The hypoxia sensitive metal transcription factor MTF-1 activates NCX1 brain promoter and participates in remote postconditioning neuroprotection in stroke

Remote limb ischemic postconditioning (RLIP) is an experimental strategy in which short femoral artery ischemia reduces brain damage induced by a previous harmful ischemic insult. Ionic homeostasis maintenance in the CNS seems to play a relevant role in mediating RLIP neuroprotection and among the effectors, the sodium-calcium exchanger 1 (NCX1) may give an important contribution, being expressed in all CNS cells involved in brain ischemic pathophysiology. The aim of this work was to investigate whether the metal responsive transcription factor 1 (MTF-1), an important hypoxia sensitive transcription factor, may (i) interact and regulate NCX1, and (ii) play a role in the neuroprotective effect mediated by RLIP through NCX1 activation. Here we demonstrated that in brain ischemia induced by transient middle cerebral occlusion (tMCAO), MTF-1 is triggered by a subsequent temporary femoral artery occlusion (FAO) and represents a mediator of endogenous neuroprotection. More importantly, we showed that MTF-1 translocates to the nucleus where it binds the metal responsive element (MRE) located at −23/−17 bp of Ncx1 brain promoter thus activating its transcription and inducing an upregulation of NCX1 that has been demonstrated to be neuroprotective. Furthermore, RLIP restored MTF-1 and NCX1 protein levels in the ischemic rat brain cortex and the silencing of MTF-1 prevented the increase of NCX1 observed in RLIP protected rats, thus demonstrating a direct regulation of NCX1 by MTF-1 in the ischemic cortex of rat exposed to tMCAO followed by FAO. Moreover, silencing of MTF-1 significantly reduced the neuroprotective effect elicited by RLIP as demonstrated by the enlargement of brain infarct volume observed in rats subjected to RLIP and treated with MTF-1 silencing. Overall, MTF-dependent activation of NCX1 and their upregulation elicited by RLIP, besides unraveling a new molecular pathway of neuroprotection during brain ischemia, might represent an additional mechanism to intervene in stroke pathophysiology

Multiplicative noise: A mechanism leading to nonextensive statistical mechanics

A large variety of microscopic or mesoscopic models lead to generic results that accommodate naturally within Boltzmann-Gibbs statistical mechanics (based on $S_1\equiv -k \int du p(u) \ln p(u)$). Similarly, other classes of models point toward nonextensive statistical mechanics (based on $S_q \equiv k [1-\int du [p(u)]^q]/[q-1]$, where the value of the entropic index $q\in\Re$ depends on the specific model). We show here a family of models, with multiplicative noise, which belongs to the nonextensive class. More specifically, we consider Langevin equations of the type $\dot{u}=f(u)+g(u)\xi(t)+\eta(t)$, where $\xi(t)$ and $\eta(t)$ are independent zero-mean Gaussian white noises with respective amplitudes $M$ and $A$. This leads to the Fokker-Planck equation $\partial_t P(u,t) = -\partial_u[f(u) P(u,t)] + M\partial_u\{g(u)\partial_u[g(u)P(u,t)]\} + A\partial_{uu}P(u,t)$. Whenever the deterministic drift is proportional to the noise induced one, i.e., $f(u) =-\tau g(u) g'(u)$, the stationary solution is shown to be $P(u, \infty) \propto \bigl\{1-(1-q) \beta [g(u)]^2 \bigr\}^{\frac{1}{1-q}}$ (with $q \equiv \frac{\tau + 3M}{\tau+M}$ and $\beta=\frac{\tau+M}{2A}$). This distribution is precisely the one optimizing $S_q$ with the constraint $_q \equiv \{\int du [g(u)]^2[P(u)]^q \}/ \{\int du [P(u)]^q \}=$constant. We also introduce and discuss various characterizations of the width of the distributions.Comment: 3 PS figure

miR-16-5p, miR-103-3p, and miR-27b-3p as Early Peripheral Biomarkers of Fetal Growth Restriction

Current tests available to diagnose fetal hypoxia in-utero lack sensitivity thus failing to identify many fetuses at risk. Emerging evidence suggests that microRNAs derived from the placenta circulate in the maternal blood during pregnancy and may be used as non-invasive biomarkers for pregnancy complications. With the intent to identify putative markers of fetal growth restriction (FGR) and new therapeutic druggable targets, we examined, in maternal blood samples, the expression of a group of microRNAs, known to be regulated by hypoxia. The expression of microRNAs was evaluated in maternal plasma samples collected from (1) women carrying a preterm FGR fetus (FGR group) or (2) women with an appropriately grown fetus matched at the same gestational age (Control group). To discriminate between early- and late-onset FGR, the study population was divided into two subgroups according to the gestational age at delivery. Four microRNAs were identified as possible candidates for the diagnosis of FGR: miR-16-5p, miR-103-3p, miR-107-3p, and miR-27b-3p. All four selected miRNAs, measured by RT-PCR, resulted upregulated in FGR blood samples before the 32nd week of gestation. By contrast, miRNA103-3p and miRNA107-3p, analyzed between the 32nd and 37th week of gestation, showed lower expression in the FGR group compared to aged matched controls. Our results showed that measurement of miRNAs in maternal blood may form the basis for a future diagnostic test to determine the degree of fetal hypoxia in FGR, thus allowing the start of appropriate therapeutic interventions to alleviate the burden of this disease

miR-16-5p, miR-103-3p, and miR-27b-3p as Early Peripheral Biomarkers of Fetal Growth Restriction

none9noCurrent tests available to diagnose fetal hypoxia in-utero lack sensitivity thus failing to identify many fetuses at risk. Emerging evidence suggests that microRNAs derived from the placenta circulate in the maternal blood during pregnancy and may be used as non-invasive biomarkers for pregnancy complications. With the intent to identify putative markers of fetal growth restriction (FGR) and new therapeutic druggable targets, we examined, in maternal blood samples, the expression of a group of microRNAs, known to be regulated by hypoxia. The expression of microRNAs was evaluated in maternal plasma samples collected from (1) women carrying a preterm FGR fetus (FGR group) or (2) women with an appropriately grown fetus matched at the same gestational age (Control group). To discriminate between early- and late-onset FGR, the study population was divided into two subgroups according to the gestational age at delivery. Four microRNAs were identified as possible candidates for the diagnosis of FGR: miR-16-5p, miR-103-3p, miR-107-3p, and miR-27b-3p. All four selected miRNAs, measured by RT-PCR, resulted upregulated in FGR blood samples before the 32nd week of gestation. By contrast, miRNA103-3p and miRNA107-3p, analyzed between the 32nd and 37th week of gestation, showed lower expression in the FGR group compared to aged matched controls. Our results showed that measurement of miRNAs in maternal blood may form the basis for a future diagnostic test to determine the degree of fetal hypoxia in FGR, thus allowing the start of appropriate therapeutic interventions to alleviate the burden of this disease.openTagliaferri S.; Cepparulo P.; Vinciguerra A.; Campanile M.; Esposito G.; Maruotti G.M.; Zullo F.; Annunziato L.; Pignataro G.Tagliaferri, S.; Cepparulo, P.; Vinciguerra, A.; Campanile, M.; Esposito, G.; Maruotti, G. M.; Zullo, F.; Annunziato, L.; Pignataro, G

Scaling detection in time series: diffusion entropy analysis

The methods currently used to determine the scaling exponent of a complex dynamic process described by a time series are based on the numerical evaluation of variance. This means that all of them can be safely applied only to the case where ordinary statistical properties hold true even if strange kinetics are involved. We illustrate a method of statistical analysis based on the Shannon entropy of the diffusion process generated by the time series, called Diffusion Entropy Analysis (DEA). We adopt artificial Gauss and L\'{e}vy time series, as prototypes of ordinary and anomalus statistics, respectively, and we analyse them with the DEA and four ordinary methods of analysis, some of which are very popular. We show that the DEA determines the correct scaling exponent even when the statistical properties, as well as the dynamic properties, are anomalous. The other four methods produce correct results in the Gauss case but fail to detect the correct scaling in the case of L\'{e}vy statistics.Comment: 21 pages,10 figures, 1 tabl

Targeted acetylation of NF-kappaB/RelA and histones by epigenetic drugs reduces post-ischemic brain injury in mice with an extended therapeutic window.

Nuclear factor-kappaB (NF-κB) p50/RelA is a key molecule with a dual effect in the progression of ischemic stroke. In harmful ischemia, but not in preconditioning insult, neurotoxic activation of p50/RelA is characterized by RelA-specific acetylation at Lys310 (K310) and deacetylation at other Lys residues. The derangement of RelA acetylation is associated with activation of Bim promoter. Objective: With the aim of producing neuroprotection by correcting altered acetylation of RelA in brain ischemia, we combined the pharmacological inhibition of histone deacetylase (HDAC) 1-3, the enzymes known to reduce global RelA acetylation, and the activation of sirtuin 1, endowed with a specific deacetylase activity on the K310 residue of RelA. To afford this aim, we tested the clinically used HDAC 1-3 inhibitor entinostat (MS-275) and the sirtuin 1 activator resveratrol. Methods: We used the mouse model of transient middle cerebral artery occlusion (MCAO) and primary cortical neurons exposed to oxygen glucose deprivation (OGD). Results: The combined use of MS-275 and resveratrol, by restoring normal RelA acetylation, elicited a synergistic neuroprotection in neurons exposed to OGD. This effect correlated with MS-275 capability to increase total RelA acetylation and resveratrol capability to reduce RelA K310 acetylation through the activation of an AMP-activated protein kinase-sirtuin 1 pathway. The synergistic treatment reproduced the acetylation state of RelA peculiar of preconditioning ischemia. Neurons exposed to the combined drugs totally recovered the optimal histone H3 acetylation.Neuroprotection was reproduced in mice subjected to MCAO and treated with MS-275 (20μg/kg and 200μg/kg) or resveratrol (6800μg/kg) individually. However, the administration of lowest doses of MS-275 (2μg/kg) and resveratrol (68μg/kg) synergistically reduced infarct volume and neurological deficits. Importantly, the treatment was effective even when administered 7h after the stroke onset. Chromatin immunoprecipitation analysis of cortices harvested from treated mice showed that the RelA binding and histone acetylation increased at the Bcl-x L promoter and decreased at the Bim promoter. Conclusion: Our study reveals that epigenetic therapy shaping acetylation of both RelA and histones may be a promising strategy to limit post-ischemic injury with an extended therapeutic window

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

Diffusion entropy and waiting time statistics of hard x-ray solar flares

We analyze the waiting time distribution of time distances $\tau$ between two nearest-neighbor flares. This analysis is based on the joint use of two distinct techniques. The first is the direct evaluation of the distribution function $\psi(\tau)$, or of the probability, $\Psi(tau)$, that no time distance smaller than a given $\tau$ is found. We adopt the paradigm of the inverse power law behavior, and we focus on the determination of the inverse power index $\mu$, without ruling out different asymptotic properties that might be revealed, at larger scales, with the help of richer statistics. The second technique, called Diffusion Entropy (DE) method, rests on the evaluation of the entropy of the diffusion process generated by the time series. The details of the diffusion process depend on three different walking rules, which determine the form and the time duration of the transition to the scaling regime, as well as the scaling parameter $\delta$. With the first two rules the information contained in the time series is transmitted, to a great extent, to the transition, as well as to the scaling regime. The same information is essentially conveyed, by using the third rules, into the scaling regime, which, in fact, emerges very quickly after a fast transition process. We show that the significant information hidden within the time series concerns memory induced by the solar cycle, as well as the power index $\mu$. The scaling parameter $\delta$ becomes a simple function of $\mu$, when memory is annihilated. Thus, the three walking rules yield a unique and precise value of $\mu$ if the memory is wisely taken under control, or cancelled by shuffling the data. All this makes compelling the conclusion that $\mu = 2.138 \pm 0.01$.Comment: 23 pages, 13 figure

A novel hyperekplexia-causing mutation in the pre-transmembrane segment 1 of the human glycine receptor alpha1 subunit reduces membrane expression and impairs gating by agonists

In this study, we have compared the functional consequences of three mutations (R218Q, V260M, and Q266H) in the 1 subunit of the glycine receptor (GlyRA1) causing hyperekplexia, an inherited neurological channelopathy. In HEK-293 cells, the agonist EC50s for glycine- activated Cl currents were increased from 26 M in wtGlyRA1, to 5747, 135, and 129 M in R218Q, V260M, and Q266H GlyRA1 channels, respectively. Cl currents elicited by -alanine and taurine, which behave as agonists at wtGlyRA1, were decreased in V260M and Q266H mutant receptors and virtually abolished in GlyRA1 R218Q receptors. Gly-gated Cl currents were similarly antagonized by low concentrations of strychnine in both wild-type (wt) and R218Q GlyRA1 channels, suggesting that the Arg-218 residue plays a crucial role in GlyRA1 channel gating, with only minor effects on the agonist/ antagonist binding site, a hypothesis supported by our molecular model of the GlyRA1 subunit. The R218Q mutation, but not the V260M or the Q266H mutation, caused a marked decrease of receptor subunit expression both in total cell lysates and in isolated plasma membrane proteins. This decreased expression does not seem to explain the reduced agonist sensitivity of GlyRA1 R218Q channels since no difference in the apparent sensitivity to glycine or taurine was observed when wtGlyRA1 receptors were expressed at levels comparable with those of R218Q mutant receptors. In conclusion, multiple mechanisms may explain the dramatic decrease in GlyR function caused by the R218Q mutation, possibly providing the molecular basis for its association with a more severe clinical phenotype

miR135a administration ameliorates brain ischemic damage by preventing TRPM7 activation during brain ischemia

Background: miRNA-based strategies have recently emerged as a promising therapeutic approach in several neurodegenerative diseases. Unregulated cation influx is implicated in several cellular mechanisms underlying neural cell death during ischemia. The brain constitutively active isoform of transient receptor potential melastatin 7 (TRPM7) represents a glutamate excitotoxicity-independent pathway that significantly contributes to the pathological Ca2+ overload during ischemia. Aims: In the light of these premises, inhibition of TRPM7 may be a reasonable strategy to reduce ischemic injury. Since TRPM7 is a putative target of miRNA135a, the aim of the present paper was to evaluate the role played by miRNA135a in cerebral ischemia. Therefore, the specific objectives of the present paper were: (1) to evaluate miR135a expression in temporoparietal cortex of ischemic rats; (2) to investigate the effect of the intracerebroventricular (icv) infusion of miR135a on ischemic damage and neurological functions; and (3) to verify whether miR135a effects may be mediated by an alteration of TRPM7 expression. Methods: miR135a expression was evaluated by RT- PCR and FISH assay in temporoparietal cortex of ischemic rats. Ischemic volume and neurological functions were determined in rats subjected to transient middle cerebral artery occlusion (tMCAo) after miR135a intracerebroventricular perfusion. Target analysis was performed by Western blot. Results: Our results demonstrated that, in brain cortex, 72 h after ischemia, miR135a expression increased, while TRPM7 expression was parallelly downregulated. Interestingly, miR135a icv perfusion strongly ameliorated the ischemic damage and improved neurological functions, and downregulated TRPM7 protein levels. Conclusions: The early prevention of TRPM7 activation is protective during brain ischemia