Caspase-1 has a critical role in blood-brain barrier injury and its inhibition contributes to multifaceted repair

International audienceExcessive inflammation might activate and injure the blood-brain barrier (BBB), a common feature of many central nervous system (CNS) disorders. We previously developed an in vitro BBB injury model in which the organophosphate paraoxon (PX) affects the BBB endothelium by attenuating junctional protein expression leading to weakened barrier integrity. The objective of this study was to investigate the inflammatory cellular response at the BBB to elucidate critical pathways that might lead to effective treatment in CNS pathologies in which the BBB is compromised. We hypothesized that caspase-1, a core component of the inflammasome complex, might have important role in BBB function since accumulating evidence indicates its involvement in brain inflammation and pathophysiology

<i>Odorant binding protein 69a</i> connects social interaction to modulation of social responsiveness in <i>Drosophila</i>

<div><p>Living in a social environment requires the ability to respond to specific social stimuli and to incorporate information obtained from prior interactions into future ones. One of the mechanisms that facilitates social interaction is pheromone-based communication. In <i>Drosophila melanogaster</i>, the male-specific pheromone cis-vaccenyl acetate (cVA) elicits different responses in male and female flies, and functions to modulate behavior in a context and experience-dependent manner. Although it is the most studied pheromone in flies, the mechanisms that determine the complexity of the response, its intensity and final output with respect to social context, sex and prior interaction, are still not well understood. Here we explored the functional link between social interaction and pheromone-based communication and discovered an odorant binding protein that links social interaction to sex specific changes in cVA related responses. <i>Odorant binding protein 69a</i> (<i>Obp69a</i>) is expressed in auxiliary cells and secreted into the olfactory sensilla. Its expression is inversely regulated in male and female flies by social interactions: cVA exposure reduces its levels in male flies and increases its levels in female flies. Increasing or decreasing Obp69a levels by genetic means establishes a functional link between Obp69a levels and the extent of male aggression and female receptivity. We show that activation of cVA-sensing neurons is sufficeint to regulate Obp69a levels in the absence of cVA, and requires active neurotransmission between the sensory neuron to the second order olfactory neuron. The cross-talk between sensory neurons and non-neuronal auxiliary cells at the olfactory sensilla, represents an additional component in the machinery that promotes behavioral plasticity to the same sensory stimuli in male and female flies.</p></div

<i>Obp69a</i> transcription is dimorphically regulated in response to male scents, and exposure to the male pheromone cVA.

<p>Total RNA extracted from heads of male and female flies exposed to male scents (<b>A,B</b>) or to cVA (<b>C,D</b>) for three days was analyzed for <i>Obp69a</i> mRNA levels by RT-qPCR. Statistical significance was determined by one-way ANOVA with Tukey post-hoc analysis. Error bars signify SEM. ***<i>P</i><0.001, (A) F(2, 6) = 28.5, (B) F(2, 6) = 44.1, (C) F(2, 6) = 24.3, (D) F(2, 6) = 28.3. n = 3 independent experiments of 15–20 fly heads/sample.</p

Obp69a links prior social interaction to modulation of social responsivity.

<p><b>A</b>. Obp69a was knocked-down by RNAi in male flies and number of lunges was scored. Statistical significance was determined by one-way ANOVA with Tukey post-hoc analysis, F(2, 69) = 25.4 ***<i>P</i><0.001, n = 24. <b>B, C</b>. Obp69a was over-expressed in male flies and the number of lunges was scored (<b>B</b>) or the time until the first lunge (latency) was measured. Statistical significance was determined by one-way ANOVA with Tukey post-hoc analysis. (B) F(2, 63) = 8.5, (C) F(2, 62) = 6.2 **<i>P</i><0.01, ***<i>P</i><0.001, n = 24 <b>D.</b> Female flies were exposed to male scents prior to the courtship assay, and the time to copulation with WT virgin male flies was measured. Statistical significance was determined by Students t-test. **P<0.01, n = 17. <b>E.</b> RNAi to Obp69a was expressed in females that were previously exposed to male scents, and the time to copulation with WT virgin males was measured. Statistical significance was determined by one-way ANOVA with Tukey post-hoc analysis, F(4, 42) = 3.55, *<i>P</i><0.05, n = 18. <b>F.</b> Obp69a-GFP was expressed in female flies, which were exposed to 1μg cVA prior to mating with WT virgin males. The time to copulation was measured. Statistical significance was determined by one-way ANOVA with Tukey post-hoc analysis, F(2, 44) = 4.9, **P<0.01, n = 34, 42 and 31 for <i>Obp69aMi-GAL4</i>, <i>UAS-Obp69a-GFP</i> and <i>Obp69aMi</i>-<i>GAL4</i>; <i>UAS-Obp69a-GFP</i> respectively.</p

Endothelial Iron Homeostasis Regulates Blood-Brain Barrier Integrity via the HIF2α—Ve-Cadherin Pathway

International audienceThe objective of this study was to investigate the molecular response to damage at the blood brain barrier (BBB) and to elucidate critical pathways that might lead to effective treatment in central nervous system (CNS) pathologies in which the BBB is compromised. We have used a human, stem-cell derived in-vitro BBB injury model to gain a better understanding of the mechanisms controlling BBB integrity. Chemical injury induced by exposure to an organophosphate resulted in rapid lipid peroxidation, initiating a ferroptosis-like process. Additionally, mitochondrial ROS formation (MRF) and increase in mitochondrial membrane permeability were induced, leading to apoptotic cell death. Yet, these processes did not directly result in damage to barrier functionality, since blocking them did not reverse the increased permeability. We found that the iron chelator, Desferal© significantly decreased MRF and apoptosis subsequent to barrier insult, while also rescuing barrier integrity by inhibiting the labile iron pool increase, inducing HIF2α expression and preventing the degradation of Ve-cadherin specifically on the endothelial cell surface. Moreover, the novel nitroxide JP4-039 significantly rescued both injury-induced endothelium cell toxicity and barrier functionality. Elucidating a regulatory pathway that maintains BBB integrity illuminates a potential therapeutic approach to protect the BBB degradation that is evident in many neurological diseases

<i>Obp69a</i> transcriptional regulation requires active neurotransmission of the information from the sensory neuron to the second order olfactory neuron.

<p>(<b>A</b>) Male flies expressing <i>UAS-Cs-Chrimson</i> and <i>UAS-Shibire</i>^<i>ts</i> in Or65a neurons where subjected to three 15 minutes long optogenetic activations as a positive control, synaptic release blocking, or both. Obp69a relative expression was then measured using RT-qPCR. (<b>B</b>) Female flies expressing <i>UAS-Cs-Chrimson</i> and <i>UAS-Shibire</i>^<i>ts</i> in Or67d neurons where subjected to three 15 minutes long optogenetic activation as a positive control, synaptic release blocking, or both. Obp69a relative expression was then measured using RT-qPCR. Statistical significance was determined by One-way ANOVA with Tukey post-hoc analysis. Error bars signify SEM. (A) F(2,6) = 21.18, (B) F(2, 6) = 7.9, *<i>P</i><0.05, **<i>P</i><0.01 n = 3 independent experiments of 10–15 fly heads/sample. (<b>C</b>) Proposed model in which exposure to cVA regulates the production of Obp69a in accessory cells oppositely in male and female flies, via a mechanism that depends on relaying the information from the sensory neurons to the second order olfactory neurons in the brain, and eventually back to Obp69a producing cells.</p

BBB opening by low pulsed electric fields, depicted by delayed-contrast MRI, enables efficient delivery of therapeutic doxorubicin doses into mice brains

Abstract Background Pharmacological treatment of CNS diseases is limited due to the presence of the blood-brain barrier (BBB). Recent years showed significant advancement in the field of CNS drug delivery enablers, with technologies such as MR-guided focused ultrasound reaching clinical trials. This have inspired researchers in the field to invent novel brain barriers opening (BBo) technologies that are required to be simple, fast, safe and efficient. One such technology, recently developed by us, is BDF (Barrier Disrupting Fields), based on low pulsed electric fields (L-PEFs) for opening the BBB in a controlled, safe, reversible and non-invasive manner. Here, we conducted an in vivo study to show that BDF is a feasible technology for delivering Doxorubicin (Doxo) into mice brain. Means for depicting BBBo levels were developed and applied for monitoring the treatment and predicting response. Overall, the goals of the presented study were to demonstrate the feasibility for delivering therapeutic Doxo doses into naïve and tumor-bearing mice brains and applying delayed–contrast MRI (DCM) for monitoring the levels of BBBo. Methods L-PEFs were applied using plate electrodes placed on the intact skull of naïve mice. L-PEFs/Sham mice were scanned immediately after the procedure by DCM (“MRI experiment”), or injected with Doxo and Trypan blue followed by delayed (4 h) perfusion and brain extraction (“Doxo experiment”). Doxo concentrations were measured in brain samples using confocal microscopy and compared to IC50 of Doxo in glioma cell lines in vitro. In order to map BBBo extent throughout the brain, pixel by pixel MR image analysis was performed using the DCM data. Finally, the efficacy of L-PEFs in combination with Doxo was tested in nude mice bearing intracranial human glioma tumors. Results Significant amount of Doxo was found in cortical regions of all L-PEFs-treated mice brains (0.50 ± 0.06 µg Doxo/gr brain) while in Sham brains, Doxo concentrations were below or on the verge of detection limit (0.03 ± 0.02 µg Doxo/gr brain). This concentration was x97 higher than IC50 of Doxo calculated in gl261 mouse glioma cells and x8 higher than IC50 of Doxo calculated in U87 human glioma cells. DCM analysis revealed significant BBBo levels in the cortical regions of L-PEFs-treated mice; the average volume of BBBo in the L-PEFs-treated mice was x29 higher than in the Sham group. The calculated BBBo levels dropped exponentially as a function of BBBo threshold, similarly to the electric fields distribution in the brain. Finally, combining non-invasive L-PEFs with Doxo significantly decreased brain tumors growth rates in nude mice. Conclusions Our results demonstrate significant BBBo levels induced by extra-cranial L-PEFs, enabling efficient delivery of therapeutic Doxo doses into the brain and reducing tumor growth. As BBBo was undetectable by standard contrast-enhanced MRI, DCM was applied to generate maps depicting the BBBo levels throughout the brain. These findings suggest that BDF is a promising technology for efficient drug delivery into the brain with important implications for future treatment of brain cancer and additional CNS diseases

<i>Odorant binding protein 69a</i> (<i>Obp69a</i>) exhibits sexually dimorphic expression levels and is regulated inversely by social conditions in male and female flies.

<p><b>A</b>. Male and female flies were housed separately in groups of 5 flies/vial for 3 days. Total RNA extracted from heads of grouped male and female flies was analyzed for mRNA levels of <i>lush</i>, <i>obp69a</i>, <i>cyp6a20</i>, <i>est-6 and obp28a</i> by RT-qPCR. Statistical significance was determined by Student’s T-test with Bonferroni correction for multiple hypothesis testing. Error bars signify SEM *<i>P</i><0.001, n.s., not significant, n = 6 independent experiments with 10–15 fly heads/sample. <b>B.</b> Schematic illustration of social conditions set-up. WT males (upper panel) or females (lower panel) were housed individually, in groups of five same sex flies/vial or in groups of five male and five female flies for 3 days. <b>C-L.</b> Total RNA extracted from heads of male and female flies under single housing, same sex group and mixed sex group was analyzed for mRNA levels of <i>lush</i>, <i>obp69a</i>, <i>cyp6a20</i>, <i>est-6</i> and <i>obp28a</i> by RT-qPCR. Statistical significance was determined by one-way ANOVA with Tukey post-hoc analysis and Bonferroni correction for multiple hypothesis testing. (D) F(2, 6) = 9.4 **<i>P</i><0.01. (E) F(2, 6) = 11.03 **<i>P</i><0.01. (I) F(2,6) = 12.2 **P<0.01. n = 6 independent experiments of 15–20 fly heads/sample.</p

Failure to mate enhances investment in behaviors that may promote mating reward and impairs the ability to cope with stressors via a subpopulation of Neuropeptide F receptor neurons.

Living in dynamic environments such as the social domain, where interaction with others determines the reproductive success of individuals, requires the ability to recognize opportunities to obtain natural rewards and cope with challenges that are associated with achieving them. As such, actions that promote survival and reproduction are reinforced by the brain reward system, whereas coping with the challenges associated with obtaining these rewards is mediated by stress-response pathways, the activation of which can impair health and shorten lifespan. While much research has been devoted to understanding mechanisms underlying the way by which natural rewards are processed by the reward system, less attention has been given to the consequences of failure to obtain a desirable reward. As a model system to study the impact of failure to obtain a natural reward, we used the well-established courtship suppression paradigm in Drosophila melanogaster as means to induce repeated failures to obtain sexual reward in male flies. We discovered that beyond the known reduction in courtship actions caused by interaction with non-receptive females, repeated failures to mate induce a stress response characterized by persistent motivation to obtain the sexual reward, reduced male-male social interaction, and enhanced aggression. This frustrative-like state caused by the conflict between high motivation to obtain sexual reward and the inability to fulfill their mating drive impairs the capacity of rejected males to tolerate stressors such as starvation and oxidative stress. We further show that sensitivity to starvation and enhanced social arousal is mediated by the disinhibition of a small population of neurons that express receptors for the fly homologue of neuropeptide Y. Our findings demonstrate for the first time the existence of social stress in flies and offers a framework to study mechanisms underlying the crosstalk between reward, stress, and reproduction in a simple nervous system that is highly amenable to genetic manipulation

Obp69a is expressed in cells within the third antennal segment and is exported to the lymph.

<p><b>A-C</b>. Relative Obp69a expression levels in male heads and bodies (<b>A</b>), and between heads without antennae or maxillary palps in males (<b>B</b>) and females (<b>C</b>). Statistical significance was determined using Student’s t-test (<b>A</b>) ***<i>P</i><0.001, or One-way ANOVA with Tukey post-hoc analysis, Error bars signify SEM. (<b>B</b>)F(3,8) = 120, (<b>C</b>)F(3,8) = 124 **P<0.01. n = 3 independent experiments of 10–15 fly heads/sample. <b>D-H.</b> Confocal images of a membrane bound GFP (mcd8-GFP) in Obp69a expressing cells (<i>Obp69a-GAL4</i>), marking the 3^rd antennal segment. Note the GFP expression in the eyes is a marker of the <i>minos</i> element. (D). Arrowheads mark individual cells in antenna. E, F. Confocal images of a membrane-bound GFP (mcd8-GFP) in Obp69a expressing cells (using <i>Obp69a-GAL4</i> and <i>Minos Obp69a-GAL4</i> accordingly), marking the 3^rd antennal segment and presumably auxiliary cells. <b>G, H.</b> Confocal images of a transgenic Obp69a fused to GFP (<i>UAS-Obp69a-GFP</i>) expressed in Obp69a-expressing cells (Obp69a-GAL4). Asterisks mark Obp69a-GFP expression within the cells, Arrowheads mark exported Obp69a-GFP in the lymph. <b>I.</b> Western blot analysis of antennae and heads of <i>Obp69a-GAL4/+; UAS-Obp69a-GFP</i> flies using anti-GFP antibodies. <b>J</b>. RNAi to Obp69a was expressed in Or67d neurons, Lush, Obp69a, and Obp28a and nompA cells. <i>Obp69a</i> mRNA levels assessed by RT-qPCR. Statistical significance between relative mRNA levels in control and KD in each cell type was determined by Student’s T-test with Bonferroni correction for multiple hypothesis testing, Error bars signify SEM. *<i>P</i><0.05, n.s., not-significant. n = 3 independent experiments of 10–15 fly heads/sample.</p