IDENTIFICATION OF MICRORNAs REGULATING IONIC HOMEOSTASIS AS DIAGNOSTIC AND THERAPEUTIC TOOLS FOR CEREBRAL ISCHEMIA

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

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

Amyloid β-Induced Upregulation of Nav1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer's Disease Mouse Model

Hyperexcitability and alterations in neuronal networks contribute to cognitive impairment in Alzheimer's Disease (AD). Voltage-gated sodium channels (NaV), which are crucial for regulating neuronal excitability, have been implicated in AD-related hippocampal hyperactivity and higher incidence of spontaneous non-convulsive seizures. Here, we show by using primary hippocampal neurons exposed to amyloid-β1-42 (Aβ1-42) oligomers and from Tg2576 mouse embryos, that the selective upregulation of NaV1.6 subtype contributes to membrane depolarization and to the increase of spike frequency, thereby resulting in neuronal hyperexcitability. Interestingly, we also found that NaV1.6 overexpression is responsible for the aberrant neuronal activity observed in hippocampal slices from 3-month-old Tg2576 mice. These findings identify the NaV1.6 channels as a determinant of the hippocampal neuronal hyperexcitability induced by Aβ1-42 oligomers. The selective blockade of NaV1.6 overexpression and/or hyperactivity might therefore offer a new potential therapeutic approach to counteract early hippocampal hyperexcitability and subsequent cognitive deficits in the early stages of AD

Synergistic association of valproate and resveratrol reduces brain injury in ischemic stroke

Histone deacetylation, together with altered acetylation of NF-κB/RelA, encompassing the K310 residue acetylation, occur during brain ischemia. By restoring the normal acetylation condition, we previously reported that sub-threshold doses of resveratrol and entinostat (MS-275), respectively, an activator of the AMP-activated kinase (AMPK)-sirtuin 1 pathway and an inhibitor of class I histone deacetylases (HDACs), synergistically elicited neuroprotection in a mouse model of ischemic stroke. To improve the translational power of this approach, we investigated the efficacy of MS-275 replacement with valproate, the antiepileptic drug also reported to be a class I HDAC blocker. In cortical neurons previously exposed to oxygen glucose deprivation (OGD), valproate elicited neuroprotection at 100 nmol/mL concentration when used alone and at 1 nmol/mL concentration when associated with resveratrol (3 nmol/mL). Resveratrol and valproate restored the acetylation of histone H3 (K9/18), and they reduced the RelA(K310) acetylation and the Bim level in neurons exposed to OGD. Chromatin immunoprecipitation analysis showed that the synergistic drug association impaired the RelA binding to the Bim promoter, as well as the promoter-specific H3 (K9/18) acetylation. In mice subjected to 60 min of middle cerebral artery occlusion (MCAO), the association of resveratrol 680 µg/kg and valproate 200 µg/kg significantly reduced the infarct volume as well as the neurological deficits. The present study suggests that valproate and resveratrol may represent a promising ready-to-use strategy to treat post-ischemic brain damage

Exosomes Derived From Schwann Cells Ameliorate Peripheral Neuropathy in Type II Diabetic Mice.

Schwann cell-derived exosomes communicate with dorsal root ganglia (DRG) neurons. The present study investigated the therapeutic effect of exosomes derived from healthy Schwann cells (SC-Exos) on diabetic peripheral neuropathy (DPN). We found that intravenous administration of SC-Exos to type II diabetic db/db mice with peripheral neuropathy remarkably ameliorated DPN by improving sciatic nerve conduction velocity and increasing thermal and mechanical sensitivity. These functional improvements were associated with the augmentation of epidermal nerve fibers, and remyelination of sciatic nerves. Quantitative RT-PCR and Western blot analysis of sciatic nerve tissues showed that the SC-Exo treatment reversed diabetes-reduced microRNA (miR)-21, -27a and -146a and diabetes-increased Semaphorin 6A (SEMA6A), Ras homolog gene family, member A (RhoA), phosphatase and tensin homolog (PTEN), and nuclear factor-κB (NF-κB). In vitro data showed that SC-Exos promoted neurite outgrowth of diabetic DRG neurons and migration of Schwann cells challenged by high glucose. Collectively, these novel data provide evidence that SC-Exos have a therapeutic effect on DPN in mice and suggest that SC-Exos modulation of miRs contribute to this therapy

Small extracellular vesicles ameliorate peripheral neuropathy and enhance chemotherapy of oxaliplatin on ovarian cancer

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

Ionic Homeostasis Maintenance in ALS: Focus on New Therapeutic Targets

Amyotrophic lateral sclerosis (ALS) is one of the most threatening neurodegenerative disease since it causes muscular paralysis for the loss of Motor Neurons 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 displays the familiar form (fALS). Approximately 20% of fALS is associated with 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

Synergistic association of valproate and resveratrol reduces brain injury in ischemic stroke.

Different studies demonstrated that histone deacetylation and modification of NF-kB/RelA acetylation occur during brain ischemia. We previously demonstrated that, sub-threshold doses of resveratrol, a sirtuin 1 activator, and MS-275, a class I HDAC inhibitor, elicited neuroprotection in a mouse model of MCAO. In the present work, we replace MS-275 with valproate, an antiepileptic drug also reported as a class I HDACs inhibitor. In cortical neurons exposed to 3h of OGD, 24h of treatment with 100 µM valproate resulted neuroprotective per se, while in association with resveratrol it was active at 1µM. In mice subjected to 60 minutes of MCAO the association of resveratrol 680 µg/kg and valproate 200 µg/kg significantly reduced the infarct volume as well as the neurological deficits. Single treatments at the same doses had no effects, while at the higher doses, resveratrol 6,8 mg/kg or valproate 20 mg/kg limited the infarct volume but did not reduce the neurological deficits. In accordance with the effect observed by combining resveratrol and MS-275, the association of resveratrol and VPA restored the acetylation levels of histone H3 (K9/18) reduced after OGD exposure. Moreover, the application of resveratrol and VPA reversed the OGD-mediated increase in the RelA(K310) acetylation. Finally, ChIP assays in cortical neurons exposed to OGD demonstrated that the addition of resveratrol (3 μM) and valproate (1 μM), totally impaired the RelA binding at the Bim promoter as well as the promoter–specific H3 (K9/18) acetylation. We can conclude that valproate and resveratrol may represent a promising ready-to-use strategy for the therapy of post-ischemic brain damage

Preconditioning, induced by sub-toxic dose of the neurotoxin L-BMAA, delays ALS progression in mice and prevents Na+/Ca2+ exchanger 3 downregulation

Preconditioning (PC) is a phenomenon wherein a mild insult induces resistance to a later, severe injury. Although PC has been extensively studied in several neurological disorders, no studies have been performed in amyotrophic lateral sclerosis (ALS). Here we hypothesize that a sub-toxic acute exposure to the cycad neurotoxin beta-methylamino-L-alanine (L-BMAA) is able to delay ALS progression in SOD1 G93A mice and that NCX3, a membrane transporter able to handle the deregulation of ionic homeostasis occurring during ALS, takes part to this neuroprotective effect. Preconditioning effect was examined on disease onset and duration, motor functions, and motor neurons in terms of functional declines and severity of histological damage in male and female mice. Our findings demonstrate that a sub-toxic dose of L-BMAA works as preconditioning stimulus and is able to delay ALS onset and to prolong ALS mice survival. Interestingly, preconditioning prevented NCX3 downregulation in SOD1 G93A mice spinal cord, leading to an increased number of motor neurons associated to a reduced astrogliosis, and reduced the denervation of neuromuscular junctions observed in SOD1 G93A mice. These protective effects were mitigated in ncx3+/-mice. This study established for the first time an animal model of preconditioning in ALS and candidates NCX3 as a new therapeutic target.SCOPUS: ar.jinfo:eu-repo/semantics/publishe