immune cell number and function, especially in CD4+ T cells. Finally, this study may contribute to the novel mechanistic studies aim of identifying, in depth, the role of the TRIM37 protein in the immune system.

Transcriptional and epigenetic regulation of ncx1 and ncx3 in the brain.

Sodium-calcium exchanger (NCX) 1 and 3, have been demonstrated to play a relevant role in controlling the intracellular homeostasis of sodium and calcium ions in physiological and patho-physiological conditions. While NCX1 and NCX3 knocking-down have been both implicated in brain ischemia, several aspects of the epigenetic regulation of these two antiporters transcription were not yet well characterized. In response to stroke, NCX1 and NCX3 transcriptional regulation occurs from specific promoter sequences. Several evidences have shown that the expression of NCX1 and NCX3 can be determined by epigenetic modifications, consisting in changes of the histone acetylation levels on their promoter sequences. An interesting issue is that histone modifications at the NCX1 and NCX3 promoters could be linked to neurodegeneration occurring after stroke. Therefore, identifying the epigenetic regulation at the NCX1 and NCX3 promoters could permit to identify new molecular targets that can open new strategies for stroke treatment. The current review reassumes the recent knowledge of histone modifications of NCX1 and NCX3 genes in brain in physiological and patho-physiological conditions.

HDAC4 and HDAC5 form a complex with DREAM that epigenetically down-regulates NCX3 gene and its pharmacological inhibition reduces neuronal stroke damage.

The histone deacetylases (HDACs)-dependent mechanisms regulating gene transcription of the Na+/Ca+ exchanger isoform 3 (ncx3) after stroke are still unknown. Overexpression or knocking-down of HDAC4/HDAC5 down-regulates or increases, respectively, NCX3 mRNA and protein. Likewise, MC1568 (class IIa HDACs inhibitor), but not MS-275 (class I HDACs inhibitor) increased NCX3 promoter activity, gene and protein expression. Furthermore, HDAC4 and HDAC5 physically interacted with the transcription factor downstream regulatory element antagonist modulator (DREAM). As MC1568, DREAM knocking-down prevented HDAC4 and HDAC5 recruitment to the ncx3 promoter. Importantly, DREAM, HDAC4, and HDAC5 recruitment to the ncx3 gene was increased in the temporoparietal cortex of rats subjected to transient middle cerebral artery occlusion (tMCAO), with a consequent histone-deacetylation of ncx3 promoter. Conversely, the tMCAO-induced NCX3 reduction was prevented by intracerebroventricular injection of siDREAM, siHDAC4, and siHDAC5. Notably, MC1568 prevented oxygen glucose deprivation plus reoxygenation and tMCAO-induced neuronal damage, whereas its neuroprotective effect was abolished by ncx3 knockdown. Collectively, we found that: (1) DREAM/HDAC4/HDAC5 complex epigenetically down-regulates ncx3 gene transcription after stroke, and (2) pharmacological inhibition of class IIa HDACs reduces stroke-induced neurodetrimental effects.

Resveratrol treatment reduces the vulnerability of SH-SY5Y cells and cortical neurons overexpressing SOD1-G93A to Thimerosal toxicity through SIRT1/DREAM/PDYN pathway.

In humans, mutation of glycine 93 to alanine of Cu++/Zn++ superoxide dismutase type-1 (SOD1-G93 A) has been associated to some familial cases of Amyotrophic Lateral Sclerosis (ALS). Several evidence proposed the involvement of environmental pollutants that like mercury could accelerate ALS symptoms. SH-SY5Y cells stably transfected with SOD1 and G93 A mutant of SOD1 constructs were exposed to non-toxic concentrations (0.01 µM) of ethylmercury thiosalicylate (thimerosal) for 24 h. Interestingly, we found that thimerosal, in SOD1-G93 A cells, but not in SOD1 cells, reduced cell survival. Furthermore, thimerosal-induced cell death occurred in a concentration dependent-manner and was prevented by the Sirtuin 1 (SIRT1) activator Resveratrol (RSV). Moreover, thimerosal decreased the protein expression of transcription factor Downstream Regulatory Element Antagonist Modulator (DREAM), but not DREAM gene. Interestingly, DREAM reduction was blocked by co-treatment with RSV, suggesting the participation of SIRT1 in determining this effect. Immunoprecipitation experiments in SOD1-G93 A cells exposed to thimerosal demonstrated that RSV increased DREAM deacetylation and reduced its polyubiquitination. In addition, RSV counteracted thimerosal-enhanced prodynorphin (PDYN) mRNA, a DREAM target gene. Furthermore, cortical neurons transiently transfected with SOD1-G93 A construct and exposed to thimerosal (0.5 µM/24 h) showed a reduction of DREAM and an up-regulation of the prodynorphin gene. Importantly, both the treatment with RSV or the transfection of siRNA against prodynorphin significantly reduced thimerosal-induced neurotoxicity, while DREAM knocking-down potentiated thimerosal-reduced cell survival. These results demonstrate the particular vulnerability of SOD1-G93 A neuronal cells to thimerosal and that RSV via SIRT1 counteracts the neurodetrimental effect of this toxicant by preventing DREAM reduction and prodynorphin up-regulation.

The miR206-JunD Circuit Mediates the Neurotoxic Effect of Methylmercury in Cortical Neurons.

Methylmercury (MeHg) causes neuronal death through different pathways. Particularly, we found that in cortical neurons it increased the expression of Repressor Element-1 Silencing Transcription Factor (REST), histone deacetylase (HDAC)4, Specificity Protein (Sp)1, Sp4, and reduced the levels of brain-derived neurotrophic factor (BDNF). Herein, in rat cortical neurons we investigated whether microRNA (miR)206 can modulate MeHg-induced cell death by regulating REST/HDAC4/Sp1/Sp4/BDNF axis. MeHg (1 µM) reduced miR206 expression after both 12 and 24 h and miR206 transfection prevented MeHg-induced neuronal death. Furthermore, miR206 reverted MeHg-induced REST and Sp4 increase and BDNF reduction at gene and protein level, and reverted HDAC4 protein increase, but not HDAC4 mRNA upregulation. Moreover, since no miR206 seed sequences were identified in the 3'-untranslated regions (3'-UTRs) of REST and SP4, we investigated the role of JunD, that presents a consensus motif on REST, Sp4, and BDNF promoters. Indeed, MeHg increased JunD mRNA and protein levels, and JunD knockdown counteracted MeHg-induced REST, Sp4 increase, but not BDNF reduction. Furthermore, we identified a miR206 binding site in the 3'-UTR of JunD mRNA (miR206/JunD) and mutagenesis of miR206/JunD site reverted JunD luciferase activity reduction induced by miR206. Finally, miR206 prevented MeHg-increased JunD binding to REST and Sp4 promoters, and MeHg-reduced BDNF expression was determined by the increase of HDAC4 binding on BDNF promoter IV. Collectively, these results suggest that miR206 downregulation induced by MeHg exposure determines an upregulation of HDAC4, that in turn downregulated BDNF, and the activation of JunD that, by binding REST and Sp4 gene promoters, increased their expression.

The neurotoxicant PCB-95 by increasing the neuronal transcriptional repressor REST down-regulates caspase-8 and increases Ripk1, Ripk3 and MLKL expression determining necroptotic neuronal death.

Our previous study showed that the environmental neurotoxicant non-dioxin-like polychlorinated biphenyl (PCB)-95 increases RE1-silencing transcription factor (REST) expression, which is related to necrosis, but not apoptosis, of neurons. Meanwhile, necroptosis is a type of a programmed necrosis that is positively regulated by receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL) and negatively regulated by caspase-8. Here we evaluated whether necroptosis contributes to PCB-95-induced neuronal death through REST up-regulation. Our results demonstrated that in cortical neurons PCB-95 increased RIPK1, RIPK3, and MLKL expression and decreased caspase-8 at the gene and protein level. Furthermore, the RIPK1 inhibitor necrostatin-1 or siRNA-mediated RIPK1, RIPK3 and MLKL expression knockdown significantly reduced PCB-95-induced neuronal death. Intriguingly, PCB-95-induced increases in RIPK1, RIPK3, MLKL expression and decreases in caspase-8 expression were reversed by knockdown of REST expression with a REST-specific siRNA (siREST). Notably, in silico analysis of the rat genome identified a REST consensus sequence in the caspase-8 gene promoter (Casp8-RE1), but not the RIPK1, RIPK3 and MLKL promoters. Interestingly, in PCB-95-treated neurons, REST binding to the Casp8-RE1 sequence increased in parallel with a reduction in its promoter activity, whereas under the same experimental conditions, transfection of siREST or mutation of the Casp8-RE1 sequence blocked PCB-95-induced caspase-8 reduction. Since RIPK1, RIPK3 and MLKL rat genes showed no putative REST binding site, we assessed whether the transcription factor cAMP Responsive Element Binding Protein (CREB), which has a consensus sequence in all three genes, affected neuronal death. In neurons treated with PCB-95, CREB protein expression decreased in parallel with a reduction in binding to the RIPK1, RIPK3 and MLKL gene promoter sequence. Furthermore, CREB overexpression was associated with reduced promoter activity of the RIPK1, RIPK3 and MLKL genes. Collectively, these results indicate that PCB-95 was associated with REST-induced necroptotic cell death by increasing RIPK1, RIPK3 and MLKL expression and reducing caspase-8 levels. In addition, since REST is involved in several neurological disorders, therapies that block REST-induced necroptosis could be a new strategy to revert the neurodetrimental effects associated to its overexpression.

p38/Sp1/Sp4/HDAC4/BDNF Axis Is a Novel Molecular Pathway of the Neurotoxic Effect of the Methylmercury.

The molecular pathways involved in methylmercury (MeHg)-induced neurotoxicity are not fully understood. Since pan-Histone deacetylases (HDACs) inhibition has been found to revert the neurodetrimental effect of MeHg, it appeared of interest to investigate whether the pattern of HDACs isoform protein expression is modified during MeHg-induced neurotoxicity and the transcriptional/transductional mechanisms involved. SH-SY5Y neuroblastoma cells treated with MeHg 1 µM for 12 and 24 h showed a significant increase of HDAC4 protein and gene expression, whereas the HDACs isoforms 1-3, 5, and 6 were unmodified. Furthermore, MeHg-induced HDAC4 increase was reverted when cells were transfected with siRNAs against specificity protein 1 (Sp1) and Sp4, that were both increased during MeHg exposure. Next we studied the role of extracellular-signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs) in MeHg-induced increase of Sp1, Sp4, and HDAC4 expression. As shown by Western Blot analysis MeHg exposure increased the phosphorylation of p38, but not of ERK and JNK. Notably, when p38 was pharmacologically blocked, MeHg-induced Sp1, Sp4 protein expression, and HDAC4 protein and gene expression was reverted. In addition, MeHg exposure increased the binding of HDAC4 to the promoter IV of the Brain-derived neurotrophic factor (BDNF) gene, determining its mRNA reduction, that was significantly counteracted by HDAC4 knocking down. Furthermore, rat cortical neurons exposed to MeHg (1 µM/24 h) showed an increased phosphorylation of p38, in parallel with an up-regulation of Sp1, Sp4, and HDAC4 and a down-regulation of BDNF proteins. Importantly, transfection of siRNAs against p38, Sp1, Sp4, and HDAC4 or transfection of vector overexpressing BDNF significantly blocked MeHg-induced cell death in cortical neurons. All these results suggest that p38/Sp1-Sp4/HDAC4/BDNF may represent a new pathway involved in MeHg-induced neurotoxicity.

Possible Therapeutic Effects of Ozone Mixture on Hypoxia in Tumor Development.

Recent literature highlights that ozone therapy could be considered a viable adjuvant therapy in oncological patients receiving radio-chemotherapy. The use of ozone therapy in these patients enhances the action of chemotherapy and at the same time reduces side-effects, such as nausea, vomiting, opportunistic infections, buccal ulcers, hair loss and fatigue. Such positive therapeutic effects of ozone therapy can cause a larger physical and mental wellbeing resulting in improved quality of life. This work reviews the recent acquisition of scientific knowledge regarding the ozone therapy and highlights the molecular and cellular pathways involved.

MC1568 Inhibits Thimerosal-Induced Apoptotic Cell Death by Preventing HDAC4 Up-Regulation in Neuronal Cells and in Rat Prefrontal Cortex.

Ethylmercury thiosalicylate (thimerosal) is an organic mercury-based compound commonly used as an antimicrobial preservative that has been found to be neurotoxic. In contrast, histone deacetylases (HDACs) inhibition has been found to be neuroprotective against several environmental contaminants, such as polychlorinated biphenyls, di-2-ethylhexyl phthalate, and methylmercury. The aim of this study was to investigate the effect of HDAC inhibition on thimerosal-induced neurotoxicity in neuroblastoma cells and cortical neurons. Interestingly, we found that thimerosal, at 0.5 µM in SH-SY5Y cells and at 1 µM in neurons, caused cell death by activation of apoptosis, which was prevented by the HDAC class IIA inhibitor MC1568 but not the class I inhibitor MS275. Furthermore, thimerosal specifically increased HDAC4 protein expression but not that of HDACs 5, 6, 7, and 9. Western blot analysis revealed that MC1568 prevented thimerosal-induced HDAC4 increase. In addition, both HDAC4 knocking-down and MC1568 inhibited thimerosal-induced cell death in SH-SY5Y cells and cortical neurons. Importantly, intramuscular injection of 12 µg/kg thimerosal on postnatal days 7, 9, 11, and 15 increased HDAC4 levels in the prefrontal cortex (PFC), which decreased histone H4 acetylation in infant male rats, in parallel increased motor activity changes. In addition, coadministration of 40 mg/kg MC1568 (intraperitoneal injection) moderated the HDAC4 increase which reduced histone H4 deacetylation and caspase-3 cleavage in the PFC. Finally, open-field testing showed that thimerosal-induced motor activity changes are reduced by MC1568. These findings indicate that HDAC4 regulates thimerosal-induced cell death in neurons and that treatment with MC1568 prevents thimerosal-induced activation of caspase-3 in the rat PFC.

MS-275 inhibits aroclor 1254-induced SH-SY5Y neuronal cell toxicity by preventing the formation of the HDAC3/REST complex on the synapsin-1 promoter.

Polychlorinated biphenyl (PCB) exposure has been associated with neurodegenerative diseases, such as Parkinson's disease, amyotrophic lateral sclerosis, and dementia. Neuronal death elicited by the PCB mixture Aroclor 1254 (A1254) has been attributed to an increase in RE-1-silencing transcription factor (REST), which, in turn, correlates with a decrease in the synapsin-1 promoter gene. Although histone deacetylase (HDAC) inhibitors are known to be neuroprotective in several neurologic disorders, the core mechanisms governing this effect are not yet understood. Here, to examine how HDAC class I [N-(2-aminophenyl)-4-[N-(pyridin-3-yl-methoxycarbonyl)aminomethyl]-benzamide (MS-275)] and HDAC class II [3-[5-(3-(3-fluorophenyl)-3-oxopropen-1-yl)-1-methyl-1H-pyrrol-2-yl]-N-hydroxy-2-propenamide (MC-1568)] inhibitors prevent A1254-induced neuronal cell death, we exposed SH-SY5Y neuroblastoma cells to A1254. Exposure to A1254 (30.6 µM) for 24 and 48 hours resulted in a time-dependent cell death. Indeed, after 48 hours, MS-275, but not MC-1568, reverted A1254-induced cell death in a dose-dependent manner. Furthermore, A1254 significantly increased HDAC3, but not HDAC1 or HDAC2. Interestingly, REST physically interacted with HDAC3 after A1254 exposure. Chromatin immunoprecipitation assays revealed that MS-275 reverted the increased levels of HDAC3 binding and decreased acetylation of histone H3 within the synapsin-1 promoter region, thus reverting synapsin-1 mRNA reduction. Moreover, REST knockdown by small interfering RNA (siRNA) prevented HDAC3 from binding to the synapsin-1 promoter. Likewise, HDAC3 siRNA significantly reduced A1254-induced cell toxicity in SH-SY5Y cells and cortical neurons. Hence, this study demonstrates that inhibition of HDAC class I attenuates A1254-induced neuronal cell death by preventing HDAC3 binding and histone deacetylation within the synapsin-1 promoter region.

Use of a non-specific immunomodulation therapy as a therapeutic vasculogenesis strategy in no-option critical limb ischemia patients.

BACKGROUND/AIMS: Inflammatory mediators contribute to the impairment of vasculogenesis by reducing endothelial progenitor cells (EPCs) mobilization in atherosclerotic vasculopathy. We tested the hypothesis that administration of an oxygen/ozone mixture (IMT) might counteract this pathophysiological mechanism and enhance limb tissue perfusion in patients with critical limb ischemia (CLI). METHODS: Randomized patients with rest pain or ischemic ulcers and transcutaneous oxygen tension (TcPO(2)) <40 mmHg and/or toe pressure <50 mmHg received placebo (n=74) or a non-specific immunomodulation therapy (IMT) (n=77), autologous blood exposed to oxygen/ozone gas mixture by intragluteal injection, on day 1, 2, 7, and once a week thereafter for at least 22 weeks. Patients were evaluated for changes in TcPO(2), levels of circulating EPCs (CD34/KDR-positive cells) and inflammation (tumor necrosis factor-alpha-TNF-alpha). RESULTS: TcPO(2) and CD34/CD133-positive cells increased at 22 weeks in IMT group (P<0.01) whereas no changes were observed in placebo group. TNF-alpha levels decreased at 6 months in IMT group (P<0.001) whereas no changes were observed in placebo group. There was a strong positive correlation between CD34/KDR-positive cells and TcPO(2) (r=0.56, P<0.01). Moreover, there was an inverse correlation between CD34/KDR-positive cells and TNF-alpha (r=-0.51, P<0.01). CONCLUSIONS: Intramuscular injection of IMT may improve wound healing and limb salvage in patients with CLI.