Primary Cultures Of Cortical Neurons Research Articles

Fc gamma receptors are expressed in the developing rat brain and activate downstream signaling molecules upon cross-linking with immune complex

BackgroundExposure of the developing brain to immune mediators, including antibodies, is postulated to increase risk for neurodevelopmental disorders and neurodegenerative disease. It has been suggested that immunoglobulin G-immune complexes (IgG-IC) activate Fc gamma receptors (FcγR) expressed on neurons to modify signaling events in these cells. However, testing this hypothesis is hindered by a paucity of data regarding neuronal FcγR expression and function.MethodsFcγR transcript expression in the hippocampus, cortex, and cerebellum of neonatal male and female rats was investigated ex vivo and in mixed cultures of primary hippocampal and cortical neurons and astrocytes using quantitative PCR analyses. Expression at the protein level in mixed cultures of primary hippocampal and cortical neurons and astrocytes was determined by immunocytochemistry, western blotting, proteotype analysis, and flow cytometry. The functionality of these receptors was assessed by measuring changes in intracellular calcium levels, Erk phosphorylation, and IgG internalization following stimulation with IgG-immune complexes.ResultsFcgrIa, FcgrIIa, FcgrIIb, FcgrIIIa, and Fcgrt transcripts were detectable in the cortex, hippocampus, and cerebellum at postnatal days 1 and 7. These transcripts were also present in primary hippocampal and cortical cell cultures, where their expression was modulated by IFNγ. Expression of FcγRIa, FcγRIIb, and FcγRIIIa, but not FcγRIIa or FcRn proteins, was confirmed in cultured hippocampal and cortical neurons and astrocytes at the single cell level. A subpopulation of these cells co-expressed the activating FcγRIa and the inhibitory FcγRIIb. Functional analyses demonstrated that exposure of hippocampal and cortical cell cultures to IgG-IC increases intracellular calcium and Erk phosphorylation and triggers FcγR-mediated internalization of IgG.ConclusionsOur data demonstrate that developing neurons and astrocytes in the hippocampus and the cortex express signaling competent FcγR. These findings suggest that IgG antibodies may influence normal neurodevelopment or function via direct interactions with FcγR on non-immune cells in the brain.

Probing synaptic connectivity and function using high-density microelectrode arrays and whole-cell patch-clamp recordings

Event Abstract Back to Event Probing synaptic connectivity and function using high-density microelectrode arrays and whole-cell patch-clamp recordings Julian Bartram1*, Manuel Schröter1, Silvia Ronchi1, Vishalini Emmenegger1, Jan Müller1 and Andreas R. Hierlemann1 1 ETH Zürich, Department of Biosystems Science and Engineering, Switzerland Motivation Synaptic efficacy and synapse number of monosynaptic connections between neurons are often regulated by the spiking activity of the respective pre- and postsynaptic cell. Progress towards a better understanding of the rules and mechanisms that underlie such modifications has been limited due to the difficulties associated with simultaneously studying plasticity at multiple synaptic inputs. Here, we provide a solution to this problem by combining cutting-edge high-density microelectrode array (HD-MEA) technology with the patch-clamp technique. While the latter allows for accurate measurement of postsynaptic currents or potentials, evoked by individual synaptic activation, the HD-MEA technology provides large-scale information about unit activity and allows for selective stimulation of neurons, including multiple presynaptic cells. The proposed approach has been applied to comprehensively examine forms of homeostatic plasticity – a collection of crucial processes acting at different temporal scales in order to stabilize neuronal firing rates. We report on a characterization of classic synaptic scaling operating in mature cortical networks and propose a novel model for the study of homeostatic plasticity during natural network states. Material and Methods In this study, we investigated homeostatic plasticity in cultures of primary cortical neurons. Many variants of this form of plasticity were originally identified in reduced preparations, but have since been confirmed in vivo [1,2], which supports the choice of such a cell culture model system. Isolation, plating on MEA chips, and treatment of primary cortical neurons were previously described [3]. A complementary-metal-oxide-semiconductor- (CMOS) based HD-MEA system with 26’400 microelectrodes, an electrode pitch of 17.5 μm and 1024 reconfigurable readout channels [4] was employed for all extracellular recording and stimulation experiments. Patch-clamp recordings in current-clamp or voltage-clamp mode were conducted in conjunction with HD-MEA stimulation and recordings or, in some cases, independently. Homeostatic plasticity was initially induced by pharmacological alteration of global network spiking starting at 14 - 21 days in vitro. Effects on network spiking, induced in this way, were investigated by performing array-wide activity scans. Some of these results were further complemented with miniature excitatory postsynaptic current (mEPSC) recordings, which provide more direct information about synaptic efficacies. Selective stimulation of cells with the HD-MEA during whole-cell patch-clamp recordings enabled the assessment of synaptic strength at multiple inputs. Finally, recording of the spiking activity of all identified presynaptic cells and the postsynaptic neuron during the induction of homeostatic plasticity will permit the examination of correlations between spike patterns and changes in synaptic strength. Results Array-wide network activity was assessed before, during and after the induction of homeostatic plasticity with 12 – 48 h of tetrodotoxin (TTX; 1 μM) treatment. The relatively limited cell migration in mature cultures facilitated the tracking of units over the multiday recording period. Clear increases in network activity could be observed following the washing out of TTX relative to controls. mEPSC recordings from before and after TTX treatment suggest that this increase was due to homeostatic adjustments of excitatory synaptic efficacy. Interestingly, while the network remained in an elevated activity state for several hours following TTX withdrawal, spiking subsequently decreased within a 24 h period. This likely reflected a homeostatic down-regulation of synaptic strength due to the high network spiking activity. Finally, using the electrical stimulation capabilities of the HD-MEA system, we have successfully measured the strength of multiple synaptic inputs. Discussion We demonstrated in this study how the combination of HD-MEA technology and the patch-clamp technique provides unique abilities to study synaptic homeostasis, and synaptic plasticity in general, by providing crucial information about spontaneous global network spiking, exquisite control over spiking of individual cells, and the ability to record subthreshold postsynaptic responses. A homeostatic down-regulation of network activity and (excitatory) synaptic efficacy, following the elevation of network spiking activity by TTX treatment, is a very attractive form of homeostatic plasticity, as no artificial stimulus or agent (e. g. TTX) is present during the induction phase. Moreover, with global spiking activity being consistent with synaptic efficacies, the network is in a more natural state. We are now attempting to link the observed changes in synaptic efficacy with the respective pre- and postsynaptic spike patterns, recorded during the induction phase, promising new insights into the rules and mechanisms underlying homeostatic plasticity. Acknowledgements This work was supported by the ERC Advanced Grant 694829 ‘neuroXscales’.

Clarified Açaí (Euterpe oleracea) Juice as an Anticonvulsant Agent: In Vitro Mechanistic Study of GABAergic Targets.

Seizures affect about 50 million people around the world. Approximately 30% of seizures are refractory to the current pharmacological arsenal, so, the pursuit of new therapeutic alternatives is essential. Clarified Euterpe oleracea (EO) juice showed anticonvulsant properties similar to diazepam in an in vivo model with pentylenetetrazol, a GABAA receptor blocker. This study investigated the effects of EO on the main GABAergic targets for anticonvulsant drugs, analyzing the effect on the GABA receptor's benzodiazepine and picrotoxinin binding sites and the GABA uptake. Primary cultures of cortical neurons and astrocytes were treated with EO (0–25%) for up to 90 min. [3H]Flunitrazepam and [3H]TBOB binding, [3H]GABA uptake, cell viability, and morphology were assayed. Nonlethal concentrations of EO increased agonist binding and decreased antagonist binding in cortical neurons. Low concentrations significantly inhibited GABA uptake, especially in astrocytes, suggesting an accumulation of endogenous GABA in the synaptic cleft. The results demonstrate, for the first time, that EO can improve GABAergic neurotransmission via interactions with GABAA receptor and modulation of GABA uptake. Understanding these molecular mechanisms will help in the treatment of seizures and epilepsy, especially in developing countries where geographic isolation and low purchasing power are the main barriers to access to adequate treatment.

Morphological alterations induced by the exposure to TiO2 nanoparticles in primary cortical neuron cultures and in the brain of rats.

Nowadays, nanoparticles (NPs) of titanium dioxide (TiO2) are abundantly produced. TiO2 NPs are present in various food products, in paints, cosmetics, sunscreens and toothpastes. However, the toxicity of TiO2 NPs on the central nervous system has been poorly investigated until now. The aim of this study was to evaluate the toxicity of TiO2 NPs on the central nervous system in vitro and in vivo. In cell cultures derived from embryonic cortical brain of rats, a significant decrease in neuroblasts was observed after 24 to 96 h of incubation with TiO2 NPs (5 to 20 μg/ml). This phenomenon resulted from an inhibition of neuroblast proliferation and a concomitant increase in apoptosis. In the same time, a gliosis, characterized by an increase in proliferation of astrocytes and the hypertrophy of microglial cells, occurred. The phagocytosis of TiO2 NPs by microgliocytes was also observed. In vivo, after intraperitoneal injection, the TiO2 NPs reached the brain through the blood brain barrier and the nanoparticles promoted various histological injuries such as cellular lysis, neuronal apoptosis, and inflammation. A reduction of astrocyte population was observed in some brain area such as plexiform zone, cerebellum and subependymal area. An oxidative stress was also detected by immunohistochemistry in neurons of hippocampus, cerebellum and in subependymal area. In conclusion, our study demonstrated clearly the toxic impact of TiO2 NPs on rat brain and neuronal cells and pointed about not yet referenced toxicity impacts of TiO2 such as the reduction of neuroblast proliferation both in vitro and in vivo.

The Role of Astaxanthin on Transcriptional Regulation of NMDA Receptors Voltage Sensitive Calcium Channels and Calcium Binding Proteins in Primary Cortical Neurons.

Calcium (Ca) is the phenomenon intracellular molecule that regulate many cellular process in neurons physiologically. Calcium dysregulation may occur in neurons due to excessive synaptic release of glutamate or other reasons related with neurodegeneration. Astaxanthin is a carotenoid that has antioxidant effect in cell. The purpose of this study was to investigate whether astaxanthin affects NMDA subunits, calcium binding proteins and L Type voltage sensitive Ca-channels (LVSCC) in primary cortical neuron cultures in order to see its role in calcium metabolism. Primary cortical neurons were prepared from embryonic day 16-Sprague Dawley rat embryos. The cultures were treated with 10 nM and 20 nM astaxanthin on day 7. NMDA subunits, LVSCC-A1C and LVSCC-A1D, calbindinD28k and parvalbumin mRNA expression levels was determined by qRT-PCR at 4, 24 and 48 hours. Our findings indicate that astaxanthin could have direct or indirect outcome on calcium homeostasis by regulating mRNA expression levels of NMDA subunits, LVSCC-A1C and LVSCC-A1D, calbindinD28k and parvalbumin by a dose and time dependent manner. Neuroprotective effects of astaxanthin as a Ca homeostasis regulator should be noted throughout neurodegenerative disorders, and neurosurgery applications.

Erratum: Investigating the Therapeutic Mechanism of Cannabidiol in a Human Induced Pluripotent Stem Cell (iPSC)-Based Model of Dravet Syndrome.

Dravet syndrome is an infantile epileptic encephalopathy primarily caused by loss-of-function variants of the gene SCN1A Standard treatment regimens have very limited efficacy to combat the life-threatening seizures in Dravet syndrome or the behavioral-cognitive comorbidities of the disease. Recently there has been encouraging progress in developing new treatments for this disorder. One of the clinical advances is cannabidiol (CBD), a compound naturally found in cannabis and shown to further reduce convulsive seizures in patients when used together with existing drug regimens. Like many other natural products, the exact therapeutic mechanism of CBD remains undefined. Previously we have established a human cellular model of Dravet syndrome by differentiating patient-derived induced pluripotent stem cells (iPSCs) into telencephalic inhibitory and excitatory neurons. Here we have applied this model to investigate the antiepileptic mechanism(s) of CBD at the cellular level. We first determined the effect of escalating the concentrations of CBD on neuronal excitability, using primary culture of rat cortical neurons. We found modulatory effects on excitability at submicromolar concentrations and toxic effects at high concentrations (15 µM). We then tested CBD at 50 nM, a concentration that corresponds to the estimated human clinical exposure, in telencephalic neurons derived from a patient iPSC line and control cell line H9. This 50 nM of CBD increased the excitability of inhibitory neurons but decreased the excitability of excitatory neurons, without changing the amplitude of sodium currents in either cell type. Our findings suggest a cell type-dependent mechanism for the therapeutic action of CBD in Dravet syndrome that is independent of sodium channel activity.

Investigating the Therapeutic Mechanism of Cannabidiol in a Human Induced Pluripotent Stem Cell (iPSC)-Based Model of Dravet Syndrome.

Dravet syndrome is an infantile epileptic encephalopathy primarily caused by loss-of-function variants of the gene SCN1A Standard treatment regimens have very limited efficacy to combat the life-threatening seizures in Dravet syndrome or the behavioral-cognitive comorbidities of the disease. Recently there has been encouraging progress in developing new treatments for this disorder. One of the clinical advances is cannabidiol (CBD), a compound naturally found in cannabis and shown to further reduce convulsive seizures in patients when used together with existing drug regimens. Like many other natural products, the exact therapeutic mechanism of CBD remains undefined. Previously we have established a human cellular model of Dravet syndrome by differentiating patient-derived induced pluripotent stem cells (iPSCs) into telencephalic inhibitory and excitatory neurons. Here we have applied this model to investigate the antiepileptic mechanism(s) of CBD at the cellular level. We first determined the effect of escalating the concentrations of CBD on neuronal excitability, using primary culture of rat cortical neurons. We found modulatory effects on excitability at submicromolar concentrations and toxic effects at high concentrations (15 µM). We then tested CBD at 50 nM, a concentration that corresponds to the estimated human clinical exposure, in telencephalic neurons derived from a patient iPSC line and control cell line H9. This 50 nM of CBD increased the excitability of inhibitory neurons but decreased the excitability of excitatory neurons, without changing the amplitude of sodium currents in either cell type. Our findings suggest a cell type-dependent mechanism for the therapeutic action of CBD in Dravet syndrome that is independent of sodium channel activity.

Multielectrode array recordings of human iPSC-derived neurons reveal differences in network activity depending on differentiation protocol and genome modification

Event Abstract Back to Event Multielectrode array recordings of human iPSC-derived neurons reveal differences in network activity depending on differentiation protocol and genome modification Sandra K. Fischer1* and Frank Gillardon1 1 Boehringer Ingelheim (Germany), Germany With increasing prevalence of neuropsychiatric disorders, there is an ever-growing need for suitable model systems to understand the underlying pathological mechanisms. Currently most data are derived from animal models, although correlations between animal and human are often weak. However, acquisition of human neuronal material is rarely possible and afflicted with ethic concerns. Human induced pluripotent stem cell (hiPSC) derived neuronal cultures offer a possibility to overcome these limitations. Synaptic activity and neurotransmission deficits have been identified as core pathophysiologies of neuropsychiatric disorders. The multi electrode array (MEA) technology allows for repeated measurements of electrical activity of functional neuronal networks over an extended period of time. We cultivated iPSC-derived neuronal cultures on the Multichannel Systems MEA multiwell platform and assessed synaptic activity over time and in response to treatment with pharmacological agents. The hiPSC-derived neurons developed electrical activity from single spikes to bursts over time indicating maturation. Dot-seeded GlutaNeurons (iCell Neurons, Cellular Dynamics International) showed their peak activity after 2-3 weeks in culture with the highest burst activity in week 3. Ngn2-induced Neurons (iCell Neurons, Cellular Dynamics International) displayed a weaker total activity with lower spike rate, less burst activity and a maximum activity after 6 weeks in culture. Treatment with the GABA antagonist Bicuculline evoked synchronized burst firing in GlutaNeuron cultures containing approx. 20% GABAergic neurons, whereas the spontaneous activity of the purely glutamatergic culture of Induced Neurons was unchanged as expected. Loss-of-function mutations in ErbB receptor tyrosine kinases have been identified as genetic risk factors for neuropsychiatric diseases. Upon pharmacological inhibition of ErbB receptor tyrosine kinases, neuronal network activity was inhibited, consistent with our MEA recordings from primary rat cortical neuron cultures. Moreover, we generated a loss-of-function mutation in a high risk gene for schizophrenia in hiPSC from a healthy donor utilizing the CRISPR/Cas9 gene editing system. Following neuronal differentiation into the forebrain lineage electrophysiological activity was monitored on the MEA multiwell system during network formation and maturation. Preliminary data indicate altered spontaneous network activity in mutant iPSC-derived neurons compared to isogenic controls. In summary, our findings demonstrate that hiPSC-derived neurons form functional neuronal networks on MEA plates which can serve as suitable system for modeling of neuropsychiatric diseases. Acknowledgements The authors thank Dr. Sabine Lange (Cellular Dynamics International) for helpful discussions. Keywords: IPSC-derived neurons, neuropsychiatric disorder, electrical network activity, multielectrode array plate, CRISPR/Cas9 gene editing Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018. Presentation Type: Poster Presentation Topic: Stem cell-derived applications Citation: Fischer SK and Gillardon F (2019). Multielectrode array recordings of human iPSC-derived neurons reveal differences in network activity depending on differentiation protocol and genome modification. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00015 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Mrs. Sandra K Fischer, Boehringer Ingelheim (Germany), Hannover, Germany, sandra.fischer@boehringer-ingelheim.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sandra K Fischer Frank Gillardon Google Sandra K Fischer Frank Gillardon Google Scholar Sandra K Fischer Frank Gillardon PubMed Sandra K Fischer Frank Gillardon Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Tetramethylpyrazine nitrone activates the BDNF/Akt/CREB pathway to promote post-ischaemic neuroregeneration and recovery of neurological functions in rats.

Neuronal regeneration from endogenous precursors is an attractive strategy for the treatment of ischaemic stroke. However, most stroke-generated newborn neurons die over time. Therefore, a drug that is both neuroprotective and pro-neurogenic may be beneficial after stroke. Here, we assessed the neurogenic and oligodendrogenic effects of tetramethylpyrazine nitrone (TBN), a neuroprotective drug candidate for stroke, in a rat model of ischaemic stroke. We used Sprague Dawley rats with middle cerebral artery occlusion (MCAO). TBN was administered by tail vein injection beginning at 3h post ischaemia. Therapeutic effect of TBN was evaluated by neurological behaviour and cerebral infarction. Promotion of neurogenesis and oligodendrogenesis was determined by double immunofluorescent staining and Western blotting analyses. Primary cultures of cortical neurons were used to assess the effect of TBN on neuronal differentiation in vitro. TBN reduced cerebral infarction, preserved and/or restored neurological function and promoted neurogenesis and oligodendrogenesis in rats after MCAO. In addition, TBN stimulated neuronal differentiation on primary culture of cortical neurons in vitro. Pro-neurogenic effects of TBN were attributed to its activation of the AKT/cAMP responsive element-binding protein through increasing brain-derived neurotrophic factor (BDNF) expression, as shown by the abolition of the effects of TBN by a specific inhibitor of BDNF receptor ANA-12 and by the PI3K inhibitor LY294002. As TBN can simultaneously provide neuroprotection and pro-neurogenic effects, it may be a promising treatment for both acute phase neuroprotection and long-term functional recovery after ischaemic stroke.

The role of caveolin-1 in morphine-induced structural plasticity in primary cultured mouse cerebral cortical neurons.

Long-term repeated application of morphine induces plasticity changes in sensitive neurons, which represents the neurobiological basis of morphine addiction. Caveolin-1 (Cav-1) is an important scaffolding protein that plays a key role in the regulation of neurostructural plasticity. However, in-depth studies focused on the role of Cav-1 in morphine-induced changes in neurostructural plasticity are lacking. The present study explored the effects of Cav-1 on the expression levels of 2 markers of neurite outgrowth, growth association protein 43 (GAP-43) and microtubule-associated protein 2 (MAP-2), during the process of morphine-induced changes in the structural plasticity. A primary mouse cerebral cortical neuron culture was established, and Cav-1 knockdown was achieved. The results showed that morphine at a concentration of 10.0μmol/L had no adverse effect on neuronal viability, but enhanced the Cav-1 and GAP-43 levels and induced the outgrowth of MAP-2-labeled neurites. The qRT-PCR data showed that 10.0μmol/L of morphine increased the Cav-1 mRNA level and promoted Cav-1 expression at the transcriptional level. Moreover, Cav-1 knockdown inhibited the morphine-induced upregulation of GAP-43 expression and the prolongation of MAP-2-labeled neurites. In conclusion, morphine was capable of inducing changes in the structural plasticity of primary cultured mouse cortical neurons, and Cav-1 played an important role in this process. Inhibition of Cav-1 expression reduced the morphine-induced increase in the neuronal growth markers GAP-43 and MAP-2. Thus, Cav-1 may be a potential molecular target for inhibiting morphine-induced neuroplasticity.

Cyclin-dependent kinase 5-mediated phosphorylation of CHIP promotes the tAIF-dependent death pathway in rotenone-treated cortical neurons.

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase. Its dysregulation has been implicated in various neurodegenerative diseases. We previously reported that phosphorylation of the C-terminus of the Hsc70-interacting protein (CHIP) by Cdk5 promotes truncated apoptosis-inducing factor (tAIF)-mediated neuronal death induced by oxidative stress. Here, we determined whether this Cdk5-dependent cell death signaling pathway is present in experimental models of Parkinson's disease. First, we showed that rotenone activates Cdk5 in primary cultures of cortical neurons and causes tAIF-dependent neuronal cell death. This event was attenuated by negative regulation of endogenous Cdk5 activity by the pharmacological Cdk5 inhibitor, roscovitine, or by lentiviral knockdown of Cdk5. Cdk5 phosphorylates CHIP at Ser20 in rotenone-treated neurons. Consequently, overexpression of CHIPS20A, but not CHIPWT, attenuates tAIF-induced cell death in rotenone-treated cortical neurons. Taken together, these results indicate that phosphorylation of CHIP at Ser20 by Cdk5 activation inhibits CHIP-mediated tAIF degradation, thereby contributing to tAIF-induced neuronal cell death following rotenone treatment.

Neuroprotective effects of orientin on oxygen-glucose deprivation/reperfusion-induced cell injury in primary culture of rat cortical neurons.

Orientin (luteolin-8-C-glucoside) is a phenolic compound found abundantly in millet, juice, and peel of passion fruit and has been shown to have antioxidant properties. In the present study, we explored the effects of orientin on oxygen-glucose deprivation/reperfusion (OGD/RP)-induced cell injury in primary culture of rat cortical neurons using an in vitro model of neonatal ischemic brain injury. The reduced cell viability and elevated lactate dehydrogenase leakage were observed after OGD/RP exposure, which were then reversed by orientin (10, 20, and 30 µM) pretreatment in a dose-dependent manner. Additionally, OGD/RP treatment resulted in significant oxidative stress, accompanied by enhanced intracellular reactive oxygen species (ROS) generation, and obvious depletion in the activities of intracellular Mn-superoxide dismutase, catalase, and glutathione peroxidase antioxidases. However, these effects were dose dependently restored by orientin pretreatment. We also found that orientin pretreatment dose dependently suppressed [Ca2+]i increase and mitochondrial membrane potential dissipation caused by OGD/RP in primary culture of rat cortical neurons. Western blot analysis showed that OGD/RP exposure induced a distinct decrease of Bcl-2 protein and a marked elevation of Bax, caspase-3, and cleaved caspase-3 proteins; whereas these effects were dose dependently reversed by orientin incubation. Both the caspase-3 activity and the apoptosis rate were increased under OGD/RP treatment, but was then dose dependently down-regulated by orientin (10, 20, and 30 µM) incubation. Moreover, orientin pretreatment dose dependently inhibited OGD/RP-induced phosphorylation of JNK and ERK1/2. Notably, JNK inhibitor SP600125 and ERK1/2 inhibitor PD98059 also dramatically attenuated OGD/RP-induced cell viability loss and ROS generation, and further, orientin failed to protect cortical neurons with the interference of JNK activator anisomycin or ERK1/2 activator FGF-2. Taken together, these results demonstrated that orientin has significant neuroprotective effects against OGD/RP-induced cell injury via JNK and ERK1/2 signaling pathways in primary culture of rat cortical neurons. Impact statement Orientin has been used in traditional eastern medicine and reported to possess antioxidant properties. However, the effects of orientin on neonatal ischemic brain injury and the underlying mechanisms involved have not been studied. Our results showed that orientin exerts significant neuroprotective effects on cell injury caused by oxygen-glucose deprivation/reperfusion via the JNK and ERK1/2 signaling pathways in primary culture of rat cortical neurons, implying the potential therapeutic application of orientin via the suppression of oxidative stress and cell apoptosis. This research suggested that orientin may be used as a therapeutic and preventive option for newborn cerebral ischemia/reperfusion injury.

Specific rescue by ortho-hydroxy atorvastatin of cortical GABAergic neurons from previous oxygen/glucose deprivation: role of pCREB.

The statin atorvastatin (ATV) given as a post-treatment has been reported beneficial in stroke, although the mechanisms involved are not well understood so far. Here, we investigated invitro the effect of post-treatment with ATV and its main bioactive metabolite ortho-hydroxy ATV (o-ATV) on neuroprotection after oxygen and glucose deprivation (OGD), and the role of the pro-survival cAMP response element-binding protein (CREB). Post-OGD treatment of primary cultures of rat cortical neurons with o-ATV, but not ATV, provided neuroprotection to a specific subset of cortical neurons that were large and positive for glutamic acid decarboxylase (large-GAD(+) neurons, GABAergic). Significantly, only these GABAergic neurons showed an increase in phosphorylated CREB (pCREB) early after neuronal cultures were treated post-OGD with o-ATV. We found that o-ATV, but not ATV, increased the neuronal uptake of glutamate from the medium; this provides a rationale for the specific effect of o-ATV on pCREB in large-GABAergic neurons, which have a higher ratio of synaptic (pCREB-promoting) vs extrasynaptic (pCREB-reducing) N-methyl-D-aspartate (NMDA) receptors (NMDAR) than that of small-non-GABAergic neurons. When we pharmacologically increased pCREB levels post-OGD in non-GABAergic neurons, through the selective activation of synaptic NMDAR, we observed as well long-lasting neuronal survival. We propose that the statin metabolite o-ATV given post-OGD boosts the intrinsic pro-survival factor pCREB in large-GABAergic cortical neurons invitro, this contributing to protect them from OGD.

Insulin-like growth factor binding protein-6 released from human mesenchymal stem cells confers neuronal protection through IGF-1R-mediated signaling

Human bone marrow-derived mesenchymal stem cells (hMSCs) are a desirable cell source for cell-based therapy to treat nervous system injuries due to their ability to differentiate into specific cell types. In addition to their multi-potency, hMSCs render the tissue microenvironment more favorable for tissue repair by secreting various growth factors. Our previous study demonstrated that hMSCs secrete several growth factors, including several insulin-like growth factor binding proteins (IGFBPs). Among these, IGFBP-6 binds with high affinity and inhibits insulin growth factor-2 (IGF-2) to inhibit the growth of IGF-2-dependent tumors. However, the function of IGFBP-6 in the nervous system remains to be fully elucidated. The present study investigated the protective effects of IGFBP-6 secreted by hMSCs on H2O2-injured primary cortical neuron cultures and lysolecithin-injured organotypic spinal cord slice cultures. Treatment of the H2O2-injured cortical neurons with conditioned media from hMSCs (hMSC-CM) increased the phosphorylation of Akt, reduced cell death and mitochondrial translocation of Bax, and regulated extracellular levels of IGF-1 and IGF-2. MTT assay, western blot analysis and ELISA were used to detect the cell viability and protein expression levels, respectively. An inhibitory antibody against IGFBP-6 eliminated this hMSC-CM-mediated neuroprotective effect in the injured cortical neuron cultures and spinal cord slice cultures. In addition, treatment with cyclolignan picropodophyllin, an inhibitor of IGF-1 receptor (IGF-1R), significantly inhibited neuronal protection by hMSC-CM. These findings demonstrated that hMSC-CM-mediated neuroprotection was attributed to IGF-1R-mediated signaling, potentiated via the inhibition of IGF-2 by IGFBP-6. The results of the present study provide insight into the mechanism by which hMSC administration may promote recovery from nerve injury.

Is trehalose an autophagic inducer? Unraveling the roles of non-reducing disaccharides on autophagic flux and alpha-synuclein aggregation

Autophagy is a pivotal intracellular process by which cellular macromolecules are degraded upon various stimuli. A failure in the degradation of autophagic substrates such as impaired organelles and protein aggregates leads to their accumulations, which are characteristics of many neurodegenerative diseases. Pharmacological activation of autophagy has thus been considered a prospective therapeutic approach for treating neurodegenerative diseases. Among a number of autophagy-inducing agents, trehalose has received attention for its beneficial effects in different disease models of neurodegeneration. However, how trehalose promotes autophagy has not been fully revealed. We investigated the influence of trehalose and other disaccharides upon autophagic flux and aggregation of α-synuclein, a protein linked to Parkinson's disease. In differentiated human neuroblastoma and primary rat cortical neuron culture models, treatment with trehalose and other disaccharides resulted in accumulation of lipidated LC3 (LC3-II), p62, and autophagosomes, whereas it decreased autolysosomes. On the other hand, addition of Bafilomycin A1 to trehalose treatments had relatively marginal effect, an indicative of autophagic flux blockage. In concordance with these results, the cells treated with trehalose exhibited an incremental tendency in α-synuclein aggregation. Secretion of α-synuclein was also elevated in the culture medium upon trehalose treatment, thereby significantly increasing intercellular transmission of this protein. Despite the substantial increase in α-synuclein aggregation, which normally leads to cell death, cell viability was not affected upon treatment with trehalose, suggesting an autophagy-independent protective function of trehalose against protein aggregates. This study demonstrates that, although trehalose has been widely considered an autophagic inducer, it may be actually a potent blocker of the autophagic flux.

Differential Molecular Targets for Neuroprotective Effect of Chlorogenic Acid and its Related Compounds Against Glutamate Induced Excitotoxicity and Oxidative Stress in Rat Cortical Neurons.

The present study has been designed to explore the molecular mechanism and signaling pathway targets of chlorogenic acid (CGA) and its main hydrolysates, caffeic (CA) and quinic acid in the protective effect against glutamate-excitotoxicity. For this purpose 8-DIV cortical neurons in primary culture were exposed to 50μM L-glutamic acid plus 10µM glycine, with or without 10-100μM tested compounds. Chlorogenic acid and caffeic acid via their antioxidant properties inhibited cell death induced by glutamate in dose depended manner. However, quinic acid slightly protects neurons at a higher dose. DCF, JC-1 and Ca2+sensitive fluorescent dye fura-2, were used to measure intracellular ROS accumulation, mitochondrial membrane potential integration and intracellular calcium concentration [Ca2+] i . Results indicate that similarly, CGA acts as a protective agent against glutamate-induced cortical neurons injury by suppressing the accumulation of endogenous ROS and restore the mitochondrial membrane potential, activate the enzymatic antioxidant system by the increase levels of SOD activity and modulate the rise of intracellular calcium levels by increasing the rise of intracellular concentrations of Ca2+caused by glutamate overstimulation. PKC signaling cascade appear to be engaged in this protective mechanism. Interseling, CGA and CA also exhibit antiapoptotic properties against glutamate-induced cleaved activation of pro-caspases; caspase 1,8 and 9 and calpain (PD 150606,Calpeptin and MDL 28170).These data suggest that neuroprotective activity of CGA ester may occurs throught its hydrolysate,the caffeic acid and its interaction with intracellular molecules suggesting that CGA exert its neuroprotection via its caffeoly acid group that might potentially be used as a therapeutic agent in neurodegeneratives disorders associated with glutamate excitotoxicity.

D-4F increases microRNA-124a and reduces neuroinflammation in diabetic stroke rats.

D-4F is an apolipoprotein-A1 mimetic peptide that promotes anti-inflammatory effects. MicroRNA-124 is the most abundant brain-specific microRNA and has anti-inflammatory effects. In this study, we investigated the therapeutic efficacy and mechanisms of D-4F treatment of stroke in type one diabetes mellitus (T1DM) rats. Male Wistar rats were induced with T1DM, subjected to embolic middle cerebral artery occlusion and treated with PBS or D-4F (1 mg/kg i.p.) at 2, 24 and 48 hours after stroke (n=8/group). A battery of function tests, brain blood barrier (BBB) integrity, white matter changes and microRNA expression were evaluated in vivo and in vitro. D-4F treatment in T1DM-stroke rats significantly improves functional outcome, decreases BBB leakage, increases tight junction protein expression, decreases white matter damage and inflammatory factor expression, while increasing anti-inflammatory M2 macrophage polarization in the ischemic brain. D-4F significantly increases microRNA-124a expression, and decreases matrix metalloproteinase-9, tumor necrosis factor-α and toll-like receptor-4 gene expression in the ischemic brain, and in primary cortical neuronal and microglial cultures. Inhibition of microRNA-124 in cultured primary cortical neurons and microglia attenuates D-4F induced anti-inflammatory effects and M2 macrophage polarization. D-4F treatment of T1DM-stroke increases microRNA-124 expression, promotes anti-inflammatory effects and M2 macrophage polarization, which may contribute to D-4F-induced improvement in neurological function, and BBB and white matter integrity.

The Progestin Receptor Interactome in the Female Mouse Hypothalamus: Interactions with Synaptic Proteins Are Isoform Specific and Ligand Dependent.

Progestins bind to the progestin receptor (PR) isoforms, PR-A and PR-B, in brain to influence development, female reproduction, anxiety, and stress. Hormone-activated PRs associate with multiple proteins to form functional complexes. In the present study, proteins from female mouse hypothalamus that associate with PR were isolated using affinity pull-down assays with glutathione S-transferase–tagged mouse PR-A and PR-B. Using complementary proteomics approaches, reverse phase protein array (RPPA) and mass spectrometry, we identified hypothalamic proteins that interact with PR in a ligand-dependent and isoform-specific manner and were confirmed by Western blot. Synaptic proteins, including synapsin-I and synapsin-II, interacted with agonist-bound PR isoforms, suggesting that both isoforms function in synaptic plasticity. In further support, synaptogyrin-III and synapsin-III associated with PR-A and PR-B, respectively. PR also interacted with kinases, including c-Src, mTOR, and MAPK1, confirming phosphorylation as an integral process in rapid effects of PR in the brain. Consistent with a role in transcriptional regulation, PR associated with transcription factors and coactivators in a ligand-specific and isoform-dependent manner. Interestingly, both PR isoforms associated with a key regulator of energy homeostasis, FoxO1, suggesting a novel role for PR in energy metabolism. Because many identified proteins in this PR interactome are synaptic proteins, we tested the hypothesis that progestins function in synaptic plasticity. Indeed, progesterone enhanced synaptic density, by increasing synapsin-I–positive synapses, in rat primary cortical neuronal cultures. This novel combination of RPPA and mass spectrometry allowed identification of PR action in synaptic remodeling and energy homeostasis and reveals unique roles for progestins in brain function and disease.

Involvement of superoxide in malaoxon-induced toxicity in primary cultures of cortical neurons

ABSTRACTOrganophosphorus compounds (OP) represent a class of insecticides that are used most globally. The neurotoxic effects are attributed mainly to acetylcholinesterase (AChE) enzyme inhibition, which is responsible for cholinergic manifestations in individuals acutely exposed to OP. However, AChE inhibition alone cannot account for the wide range of symptoms that were reported following OP exposures. In agreement with this, evidence shows that non-cholinergic events may be mechanistically linked to OP-induced neurotoxicity. The aim of this study was to investigate the potential occurrence of oxidative stress as a critical step in the toxicity induced by the OP malaoxon(MAL) using primary cultures of mouse cortical neurons, as well as to distinguish MAL-induced oxidative stress and cell toxicity from an action on AChE blockade. Primary cultures of mouse cortical neurons were treated with MAL (0.01; 0.1; 1; 10; or 100 µM) at varying time points (1, 3, 6, 24, 48, or 144 hr) and the following biochemical parameters determined including cell viability, AChE activity, and superoxide production. MAL significantly reduced cell viability in a concentration- and time-dependent manner. Of note, 1 µM MAL significantly inhibited (approximately 75%) AChE activity after 48 hr incubation. Pralidoxime (PRAL) (600 µM), a classical AChE reactivator, significantly protected against MAL-induced AChE blockade; however, PRAL did not affect MAL-mediated fall in cellular viability, indicating that AChE inhibition is not necessarily correlated with insecticide-induced decrease in cell survival. MAL-induced diminished cell viability was preceded by a significant increase in superoxide anion production. The antioxidant agent ascorbic acid (AA) (200 µM), which significantly protected against MAL-induced superoxide anion production, did not alter MAL-induced AChE inhibition and significantly prevented insecticide-mediated fall in cell survival. Data show that increased superoxide anion production is an event that precedes MAL-induced cell toxicity in primary cultures of mouse cortical neurons. Based on the preventative effects of AA against MAL-mediated superoxide anion production and reduced cell viability, evidence indicates that oxidative stress represents an important step mediating MAL-induced toxicity in neurons and that AChE inhibition is not necessarily correlated with lowered cell survival noted in insecticide-exposed cells.

Reactive Oxygen Species Produced by a Photodynamic Effect Induced Calcium Signal in Neurons and Astrocytes.

Photodynamic therapy (PDT) leads to production of reactive oxygen species (ROS) and cell destruction due to oxidative stress. We used photodynamic effect of photosensitizer radachlorin to unravel the effect of photo-induced oxidative stress on the calcium signal and lipid peroxidation in primary culture of cortical neurons and astrocytes using live cell imaging. We have found that irradiation in presence of 200nM of radachlorin induces calcium signal in primary neurons and astrocytes. Photo-induced neuronal calcium signal depends on internal calcium stores as it was still observed in calcium-free medium and could be blocked by depletion of endoplasmic reticulum (ER) stores with inhibitor of sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) thapsigargin. Both inhibitors of phospholipase C activity U73122 and water-soluble analogue of vitamin E Trolox suppressed calcium response activated by PDT. We have also observed that the photodynamic effect of radachlorin induces lipid peroxidation in neurons and astrocytes. This data demonstrate that lipid peroxidation induced by PDT in neurons and astrocytes leads to activation of phospholipase C that results in production of inositol 1,4,5-trisphosphate (IP3).