1,273 research outputs found

    Effekte pathogener Anti-NMDAR-Antikörper auf die Funktionalität von Synapsen und neuronalen Netzwerken

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    N-methyl-D-aspartate (NMDA) receptors lay at the core of excitatory glutamatergic transmission and their dysfunction has been implicated in a number of neurological and psychiatric disorders. One such recently described disease is anti-NMDAR encephalitis, characterized by prominent psychiatric, cognitive and autonomic symptoms, which are linked to presence of autoantibodies targeting the NMDARs. To date, the majority of mechanistic studies have focused on antibodies’ action in the hippocampus, where they cause receptor cross-linking and internalization. However, little is known what is the specific contribution of individual antibodies and what are their effects in other brain regions such as cortex, which could help explain dysfunction on higher cognitive level. Here, we employed recently developed monoclonal anti-NMDAR autoantibodies and studied their effects on in vitro rodent neuronal cultures, using electrophysiological and imaging techniques. We report that both affinity-matured and germline, “naïve” NMDAR autoantibodies can pose pathogenic effects and impair NMDAR transmission. Moreover, these autoantibodies show brain regional specificity, exerting different effects in hippocampal versus cortical neurons. While in hippocampus they impair NMDAR currents of excitatory neurons, in cultures from cortex they selectively decrease NMDA currents and synaptic output of inhibitory, but not excitatory, neurons. Consequently, decreased inhibitory drive leads to disinhibition of networks from cortical neurons, bringing them into a hyper-excitable state. This is further associated with lowered levels of crucial pre-synaptic inhibitory proteins, specifically in inhibitory-to-excitatory neuron synapses. Together, these findings deepen our understanding of the pathology of autoimmune encephalitis by showing pathogenic potential of both matured and naïve autoantibodies and providing a novel, cortex specific mechanism of antibody-induced network hyper-excitability. Of note, similar mechanisms of NMDA-mediated cortical disinhibition have been suggested to underlie the etiology of schizophrenia, therefore there is an emerging framework for common mechanisms across neuropsychiatric disorders.N-Methyl-D-Aspartat-Rezeptoren (NMDA-Rezeptoren) sind das Herzstück der exzitatorischen glutamatergen Signalübertragung, und ihre Fehlfunktion wird mit einer Reihe von neurologischen und psychiatrischen Erkrankungen in Verbindung gebracht. Eine solche kürzlich beschriebene Krankheit ist die Anti-NMDAR-Enzephalitis, die durch auffällige psychiatrische, kognitive und andere autonome Symptome gekennzeichnet ist, alle werden mit dem Vorhandensein von Autoantikörpern gegen NMDARs in Verbindung gebracht. Bisher haben sich die meisten mechanistischen Studien auf die Wirkung der Antikörper im Hippocampus konzentriert, wo sie eine Vernetzung und Internalisierung der Rezeptoren verursachen. Es ist jedoch nur wenig darüber bekannt, welchen spezifischen Beitrag einzelne Antikörper leisten und welche Auswirkungen sie in anderen Hirnregionen wie zum Beispiel dem Kortex haben. Effekte der Autoantikörper im Kortex könnten eine Erklärung für die beobachtete Dysfunktion auf höherer kognitiver Ebene liefern. In dieser Studie haben wir kürzlich entwickelte monoklonale Anti-NMDAR-Autoantikörper eingesetzt und ihre Auswirkungen auf neuronale In-vitro-Kulturen von Nagetieren mit Hilfe bildgebender und elektrophysiologischer Verfahren untersucht. Wir berichten, dass sowohl affinitätsgereifte als auch keimbahnspezifische, "naive" NMDAR-Autoantikörper pathogen wirken und die NMDAR-Signalübertragung beeinträchtigen können. Darüber hinaus weisen diese Autoantikörper eine hirnregionale Spezifität auf, indem sie in hippocampalen und kortikalen Neuronen unterschiedliche Wirkungen entfalten. Während sie im Hippocampus die NMDAR-Ströme exzitatorischer Neuronen beeinträchtigen, vermindern sie in kortikalen Kulturen selektiv die NMDA-Ströme und die synaptische Übertragung inhibitorischer, aber nicht exzitatorischer Neuronen. Infolgedessen führt die verringerte hemmende Wirkung zu einer generellen Enthemmung kortikaler neuronaler Netzwerke und was diese in einen übererregbaren Zustand versetzt. Dies geht zusätzlich einher mit einer Abnahme wichtiger präsynaptischer inhibitorischer Proteine, insbesondere in Synapsen zwischen inhibitorischen und erregenden Neuronen. Zusammengenommen vertiefen diese Ergebnisse unser Verständnis der Pathologie der Autoimmunenzephalitis, indem sie das pathogene Potenzial sowohl gereifter als auch naiver Autoantikörper aufzeigen und einen neuen, Kortex-spezifischen Mechanismus der antikörperinduzierten Hypererregbarkeit von neuronalen Netzwerken liefern. Es ist bemerkenswert, dass ähnliche Mechanismen der NMDA-vermittelten kortikalen Enthemmung auch für die Pathologie der Schizophrenie verantwortlich gemacht werden, so dass sich gemeinsame, grundlegende Mechanismen bei neuropsychiatrischen Störungen abzeichnen

    The role of AD protective variant PLCγ2P522R in modulating microglia mediated clearance and synaptic pruning

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    PLCG2-P522R, a rare coding variant in the Phospholipase C gamma-2 (PLCG2) gene, has been found to be protective against late onset Alzheimer's disease (AD). Within the central nervous system, PLCγ2 is most abundantly expressed in microglia, and microglial mediated neuroinflammatory system has emerged as a major contributor to the molecular and phenotypic changes observed in the AD brain. However, the mechanism by which the P522R variant of PLCγ2 reduces AD pathology is still unknown. BV2 (mouse microglia) cells and human induced pluripotent stem-cells (hiPSC) derived microglia were used in this thesis work to evaluate the role of PLCγ2 in modifying various disease-relevant microglia functions. PLCγ2WT and PLCγ2P522R expression constructs were transfected into BV2 cells to examine the effects of PLCγ2 overexpression on various microglia functions including amyloid beta (Aβ) clearance and synaptic targeting, and various transcriptional changes linked to AD. hiPSCs were genome edited using CRISPR/Cas9 to generate both heterozygous and homozygous forms of the PLCG2_P522R variant in healthy controls. These hiPSC derived microglia were used to explore the effects of the PLCγ2P522R basal level on disease-relevant processes, such as microglial capacity to uptake Aβ and synapses. Microglia transcriptional changes were examined using targeted qPCR analysis to investigate changes in expression of key microglial genes. Mitochondrial function and calcium level changes were also investigated in these microglia cells to determine their metabolic fitness. In addition, the microglia were subjected to acute and chronic treatment of oligomeric Aβ to examine the impact of PLCγ2P522R on microglia's ability to respond to acute and chronic stress. As a result, the effects of Aβ oligomers on lysosomal biogenesis and phagocytic capacities of these microglia were examined further. As a result of PLCγ2 overexpression, Aβ uptake and other immune- provoking cargoes like zymosan were significantly increased. In contrast, the uptake of synaptosomes in BV2 cells overexpressing PLCγ2 was considerably reduced. Similarly, microglia generated from hiPSCs also showed enhanced clearance of Aβ and preservation of synapses by PLCγ2P522R variants. In the PLCγ2P522R microglia variants, the expression of multiple genes, including IL-10 and CX3CR1, as well as mitochondrial function, cytoplasmic calcium flux, and cellular motility were all increased. It was found that the protective effect of PLCγ2P522R was vitally dependent on 'allelic-dose', as homozygous cells displayed a lower preservation of synapse and a distinct gene expression profile compared to heterozygous cells. Similarly, microglia with the protective mutation PLCγ2P522R displayed higher inflammatory cytokine IL-1β level, and better response to acute treatment with Aβ oligomers. PLCγ2P522R appeared to resist the quiescence that was seen in WT microglia variants, by increasing cytokine production and lysosomal biogenesis. My findings suggest that the P522R variant in PLCγ2 increases microglia capacity to clear toxic aggregates such as Aβ while preserving synapses. Furthermore, my findings suggests that PLCγ2P522R contributes to greater microglial surveillance, as well as microglia priming towards a pro-inflammatory state, along with an increased capacity to adapt to growing energy demands. This, however, also shows the delicate balance of this system, as increasing the 'dosage' of PLCγ2P522R may result in diminished favourable benefits

    Study of neural circuits using multielectrode arrays in movement disorders

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    Treballs Finals de Grau d'Enginyeria Biomèdica. Facultat de Medicina i Ciències de la Salut. Universitat de Barcelona. Curs: 2022-2023. Tutor/Director: Rodríguez Allué, Manuel JoséNeurodegenerative movement-related disorders are characterized by a progressive degeneration and loss of neurons, which lead to motor control impairment. Although the precise mechanisms underlying these conditions are still unknown, an increasing number of studies point towards the analysis of neural networks and functional connectivity to unravel novel insights. The main objective of this work is to understand cellular mechanisms related to dysregulated motor control symptoms in movement disorders, such as Chorea-Acanthocytosis (ChAc), by employing multielectrode arrays to analyze the electrical activity of neuronal networks in mouse models. We found no notable differences in cell viability between neurons with and without VPS13A knockdown, that is the only gene known to be implicated in the disease, suggesting that the absence of VPS13A in neurons may be partially compensated by other proteins. The MEA setup used to capture the electrical activity from neuron primary cultures is described in detail, pointing out its specific characteristics. At last, we present the alternative backup approach implemented to overcome the challenges faced during the research process and to explore the advanced algorithms for signal processing and analysis. In this report, we present a thorough account of the conception and implementation of our research, outlining the multiple limitations that have been encountered all along the course of the project. We provide a detailed analysis on the project’s economical and technical feasibility, as well as a comprehensive overview of the ethical and legal aspects considered during the execution

    The role of the brain extracellular space in diffusion and cell signalling

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    134 p.The extracellular space (ECS) is a highly complex space consisting of narrow interconnected channels and reservoirs. The ECS substructures are usually few nanometers wide and consequently, they are very difficult to visualize. In addition, the brain ECS is a very dynamic structure, that changes at different temporal scales. These structural changes can be physiological or they can have a pathological cause. In fact, astrocytic swelling at the expense of the ECS volume is one of the hallmarks of epilepsy. Particularly, we are interested in how ECS volume changes affect GABAergic inhibition, the main source of inhibition in the brain and one of the most studied processes in the onset of epileptogenesis.On the other hand, most intercellular signalling in the brain occurs by diffusion of particles through the ECS channels. Understanding how diffusion is regulated by the fine geometry of the brain neuropil is becoming the focus of interest for researchers. However, progress in this field is limited by the difficulty to access local ECS diffusion with experimental techniques. Recently developed techniques, such as super-resolution shadow imaging (SUSHI), are opening the doors to understand diffusion of molecules through the brain sub-micron ECS structures. In this study, we aim to investigate how the nano-scale ECS geometry of the live brain tissue shapes the diffusion of transmitters and its impact on cellular communication. To attain this goal, we have developed a novel computational model, based on SUSHI images

    Type 3 adenylyl cyclase, neuronal primary cilia, and hippocampus-dependent memory formation

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    Primary cilia are microtubule-based cellular antennae present in most vertebrate cells including neurons. Neuronal primary cilia have abundant expression of G-protein coupled receptors (GPCRs) and downstream cAMP signaling components such as type 3 adenylyl cyclase (AC3). The deflects of neuronal cilia is associated with many memory-related disorders, such as intellectual disability. Thus far, little is known about how neuronal primary cilia regulate neuronal activity and affect hippocampal memory formation. Episodic memory is thought to be encoded by sparsely distributed memory-eligible neurons in the hippocampus and neocortex. However, it is not clear how memory-eligible neurons interact with one another to form and retrieve a memory. The objectives of my dissertation are to determine the roles of AC3 in regulating cortical protein phosphorylation, to examine the cellular mechanism of episodic memory formation, and to examine how neuronal primary cilia regulate trace fear memory formation. Project 1: Compare protein phosphorylation levels in the prefrontal cortex between AC3 knockout (KO) and wildtype (WT) mice. AC3 represents a key enzyme mediating ciliary cAMP signaling in neurons and is genetically associated with major depressive disorder (MDD) and autism spectrum disorders (ASD). The major downstream effector protein of cAMP in cells is protein kinase A (PKA), whose activation leads to the phosphorylation of numerous proteins to propagate the signaling downstream. In my mass spectrometry-based phosphoproteomic study using conditional AC3 KO mice, I identified thousands of peptides from prefrontal cortical tissues, some of which are differentially phosphorylated in AC3 WT and KO samples. In addition, this effort led to identification of over two hundred proteins, whose phosphorylation were sex-biased. Surprisingly, a high percentage of these targets (31%) are autism-associated proteins/genes. Hence, this study provides the first phosphoproteomic evidence suggesting that sex-biased protein phosphorylation may contribute to the sexual dimorphism of autism. Project 2: Investigate how hippocampal neurons are recruited to interact with each other to encode a trace fear memory. Using in vivo calcium imaging in freely behaving mice, I found that a small portion of highly active hippocampal neurons (termed primed neurons) are actively engaged in memory formation and retrieval. I found that induction of activity synchronization among primed neurons from random dynamics is critical for trace memory formation and retrieval. My work has provided direct in vivo evidence to challenge the long-held paradigm that activation and re-activation of memory cells encodes and retrieves memory, respectively. These findings support a new mechanistic model for associative memory formation, in that primed neurons connect with each other to forge a new circuit, bridging a conditional stimulus with an unconditional stimulus. Project 3: Develop an analytical method to identify primed neurons and determine the roles of neuronal primary cilia on hippocampal neuronal priming and trace memory formation. Neuronal primary cilia are “cellular antennae” which sense and transduce extracellular signals into neuronal soma. However, to date little is known about how neuronal primary cilia influence neuronal functions and hippocampal memory. I utilized conditional Ift88 knockout mice (to ablate cilia) as loss-of-function models. I found that inducible conditional Ift88 KOs display more severe learning deficits compared to their littermate controls. Cilia-ablated mice showed reduced overall neuronal activity, decreased number of primed neurons, and failed to form burst synchronization. These data support the conclusion that alteration of neuronal primary cilia impairs trace fear memory by decreasing hippocampal neuronal priming and the formation of burst synchronization. This study also provides evidence to support the importance of burst synchronization among primed neurons on memory formation and retrieval

    Combining Tumour-Treating Fields with DNA damage response inhibitors for the improved treatment of glioblastoma

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    Glioblastoma is the most common and deadliest type of primary brain cancer, taking over 2,500 lives each year in the UK. glioblastoma has a median overall survival of 10-16 months, despite treatment consisting of maximal surgical resection followed by chemo- and radio-therapy. glioblastoma survival rates have seen little improvement over the past 40 years and given this devastating prognosis, new treatment options for the management of glioblastoma are urgently needed. Recently, Tumour Treating Fields (TTFields) has emerged as a novel fourth modality for the treatment of high-grade gliomas following its success in clinical trials, where the addition of TTFields to standard care temozolomide was shown to increase median progression-free survival (6.7 versus 4.0 months) and overall survival (20.9 vs 16.0 months) of newly-diagnosed glioblastoma patients compared to temozolomide alone. TTFields are primarily thought to mediate their anti-cancer effects by disrupting tubulin dimer alignment during mitosis, resulting in abnormal chromosomal segregation and mitotic cell death. In addition, recent data suggests that TTFields affect a number of other cellular processes – 1- cell membrane and blood-brain barrier (BBB) permeability, 2- cell migration and invasion, 3- anti�tumour immunity, 4- autophagy, and 5- replication stress and DNA damage repair, the latter of which will be the focus of this project. TTFields has also been shown to downregulate DNA damage response (DDR) proteins and delay the repair of radiotherapy- and chemotherapy-induced DNA lesions, an effect that is thought to be mediated through reduced homologous recombination repair efficiency and induction of a ‘BRCAness’ phenotype. Such vulnerabilities within DNA damage repair pathways provides a rational for the use of TTFields in combinational therapeutic approaches that target the DDR. We therefore aim to assess whether combining TTFields with DDR inhibitors (PARPi, ATMi, ATRi and WEE1i) can enhance the efficacy of TTFields in clinically relevant glioblastoma stem-like cultures (GSCs) using a number of established cell survival assays. Additionally, we aim to investigate the mechanisms by which combination treatments of DDR inhibitors and TTFields affect the DNA damage response. In this thesis we show that combining TTFields with radiation and clinically approved PARP inhibitor therapy leads to significantly increased amounts of DNA damage with concurrent decreased clonogenic survival in GSCs. Furthermore, we have shown similar impressive potency when TTFields treatments are combined with BBB-penetrant ATR inhibitors that are currently being assessed in various global clinical trials for glioblastoma as well as other cancers. Overall, these exciting findings support further assessment of TTFields and DDRi combinations to underpin future clinical trials combining TTFields with clinically approved DDRi to improve outcomes for patients with currently incurable high-grade gliomas

    Investigating small extracellular vesicle miRNA as biomarkers for Alzheimer’s disease

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    Alzheimer’s disease (AD) is the leading cause of dementia, a syndrome impacting over 900,000 people in the UK alone. There are currently no disease modifying treatments for AD, which is largely attributable to the heterogenous basis of the disease which is known to have multiple genetic and environmental contributors. Early identification of the pathogenic drivers of disease could help with both the diagnosis of specific dementia subtypes and the development of more targeted, personalised, therapeutic interventions.Extracellular vesicles (EVs) can cross the blood-brain-barrier and have been shown to carry AD associated cargoes, including amyloid-β and tau. EV miRNA presents a promising avenue for biomarkers for AD. Within this project, EVs were isolated from fibroblasts, hydrogen peroxide treated SH-SY5Y cells and human brain tissue, by sequential centrifugation and separation by size exclusion chromatography. Isolated EVs were characterised using western blotting, fluorescence nanoparticle tracking analysis, and transmission electron microscopy. MiRNA analysis was performed using qPCR and small RNA sequencing.Isolated EVs displayed size ranges in line with small EVs (< 150 nm) and expressed EV associated proteins, including tetraspanins CD9, CD63 and CD81, while not expressing cellular associated markers. Small RNA sequencing identified a panel of upregulated (miR-203a, miR-141, miR-361, miR-30a, and miR-125b-1) and downregulated (miR-582 and miR-1248) miRNAs in brain derived EVs (BDEVs) in AD. In fibroblast derived EVs, miR-146, miR-92a and miR-134 were upregulated in both qPCR and RNA sequencing, while miR-134 was downregulated in SH-SY5Y EVs. When stratified for females, miR-27a and miR-668 displayed increased dysregulation in BDEVs in AD. miR-185, miR-132 and miR-660 showed converse patterns of dysregulation in AD, between fibroblast derived and brain derived EVs. In both, fibroblast derived and brain derived EVs, miR-660 was inversely dysregulated in AD between males and females.Combined we highlight a panel of EV miRNAs that show promise as biomarkers for AD that express centrally and peripherally, that can support early intervention of disease.Key words: Alzheimer’s disease, extracellular vesicles, miRNA, fibroblast, brain tissue, SH-SY5Y, biomarker

    Neuroinflammatory and protein responses to TBI

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    Background: Traumatic brain injury (TBI) is a leading cause of death and disability world-wide, affecting approximately 69 million individuals each year. It is also recognised as one of the major, modifiable risk factors in the development of neurodegenerative disease in later life, with exposure to moderate to severe single TBI and repetitive, mild TBI both recognised as contributing factors in the development of dementia. However, the neuropathological mechanisms contributing to late neurodegeneration remain uncertain. The complex polypathology emerging from the initial biomechanical injury mirror other neurodegenerative disease and include abnormal aggregation of proteins such as tau and Aβ and neuroinflammation, which will be explored in this thesis. Methodology: Utilising material from the Glasgow TBI archive, in cohorts of patients aged ≤60 exposed to acute and long-term survival following moderate to severe, single TBI and appropriately age-matched controls, the role of the cellular adaptive immune response to injury was assessed through immunocytochemistry for T- and B-lymphocyte populations. These cohorts as well as tissue from patients exposed to repetitive mild TBI (rTBI) and appropriately matched controls were studied to characterise the differential astroglial response to injury across injury subgroups through measurement of GFAP, AQP4 and NQO1 immunoreactivity. A cohort of patients aged ≥60 years who survived acutely following moderate to severe, single TBI compared to age-matched, uninjured controls with no history of neurodegenerative disease, were examined to evaluate the effect of age and TBI using modifications of clinically recognised, standardised, semi-quantitative scoring systems of tau and Aβ immunoreactivity. Lastly, a protocol for the assessment of our archival, FFPE tissue was devised to allow comparison of proteomes of cohorts of patients exposed to mild rTBI, AD patients and appropriately matched controls, using liquid-chromatography mass-spectrometry. Results: There is no histological evidence of a significant cellular response from the adaptive immune system following exposure to moderate to severe, single TBI in patients surviving acutely or long-term after injury, with no increase in T- or B-lymphocytes. There was, unexpectedly, a decrease in T-lymphocytes in long-term survivors of TBI. Contrastingly, there was an increase in reactive astrogliosis following exposure to TBI; demonstrated by an increase in AQP4-immunoractive astrocytes in acutely surviving patients and increase in GFAP expression in long-term surviving patients and an increase in NQO1 expression in rTBI patients compared to age-matched uninjured controls. There was also an increase in both AQP4 and GFAP expression in elderly uninjured controls compared to younger uninjured controls. Examining the expression of Aβ and tau in elderly patients exposed to single, moderate to severe TBI showed an increase in neuritic Aβ plaque and in the regional distribution of all plaque compared to uninjured, elderly controls, but no increase in expression or distribution of NFTs. Finally, a protocol for processing archival FFPE resulted in identification of 267 proteins from across rTBI, AD and uninjured, non-NND controls with significantly differential expression in 84 of them. Conclusions: Together, these findings increase understanding of the neuropathological changes occurring following moderate to severe, single TBI and repetitive TBI. These data demonstrate that there is an astrogliotic but not adaptive cellular response after moderate to severe single TBI whilst also showing that age is correlated with an increase in reactive astrogliosis for several markers. Age was also examined in the examination of proteinopathic changes following acute survival after moderate to severe injury and changes suggest that TBI may occur as a result of increased amyloid pathology as neuritic plaque was observed > 2 weeks after injury. Lastly, that archival FFPE samples were successfully processed to allow identification of proteins from across a range of cohorts, revealing differences in protein expression that underpin the neuropathological changes which contribute to the long-term, post-TBI neurodegenerative process

    KATP channels are necessary for glucose-dependent increases in amyloid-β and Alzheimer\u27s disease-related pathology

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    Elevated blood glucose levels, or hyperglycemia, can increase brain excitability and amyloid-β (Aβ) release, offering a mechanistic link between type 2 diabetes and Alzheimer\u27s disease (AD). Since the cellular mechanisms governing this relationship are poorly understood, we explored whether ATP-sensitive potassium (KATP) channels, which couple changes in energy availability with cellular excitability, play a role in AD pathogenesis. First, we demonstrate that KATP channel subunits Kir6.2/KCNJ11 and SUR1/ABCC8 were expressed on excitatory and inhibitory neurons in the human brain, and cortical expression of KCNJ11 and ABCC8 changed with AD pathology in humans and mice. Next, we explored whether eliminating neuronal KATP channel activity uncoupled the relationship between metabolism, excitability, and Aβ pathology in a potentially novel mouse model of cerebral amyloidosis and neuronal KATP channel ablation (i.e., amyloid precursor protein [APP]/PS1 Kir6.2-/- mouse). Using both acute and chronic paradigms, we demonstrate that Kir6.2-KATP channels are metabolic sensors that regulate hyperglycemia-dependent increases in interstitial fluid levels of Aβ, amyloidogenic processing of APP, and amyloid plaque formation, which may be dependent on lactate release. These studies identify a potentially new role for Kir6.2-KATP channels in AD and suggest that pharmacological manipulation of Kir6.2-KATP channels holds therapeutic promise in reducing Aβ pathology in patients with diabetes or prediabetes

    The Critical Role of Spreading Depolarizations in Early Brain Injury: Consensus and Contention

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    Background: When a patient arrives in the emergency department following a stroke, a traumatic brain injury, or sudden cardiac arrest, there is no therapeutic drug available to help protect their jeopardized neurons. One crucial reason is that we have not identified the molecular mechanisms leading to electrical failure, neuronal swelling, and blood vessel constriction in newly injured gray matter. All three result from a process termed spreading depolarization (SD). Because we only partially understand SD, we lack molecular targets and biomarkers to help neurons survive after losing their blood flow and then undergoing recurrent SD. Methods: In this review, we introduce SD as a single or recurring event, generated in gray matter following lost blood flow, which compromises the Na/K pump. Electrical recovery from each SD event requires so much energy that neurons often die over minutes and hours following initial injury, independent of extracellular glutamate. Results: We discuss how SD has been investigated with various pitfalls in numerous experimental preparations, how overtaxing the Na/K ATPase elicits SD. Elevated K or glutamate are unlikely natural activators of SD. We then turn to the properties of SD itself, focusing on its initiation and propagation as well as on computer modeling. Conclusions: Finally, we summarize points of consensus and contention among the authors as well as where SD research may be heading. In an accompanying review, we critique the role of the glutamate excitotoxicity theory, how it has shaped SD research, and its questionable importance to the study of early brain injury as compared with SD theory.This work was supported by grants from the Heart and Stroke Foundation of Canada and the National Science and Engineering Research Council of Canada to RDA, an NIH grant (NS106901) to CWS, a National Research, Development and Innovation Office of Hungary grant (K1343777) and EU Horizon 2020 research and innovation program (739953) to EF and from DFG Deutsche Forschungsgemeinschaft (German Research Council) (DFG DR 323/5-1), DFG DR 323/10-1, and BMBF Bundesministerium fuer Bildung und Forschung (EraNet Neuron EBio2, with funds from BMBF 01EW2004) to JPD
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