Oligodendrogenesis from neural stem cells: Perspectives for remyelinating strategies

Mobilization of remyelinating cells spontaneously occurs in the adult brain. These cellular resources are specially active after demyelinating episodes in early phases of multiple sclerosis (MS). Indeed, oligodendrocyte precursor cells (OPCs) actively proliferate, migrate to and repopulate the lesioned areas. Ultimately, efficient remyelination is accomplished when new oligodendrocytes reinvest nude neuronal axons, restoring the normal properties of impulse conduction. As the disease progresses this fundamental process fails. Multiple causes seem to contribute to such transient decline, including the failure of OPCs to differentiate and enwrap the vulnerable neuronal axons. Regenerative medicine for MS has been mainly centered on the recruitment of endogenous self-repair mechanisms, or on transplantation approaches. The latter commonly involves grafting of neural precursor cells (NPCs) or neural stem cells (NSCs), with myelinogenic potential, in the injured areas. Both strategies require further understanding of the biology of oligodendrocyte differentiation and remyelination. Indeed, the success of transplantation largely depends on the pre-commitment of transplanted NPCs or NSCs into oligodendroglial cell type, while the endogenous differentiation of OPCs needs to be boosted in chronic stages of the disease. Thus, much effort has been focused on finding molecular targets that drive oligodendrocytes commitment and development. The present review explores several aspects of remyelination that must be considered in the design of a cell-based therapy for MS, and explores more deeply the challenge of fostering oligodendrogenesis. In this regard, we discuss herein a tool developed in our research group useful to search novel oligodendrogenic factors and to study oligodendrocyte differentiation in a time- and cost-saving manner

Phytochemical and antioxidant characterization of Hypericum perforatum alcoholic extracts

The antioxidant potentials of a total ethanolic extract of Hypericum perforatum (TE) and fractions were evaluated and correlated with their phenolic contents. The extracts were fully characterised by HPLC-DAD-MS-MS. Kaempferol 3-rutinoside and rutin-acetyl were identified for the first time in TE extracts. The free radical-scavenging properties of TE (EC50=21 [mu]g dwb/ml) and fractions were studied using DPPH. Fractions containing flavonoids and/or caffeoylquinic acids were found to be the main contributors to the free radical-scavenging activity of the TE. Lipid peroxidation, induced with ascorbate/Fe2+, was significantly reduced in the presence of the TE (EC50=26 [mu]g dwb/ml) and fractions containing flavonoids and/or caffeoylquinic acids. The fraction containing flavonoid aglycones was found to be responsible for a major part of the TE protection against lipid peroxidation. Hypericins and hyperforins made no significant contributions to the antioxidant properties of TE. Human consumption of H. perforatum extract or fractions could be beneficial.http://www.sciencedirect.com/science/article/B6T6R-4CG2JK5-3/1/ae503f0537dcaaab2ede0770605a66c

Tumor necrosis factor-alpha modulates survival, proliferation, and neuronal differentiation in neonatal subventricular zone cell cultures

Tumor necrosis factor (TNF)- has been reported to modulate brain injury, but remarkably, little is known about its effects on neurogenesis. We report that TNF- strongly influences survival, proliferation, and neuronal differentiation in cultured subventricular zone (SVZ) neural stem/progenitor cells derived from the neonatal P1-3 C57BL/6 mice. By using single-cell calcium imaging, we developed a method, based on cellular response to KCl and/or histamine, that allows the functional evaluation of neuronal differentiation. Exposure of SVZ cultures to 1 and 10 ng/ml mouse or 1 ng/ml human recombinant TNF- resulted in increased differentiation of cells displaying a neuronal-like profile of [Ca2+]i responses, compared with the predominant profile of immature cells observed in control, nontreated cultures. Moreover, by using neutralizing antibodies for each TNF- receptor, we found that the proneurogenic effect of 1 ng/ml TNF- is mediated via tumor necrosis factor receptor 1 activation. Accordingly, the percentage of neuronal nuclear protein-positive neurons was increased following exposure to mouse TNF-. Interestingly, exposure of SVZ cultures to 1 ng/ml TNF- induced cell proliferation, whereas 10 and 100 ng/ml TNF- induced apoptotic cell death. Moreover, we found that exposure of SVZ cells to TNF- for 15 minutes or 6 hours caused an increase in the phospho-stress-activated protein kinase/c-Jun N-terminal kinase immunoreactivity initially in the nucleus and then in growing axons, colocalizing with tau, consistent with axonogenesis. Taken together, these results show that TNF- induces neurogenesis in neonatal SVZ cell cultures of mice. TNF-, a proinflammatory cytokine and a proneurogenic factor, may play a central role in promoting neurogenesis and brain repair in response to brain injury and infectio

Neuropeptide Y inhibits interleukin-1β-induced phagocytosis by microglial cells

<p>Abstract</p> <p>Background</p> <p>Neuropeptide Y (NPY) is emerging as a modulator of communication between the brain and the immune system. However, in spite of increasing evidence that supports a role for NPY in the modulation of microglial cell responses to inflammatory conditions, there is no consistent information regarding the action of NPY on microglial phagocytic activity, a vital component of the inflammatory response in brain injury. Taking this into consideration, we sought to assess a potential new role for NPY as a modulator of phagocytosis by microglial cells.</p> <p>Methods</p> <p>The N9 murine microglial cell line was used to evaluate the role of NPY in phagocytosis. For that purpose, an IgG-opsonized latex bead assay was performed in the presence of lipopolysaccharide (LPS) and an interleukin-1β (IL-1β) challenge, and upon NPY treatment. A pharmacological approach using NPY receptor agonists and antagonists followed to uncover which NPY receptor was involved. Moreover, western blotting and immunocytochemical studies were performed to evaluate expression of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27), in an inflammatory context, upon NPY treatment.</p> <p>Results</p> <p>Here, we show that NPY inhibits phagocytosis of opsonized latex beads and inhibits actin cytoskeleton reorganization triggered by LPS stimulation. Co-stimulation of microglia with LPS and adenosine triphosphate also resulted in increased phagocytosis, an effect inhibited by an interleukin-1 receptor antagonist, suggesting involvement of IL-1β signaling. Furthermore, direct application of LPS or IL-1β activated downstream signaling molecules, including p38 MAPK and HSP27, and these effects were inhibited by NPY. Moreover, we also observed that the inhibitory effect of NPY on phagocytosis was mediated <it>via </it>Y₁receptor activation.</p> <p>Conclusions</p> <p>Altogether, we have identified a novel role for NPY in the regulation of microglial phagocytic properties, in an inflammatory context.</p

Activation of neuropeptide Y receptors is neuroprotective against excitotoxicity in organotypic hippocampal slice cultures

Glutamate and NPY have been implicated in hippocampal neuropathology in temporal lobe epilepsy. Thus, we investigated the involvement of NPY receptors in mediating neuroprotection against excitotoxic insults in organotypic cultures of rat hippocampal slices. Exposure of hippocampal slice cultures to 2 μM AMPA (α-amino-3-hydroxy-5-methyl-isoxazole-4- propionate) induced neuronal degeneration, monitored by propidium iodide uptake, of granule cells and CA1 pyramidal cells. For dentate granule cells, selective activation of Y1, Y2, or Y5 receptors with 1 μM [Leu31,Pro34]NPY, 300 nM NPY13–36 or 1 μM 500 nM NPY(19–23)- 1 3 4 6 31 32 34 (Gly ,Ser ,Gln ,Thr ,Ala ,Aib ,Gln )-PP, respectively, had a neuroprotective effect against AMPA, whereas only the activation of Y2 receptors was effective for CA1 pyramidal cells. When the slice cultures were exposed to 6 μM kainate, the CA3 pyramidal cells displayed significant degeneration, and in this case the activation of Y1, Y2, and Y5 receptors was neuroprotective. For the kainic acid-induced degeneration of CA1 pyramidal cells, it was again found that only the Y2 receptor activation was effective. Based on the present findings, it was concluded that Y1, Y2, and Y5 receptors effectively can modify glutamate receptor-mediated neurodegeneration in the hippocampu

Effect of Hypoproteic and High-Fat Diets on Hippocampal Blood-Brain Barrier Permeability and Oxidative Stress

Worldwide, millions of people are exposed to dietary imbalance that impacts in health and quality of life. In developing countries, like in Brazil, in poor settings, dietary habits, traditionally hypoproteic, are changing rapidly to western-type high-fat foods. These rapidly changing dietary habits are imposing new challenges to human health and there are many questions in the field that remain to be answered. Accordingly, we currently do not know if chronic consumption of hypoproteic (regional basic diet, RBD) or high-fat diets (HFD) may impact the brain physiology, contributing to blood-brain barrier (BBB) dysfunction and neuroinflammatory events. To address this issue, mice were challenged by breastfeeding from dams receiving standard, RBD or HFD from suckling day 10 until weaning. Immediately after weaning, mice continued under the same diets until post-natal day 52. Herein, we show that both RBD and HFD cause not only a peripheral but also a consistent central neuroinflammatory response, characterized by an increased production of Reactive Oxygen Species (ROS) and pro-inflammatory cytokines. Additionally, BBB hyperpermeability, accounted by an increase in hippocampal albumin content, a decrease in claudin-5 protein levels and collagen IV immunostaining, was also observed together with an upregulation of vascular cell adhesion molecule 1 (VCAM-1). Interestingly, we also identified a significant astrogliosis, manifested by upregulation of GFAP and S100β levels and an intensification of arbor complexity of these glial cells. In sum, our data show that dietary imbalance, related with hypoproteic or high-fat content, impairs BBB properties potentially favoring the transmigration of peripheral immune cells and induces both a peripheral and central neuroinflammatory status. Noteworthy, neuroinflammatory events in the hippocampus may cause neuronal malfunction leading to cognitive deficits and long-term persistence of this phenomenon may contribute to age-related neurodegenerative diseases

Activation of Type 1 Cannabinoid Receptor (CB1R) promotes neurogenesis in murine subventricular zone cell cultures

The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here, we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal, proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive), neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells, as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs, an effect blocked by Notch pathway inhibition. Moreover, R-m-AEA treatment for 48 h, increased proliferation as assessed by BrdU incorporation assay, an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly, stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation), at 7 days, as shown by counting the number of NeuN-positive neurons in the cultures. Moreover, by monitoring intracellular calcium concentrations ([Ca2+](i)) in single cells following KCl and histamine stimuli, a method that allows the functional evaluation of neuronal differentiation, we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251, for 7 days, thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation, R-m-AEA also increased neurite growth, as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together, these results demonstrate that CB1R activation induces proliferation, self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures.Fundacao para a Ciencia e a Tecnologia - Portugal [POCTI/SAU-NEU/68465/2006, PTDC/SAU-NEU/104415/2008, PTDC/SAU-NEU/101783/2008, POCTI/SAU-NEU/110838/2009]; Fundacao Calouste Gulbenkian [96542]; Fundacao para a Ciencia e Tecnologiainfo:eu-repo/semantics/publishedVersio

ARIA 2016 : Care pathways implementing emerging technologies for predictive medicine in rhinitis and asthma across the life cycle

The Allergic Rhinitis and its Impact on Asthma (ARIA) initiative commenced during a World Health Organization workshop in 1999. The initial goals were (1) to propose a new allergic rhinitis classification, (2) to promote the concept of multi-morbidity in asthma and rhinitis and (3) to develop guidelines with all stakeholders that could be used globally for all countries and populations. ARIA-disseminated and implemented in over 70 countries globally-is now focusing on the implementation of emerging technologies for individualized and predictive medicine. MASK [MACVIA (Contre les Maladies Chroniques pour un Vieillissement Actif)-ARIA Sentinel NetworK] uses mobile technology to develop care pathways for the management of rhinitis and asthma by a multi-disciplinary group and by patients themselves. An app (Android and iOS) is available in 20 countries and 15 languages. It uses a visual analogue scale to assess symptom control and work productivity as well as a clinical decision support system. It is associated with an inter-operable tablet for physicians and other health care professionals. The scaling up strategy uses the recommendations of the European Innovation Partnership on Active and Healthy Ageing. The aim of the novel ARIA approach is to provide an active and healthy life to rhinitis sufferers, whatever their age, sex or socio-economic status, in order to reduce health and social inequalities incurred by the disease.Peer reviewe

Understanding the physiology of glutamate receptors by use of a protocol for neuronal staining

Teaching students about the physiology of neurotransmitter receptors usually requires practical lessons with the use of sophisticated equipment and complex analysis of data. Here, we report our experience in teaching medical students with a simple, practical protocol that transforms the physiology of glutamate receptors into neuronal staining, observable under bright-field microscopy. Essentially, the students were challenged to selectively stain a subpopulation of cultured neurons expressing Ca(2+)-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors (a subgroup of ionotropic glutamate receptors). Neurons expressing this type of receptors were loaded with Co(2+) (in substitution for Ca(2+)) after nondesensitizing activation of AMPA receptors. After precipitation, the Co(2+) was revealed after treatment with silver. At the end of the procedure, the neurons expressing Ca(2+)-permeable AMPA receptors were visually identified under bright-field microscopy. The procedure allowed the visualization of the complete dendritic network of the stained neurons and allowed the students to learn very efficiently about the physiology of glutamate receptor

Presynaptic kainate receptors modulating glutamatergic transmission in the rat hippocampus are inhibited by arachidonic acid

Kainate receptors are ionotropic glutamate receptors located postsynaptically, mediating frequency-dependent transmission, and presynaptically, modulating transmitter release. In contrast to the excitatory postsynaptic kainate receptors, presynaptic kainate receptor can also be inhibitory and their effects may involve a metabotropic action. Arachidonic acid (AA) modulates most ionotropic receptors, in particular postsynaptic kainate receptor-mediated currents. To further explore differences between pre- and postsynaptic kainate receptors, we tested if presynaptic kainate receptors are affected by AA. Kainate (0.3-3 [mu]M) and the kainate receptor agonist, domoate (60-300 nM), inhibited by 19-54% the field excitatory postsynaptic potential (fEPSP) slope in rat CA1 hippocampus, and increased by 12-32% paired-pulse facilitation (PPF). AA (10 [mu]M) attenuated by 37-72% and by 62-66% the domoate (60-300 nM)-induced fEPSP inhibition and paired-pulse facilitation increase, respectively. This inhibition by AA was unaffected by cyclo- and lipo-oxygenase inhibitors, indomethacin (20 [mu]M) and nordihydroguaiaretic acid (NDGA, 50 [mu]M) or by the free radical scavenger, N-acetyl--cysteine (0.5 mM). The K+ (20 mM)-evoked release of [3H]glutamate from superfused hippocampal synaptosomes was inhibited by 18-39% by domoate (1-10 [mu]M), an effect attenuated by 35-63% by AA (10 [mu]M). Finally, the KD (40-55 nM) of the kainate receptor agonist [3H]-(2S,4R)-4-methylglutamate ([3H]MGA) (0.3-120 nM) binding to hippocampal synaptosomal membranes was increased by 151-329% by AA (1-10 [mu]M). These results indicate that AA directly inhibits presynaptic kainate receptor controlling glutamate release in the CA1 area of the rat hippocampus.http://www.sciencedirect.com/science/article/B6T0B-49JHH14-1/1/306d6b018d3a5d33b6a0973186ea4ed