AMP-Activated Protein Kinase (AMPK) Activation and Signalling Following Neuronal Excitotoxicity

Toxic stimulation of neurons, excitotoxicity, is a pathological process implicated in ischaemic stroke and neurodegenerative diseases. Excess glutamate in the synaptic cleft leads to severe ionic influx into the neuron, energetic stress and the activation of the AMP-activated protein Kinase (AMPK), a key regulator of energetic homeostasis. Interestingly, AMPK activation during excitotoxicity can be both cytoprotective and cytotoxic, but the molecular switches that determine whether neurons undergo necrosis or apoptosis or tolerate an excitotoxic insult are not well understood. Using an interdisciplinary approach combining biochemistry, single-cell imaging and computational modelling, we here investigated some of the mechanisms governing neuronal fate following transient glutamate excitotoxicity. We characterised neuronal bioenergetics in rat primary neurons at single-cell level using novel fluorescent sensors for intracellular glucose, ATP and AMPK activity. We identified ATP depletion and recovery to energetic homeostasis, along with AMPK activation, as acute, surprisingly rapid responses following the onset and termination of excitotoxicity. Interestingly, glutamate exposure also induced an accumulation of intracellular glucose, providing an additional source of energy during and after glutamate-induced bioenergetic stress. Surprisingly, cells that more quickly recovered their glucose levels to baseline survived longer, indicating that the ability to regulate the glutamate-mediated glucose accumulation may be beneficial for neuronal viability. Employing computational modelling of neuronal bioenergetics and AMPKmediated survival signalling in excitotoxicity, we correctly resembled the rapid single-cell kinetics of ATP levels and AMPK activity, but failed to render glucose dynamics, indicating the presence of additional glucose regulatory mechanisms during excitotoxicity. Further single-cell experiments revealed that neither inhibition of AMPK, inhibition of glucose transport, nor removal of extracellular substrate completely abolished the glucose increase, suggesting the presence of an intracellular glucose source in neurons, releasable during excitotoxic stress and independent of glucose transport and AMPK activity. Development of a second computational model, of AMPK-mediated apoptotic signalling activated during excitotoxicity, revealed a network motif (coherent feed-forward loop) capable of filtering the effects of short-term AMPK activity on the expression of the pro-apoptotic protein Bim. This motif may prevent unwanted Bim expression and apoptosis during transient or physiological bioenergetic stress, allowing AMPK to mediate its pro-survival effects. Biochemistry experiments validated model predictions that Bim expression was determined by the duration of AMPK activity following transient neuronal excitotoxicity. We conclude that our research increased insight into the molecular mechanisms that govern neuronal fate following transient excitotoxicity, which may eventually help to predict potential therapeutic targets capable of reducing neuronal damage in stroke and neurodegenerative diseases

Single-Cell Imaging of Bioenergetic Responses to Neuronal Excitotoxicity and Oxygen and Glucose Deprivation

Excitotoxicity is a condition occurring during cerebral ischemia, seizures, and chronic neurodegeneration. It is characterized by overactivation of glutamate receptors, leading to excessive Ca2+/Na+ influx into neurons, energetic stress, and subsequent neuronal injury.We and others have previously investigated neuronal populations to study how bioenergetic parameters determine neuronal injury; however, such experiments are often confounded by population-based heterogeneity and the contribution of effects of non-neuronal cells. Hence, we here characterized bioenergetics during transient excitotoxicity in rat and mouse primary neurons at the single-cell level using fluorescent sensors for intracellular glucose, ATP, and activation of the energy sensor AMP-activated protein kinase (AMPK). We identified ATP depletion and recovery to energetic homeostasis, along withAMPKactivation, as surprisingly rapid and plastic responses in two excitotoxic injury paradigms. We observed rapid recovery of neuronal ATP levels also in the absence of extracellular glucose, or when glycolytic ATP production was inhibited, but found mitochondria to be critical for fast and complete energetic recovery. Using an injury model of oxygen and glucose deprivation, we identified a similarly rapid bioenergetics response, yet with incompleteATPrecovery and decreasedAMPKactivity. Interestingly, excitotoxicity also induced an accumulation of intracellular glucose, providing an additional source of energy during and after excitotoxicity-induced energy depletion. We identified this to originate from extracellular, AMPKdependent glucose uptake and from intracellular glucose mobilization. Surprisingly, cells recovering their elevated glucose levels faster to baseline survived longer, indicating that the plasticity of neurons to adapt to bioenergetic challenges is a key indicator of neuronal viability

Urachal endometrioma: a case report

<p>Abstract</p> <p>Introduction</p> <p>We discuss a rare presentation of an unusual case of endometrioma.</p> <p>Case presentation</p> <p>A 40-year-old Caucasian woman presented with subacute abdominal pain and a suprapubic mass. A final diagnosis was made after the mass was resected and histopathology confirmed an endometrioma originating from an urachal remnant. Select imaging studies and histopathology are presented in this case report.</p> <p>Conclusion</p> <p>While endometriomata are well known to arise from abdominal scars, the condition described in this case report is a rare example of an endometrioma arising from the urachus. A review of the pathological complications of the urachus is also included.</p

Genome-wide microRNA profiling of plasma from three different animal models identifies biomarkers of temporal lobe epilepsy

Epilepsy diagnosis is complex, requires a team of specialists and relies on in-depth patient and family history, MRI-imaging and EEG monitoring. There is therefore an unmet clinical need for a non-invasive, molecular-based, biomarker to either predict the development of epilepsy or diagnose a patient with epilepsy who may not have had a witnessed seizure. Recent studies have demonstrated a role for microRNAs in the pathogenesis of epilepsy. MicroRNAs are short non-coding RNA molecules which negatively regulate gene expression, exerting profound influence on target pathways and cellular processes. The presence of microRNAs in biofluids, ease of detection, resistance to degradation and functional role in epilepsy render them excellent candidate biomarkers. Here we performed the first multi-model, genome-wide profiling of plasma microRNAs during epileptogenesis and in chronic temporal lobe epilepsy animals. From video-EEG monitored rats and mice we serially sampled blood samples and identified a set of dysregulated microRNAs comprising increased miR-93-5p, miR-142-5p, miR-182-5p, miR-199a-3p and decreased miR-574-3p during one or both phases. Validation studies found miR-93-5p, miR-199a-3p and miR-574-3p were also dysregulated in plasma from patients with intractable temporal lobe epilepsy. Treatment of mice with common anti-epileptic drugs did not alter the expression levels of any of the five miRNAs identified, however administration of an anti-epileptogenic microRNA treatment prevented dysregulation of several of these miRNAs. The miRNAs were detected within the Argonuate2-RISC complex from both neurons and microglia indicating these miRNA biomarker candidates can likely be traced back to specific brain cell types. The current studies identify additional circulating microRNA biomarkers of experimental and human epilepsy which may support diagnosis of temporal lobe epilepsy via a quick, cost-effective rapid molecular-based test

MicroRNA-335-5p suppresses voltage-gated sodium channel expression and may be a target for seizure control

There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target-miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking miR-335-5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy

GABA Regulation of Burst Firing in Hippocampal Astrocyte Neural Circuit: A Biophysical Model

It is now widely accepted that glia cells and gamma-aminobutyric acidergic (GABA) interneurons dynamically regulate synaptic transmission and neuronal activity in time and space. This paper presents a biophysical model that captures the interaction between an astrocyte cell, a GABA interneuron and pre/postsynaptic neurons. Specifically, GABA released from a GABA interneuron triggers in astrocytes the release of calcium (Ca2+) from the endoplasmic reticulum via the inositol 1, 4, 5-trisphosphate (IP3) pathway. This results in gliotransmission which elevates the presynaptic transmission probability rate (PR) causing weight potentiation and a gradual increase in postsynaptic neuronal firing, that eventually stabilizes. However, by capturing the complex interactions between IP3, generated from both GABA and the 2-arachidonyl glycerol (2-AG) pathway, and PR, this paper shows that this interaction not only gives rise to an initial weight potentiation phase but also this phase is followed by postsynaptic bursting behavior. Moreover, the model will show that there is a presynaptic frequency range over which burst firing can occur. The proposed model offers a novel cellular level mechanism that may underpin both seizure-like activity and neuronal synchrony across different brain regions

A systems approach delivers a functional microRNA catalog and expanded targets for seizure suppression in temporal lobe epilepsy

Temporal lobe epilepsy is the most common drug-resistant form of epilepsy in adults. The reorganization of neural networks and the gene expression landscape underlying pathophysiologic network behavior in brain structures such as the hippocampus has been suggested to be controlled, in part, by microRNAs. To systematically assess their significance, we sequenced Argonaute-loaded microRNAs to define functionally engaged microRNAs in the hippocampus of three different animal models in two species and at six time points between the initial precipitating insult through to the establishment of chronic epilepsy. We then selected commonly up-regulated microRNAs for a functional in vivo therapeutic screen using oligonucleotide inhibitors. Argonaute sequencing generated 1.44 billion small RNA reads of which up to 82% were microRNAs, with over 400 unique microRNAs detected per model. Approximately half of the detected microRNAs were dysregulated in each epilepsy model. We prioritized commonly up-regulated microRNAs that were fully conserved in humans and designed custom antisense oligonucleotides for these candidate targets. Antiseizure phenotypes were observed upon knockdown of miR-10a-5p, miR-21a-5p, and miR-142a-5p and electrophysiological analyses indicated broad safety of this approach. Combined inhibition of these three microRNAs reduced spontaneous seizures in epileptic mice. Proteomic data, RNA sequencing, and pathway analysis on predicted and validated targets of these microRNAs implicated derepressed TGF-\u3b2 signaling as a shared seizure-modifying mechanism. Correspondingly, inhibition of TGF-\u3b2 signaling occluded the antiseizure effects of the antagomirs. Together, these results identify shared, dysregulated, and functionally active microRNAs during the pathogenesis of epilepsy which represent therapeutic antiseizure targets

Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling

Background: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. Results: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. Conclusion: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases

Difficult Life Events, Selective Migration and Spatial Inequalities in Mental Health in the UK

Objective: Research has indicated that people moving towards neighbourhoods with disadvantaged socio-economic status have poor health, in particular mental health, but the reasons for this are unclear. This study aims to assess why people moving towards more socio-economically deprived areas have poor mental health. It focuses upon the role of difficult life events that may both trigger moves and damage mental health. This study investigates how mental health and socio-spatial patterns of mobility vary between people moving following difficult life events and for other reasons.&lt;p&gt;&lt;/p&gt; Methods: Longitudinal analysis of British Household Panel Survey data describing adults’ moves between annual survey waves, pooled over ten years, 1996-2006 (N=122,892 observations). Respondents were defined as ‘difficult life event movers’ if they had experienced relationship breakdown, housing eviction/repossession, or job loss between waves. Respondents were categorised as moving to more or less deprived quintiles using their Census Area Statistic residential ward Carstairs score. Mental health was indicated by self-reported mental health problems. Binary logistic regression models of weighted data were adjusted for age, sex, education and social class.&lt;p&gt;&lt;/p&gt; Results: The migration rate over one year was 8.5%; 14.1% of movers had experienced a difficult life event during this time period. Adjusted regression model odds of mental health problems among difficult life event movers were 1.67 (95% CI 1.35-2.07) relative to other movers. Odds of difficult life events movers, compared to other movers, moving to a less deprived area, relative to an area with a similar level of deprivation, were 0.70 (95% CI 0.58-0.84). Odds of mental health problems among difficult life event movers relocating to more deprived areas were highly elevated at 2.40 (95% CI 1.63-3.53), relative to stayers.&lt;p&gt;&lt;/p&gt; Conclusion: Difficult life events may influence health selective patterns of migration and socio-spatial trajectories, reducing moves to less deprived neighbourhoods among people with mental illness

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570