Neurotoxic Potential of Deoxynivalenol in Murine Brain Cell Lines and Primary Hippocampal Cultures

Chronic exposure to the mycotoxin deoxynivalenol (DON) from grain-based food and feed affects human and animal health. Known consequences include entereopathogenic and immunotoxic defects; however, the neurotoxic potential of DON has only come into focus more recently due to the observation of behavioural disorders in exposed farm animals. DON can cross the blood-brain barrier and interfere with the homeostasis/functioning of the nervous system, but the underlying mechanisms of action remain elusive. Here, we have investigated the impact of DON on mouse astrocyte and microglia cell lines, as well as on primary hippocampal cultures by analysing different toxicological endpoints. We found that DON has an impact on the viability of both glial cell types, as shown by a significant decrease of metabolic activity, and a notable cytotoxic effect, which was stronger in the microglia. In astrocytes, DON caused a G1 phase arrest in the cell cycle and a decrease of cyclic-adenosine monophosphate (cAMP) levels. The pro-inflammatory cytokine tumour necrosis factor (TNF)-α was secreted in the microglia in response to DON exposure. Furthermore, the intermediate filaments of the astrocytic cytoskeleton were disturbed in primary hippocampal cultures, and the dendrite lengths of neurons were shortened. The combined results indicated DON’s considerable potential to interfere with the brain cell physiology, which helps explain the observed in vivo neurotoxicological effects.

Biocompatibility of Platinum Nanoparticles in Brain ex vivo Models in Physiological and Pathological Conditions

Platinum nanoparticles (PtNPs) have unique physico-chemical properties that led to their use in many branches of medicine. Recently, PtNPs gathered growing interest as delivery vectors for drugs, biosensors and as surface coating on chronically implanted biomedical devices for improving electrochemical properties. However, there are contradictory statements about their biocompatibility and impact on target organs such as the brain tissue, where these NPs are finding many applications. Furthermore, many of the reported studies are conducted in homeostasis conditions and, consequently, neglect the impact of the pathologic conditions on the tissue response. To expand our knowledge on the effects of PtNPs on neuronal and glial cells, we investigated the acute effects of monodisperse sodium citrate-coated PtNPs on rat organotypic hippocampal cultures in physiological or neuronal excitotoxic conditions induced by kainic acid (KA). The cellular responses of the PtNPs were evaluated through cytotoxic assays and confocal microscopy analysis. To mimic a pathologic scenario, 7-day organotypic hippocampal cultures were exposed to KA for 24 h. Subsequently, PtNPs were added to each slice. We show that incubation of the slices with PtNPs for 24 h, does not severely impact cell viability in normal conditions, with no significant differences when comparing the dentate gyrus (DG), as well as CA3 and CA1 pyramidal cell layers. Such effects are not exacerbated in KA-treated slices, where the presence of PtNPs does not cause additional neuronal propidium iodide (PI) uptake in CA3 and CA1 pyramidal cell layers. However, PtNPs cause microglial cell activation and morphological alterations in CA3 and DG regions indicating the establishment of an inflammatory reaction. Morphological analysis revealed that microglia acquire activated ameboid morphology with loss of ramifications, as a result of their response to PtNPs contact. Surprisingly, this effect is not increased in pathological conditions. Taken together, these results show that PtNPs cause microglia alterations in short-term studies. Additionally, there is no worsening of the tissue response in a neuropathological induced scenario. This work highlights the need of further research to allow for the safe use of PtNPs. Also, it supports the demand of the development of novel and more biocompatible NPs to be applied in the brain.

Control of neurotransmitter release by two distinctmembrane-binding faces of the Munc13-1 C­1C2B region

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C1 domain, a Ca2+/PIP2-binding C2B domain, a MUN domain and a C2C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane through distinct interactions of the C­1C2B region with the plasma membrane: i) one involving a polybasic face that is expected to yield a perpendicular orientation of Munc13-1 and hinder release; and ii) another involving the DAG-Ca2+-PIP2-binding face that is predicted to result in a slanted orientation and facilitate release. Here we have tested this model and investigated the role of the C­1C2B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair Ca2+-independent liposome bridging and fusion in in vitro reconstitution assays, and synaptic vesicle priming in primary murine hippocampal cultures. A K720E mutation in the polybasic face and a K706E mutation in the C2B domain Ca2+-binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca2+-evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C1-C2B region of Munc13-1 plays a central role in vesicle priming and support the notion that two distinct faces of this region control neurotransmitter release and short-term presynaptic plasticity.

The N-Formyl Peptide Receptor 2 (FPR2) Agonist MR-39 Exhibits Anti-Inflammatory Activity in LPS-Stimulated Organotypic Hippocampal Cultures

Accumulating evidence indicates a pivotal role for chronic inflammatory processes in the pathogenesis of neurodegenerative and psychiatric disorders. G protein-coupled formyl peptide receptor 2 (FPR2) mediates pro-inflammatory or anti-/pro-resolving effects upon stimulation with biased agonists. We aimed to evaluate the effects of a new FPR2 ureidopropanamide agonist, compound MR-39, on neuroinflammatory processes in organotypic hippocampal cultures (OHCs) derived from control (WT) and knockout FPR2−/− mice (KO) exposed to bacterial endotoxin (lipopolysaccharide; LPS). Higher LPS-induced cytokine expression and basal release were observed in KO FPR2 cultures than in WT cultures, suggesting that a lack of FPR2 enhances the OHCs response to inflammatory stimuli. Pretreatment with MR-39 abolished some of the LPS-induced changes in the expression of genes related to the M1/M2 phenotypes (including Il-1β, Il-6, Arg1, Il-4, Cd74, Fizz and Cx3cr1) and TNF-α, IL-1β and IL-4 release in tissue derived from WT but not KO mice. Receptor specificity was confirmed by adding the FPR2 antagonist WRW4, which abolished the abovementioned effects of MR-39. Further biochemical data showed an increase in the phospho-p65/total p65 ratio after LPS stimulation in hippocampal tissues from both WT and KO mice, and MR-39 only reversed this effect on WT OHCs. LPS also increased TRAF6 levels, which are critical for the TLR4-mediated NF-κB pro-inflammatory responses. MR-39 attenuated the LPS-evoked increase in the levels of the NLRP3 and caspase-1 proteins in WT but not KO hippocampal cultures. Since NLRP3 may be involved in the pyroptosis, a lytic type of programmed cell death in which the main role is played by Gasdermin D (GSDMD), we examined the effects of LPS and/or MR-39 on the GSDMD protein level. LPS only increased GSDMD production in the WT tissues, and this effect was ameliorated by MR-39. Collectively, this study indicates that the new FPR2 agonist efficiently abrogates LPS-induced neuroinflammation in an ex vivo model, as evidenced by a decrease in pro-inflammatory cytokine expression and release as well as the downregulation of NLRP3 inflammasome-related pathways.

Neuronal circuits overcome imbalance in excitation and inhibition by adjusting connection numbers

The interplay between excitation and inhibition is crucial for neuronal circuitry in the brain. Inhibitory cell fractions in the neocortex and hippocampus are typically maintained at 15 to 30%, which is assumed to be important for stable dynamics. We have studied systematically the role of precisely controlled excitatory/inhibitory (E/I) cellular ratios on network activity using mice hippocampal cultures. Surprisingly, networks with varying E/I ratios maintain stable bursting dynamics. Interburst intervals remain constant for most ratios, except in the extremes of 0 to 10% and 90 to 100% inhibitory cells. Single-cell recordings and modeling suggest that networks adapt to chronic alterations of E/I compositions by balancing E/I connectivity. Gradual blockade of inhibition substantiates the agreement between the model and experiment and defines its limits. Combining measurements of population and single-cell activity with theoretical modeling, we provide a clearer picture of how E/I balance is preserved and where it fails in living neuronal networks.

Human Wharton’s jelly mesenchymal stem cells protect neural cells from oxidative stress through paracrine mechanisms

Aim: Mesenchymal stem cells (MSCs) have neuroprotective and immunomodulatory properties, which are partly mediated by extracellular vesicles (EVs) secretion. We aimed to evaluate the effects of human Wharton’s jelly-derived MSCs (WJ-MSCs) and their EVs on rat hippocampal cultures subjected to hydrogen peroxide (H2O2). Materials & methods: Hippocampal dissociated cultures were either co-cultured with WJ-MSCs or treated with their EVs prior to H2O2 exposure and reactive oxygen species levels and cell viability were evaluated. Results: Coculture with WJ-MSCs or pre-incubation with EVs prior to the insult reduced reactive oxygen species after H2O2 exposure. Cell viability was improved only when coculture was maintained following the insult, while EVs did not significantly improve cell viability. Conclusion: WJ-MSCs have potential antioxidant and neuroprotective effects on hippocampal cultures which might be partially mediated by EVs.