Paracrine effect of carbon monoxide - astrocytes promote neuroprotection through purinergic signaling in mice

© 2016. Published by The Company of Biologists Ltd.The neuroprotective role of carbon monoxide (CO) has been studied in a cell-autonomous mode. Herein, a new concept is disclosed - CO affects astrocyte-neuron communication in a paracrine manner to promote neuroprotection. Neuronal survival was assessed when co-cultured with astrocytes that had been pre-treated or not with CO. The CO-pre-treated astrocytes reduced neuronal cell death, and the cellular mechanisms were investigated, focusing on purinergic signaling. CO modulates astrocytic metabolism and extracellular ATP content in the co-culture medium. Moreover, several antagonists of P1 adenosine and P2 ATP receptors partially reverted CO-induced neuroprotection through astrocytes. Likewise, knocking down expression of the neuronal P1 adenosine receptor A2A-R (encoded by Adora2a) reverted the neuroprotective effects of CO-exposed astrocytes. The neuroprotection of CO-treated astrocytes also decreased following prevention of ATP or adenosine release from astrocytic cells and inhibition of extracellular ATP metabolism into adenosine. Finally, the neuronal downstream event involves TrkB (also known as NTRK2) receptors and BDNF. Pharmacological and genetic inhibition of TrkB receptors reverts neuroprotection triggered by CO-treated astrocytes. Furthermore, the neuronal ratio of BDNF to pro-BDNF increased in the presence of CO-treated astrocytes and decreased whenever A2A-R expression was silenced. In summary, CO prevents neuronal cell death in a paracrine manner by targeting astrocytic metabolism through purinergic signaling.publishersversionpublishe

Carbon Monoxide Modulation of Microglia-Neuron Communication: Anti-Neuroinflammatory and Neurotrophic Role

This work was financed by FEDER-Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020-Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT-Fundação para a Ciência e a Tecnologia/Ministério da Ciência (FCT), Tecnologia e Ensino Superior in the framework of FCT-ANR/NEU-NMC/0022/2012 grant, PTDC/MEC-NEU/28750/2017 grant, Applied Molecular Biosciences Unit-UCIBIO (UID/Multi/04378/2019) grant; LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy; and FCT provided individual financial support to NLS (PD/BD/127819/2016), BFM (PD/BD/128336/2017) and HLAV (IF/00185/2012).Microglia, the ‘resident immunocompetent cells’ of the central nervous system (CNS), are key players in innate immunity, synaptic refinement and homeostasis. Dysfunctional microglia contribute heavily to creating a toxic inflammatory milieu, a driving factor in the pathophysiology of several CNS disorders. Therefore, strategies to modulate the microglial function are required to tackle exacerbated tissue inflammation. Carbon monoxide (CO), an endogenous gaseous molecule produced by the degradation of haem, has anti-inflammatory, anti-apoptotic, and pro-homeostatic and cytoprotective roles, among others. ALF-826A, a novel molybdenum-based CO-releasing molecule, was used for the assessment of neuron-microglia remote communication. Primary cultures of rat microglia and neurons, or the BV-2 microglial and CAD neuronal murine cell lines, were used to study the microglia-neuron interaction. An approach based on microglial-derived conditioned media in neuronal culture was applied. Medium derived from CO-treated microglia provided indirect neuroprotection against inflammation by limiting the lipopolysaccharide (LPS)-induced expression of reactivity markers (CD11b), the production of reactive oxygen species (ROS) and the secretion of inflammatory factors (TNF-α, nitrites). This consequently prevented neuronal cell death and maintained neuronal morphology. In contrast, in the absence of inflammatory stimulus, conditioned media from CO-treated microglia improved neuronal morphological complexity, which is an indirect manner of assessing neuronal function. Likewise, the microglial medium also prevented neuronal cell death induced by pro-oxidant tert-Butyl hydroperoxide (t-BHP). ALF-826 treatment reinforced microglia secretion of Interleukin-10 (IL-10) and adenosine, mediators that may protect against t-BHP stress in this remote communication model. Chemical inhibition of the adenosine receptors A2A and A1 reverted the CO-derived neuroprotective effect, further highlighting a role for CO in regulating neuron-microglia communication via purinergic signalling. Our findings indicate that CO has a modulatory role on microglia-to-neuron communication, promoting neuroprotection in a non-cell autonomous manner. CO enhances the microglial release of neurotrophic factors and blocks exacerbated microglial inflammation. CO improvement of microglial neurotrophism under non-inflammatory conditions is here described for the first time.publishersversionepub_ahead_of_prin

Carbon Monoxide Targeting Mitochondria

Mitochondria present two key roles on cellular functioning: (i) cell metabolism, being the main cellular source of energy and (ii) modulation of cell death, by mitochondrial membrane permeabilization. Carbon monoxide (CO) is an endogenously produced gaseoustransmitter, which presents several biological functions and is involved in maintaining cell homeostasis and cytoprotection. Herein, mitochondrion is approached as the main cellular target of carbon monoxide (CO). In this paper, two main perspectives concerning CO modulation of mitochondrial functioning are evaluated. First, the role of CO on cellular metabolism, in particular oxidative phosphorylation, is discussed, namely, on: cytochrome c oxidase activity, mitochondrial respiration, oxygen consumption, mitochondrial biogenesis, and general cellular energetic status. Second, the mitochondrial pathways involved in cell death inhibition by CO are assessed, in particular the control of mitochondrial membrane permeabilization

Carbon monoxide effect in hippocampus after perinatal hypoxia-ischemia – apoptotic profiles.

<p>Whereas contralateral hippocampus displayed a preserved morphology (<b>A</b>) following HI, diffuse tissue disruption was detected in the hippocampus ipsilateral to the occlusion (<b>B</b>). C–E are representative pictures of ischemic hippocampus, where diffuse apoptosis was documented; with peculiar morphological features including pyknotic nuclei (<b>C</b>), indicating early stage of apoptosis, progressive nuclear fragmentation (<b>D</b>) and karyorrhexis as confirmed by detectable apoptotic bodies (<b>E</b>). Compared to HI group, the number of apoptotic profiles was significantly lower when animals were exposed to CO prior to HI (<b>F</b>). All values are mean ± SD (error bars); *<i>p</i><0.05 compared to Control group for the corresponding side and **<i>p</i><0.05 compared to HI group ischemic hippocampus. (<b>G</b>) For each group there is no significant difference in cytotoxic edema volume (mm³) between the ipsi- and the contralateral hippocampus.</p

Effect of carbon monoxide treatment on neuronal apoptosis.

<p>(<b>A</b>) Representative micrographs of neurons treated or not with 20 µM of glutamate and 10 µM of CO. Apoptotic hallmarks were analyzed by fluorescent microscopy. <i>Upper panel</i>, for the photos taken with the filter for phase contrast, <i>middle panel</i>, for Hoechst (white arrows for nuclei with condensed chromatin) and <i>lower panel</i>, for propidium iodide (white arrows for cells which membrane integrity was lost). (<b>B</b>) Primary cultures of neuronal cells were pre-treated with 10 µM CO, followed by 24 h of glutamate (10–30 µM) treatment. Cell viability was assessed by counting cells containing normal nuclei and plasmatic membrane integrity. For each coverslip, at least 1500 cells were counted. All values are mean ± SD (error bars), n = 5; *<i>p</i><0.05 compared to control. (<b>C</b>) The effect of 10 µM CO treatment on Bcl-2 expression was assessed by its mRNA quantification.</p

Experimental groups and time-points schematic representation.

<p><b>Control group</b>, <i>n = 22</i>, untreated animals that did not suffer any treatment; <b>Carbon Monoxide (CO) group</b>, <i>n = 16</i>, subjected to 3 exposures of 250 ppm, for 1 h at P4, P5 and P6; <b>Hypoxia-Ischemia (HI) group</b>, <i>n = 17</i>, animals that underwent surgery and hypoxia (8% of O₂ in nitrogen) exposure for 75 minutes; <b>CO+HI group</b>, <i>n = 19</i>, CO treatment plus hypoxia-ischemia. Animals were euthanized at 6 and 24 h <i>post</i>-HI. Brains were collected and analyzed for lesion volume and cell death markers, as described in the methods section. <i>Histo</i>, for brains analyzed by histological methods; <i>WB</i>, for brains collected and processed for western blot analysis.</p

Carbon monoxide effect in hippocampus after perinatal hypoxia-ischemia – cleaved caspase 3 expression.

<p>Low (scale bar = 100 µm) magnification CLSM photographs of the hippocampus of HI (A, B), CO-HI (C, D) and CO sham operated (E, F) rat pups. In blue, DAPI-stained nuclei; in red, cleaved caspase 3-positive cells. Caspase 3-positive profiles following HI were particularly frequent in CA1–2 and in the dentate gyrus, and were decreased in number following CO preconditioning. CO preconditioning alone did not induce caspase 3 activation in sham operated animals.</p