Models and methods for conditioning the ischemic brain

Abstract Background In the last decades the need to find new neuroprotective targets has addressed the researchers to investigate the endogenous molecular mechanisms that brain activates when exposed to a conditioning stimulus. Indeed, conditioning is an adaptive biological process activated by those interventions able to confer resistance to a deleterious brain event through the exposure to a sub-threshold insult. Specifically, preconditioning and postconditioning are realized when the conditioning stimulus is applied before or after, respectively, the harmul ischemia. Aims and Results The present review will describe the most common methods to induce brain conditioning, with particular regards to surgical, physical exercise, temperature-induced and pharmacological approaches. It has been well recognized that when the subliminal stimulus is delivered after the ischemic insult, the achieved neuroprotection is comparable to that observed in models of ischemic preconditioning. In addition, subjecting the brain to both preconditioning as well as postconditioning did not cause greater protection than each treatment alone. Conclusions The last decades have provided fascinating insights into the mechanisms and potential application of strategies to induce brain conditioning. Since the identification of intrinsic cell‐survival pathways should provide more direct opportunities for translational neuroprotection trials, an accurate examination of the different models of preconditioning and postconditioning is mandatory before starting any new project

Resveratrol via sirtuin-1 downregulates RE1-silencing transcription factor (REST) expression preventing PCB-95-induced neuronal cell death

Resveratrol (3,5,4'-trihydroxystilbene) (RSV), a polyphenol widely present in plants, exerts a neuroprotective function in several neurological conditions; it is an activator of class III histone deacetylase sirtuin1 (SIRT1), a crucial regulator in the pathophysiology of neurodegenerative diseases. By contrast, the RE1-silencing transcription factor (REST) is involved in the neurotoxic effects following exposure to polychlorinated biphenyl (PCB) mixture A1254. The present study investigated the effects of RSV-induced activation of SIRT1 on REST expression in SH-SY5Y cells. Further, we investigated the possible relationship between the non-dioxin-like (NDL) PCB-95 and REST through SIRT1 to regulate neuronal death in rat cortical neurons. Our results revealed that RSV significantly decreased REST gene and protein levels in a dose- and time-dependent manner. Interestingly, overexpression of SIRT1 reduced REST expression, whereas EX-527, an inhibitor of SIRT1, increased REST expression and blocked RSV-induced REST downregulation. These results suggest that RSV downregulates REST through SIRT1. In addition, RSV enhanced activator protein 1 (AP-1) transcription factor c-Jun expression and its binding to the REST promoter gene. Indeed, c-Jun knockdown reverted RSV-induced REST downregulation. Intriguingly, in SH-SY5Y cells and rat cortical neurons the NDL PCB-95 induced necrotic cell death in a concentration-dependent manner by increasing REST mRNA and protein expression. In addition, SIRT1 knockdown blocked RSV-induced neuroprotection in rat cortical neurons treated with PCB-95. Collectively, these results indicate that RSV via SIRT1 activates c-Jun, thereby reducing REST expression in SH-SY5Y cells under physiological conditions and blocks PCB-95-induced neuronal cell death by activating the same SIRT1/c-Jun/REST pathway

resveratrol treatment reduces the vulnerability of sh sy5y cells and cortical neurons overexpressing sod1 g93a to thimerosal toxicity through sirt1 dream pdyn pathway

Abstract 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

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

Methylmercury upregulates RE-1 silencing transcription factor (REST) in SH-SY5Y cells and mouse cerebellum

Methylmercury (MeHg) is a highly neurotoxic compound that, in adequate doses, can cause damage to the brain, including developmental defects and in severe cases cell death. The RE-1-silencing transcription factor (REST) has been found to be involved in the neurotoxic effects of environmental pollutants such as polychlorinated biphenyls (PCBs). In this study, we investigated the effects of MeHg treatment on REST expression and its role in MeHg-induced neurotoxicity in neuroblastoma SH-SY5Y cells. We found that MeHg exposure caused a dose- and time- dependent apoptotic cell death, as evidenced by the appearance of apoptotic hallmarks including caspase-3 processing and annexin V uptake. Moreover, MeHg increased REST gene and gene product expression. MeHg-induced apoptotic cell death was completely abolished by REST knockdown. Interestingly, MeHg (1. μM/24. h) increased the expression of REST Corepressor (Co-REST) and its binding with REST whereas the other REST cofactor mammalian SIN3 homolog A transcription regulator (mSin3A) was not modified. In addition, we demonstrated that the acetylation of histone protein H4 was reduced after MeHg treatment and was critical for MeHg-induced apoptosis. Accordingly, the pan-histone deacetylase inhibitor trichostatin-A (TSA) prevented MeHg-induced histone protein H4 deacetylation, thereby reverting MeHg-induced neurotoxic effect. Male mice subcutaneously injected with 10 mg/kg of MeHg for 10 days showed an increase in REST expression in the granule cell layer of the cerebellum together with a decrease in histone H4 acetylation. Collectively, we demonstrated that methylmercury exposure can cause neurotoxicity by activating REST gene expression and H4 deacetylation

Prolonged NCX activation prevents SOD1 accumulation, reduces neuroinflammation, ameliorates motor behavior and prolongs survival in a ALS mouse model.

Abstract Imbalance in cellular ionic homeostasis is a hallmark of several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS). Sodium-calcium exchanger (NCX) is a membrane antiporter that, operating in a bidirectional way, couples the exchange of Ca2+ and Na + ions in neurons and glial cells, thus controlling the intracellular homeostasis of these ions. Among the three NCX genes, NCX1 and NCX2 are widely expressed within the CNS, while NCX3 is present only in skeletal muscles and at lower levels of expression in selected brain regions. ALS mice showed a reduction in the expression and activity of NCX1 and NCX2 consistent with disease progression, therefore we aimed to investigate their role in ALS pathophysiology. Notably, we demonstrated that the pharmacological activation of NCX1 and NCX2 by the prolonged treatment of SOD1G93A mice with the newly synthesized compound neurounina: (1) prevented the reduction in NCX activity observed in spinal cord; (2) preserved motor neurons survival in the ventral spinal horn of SOD1G93A mice; (3) prevented the spinal cord accumulation of misfolded SOD1; (4) reduced astroglia and microglia activation and spared the resident microglia cells in the spinal cord; (5) improved the lifespan and mitigated motor symptoms of ALS mice. The present study highlights the significant role of NCX1 and NCX2 in the pathophysiology of this neurodegenerative disorder and paves the way for the design of a new pharmacological approach for ALS

Identification of epigenetic mechanisms regulating the isoform 1 of sodium calcium exchanger (NCX1) in in vitro and in vivo models of brain ischemia and brain ischemic preconditioning

The Na+-Ca2+ exchanger 1 (NCX1) is reduced in stroke by the RE1-silencing transcription factor (REST), whereas it is increased in Ischemic Brain Preconditioning (PC) by Hypoxia-Inducible Factor 1 (HIF-1). Since ncx1 brain promoter (ncx1-Br) has five putative consensus sequences for the Specificity protein (Sp) family of transcription factors (Sp1, Sp2, Sp3, Sp4), named Sp1 A-E, we investigated the role of this family in regulating ncx1 transcription in cortical neurons. Here we found that Sp1 is a transcriptional activator, whereas Sp3 is a transcriptional repressor of ncx1, and that both bind ncx1 brain promoter (ncx1-Br) in a sequence-specific manner, modulating ncx1 transcription through the Sp1 sites C-E. Furthermore, in brain ischemia (tMCAO) the transcriptional repressors Sp3 and REST colocalize with histone-deacetylases (HDACs) HDAC1-2 at the ncx1 promoter, with a consequent hypoacetylation. By contrast, in PC+tMCAO the transcriptional activators Sp1 and HIF-1 colocalize with Histone-Acetyltransferase p300 (p300) on ncx1 promoter sequence with a consequent hyperacetylation. Moreover, when brain ischemia-induced REST and Sp3 up-regulation was prevented by intracerebroventricular (icv) injection of siRNAs for REST and Sp3, NCX1 downregulation was reverted. Interestingly, also siRNAs for HDAC1-2 was able to completely revert NCX1 reduction. On the contrary, siRNA for Sp1 and HIF-1, during PC+tMCAO, blocked NCX1 increase, that was almost completely reverted by double siRNA for HIF-1 and Sp1 or by siRNA for p300. In addition, in neurons transfected for siNCX1, and subjected to Oxygen Glucose Deprivation (OGD) (3 hours) plus Reoxygenation (RX) (24 hours), the protective effect of class I HDAC inhibitor MS-275 was counteracted, whereas neurons overexpressing NCX1 and subjected to ischemic preconditioning (PC+OGD\Rx), the detrimental effect of p300 inhibitor C646 was prevented. Collectively, these results demonstrate that NCX1 expression is regulated by Sp3\REST\HDAC1-2 complex in tMCAO and by the Sp1\HIF-1\p300 complex in PC+tMCAO, and that epigenetic therapy modulating the acetylation of ncx1 gene promoter may be a new strategy to reduce the neurodetrimental effect of stroke

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 regio

The NCX3 isoform of the Na+/Ca2+ exchanger contributes to neuroprotection elicited by ischemic postconditioning

It has been recently shown that a short sublethal brain ischemia subsequent to a prolonged harmful ischemic episode may confer ischemic neuroprotection, a phenomenon termed ischemic postconditioning. Na+/Ca2+ exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, are plasma membrane ionic transporters widely distributed in the brain and involved in the control of Na+ and Ca2+ homeostasis and in the progression of stroke damage. The objective of this study was to evaluate the role of these three proteins in the postconditioning-induced neuroprotection. The NCX protein and mRNA expression was evaluated at different time points in the ischemic temporoparietal cortex of rats subjected to tMCAO alone or to tMCAO plus ischemic postconditioning. The results of this study showed that NCX3 protein and ncx3 mRNA were upregulated in those brain regions protected by postconditioning treatment. These changes in NCX3 expression were mediated by the phosphorylated form of the ubiquitously expressed serine/threonine protein kinase p-AKT, as the p-AKT inhibition prevented NCX3 upregulation. The relevant role of NCX3 during postconditioning was further confirmed by results showing that NCX3 silencing, induced by intracerebroventricular infusion of small interfering RNA (siRNA), partially reverted the postconditioning-induced neuroprotection. The results of this study support the idea that the enhancement of NCX3 expression and activity might represent a reasonable strategy to reduce the infarct extension after stroke

Erratum: Silencing or knocking out the Na+/Ca2+ exchanger-3 (NCX3) impairs oligodendrocyte differentiation (Cell Death and Differentiation (2013) 20 (184) DOI: 10.1038/cdd.2012.139)

SCOPUS: er.jinfo:eu-repo/semantics/publishe