Pathophysiological role of brain xenobiotic nuclear receptors and P450 metabolic enzymes

Les récepteurs nucléaires des xénobiotiques et les enzymes métaboliques P450 (CYP) constituent les principaux éléments contrôlant la biotransformation des médicaments, ainsi que le maintien de des barrières physiologiques au niveau périphérique, plus particulièrement, dans le foie et dans l’intestin. Plusieurs études ont mis en évidence la présence des CYP ainsi que les récepteurs nucléaires contrôlant leur expression, tels que le Constitutive Androstane Receptor et le Pregnane Xenobiotic Receptor (CAR et PXR). Des résultats précédant indiquent la surexpression des CYP2E1 et CYP3A4 dans des tissus et des cellules isolées du cerveau de patients épileptiques pharmacorésistants. L’importance de ces résultats réside dans le rôle du CYP2E1 et CYP3A4 dans la biotransformation de plusieurs médicaments antiépileptiques (MAE) suggérant ainsi un mécanisme de pharmacorésistance aux médicaments. Contrairement aux autres récepteurs nucléaires, les fonctions physiologiques des récepteurs nucléaires des xénobiotiques au niveau vasculaire sont mal connues. Nos résultats montrent des changements spatio-temporaux de l’expression des CYP2E1 et CYP3A4 dans le cerveau après une crise aigüe et pendant le processus d’épileptogenèse chez la souris. Une exposition in vivo et in vitro au MAE Phénytoïne induit une augmentation du niveau du CYP2E1. Le Phénobarbital et la Carbamazépine n’ont pas eu d’effet. Les souris privées des récepteurs nucléaires des xénobiotiques (PXR KO et CAR KO) ne présentent pas de changement de niveau basal des CYP dans le cerveau. Cependant, les souris CAR KO présentent des dysfonctionnements neuronaux (altération de la mémoire, comportement anxieux et une diminution de l’intensité des rythmes EEG entre 3.5-7 Hz) et des modifications caractérisées par une augmentation de perméabilité vasculaire et une dispersion des neurones hippocampiques. L’ensemble des résultats indique une régulation dynamique des CYP dans le cerveau avec une extension de l’impact des récepteurs des xénobiotiques à des fonctions neurovasculaires.Xenobiotic nuclear receptors and P450 metabolic enzymes (CYP) are pivotal controllers of drug biotransformation and barrier functions in peripheral organs, including the intestine and the liver. Accumulating evidence suggested that, in human, central nervous system cells express significant levels of P450 enzymes and their upstream regulators e.g. Constitutive Androstane and Pregnane Xenobiotic Receptors (CAR, PXR). We previously showed the increased and ectopic CYP2E1 and CYP3A4 expression in brain specimens or cells obtained from drug resistant epileptic patient. These results are significant as CYP2E1 and CYP3A4 are responsible for the metabolism of several antiepileptic drugs (AED), therefore, suggesting a possible new mechanism of drug resistance. However, the exact determinants of CYP expression in the epileptic brain remain unknown. In addition, the exact role of nuclear xenobiotic receptor in the brain is understudied. The latter represents a significant knowledge gap as nuclear receptors other than the xenobiotic ones were shown to contribute to physiological neurovascular functions. Our results show spatio-temporal changes of CYP2E1 and CYP3A4 brain expression occuring after status epilepticus and during epileptogenesis in mice. Exposure to the AED phenytoin, phenobarbital, but not carbamazepine, increased brain CYP2E1 expression in vivo and in vitro. Lack of the specific xenobiotic receptors CAR and PXR did not impact basal CYP brain levels. However, we found an unexpected contribution of CAR to neuronal dysfunctions (memory impairment, anxiety like-behavior and decrease 3.5-7 Hz EEG waves) and neurovascular development, as indicated by increase vascular permeability and hippocampal neuronal dispersion. These results depict a dynamic regulation of P450 enzymes in the brain also expanding the impact of xenobiotic receptors to previously unexplored neurovascular functions

Pregnane X receptor deletion modifies recognition memory and electroencephalographic activity

Nuclear receptors (NR) are emerging as key players in the central nervous system (CNS) with reported implications in physiological and pathophysiological conditions. While other NR have been studied, it is unknown whether invalidation of the pregnane xenobiotic receptor (PXR, NR1I2) corresponds to neurological modifications in the adult brain. PXR-/- C57BL/6j and wild type mice were used to investigate: i) recognition memory, motor coordination, and anxiety-like behaviors; ii) longitudinal video-electroencephalographic (EEG) recordings and frequency wave analysis; iii) neurovascular structures by histological evaluation and expression of the cerebrovascular tight junctions ZO1 and CLDN5. Absence of PXR was associated with anxiety-like behavior and recognition memory impairment in adult mice. The latter was simultaneous to an electroencephalographic signature of lower theta frequency during sleep and abnormal delta waves. Neurophysiological changes did not correspond to significant structural changes in the adult brain, expect for a localized and minor increase in the fronto-parietal neurovascular density and reduced ZO1, but not CLDN5, expression in isolated brain capillaries. Our results converge with existing evidence supporting a link between NR expression and brain physiology. Although the exact modalities remain to be elucidated, the possibility that extra-physiological modulation of PXR may constitute a pathophysiological entry point or a molecular target for brain diseases is proposed

Constitutive Androstane Receptor: A Peripheral and a Neurovascular Stress or Environmental Sensor

International audienceXenobiotic nuclear receptors (NR) are intracellular players involved in an increasing number of physiological processes. Examined and characterized in peripheral organs where they govern metabolic, transport and detoxification mechanisms, accumulating data suggest a functional expression of specific NR at the neurovascular unit (NVU). Here, we focus on the Constitutive Androstane Receptor (CAR), expressed in detoxifying organs such as the liver, intestines and kidneys. By direct and indirect activation, CAR is implicated in hepatic detoxification of xenobiotics, environmental contaminants, and endogenous molecules (bilirubin, bile acids). Importantly, CAR participates in physiological stress adaptation responses, hormonal and energy homeostasis due to glucose and lipid sensing. We next analyze the emerging evidence supporting a role of CAR in NVU cells including the blood-brain barrier (BBB), a key vascular interface regulating communications between the brain and the periphery. We address the emerging concept of how CAR may regulate specific P450 cytochromes at the NVU and the associated relevance to brain diseases. A clear understanding of how CAR engages during pathological conditions could enable new mechanistic, and perhaps pharmacological, entry-points within a peripheral-brain axis

Evidence for Status Epilepticus and Pro-Inflammatory Changes after Intranasal Kainic Acid Administration in Mice.

Kainic acid (KA) is routinely used to elicit status epilepticus (SE) and epileptogenesis. Among the available KA administration protocols, intranasal instillation (IN) remains understudied. Dosages of KA were instilled IN in mice. Racine Scale and Video-EEG were used to assess and quantify SE onset. Time spent in SE and spike activity was quantified for each animal and confirmed by power spectrum analysis. Immunohistochemistry and qPCR were performed to define brain inflammation occurring after SE, including activated microglial phenotypes. Long term video-EEG recording was also performed. Titration of IN KA showed that a dose of 30 mg/kg was associated with low mortality while eliciting SE. IN KA provoked at least one behavioral and electrographic SE in the majority of the mice (>90%). Behavioral and EEG SE were accompanied by a rapid and persistent microglial-astrocytic cell activation and hippocampal neurodegeneration. Specifically, microglial modifications involved both pro- (M1) and anti-inflammatory (M2) genes. Our initial long-term video-EEG exploration conducted using a small cohort of mice indicated the appearance of spike activity or SE. Our study demonstrated that induction of SE is attainable using IN KA in mice. Typical pro-inflammatory brain changes were observed in this model after SE, supporting disease pathophysiology. Our results are in favor of the further development of IN KA as a means to study seizure disorders. A possibility for tailoring this model to drug testing or to study mechanisms of disease is offered

Topographic Reorganization of Cerebrovascular Mural Cells under Seizure Conditions

Summary: Reorganization of the neurovascular unit has been suggested in the epileptic brain, although the dynamics and functional significance remain unclear. Here, we tracked the in vivo dynamics of perivascular mural cells as a function of electroencephalogram (EEG) activity following status epilepticus. We segmented the cortical vascular bed to provide a size- and type-specific analysis of mural cell plasticity topologically. We find that mural cells are added and removed from veins, arterioles, and capillaries after seizure induction. Loss of mural cells is proportional to seizure severity and vascular pathology (e.g., rigidity, perfusion, and permeability). Treatment with platelet-derived growth factor subunits BB (PDGF-BB) reduced mural cell loss, vascular pathology, and epileptiform EEG activity. We propose that perivascular mural cells play a pivotal role in seizures and are potential targets for reducing pathophysiology. : Arango-Lievano et al. follow how status epilepticus changes the dynamics of mural cell turnover at the cortical vasculature, causing vessel damage. PDGF-BB, a growth factor promoting the assembly of mural cells at the vascular unit, ameliorates vessel function and reduces spontaneous epileptiform activity. Keywords: neurovascular, epilepsy, pericyte, blood flow, PDGF-BB, PDGFR-B, in vivo microscop

Redistribution of PDGFRbeta cells and NG2DsRed pericytes at the cerebrovasculature after status epilepticus

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Time-dependent microgliosis and astrogliosis following IN KA induced seizures.

<p>Analysis performed in mice experiencing stage 5 or higher. <b>A-D)</b> Representative images of IBA1 immunostaining (upper panels) and GFP fluorescence (C57BL6/J CX3CR1^+/eGFP mice; lower panels) in CA1 region in control mice (A), and 24h (B), 72h (C) and 15 days (D) after IN KA. Scale bar = 50 μm. Insets depict enlarged image of individual microglial cells. Scale bar = 10 μm <b>E-F)</b> Quantitative analysis of IBA1 immunostaining in the hippocampus 24h, 72h and 15 days after IN KA-induced SE shows significant microglial activation, including increased cell number and size. <b>G-I)</b> Representative images of GFAP immunostaining in CA1 region in control mice (G), 72h (H) and 15 days (I) after IN KA. Scale bar = 20 μm. <b>J)</b> Quantitative analysis of GFAP immunostaining in the hippocampus shows astrogliosis 72h after IN KA. Results are represented as mean ± SEM (n ≥ 6 per group). Statistical analysis was performed using a non-parametric Kruskal-Wallis one-way analysis of variance followed by Dunn’s post-test. *: p<0.05; **: p<0.01; ***: p<0.001 compared to control condition.</p

Number of mice included in the different analyses (stage≥5 Racine).

<p>Number of mice included in the different analyses (stage≥5 Racine).</p

Neurodegeneration and neuroinflammatory gene profile after IN KA induced seizures.

<p><b>A-C)</b> Fluoro-Jade C staining is observed 24h after IN KA, diminishing at 72h. Correspondence existed between presence of FJC positive neurons and behavioral score after IN KA (B: Mouse 9, mean-score = 5.8; C: Mouse 28, mean-score = 3.8). Scale bar 50 μm. <b>D-F)</b> qPCR analysis and changes of inflammatory gene levels in mice experiencing stage 5 or higher. Analysis was performed 24h (white bars) and 72h (grey bars) after IN KA. Panels (D) and (E) represent mRNA changes for M1 and M2 microglial markers. Red bars indicate genes that are not detectable under control conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150793#sec002" target="_blank">materials and methods</a> section). Results are presented as mean ± SEM (n ≥ 7 / group). Statistical analysis was performed using a non-parametric Mann-Whitney test between control and KA-treated conditions. *: p<0.05; ** p<0.01; ***: p<0.001 compared to controls.</p

Video-EEG analysis during seizure progression.

<p><b>A)</b> Heat map (blue to red) and raw numbers indicate the sum of durations (in seconds) of spike activity recorded for each animal in the given recording session. <b>B-C)</b> Examples of EEG recordings during the chronic phase (<i>e</i>.<i>g</i>., SE in B and spike activity in C).</p