Objective: Current tests available to diagnose fetal hypoxia in-utero lack sensitivity thus un-diagnosing many fetuses at risk.microRNAs derived from the placenta circulate in the maternal blood during pregnancy and may be used as biomarkers for pregnancy complications.To identify putative markers of fetal growth restriction (FGR) and new therapeutic druggable targets, we examined, in maternal blood samples, the expression of a cluster of microRNAs, known to be regulated by hypoxia.Population: Pregnant Caucasian women between 18 and 46 years old hospitalized.Design and Setting: To discriminate between
To date, the only effective pharmacological treatment for ischemic stroke is limited to the clinical use of recombinant tissue plasminogen activator (rtPA), although endovascular therapy has also emerged as an effective treatment for acute ischemic stroke. Unfortunately, the benefit of this treatment is limited to a 4.5-h time window. Most importantly, the use of rtPA is contraindicated in the case of hemorrhagic stroke. Therefore, the identification of a reliable biomarker to distinguish hemorrhagic from ischemic stroke could provide several advantages, including an earlier diagnosis, a better treatment, and a faster decision on ruling out hemorrhage so that tPA may be administered earlier. microRNAs (miRNAs) are stable non-coding RNAs crucially involved in the downregulation of gene expression via mRNA cleavage or translational repression. In the present paper, taking advantage of three preclinical animal models of stroke, we compared the miRNA blood levels of animals subjected to permanent or transient middle cerebral artery occlusion (MCAO) or to collagenase-induced hemorrhagic stroke. Preliminarily, we examined the rat miRNome in the brain tissue of ischemic and sham-operated rats; then, we selected those miRNAs whose expression was significantly modulated after stroke to create a list of miRNAs potentially involved in stroke damage. These selected miRNAs were then evaluated at different time intervals in the blood of rats subjected to permanent or transient focal ischemia or to hemorrhagic stroke. We found that four miRNAs-miR-16-5p, miR-101a-3p, miR-218-5p, and miR-27b-3p-were significantly upregulated in the plasma of rats 3 h after permanent MCAO, whereas four other different miRNAs-miR-150-5p, let-7b-5p, let-7c-5p, and miR-181b-5p-were selectively upregulated by collagenase-induced hemorrhagic stroke. Collectively, our study identified some selective miRNAs expressed in the plasma of hemorrhagic rats and pointed out the importance of a precise time point measurement to render more reliable the use of miRNAs as stroke biomarkers.
Ischemic stroke is a multifaced pathology that involves gene reprogramming. Among those genes whose expression is influenced by cerebral ischemia can be included the plasmamembrane protein sodium-calcium exchanger-1 (NCX1), whose activity is tightly related to stroke outcome. We have recently identified a microRNA (miR-103-1) able to selectively modulate NCX1 expression in brain during stroke and whose inhibition by anti-miR-103 causes brain damage reduction accompanied by NCX1 upregulation.
Furthermore, it has been recently demonstrated that a short occlusion of an artery in a separate district of the body is able to protect the brain from a previous harmful ischemic insult: a phenomenon termed “remote ischemic postconditioning” (RIPO). Little is known about neural pathways and humoral mediators that are triggered by this neuroprotective approach. In this work we hypothesize that miRNAs released in biofluids within exosomes, small microvesicles of endosomal origin important in cell-to-cell communication, may serve as messengers from blood to CNS. To this aim, we performed a screening of rat miRNome, in order to identify miRNAs that are modulated in brain after stroke and RIPO treatment. Finally, in order to verify whether NCX is involved in RIPO mechanisms able to restore ionic homeostasis, we investigated whether NCX expression was modulated after RIPO through miR-103.
Remote limb ischemic postconditioning (RLIP) is a well-established neuroprotective strategy able to protect the brain from a previous harmful ischemic insult through a sub-lethal occlusion of the femoral artery. Neural and humoral mechanisms have been proposed as mediators required to transmit the peripheral signal from limb to brain. Moreover, different studies suggest that protection observed at brain level is associated to a general genetic reprogramming involving also microRNAs (miRNAs) intervention. Methods: Brain ischemia was induced in male rats by transient occlusion of the middle cerebral artery (tMCAO), whereas RLIP was achieved by one cycle of temporary occlusion of the ipsilateral femoral artery after tMCAO. The expression profile of 810 miRNAs was evaluated in ischemic brain samples from rats subjected either to tMCAO or to RLIP. Among all analyzed miRNAs, there were four whose expression were upregulated after stroke and returned to basal level after RLIP, thus suggesting a possible involvement in RLIP-induced neuroprotection. These selected miRNAs were intracerebroventricularly infused in rats subjected to remote ischemic postconditioning, and their effect was evaluated in terms of brain damage, neurological deficit scores and expression of putative targets. Results: Twenty-one miRNAs, whose expression was significantly affected by tMCAO and by tMCAO plus RLIP, were selected based on microarray microfluidic profiling. Our data showed that: (1) stroke induced an up-regulation of let-7a and miR-143 (2) these two miRNAs were involved in the protective effects induced by RLIP and (3) HIF1-α contributes to their protective effect. Indeed, their expression was reduced after RLIP and the exogenous intracerebroventricularly infusion of let-7a and miR-143 mimics prevented neuroprotection and HIF1-α overexpression induced by RLIP. Conclusions: Prevention of cerebral let-7a and miR-143 overexpression induced by brain ischemia emerges as new potential strategy in stroke intervention.
The remodelling of neuronal ionic homeostasis by altered channels and transporters is a critical feature of the Alzheimer's disease (AD) pathogenesis. Different reports converge on the concept that the Na+/Ca2+ exchanger (NCX), as one of the main regulators of Na+ and Ca2+ concentrations and signalling, could exert a neuroprotective role in AD. The activity of NCX has been found to be increased in AD brains, where it seemed to correlate with an increased neuronal survival. Moreover, the enhancement of the NCX3 currents (INCX) in primary neurons treated with the neurotoxic amyloid β 1-42 (Aβ1-42) oligomers prevented the endoplasmic reticulum (ER) stress and neuronal death. The present study has been designed to investigate any possible modulation of the INCX, the functional interaction between NCX and the NaV1.6 channel, and their impact on the Ca2+ homeostasis in a transgenic in vitro model of AD, the primary hippocampal neurons from the Tg2576 mouse, which overproduce the Aβ1-42 peptide. Electrophysiological studies, carried in the presence of siRNA and the isoform-selective NCX inhibitor KB-R7943, showed that the activity of a specific NCX isoform, NCX3, was upregulated in its reverse, Ca2+ influx mode of operation in the Tg2576 neurons. The enhanced NCX activity contributed, in turn, to increase the ER Ca2+ content, without affecting the cytosolic Ca2+ concentrations of the Tg2576 neurons. Interestingly, our experiments have also uncovered a functional coupling between NCX3 and the voltage-gated NaV1.6 channels. In particular, the increased NaV1.6 currents appeared to be responsible for the upregulation of the reverse mode of NCX3, since both TTX and the Streptomyces griseolus antibiotic anisomycin, by reducing the NaV1.6 currents, counteracted the increase of the INCX in the Tg2576 neurons. In agreement, our immunofluorescence analyses revealed that the NCX3/NaV1.6 co-expression was increased in the Tg2576 hippocampal neurons in comparison with the WT neurons. Collectively, these findings indicate that NCX3 might intervene in the Ca2+ remodelling occurring in the Tg2576 primary neurons thus emerging as a molecular target with a neuroprotective potential, and provide a new outcome of the NaV1.6 upregulation related to the modulation of the intracellular Ca2+ concentrations in AD neurons.
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.
Background: Neuronal circuitry remodeling which compromises excitatory and inhibitory neurons is critical to improve neurological outcome after stroke. Cerebral endothelial cell (CEC) generated small extracellular vesicles (CEC-sEVs) have a therapeutic effect on stroke recovery. However, it remains challenge to use sEVs for specifically targeting individual neurons for enhancement of the circuitry after stroke. Using a chemogenetic approach, we tested the hypothesis that Designer Receptors Exclusively Activated by Designer Drugs (DREADD) activated peri-infarct cortical interneurons preferentially take up exogenous CEC-sEVs to enhance neuronal remodeling and functional recovery. Methods: Adeno-associated viruses (AAVs) carrying Gq-DREADD and Tomato under the control of the transcription factor distal-less homeobox (Dlx) enhancer element (AAV-hDlx-Gq, 500 nL) were injected into the cortex. The mice were then subjected to middle cerebral artery occlusion (MCAO). CEC-sEVs (1x10 11 particles, i.v.) were administered following interneuron activation by Clozapine-N-oxide (CNO, 5mg/kg, i.p). The circuitry was assayed by Golgi-Cox staining and biotinylated dextran amine labeled axons in the corticospinal tract (CST). Transmission electron microscope (TEM) was used to measure CEC-sEVs in neurons and neuronal mitochondrial (Mito) integrity. Results: The adjunct therapy of CEC-sEVs and CNO increased CEC-sEV uptake by peri-infarct cortical neurons and improved somatosensory functional outcome by ~70% vs CEC-sEVs or CNO alone (p<0.05, n=10/group). The adjunct therapy (p<0.05) augmented peri-infarct cortical axonal/dendritic outgrowth and CST axonal remodeling but did not reduce infarct volume. TEM analysis revealed that the neurons with uptake of CEC-sEVs (p<0.05) increased their Mito with intact cristae. Western blot showed that adjunct CEC-sEVs with CNO (p<0.05) decreased ischemia-increased Drp1 and Mff, and elevated ischemia-reduced Mito oxidative phosphorylation (OXPHOS) protein, COX5A, vs CEC-sEVs or CNO alone. Increases of Mito Drp1 and Mff impair Mito crista integrity, which reduces OXPHOS-induced ATP production. Conclusions: Our data demonstrate that activated cortical interneurons preferentially increased the uptake of CEC-sEVs, leading to the improvement of functional outcome after stroke. The increased CEC-sEV uptake by activated interneurons reduced ischemic neuronal Mito damage, which may contribute to enhanced neuronal circuitry remodeling.
Amyotrophic lateral sclerosis (ALS) is one of the most threatening neurodegenerative disease since it causes muscular paralysis for the loss of Motor Neurons (MN) in the spinal cord, brainstem and motor cortex. Up until now, no effective pharmacological treatment is available. Two forms of ALS have been described so far: 90% of the cases presents the sporadic form (sALS) whereas the remaining 10% of the cases present the familiar form (fALS). Approximately 20% of fALS are associated to inherited mutations in the Cu, Zn-superoxide dismutase 1 (SOD1) gene. In the last decade, ionic homeostasis dysregulation has been proposed as the main trigger of the pathological cascade that brings to motor-neurons loss. In the light of these premises, the present review will analyze the involvement in ALS pathophysiology of the most well studied metal ions, i.e. calcium, sodium, iron, copper and zinc, with particular focus to the role of ionic channels and transporters able to contribute in the regulation of ionic homeostasis, in order to propose new putative molecular targets for future therapeutic strategies to ameliorate the progression of this devastating neurodegenerative disease.
Abstract There are no effective treatments for chemotherapy induced peripheral neuropathy (CIPN). Small extracellular vesicles (sEVs) facilitate intercellular communication and mediate nerve function and tumour progression. We found that the treatment of mice bearing ovarian tumour with sEVs derived from cerebral endothelial cells (CEC‐sEVs) in combination with a chemo‐drug, oxaliplatin, robustly reduced oxaliplatin‐induced CIPN by decreasing oxaliplatin‐damaged myelination and nerve fibres of the sciatic nerve and significantly amplified chemotherapy of oxaliplatin by reducing tumour size. The combination therapy substantially increased a set of sEV cargo‐enriched miRNAs, but significantly reduced oxaliplatin‐increased proteins in the sciatic nerve and tumour tissues. Bioinformatics analysis revealed the altered miRNAs and proteins formed two distinct networks that regulate neuropathy and tumour growth, respectively. Intravenously administered CEC‐sEVs were internalized by axons of the sciatic nerve and cancer cells. Reduction of CEC‐sEV cargo miRNAs abolished the effects of CEC‐sEVs on oxaliplatin‐inhibited axonal growth and on amplification of the anti‐cancer effect in ovarian cancer cells, suggesting that alterations in the networks of miRNAs and proteins in recipient cells contribute to the therapeutic effect of CEC‐sEVs on CIPN. Together, the present study demonstrates that CEC‐sEVs suppressed CIPN and enhanced chemotherapy of oxaliplatin in the mouse bearing ovarian tumour.
