Cognitive Deficits Found in a Pro-inflammatory State are Independent of ERK1/2 Signaling in the Murine Brain Hippocampus Treated with Shiga Toxin 2 from Enterohemorrhagic Escherichia coli.

Shiga toxin 2 (Stx2) from enterohemorrhagic Escherichia coli (EHEC) produces hemorrhagic colitis, hemolytic uremic syndrome (HUS), and acute encephalopathy. The mortality rate in HUS increases significantly when the central nervous system (CNS) is involved. Besides, EHEC also releases lipopolysaccharide (LPS). Many reports have described cognitive dysfunctions in HUS patients, the hippocampus being one of the brain areas targeted by EHEC infection. In this context, a translational murine model of encephalopathy was employed to establish the deleterious effects of Stx2 and the contribution of LPS in the hippocampus. The purpose of this work is to elucidate the signaling pathways that may activate the inflammatory processes triggered by Stx2, which produces cognitive alterations at the level of the hippocampus. Results demonstrate that Stx2 produced depression-like behavior, pro-inflammatory cytokine release, and NF-kB activation independent of the ERK1/2 signaling pathway, while co-administration of Stx2 and LPS reduced memory index. On the other hand, LPS activated NF-kB dependent on ERK1/2 signaling pathway. Cotreatment of Stx2 with LPS aggravated the pathologic state, while dexamethasone treatment succeeded in preventing behavioral alterations. Our present work suggests that the use of drugs such as corticosteroids or NF-kB signaling inhibitors may serve as neuroprotectors from EHEC infection.

Brain angiotensin converting enzyme-2 in central cardiovascular regulation.

The brain renin-angiotensin system (RAS) plays an important role in the regulation of autonomic and neuroendocrine functions, and maintains cardiovascular homeostasis. Ang-II is the major effector molecule of RAS and exerts most of its physiological functions, including blood pressure (BP) regulation, via activation of AT1 receptors. Dysregulation of brain RAS in the central nervous system results in increased Ang-II synthesis that leads to sympathetic outflow and hypertension. Brain angiotensin (Ang) converting enzyme-2 (ACE2) was discovered two decades ago as an RAS component, exhibiting a counter-regulatory role and opposing the adverse cardiovascular effects produced by Ang-II. Studies using synthetic compounds that can sustain the elevation of ACE2 activity or genetically overexpressed ACE2 in specific brain regions found various beneficial effects on cardiovascular function. More recently, ACE2 has been shown to play critical roles in neuro-inflammation, gut dysbiosis and the regulation of stress and anxiety-like behaviors. In the present review, we aim to highlight the anatomical locations and functional implication of brain ACE2 related to its BP regulation via modulation of the sympathetic nervous system and discuss the recent developments and future directions in the ACE2-mediated central cardiovascular regulation.

Shiga toxin 2 from enterohemorrhagic Escherichia coli induces reactive glial cells and neurovascular disarrangements including edema and lipid peroxidation in the murine brain hippocampus.

BACKGROUND: Shiga toxin 2 from enterohemorrhagic Escherichia coli is the etiologic agent of bloody diarrhea, hemolytic uremic syndrome and derived encephalopathies that may result to death in patients. Being a Gram negative bacterium, lipopolysaccharide is also released. Particularly, the hippocampus has been found affected in patients intoxicated with Shiga toxin 2. In the current work, the deleterious effects of Shiga toxin 2 and lipopolysaccharide are investigated in detail in hippocampal cells for the first time in a translational murine model, providing conclusive evidences on how these toxins may damage in the observed clinic cases. METHODS: Male NIH mice (25 g) were injected intravenously with saline solution, lipopolysaccharide, Shiga toxin 2 or a combination of Shiga toxin 2 with lipopolysaccharide. Brain water content assay was made to determine brain edema. Another set of animals were intracardially perfused with a fixative solution and their brains were subjected to immunofluorescence with lectins to determine the microvasculature profile, and anti-GFAP, anti-NeuN, anti-MBP and anti-Iba1 to study reactive astrocytes, neuronal damage, myelin dysarrangements and microglial state respectively. Finally, the Thiobarbituric Acid Reactive Substances Assay was made to determine lipid peroxidation. In all assays, statistical significance was performed using the One-way analysis of variance followed by Bonferroni post hoc test. RESULTS: Systemic sublethal administration of Shiga toxin 2 increased the expressions of astrocytic GFAP and microglial Iba1, and decreased the expressions of endothelial glycocalyx, NeuN neurons from CA1 pyramidal layer and oligodendrocytic MBP myelin sheath from the fimbria of the hippocampus. In addition, increased interstitial fluids and Thiobarbituric Acid Reactive Substances-derived lipid peroxidation were also found. The observed outcomes were enhanced when sublethal administration of Shiga toxin 2 was co-administered together with lipopolysaccharide. CONCLUSION: Systemic sublethal administration of Shiga toxin 2 produced a deterioration of the cells that integrate the vascular unit displaying astrocytic and microglial reactive profiles, while edema and lipid peroxidation were also observed. The contribution of lipopolysaccharide to pathogenicity caused by Shiga toxin 2 resulted to enhance the observed hippocampal damage.

Anti-inflammatory agents reduce microglial response, demyelinating process and neuronal toxin uptake in a model of encephalopathy produced by Shiga Toxin 2.

Infections by Enterohemorrhagic Escherichia coli may cause in addition to hemolytic uremic syndrome neurological disorders which may lead to fatal outcomes in patients. The brain striatum is usually affected during this outcome. The aim of this study was to determine in this area the role of the microglia in pro-inflammatory events that may occur during Shiga toxin 2 intoxication and consequently to this, whether oligodendrocytes were being affected. In the present paper we demonstrated that anti-inflammatory treatments reduced deleterious effects in brain striatal cells exposed to Shiga toxin 2 and LPS. While dexamethasone treatment decreased microglial activation and recovered myelin integrity in the mice striatum, etanercept treatment decreased neuronal uptake of Stx2 in rat striatal neurons, improving the affected area from toxin-derived injury. In conclusion, microglial activation is related to pro-inflammatory events that may deteriorate the brain function during intoxication with Stx2 and LPS. Consequently, the role of anti-inflammatory agents in the treatment of EHEC-derived encephalopathy should be studied in clinical trials.

Maternal Western diet programs cardiometabolic dysfunction and hypothalamic inflammation via epigenetic mechanisms predominantly in the male offspring.

OBJECTIVE: Maternal exposure during pregnancy is a strong determinant of offspring health outcomes. Such exposure induces changes in the offspring epigenome resulting in gene expression and functional changes. In this study, we investigated the effect of maternal Western hypercaloric diet (HCD) programming during the perinatal period on neuronal plasticity and cardiometabolic health in adult offspring. METHODS: C57BL/6J dams were fed HCD for 1 month prior to mating with regular diet (RD) sires and kept on the same diet throughout pregnancy and lactation. At weaning, offspring were maintained on either HCD or RD for 3 months resulting in 4 treatment groups that underwent cardiometabolic assessments. DNA and RNA were extracted from the hypothalamus to perform whole genome methylation, mRNA, and miRNA sequencing followed by bioinformatic analyses. RESULTS: Maternal programming resulted in male-specific hypertension and hyperglycemia, with both males and females showing increased sympathetic tone to the vasculature. Surprisingly, programmed male offspring fed HCD in adulthood exhibited lower glucose levels, less insulin resistance, and leptin levels compared to non-programmed HCD-fed male mice. Hypothalamic genes involved in inflammation and type 2 diabetes were targeted by differentially expressed miRNA, while genes involved in glial and astrocytic differentiation were differentially methylated in programmed male offspring. These data were supported by our findings of astrogliosis, microgliosis and increased microglial activation in programmed males in the paraventricular nucleus (PVN). Programming induced a protective effect in male mice fed HCD in adulthood, resulting in lower protein levels of hypothalamic TGFß2, NF-κB2, NF-κBp65, Ser-pIRS1, and GLP1R compared to non-programmed HCD-fed males. Although TGFß2 was upregulated in male mice exposed to HCD pre- or post-natally, only blockade of the brain TGFß receptor in RD-HCD mice improved glucose tolerance and a trend to weight loss. CONCLUSIONS: Our study shows that maternal HCD programs neuronal plasticity in the offspring and results in male-specific hypertension and hyperglycemia associated with hypothalamic inflammation in mechanisms and pathways distinct from post-natal HCD exposure. Together, our data unmask a compensatory role of HCD programming, likely via priming of metabolic pathways to handle excess nutrients in a more efficient way.

Ahnak in the prefrontal cortex mediates behavioral correlates of stress resilience and rapid antidepressant action in mice.

The prefrontal cortex (PFC) is a key neural node mediating behavioral responses to stress and the actions of ketamine, a fast-acting antidepressant. The molecular mechanisms underlying these processes, however, are not fully understood. Our recent study revealed a pivotal role of hippocampal Ahnak as a regulator of cellular and behavioral adaptations to chronic stress. However, despite its significant expression in the PFC, the contribution of cortical Ahnak to behavioral responses to stress and antidepressants remains unknown. Here, using a mouse model for chronic social stress, we find that Ahnak expression in the PFC is significantly increased in stress-resilient mice and positively correlated with social interaction after stress exposure. Conditional deletion of Ahnak in the PFC or forebrain glutamatergic neurons facilitates stress susceptibility, suggesting that Ahnak is required for behavioral resilience. Further supporting this notion, Ahnak expression in the PFC is increased after the administration of ketamine or its metabolite (2R, 6R)-hydroxynorketamine (HNK). Moreover, Ahnak deletion in forebrain glutamatergic neurons blocks the restorative behavioral effects of ketamine or HNK in stress-susceptible mice. This forebrain excitatory neuron-specific Ahnak deletion reduces the frequency of mini excitatory postsynaptic currents in layer II/III pyramidal neurons, suggesting that Ahnak may induce its behavioral effects via modulation of glutamatergic transmission in the PFC. Altogether, these data suggest that Ahnak in glutamatergic PFC neurons may be critical for behavioral resilience and antidepressant actions of ketamine or HNK in chronic social stress-exposed mice.

UBR1 Promotes Sex-Dependent ACE2 Ubiquitination in Hypertension.

Background: Angiotensin (Ang)-II impairs the function of the antihypertensive enzyme ACE2 by promoting its internalization, ubiquitination and degradation thus contributing to hypertension. However, few ACE2 ubiquitination partners have been identified and their role in hypertension remains unknown. Methods: Proteomics and bioinformatic analysis were used to identify ACE2 ubiquitination partners in the brain, heart, and kidney from Ang-II-infused C57BL6/J mice from both sexes and validated the interaction between UBR1 and ACE2 in cells. Central and peripheral UBR1 knockdown was then performed in male mice to investigate its role in the maintenance of hypertension. Results: Proteomics analysis from hypothalamus identified UBR1 as a potential E3 ligase promoting ACE2 ubiquitination. Enhanced UBR1 expression, associated with ACE2 reduction, was confirmed in various tissues from hypertensive male mice and human samples. Treatment of endothelial and smooth muscle cells with testosterone, but not 17ß-estradiol, confirmed a sex-specific regulation of UBR1. In vivo silencing of UBR1 using chronic administration of small interference RNA resulted in the restoration of ACE2 levels in hypertensive males. A transient decrease in blood pressure following intracerebroventricular, but not systemic, infusion was also observed. Interestingly, UBR1 knockdown increased the brain activation of Nedd4-2, an E3 ligase promoting ACE2 ubiquitination and reduced expression of SGK1, the kinase inactivating Nedd4-2. Conclusions: These data demonstrate that UBR1 is a novel ubiquitin ligase targeting ACE2 in hypertension. UBR1 and Nedd4-2 E3 ligases appear to work synergistically to ubiquitinate ACE2. Targeting of these ubiquitin ligases may represent a novel strategy to restore ACE2 compensatory activity in hypertension.

Nedd4-2 up-regulation is associated with ACE2 ubiquitination in hypertension.

AIMS: Angiotensin-converting enzyme 2 (ACE2) is a critical component of the compensatory renin-angiotensin system that is down-regulated during the development of hypertension, possibly via ubiquitination. However, little is known about the mechanisms involved in ACE2 ubiquitination in neurogenic hypertension. This study aimed at identifying ACE2 ubiquitination partners, establishing causal relationships and clinical relevance, and testing a gene therapy strategy to mitigate ACE2 ubiquitination in neurogenic hypertension. METHODS AND RESULTS: Bioinformatics and proteomics were combined to identify E3 ubiquitin ligases associated with ACE2 ubiquitination in chronically hypertensive mice. In vitro gain/loss of function experiments assessed ACE2 expression and activity to validate the interaction between ACE2 and the identified E3 ligase. Mutation experiments were further used to generate a ubiquitination-resistant ACE2 mutant (ACE2-5R). Optogenetics, blood pressure telemetry, pharmacological blockade of GABAA receptors in mice expressing ACE2-5R in the bed nucleus of the stria terminalis (BNST), and capillary western analysis were used to assess the role of ACE2 ubiquitination in neurogenic hypertension. Ubiquitination was first validated as leading to ACE2 down-regulation, and Neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) was identified as a E3 ligase up-regulated in hypertension and promoting ACE2 ubiquitination. Mutation of lysine residues in the C-terminal of ACE2 was associated with increased activity and resistance to angiotensin (Ang)-II-mediated degradation. Mice transfected with ACE2-5R in the BNST exhibited enhanced GABAergic input to the paraventricular nucleus (PVN) and a reduction in hypertension. ACE2-5R expression was associated with reduced Nedd4-2 levels in the BNST. CONCLUSION: Our data identify Nedd4-2 as the first E3 ubiquitin ligase involved in ACE2 ubiquitination in Ang-II-mediated hypertension. We demonstrate the pivotal role of ACE2 on GABAergic neurons in the maintenance of an inhibitory tone to the PVN and the regulation of pre-sympathetic activity. These findings provide a new working model where Nedd4-2 could contribute to ACE2 ubiquitination, leading to the development of neurogenic hypertension and highlighting potential novel therapeutic strategies.

Cytokines expression from altered motor thalamus and behavior deficits following sublethal administration of Shiga toxin 2a involve the induction of the globotriaosylceramide receptor.

Encephalopathy associated with hemolytic uremic syndrome is produced by enterohemorrhagic E. coli (EHEC) infection, which releases the virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS). Neurological compromise is a poor prognosis and mortality factor of the disease, and the thalamus is one of the brain areas most frequently affected. We have previously demonstrated the effectiveness of anti-inflammatory drugs to ameliorate the deleterious effects of these toxins. However, the thalamic production of cytokines involved in pro-inflammatory processes has not yet been acknowledged. The aim of this work attempts to determine whether systemic sublethal Stx2a or co-administration of Stx2a with LPS are able to rise a proinflammatory profile accompanying alterations of the neurovascular unit in anterior and lateral ventral nuclei of the thalamus (VA-VL) and motor behavior in mice. After 4 days of treatment, Stx2a affected the lectin-bound microvasculature distribution while increasing the expression of GFAP in reactive astrocytes and producing aberrant NeuN distribution in degenerative neurons. In addition, increased swimming latency was observed in a motor behavioral test. All these alterations were heightened when Stx2a was co-administered with LPS. The expression of pro-inflammatory cytokines TNFα, INF-γ and IL-2 was detected in VA-VL. All these effects were concomitant with increased expression of the Stx receptor globotriaosylceramide (Gb3), which hints at receptor involvement in the neuroinflammatory process as a key finding of this study. In conclusion, Stx2a to Gb3 may be determinant in triggering a neuroinflammatory event, which may resemble clinical outcomes and should thus be considered in the development of preventive strategies.

Environmental Cues Modulate Microglial Cell Behavior Upon Shiga Toxin 2 From Enterohemorrhagic Escherichia coli Exposure.

Shiga toxin (Stx) produced by enterohemorrhagic E. coli produces hemolytic uremic syndrome and encephalopathies in patients, which can lead to either reversible or permanent neurological abnormalities, or even fatal cases depending on the degree of intoxication. It has been observed that the inflammatory component plays a decisive role in the severity of the disease. Therefore, the objective of this work was to evaluate the behavior of microglial cell primary cultures upon Stx2 exposure and heat shock or lipopolysaccharide challenges, as cues which modulate cellular environments, mimicking fever and inflammation states, respectively. In these contexts, activated microglial cells incorporated Stx2, increased their metabolism, phagocytic capacity, and pro-inflammatory profile. Stx2 uptake was associated to receptor globotriaosylceramide (Gb3)-pathway. Gb3 had three clearly distinguishable distribution patterns which varied according to different contexts. In addition, toxin uptake exhibited both a Gb3-dependent and a Gb3-independent binding depending on those contexts. Altogether, these results suggest a fundamental role for microglial cells in pro-inflammatory processes in encephalopathies due to Stx2 intoxication and highlight the impact of environmental cues.