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In human stroke, brain swelling is an important predictor of neurological outcome and mortality, yet treatments to reduce or prevent brain swelling are extremely limited, due in part to an inadequate understanding of mechanisms. In preclinical studies on cerebroprotection in animal models of stroke, historically, the focus has been on reducing infarct size, and in most studies, a reduction in infarct size has been associated with a corresponding reduction in brain swelling. Unfortunately, such findings on brain swelling have little translational value for treating brain swelling in patients with stroke. This is because, in humans, brain swelling usually becomes evident, either symptomatically or radiologically, days after the infarct size has stabilized, requiring that the prevention or treatment of brain swelling target mechanism(s) that are independent of a reduction in infarct size. In this problematizing review, we highlight the often-neglected concept that brain edema and brain swelling are not simply secondary, correlative phenomena of stroke but distinct pathological entities with unique molecular and cellular mechanisms that are worthy of direct targeting. We outline the advances in approaches for the study of brain swelling that are independent of a reduction in infarct size. Although straightforward, the approaches reviewed in this study have important translational relevance for identifying novel treatment targets for post-ischemic brain swelling.
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The COVID-19 pandemic caused by SARS-CoV-2 has posed unparalleled challenges due to its rapid transmission, ability to mutate, high mortality and morbidity, and enduring health complications. Vaccines have exhibited effectiveness, but their efficacy diminishes over time while new variants continue to emerge. Antiviral medications offer a viable alternative, but their success has been inconsistent. Therefore, there remains an ongoing need to identify innovative antiviral drugs for treating COVID-19 and its post-infection complications. The ORF3a (open reading frame 3a) protein found in SARS-CoV-2, represents a promising target for antiviral treatment due to its multifaceted role in viral pathogenesis, cytokine storms, disease severity, and mortality. ORF3a contributes significantly to viral pathogenesis by facilitating viral assembly and release, essential processes in the viral life cycle, while also suppressing the body's antiviral responses, thus aiding viral replication. ORF3a also has been implicated in triggering excessive inflammation, characterized by NF-κB-mediated cytokine production, ultimately leading to apoptotic cell death and tissue damage in the lungs, kidneys, and the central nervous system. Additionally, ORF3a triggers the activation of the NLRP3 inflammasome, inciting a cytokine storm, which is a major contributor to the severity of the disease and subsequent mortality. As with the spike protein, ORF3a also undergoes mutations, and certain mutant variants correlate with heightened disease severity in COVID-19. These mutations may influence viral replication and host cellular inflammatory responses. While establishing a direct link between ORF3a and mortality is difficult, its involvement in promoting inflammation and exacerbating disease severity likely contributes to higher mortality rates in severe COVID-19 cases. This review offers a comprehensive and detailed exploration of ORF3a's potential as an innovative antiviral drug target. Additionally, we outline potential strategies for discovering and developing ORF3a inhibitor drugs to counteract its harmful effects, alleviate tissue damage, and reduce the severity of COVID-19 and its lingering complications.
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Brain swelling is a major cause of death and disability in ischemic stroke. Drugs of the gliflozin class, which target the Na+-coupled D-glucose cotransporter, SGLT2, are approved for type 2 diabetes mellitus (T2DM) and may be beneficial in other conditions, but data in cerebral ischemia are limited. We studied murine models of cerebral ischemia with middle cerebral artery occlusion/reperfusion (MCAo/R). Slc5a2/SGLT2 mRNA and protein were upregulated de novo in astrocytes. Live cell imaging of brain slices from mice following MCAo/R showed that astrocytes responded to modest increases in D-glucose by increasing intracellular Na+ and cell volume (cytotoxic edema), both of which were inhibited by the SGLT2 inhibitor, canagliflozin. The effect of canagliflozin was studied in three mouse models of stroke: non-diabetic and T2DM mice with a moderate ischemic insult (MCAo/R, 1/24 h) and non-diabetic mice with a severe ischemic insult (MCAo/R, 2/24 h). Canagliflozin reduced infarct volumes in models with moderate but not severe ischemic insults. However, canagliflozin significantly reduced hemispheric swelling and improved neurological function in all models tested. The ability of canagliflozin to reduce brain swelling regardless of an effect on infarct size has important translational implications, especially in large ischemic strokes.
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Edema Encefálico , Isquemia Encefálica , Diabetes Mellitus Tipo 2 , Accidente Cerebrovascular Isquémico , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Animales , Ratones , Canagliflozina/farmacología , Canagliflozina/uso terapéutico , Edema Encefálico/tratamiento farmacológico , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Astrocitos , Transportador 2 de Sodio-Glucosa , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Glucosa , Iones , Isquemia Encefálica/tratamiento farmacológico , InfartoRESUMEN
The transformation of astrocytes into reactive states constitutes a biological response of the central nervous system under a variety of pathological insults. Astrocytes display diverse homeostatic identities that are developmentally predetermined and regionally specified. Upon transformation into reactive states associated with neurodegenerative diseases and other neurological disorders, astrocytes acquire diverse reactive phenotypes. However, it is not clear whether their reactive phenotypes are dictated by region-specific homeostatic identity or by the nature of an insult. To address this question, region-specific gene expression profiling was performed for four brain regions (cortex, hippocampus, thalamus, and hypothalamus) in mice using a custom NanoString panel consisting of selected sets of genes associated with astrocyte functions and their reactivity for five conditions: prion disease, traumatic brain injury, brain ischemia, 5XFAD Alzheimer's disease model and normal aging. Upon transformation into reactive states, genes that are predominantly associated with astrocytes were found to respond to insults in a region-specific manner. Regardless of the nature of the insult or the insult-specificity of astrocyte response, strong correlations between undirected GSA (gene set analysis) scores reporting on astrocyte reactivity and on their homeostatic functions were observed within each individual brain region. The insult-specific gene expression signatures did not separate well from each other and instead partially overlapped, forming continuums. The current study demonstrates that region-specific homeostatic identities of astrocytes are important for defining their response to pathological insults. Within region-specific populations, reactive astrocytes show continuums of gene expression signatures, partially overlapping between individual insults.
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Lesiones Traumáticas del Encéfalo , Isquemia Encefálica , Animales , Ratones , Astrocitos , Sistema Nervioso Central , EnvejecimientoRESUMEN
Over 38 million people worldwide are living with HIV/AIDS, and more than half of them are affected by HIV-associated neurocognitive disorders (HAND). Such disorders are characterized by chronic neuroinflammation, neurotoxicity, and central nervous system deterioration, which lead to short- or long-term memory loss, cognitive impairment, and motor skill deficits that may show gender disparities. However, the underlying mechanisms remain unclear. Our previous study suggested that HIV-1 infection and viral protein R (Vpr) upregulate the SUR1-TRPM4 channel associated with neuroinflammation, which may contribute to HAND. The present study aimed to explore this relationship in a mouse model of HAND. This study employed the HIV transgenic Tg26 mouse model, comparing Tg26 mice with wildtype mice in various cognitive behavioral and memory tests, including locomotor activity tests, recognition memory tests, and spatial learning and memory tests. The study found that Tg26 mice exhibited impaired cognitive skills and reduced learning abilities compared to wildtype mice, particularly in spatial memory. Interestingly, male Tg26 mice displayed significant differences in spatial memory losses (p < 0.001), while no significant differences were identified in female mice. Consistent with our early results, SUR1-TRPM4 channels were upregulated in Tg26 mice along with glial fibrillary acidic protein (GFAP) and aquaporin 4 (AQP4), consistent with reactive astrocytosis and neuroinflammation. Corresponding reductions in neurosynaptic responses, as indicated by downregulation of Synapsin-1 (SYN1) and Synaptophysin (SYP), suggested synaptopathy as a possible mechanism underlying cognitive and motor skill deficits. In conclusion, our study suggests a possible relationship between SUR1-TRPM4-mediated neuroinflammation and synaptopathy with impairments of learning and memory in mice with HAND. These findings could help to develop new therapeutic strategies for individuals living with HAND.
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Brain swelling causes morbidity and mortality in various brain injuries and diseases but lacks effective treatments. Brain swelling is linked to the influx of water into perivascular astrocytes through channels called aquaporins. Water accumulation in astrocytes increases their volume, which contributes to brain swelling. Using a mouse model of severe ischemic stroke, we identified a potentially targetable mechanism that promoted the cell surface localization of aquaporin 4 (AQP4) in perivascular astrocytic endfeet, which completely ensheathe the brain's capillaries. Cerebral ischemia increased the abundance of the heteromeric cation channel SUR1-TRPM4 and of the Na+/Ca2+ exchanger NCX1 in the endfeet of perivascular astrocytes. The influx of Na+ through SUR1-TRPM4 induced Ca2+ transport into cells through NCX1 operating in reverse mode, thus raising the intra-endfoot concentration of Ca2+. This increase in Ca2+ stimulated calmodulin-dependent translocation of AQP4 to the plasma membrane and water influx, which led to cellular edema and brain swelling. Pharmacological inhibition or astrocyte-specific deletion of SUR1-TRPM4 or NCX1 reduced brain swelling and improved neurological function in mice to a similar extent as an AQP4 inhibitor and was independent of infarct size. Thus, channels in astrocyte endfeet could be targeted to reduce postischemic brain swelling in stroke patients.
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Edema Encefálico , Accidente Cerebrovascular Isquémico , Canales Catiónicos TRPM , Humanos , Edema Encefálico/genética , Edema Encefálico/metabolismo , Astrocitos/metabolismo , Acuaporina 4/genética , Acuaporina 4/metabolismo , Accidente Cerebrovascular Isquémico/metabolismo , Agua/metabolismo , Cationes/metabolismo , Canales Catiónicos TRPM/metabolismoRESUMEN
Traumatic brain injury (TBI) is a common and often devastating illness, with wide-ranging public health implications. In addition to the primary injury, victims of TBI are at risk for secondary neurological injury by numerous mechanisms. Current treatments are limited and do not target the profound immune response associated with injury. This immune response reflects a convergence of peripheral and central nervous system-resident immune cells whose interaction is mediated in part by a disruption in the blood-brain barrier (BBB). The diverse family of cytokines helps to govern this communication and among these, Interleukin (IL)-6 is a notable player in the immune response to acute neurological injury. It is also a well-established pharmacological target in a variety of other disease contexts. In TBI, elevated IL-6 levels are associated with worse outcomes, but the role of IL-6 in response to injury is double-edged. IL-6 promotes neurogenesis and wound healing in animal models of TBI, but it may also contribute to disruptions in the BBB and the progression of cerebral edema. Here, we review IL-6 biology in the context of TBI, with an eye to clarifying its controversial role and understanding its potential as a target for modulating the immune response in this disease.
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Edema Encefálico , Lesiones Traumáticas del Encéfalo , Animales , Interleucina-6 , Barrera Hematoencefálica , Citocinas , Modelos Animales de EnfermedadRESUMEN
Endoplasmic reticulum (ER) stress and mitochondrial dysfunction are found in lesions of multiple sclerosis (MS) and animal models of MS such as experimental autoimmune encephalomyelitis (EAE), and may contribute to the neuronal loss that underlies permanent impairment. We investigated whether glatiramer acetate (GA) can reduce these changes in the spinal cords of chronic EAE mice by using routine histology, immunostaining, and electron microscopy. EAE spinal cord tissue exhibited increased inflammation, demyelination, mitochondrial dysfunction, ER stress, downregulation of NAD+ dependent pathways, and increased neuronal death. GA reversed these pathological changes, suggesting that immunomodulating therapy can indirectly induce neuroprotective effects in the CNS by mediating ER stress.
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Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Ratones , Animales , Acetato de Glatiramer/farmacología , Acetato de Glatiramer/uso terapéutico , Péptidos/farmacología , Inmunomodulación , Estrés del Retículo Endoplásmico , Mitocondrias/metabolismo , Ratones Endogámicos C57BL , Modelos Animales de EnfermedadRESUMEN
OBJECTIVES: Addressing traumatic brain injury (TBI) heterogeneity is increasingly recognized as essential for therapy translation given the long history of failed clinical trials. We evaluated differential effects of a promising treatment (glibenclamide) based on dose, TBI type (patient selection), and imaging endophenotype (outcome selection). Our goal to inform TBI precision medicine is contextually timely given ongoing phase 2/planned phase 3 trials of glibenclamide in brain contusion. DESIGN: Blinded randomized controlled preclinical trial of glibenclamide on MRI endophenotypes in two established severe TBI models: controlled cortical impact (CCI, isolated brain contusion) and CCI+hemorrhagic shock (HS, clinically common second insult). SETTING: Preclinical laboratory. SUBJECTS: Adult male C57BL/6J mice (n = 54). INTERVENTIONS: Mice were randomized to naïve, CCI±HS with vehicle/low-dose (20 µg/kg)/high-dose glibenclamide (10 µg/mouse). Seven-day subcutaneous infusions (0.4 µg/hr) were continued. MEASUREMENTS AND MAIN RESULTS: Serial MRI (3 hr, 6 hr, 24 hr, and 7 d) measured hematoma and edema volumes, T2 relaxation (vasogenic edema), apparent diffusion coefficient (ADC, cellular/cytotoxic edema), and 7-day T1-post gadolinium values (blood-brain-barrier [BBB] integrity). Linear mixed models assessed temporal changes. Marked heterogeneity was observed between CCI versus CCI+HS in terms of different MRI edema endophenotypes generated (all p < 0.05). Glibenclamide had variable impact. High-dose glibenclamide reduced hematoma volume ~60% after CCI (p = 0.0001) and ~48% after CCI+HS (p = 4.1 × 10-6) versus vehicle. Antiedema benefits were primarily in CCI: high-dose glibenclamide normalized several MRI endophenotypes in ipsilateral cortex (all p < 0.05, hematoma volume, T2, ADC, and T1-post contrast). Acute effects (3 hr) were specific to hematoma (p = 0.001) and cytotoxic edema reduction (p = 0.0045). High-dose glibenclamide reduced hematoma volume after TBI with concomitant HS, but antiedema effects were not robust. Low-dose glibenclamide was not beneficial. CONCLUSIONS: High-dose glibenclamide benefitted hematoma volume, vasogenic edema, cytotoxic edema, and BBB integrity after isolated brain contusion. Hematoma and cytotoxic edema effects were acute; longer treatment windows may be possible for vasogenic edema. Our findings provide new insights to inform interpretation of ongoing trials as well as precision design (dose, sample size estimation, patient selection, outcome selection, and Bayesian analysis) of future TBI trials of glibenclamide.
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Contusión Encefálica , Edema Encefálico , Lesiones Traumáticas del Encéfalo , Lesiones Encefálicas , Animales , Masculino , Ratones , Teorema de Bayes , Contusión Encefálica/complicaciones , Contusión Encefálica/tratamiento farmacológico , Edema Encefálico/diagnóstico por imagen , Edema Encefálico/tratamiento farmacológico , Edema Encefálico/etiología , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Traumáticas del Encéfalo/diagnóstico por imagen , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Lesiones Traumáticas del Encéfalo/complicaciones , Modelos Animales de Enfermedad , Endofenotipos , Gliburida/farmacología , Gliburida/uso terapéutico , Imagen por Resonancia Magnética , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Pathophysiological consequences of traumatic brain injury (TBI) mediated secondary injury remain incompletely understood. In particular, the impact of TBI on the differentiation and maintenance of dendritic cells (DCs), which are regarded as the most professional antigen presenting cells of the immune system, remains completely unknown. Here, we report that DC-differentiation, maintenance and functions are altered on day 3 and day 7 after TBI. METHODS: Long bones, spleen, peripheral lymph nodes (pLNs), mesenteric lymph nodes (mLNs), liver, lungs, skin and blood were collected from mice with either moderate-level cortical impact (CCI) or sham on day 1, day 3 or day 7 after TBI. Bone marrow cells were isolated from the tibias and femurs of hind limb through flushing. Tissues were digested with Collagenase-D and DNase I. Skin biopsies were digested in the presence of liberase + DNase I. Single cell suspensions were made, red blood cells were lysed with Ammonium chloride (Stem Cell Technology) and subsequently filtered using a 70 µM nylon mesh. DC subsets of the tissues and DC progenitors of the BM were identified through 10-color flow cytometry-based immunophenotyping studies. Intracellular reactive oxygen species (ROS) were identified through H2DCFDA staining. RESULTS: Our studies identify that; (1) frequencies and absolute numbers of DCs in the spleen and BM are altered on day 3 and day 7 after TBI; (2) surface expression of key molecules involved in antigen presentation of DCs were affected on day 3 and day 7 after TBI; (3) distribution and functions of tissue-specific DC subsets of both circulatory and lymphatic systems were imbalanced following TBI; (4) early differentiation program of DCs, especially the commitment of hematopoietic stem cells to common DC progenitors (CDPs), were deregulated after TBI; and (5) intracellular ROS levels were reduced in DC progenitors and differentiated DCs on day 3 and day 7 after TBI. CONCLUSIONS: Our data demonstrate, for the first time, that TBI affects the distribution pattern of DCs and induces an imbalance among DC subsets in both lymphoid and non-lymphoid organs. In addition, the current study demonstrates that TBI results in reduced levels of ROS in DCs on day 3 and day 7 after TBI, which may explain altered DC differentiation paradigm following TBI. A deeper understanding on the molecular mechanisms that contribute to DC defects following TBI would be essential and beneficial in treating infections in patients with acute central nervous system (CNS) injuries, such as TBI, stroke and spinal cord injury.
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Lesiones Traumáticas del Encéfalo , Células Dendríticas , Cloruro de Amonio/metabolismo , Animales , Lesiones Traumáticas del Encéfalo/metabolismo , Diferenciación Celular , Desoxirribonucleasa I/metabolismo , Células Madre Hematopoyéticas/metabolismo , Ratones , Nylons/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Spinal cord injury (SCI) is a devastating neurological condition with no effective treatment. Hypothermia induced by physical means (cold fluid) is established as an effective therapy in animal models of SCI, but its clinical translation to humans is hampered by several constraints. Hypothermia induced pharmacologically may be noninferior or superior to physically induced hypothermia for rapid, convenient systemic temperature reduction, but it has not been investigated previously in animal models of SCI. We used a rat model of SCI to compare outcomes in three groups: (1) normothermic controls; (2) hypothermia induced by conventional physical means; (3) hypothermia induced by intravenous (IV) dihydrocapsaicin (DHC). Male rats underwent unilateral lower cervical SCI and were treated after a 4-hour delay with physical cooling or IV DHC (â¼0.60 mg/kg total) cooling (both 33.0 ± 1.0°C) lasting 4 hours; controls were kept normothermic. Telemetry was used to monitor temperature and heart rate during and after treatments. In two separate experiments, one ending at 48 hours, the other at 6 weeks, "blinded" investigators evaluated rats in the three groups for neurological function followed by histopathological evaluation of spinal cord tissues. DHC reliably induced systemic cooling to 32-33°C. At both the time points examined, the two modes of hypothermia yielded similar improvements in neurological function and lesion size compared with normothermic controls. Our results indicate that DHC-induced hypothermia may be comparable with physical hypothermia in efficacy, but more clinically feasible to administer than physical hypothermia.
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Hipotermia Inducida , Hipotermia , Traumatismos de la Médula Espinal , Animales , Capsaicina/análogos & derivados , Hipotermia/terapia , Hipotermia Inducida/métodos , Masculino , Ratas , Ratas Sprague-Dawley , Médula Espinal/patología , Traumatismos de la Médula Espinal/terapiaRESUMEN
The ongoing SARS-CoV-2 pandemic has shocked the world due to its persistence, COVID-19-related morbidity and mortality, and the high mutability of the virus. One of the major concerns is the emergence of new viral variants that may increase viral transmission and disease severity. In addition to mutations of spike protein, mutations of viral proteins that affect virulence, such as ORF3a, also must be considered. The purpose of this article is to review the current literature on ORF3a, to summarize the molecular actions of SARS-CoV-2 ORF3a, and its role in viral pathogenesis and COVID-19. ORF3a is a polymorphic, multifunctional viral protein that is specific to SARS-CoV/SARS-CoV-2. It was acquired from ß-CoV lineage and likely originated from bats through viral evolution. SARS-CoV-2 ORF3a is a viroporin that interferes with ion channel activities in host plasma and endomembranes. It is likely a virion-associated protein that exerts its effect on the viral life cycle during viral entry through endocytosis, endomembrane-associated viral transcription and replication, and viral release through exocytosis. ORF3a induces cellular innate and pro-inflammatory immune responses that can trigger a cytokine storm, especially under hypoxic conditions, by activating NLRP3 inflammasomes, HMGB1, and HIF-1α to promote the production of pro-inflammatory cytokines and chemokines. ORF3a induces cell death through apoptosis, necrosis, and pyroptosis, which leads to tissue damage that affects the severity of COVID-19. ORF3a continues to evolve along with spike and other viral proteins to adapt in the human cellular environment. How the emerging ORF3a mutations alter the function of SARS-CoV-2 ORF3a and its role in viral pathogenesis and COVID-19 is largely unknown. This review provides an in-depth analysis of ORF3a protein's structure, origin, evolution, and mutant variants, and how these characteristics affect its functional role in viral pathogenesis and COVID-19.
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Therapeutic inhibition of critical viral functions is important for curtailing coronavirus disease-2019 (COVID-19). We sought to identify antiviral targets through genome-wide characterization of SARS-CoV-2 proteins that are crucial for viral pathogenesis and that cause harmful cytopathic effects. All twenty-nine viral proteins were tested in a fission yeast cell-based system using inducible gene expression. Twelve proteins including eight non-structural proteins (NSP1, NSP3, NSP4, NSP5, NSP6, NSP13, NSP14 and NSP15) and four accessory proteins (ORF3a, ORF6, ORF7a and ORF7b) were identified that altered cellular proliferation and integrity, and induced cell death. Cell death correlated with the activation of cellular oxidative stress. Of the twelve proteins, ORF3a was chosen for further study in mammalian cells. In human pulmonary and kidney epithelial cells, ORF3a induced cellular oxidative stress associated with apoptosis and necrosis, and caused activation of pro-inflammatory response with production of the cytokines TNF-α, IL-6, and IFN-ß1, possibly through the activation of NF-κB. To further characterize the mechanism, we tested a natural ORF3a Beta variant, Q57H, and a mutant with deletion of the highly conserved residue, ΔG188. Compared to wild type ORF3a, the ΔG188 variant yielded more robust activation of cellular oxidative stress, cell death, and innate immune response. Since cellular oxidative stress and inflammation contribute to cell death and tissue damage linked to the severity of COVID-19, our findings suggest that ORF3a is a promising, novel therapeutic target against COVID-19.
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INTRODUCTION: Cerebral edema is a key contributor to death and disability in several forms of brain injury. Current treatment options are limited, reactive, and associated with significant morbidity. Targeted therapies are emerging based on a growing understanding of the molecular underpinnings of cerebral edema. AREAS COVERED: We review the pathophysiology and relationships between different cerebral edema subtypes to provide a foundation for emerging therapies. Mechanisms for promising molecular targets are discussed, with an emphasis on those advancing in clinical trials, including ion and water channels (AQP4, SUR1-TRPM4) and other proteins/lipids involved in edema signaling pathways (AVP, COX2, VEGF, and S1P). Research on novel treatment modalities for cerebral edema [including recombinant proteins and gene therapies] is presented and finally, insights on reducing secondary injury and improving clinical outcome are offered. EXPERT OPINION: Targeted molecular strategies to minimize or prevent cerebral edema are promising. Inhibition of SUR1-TRPM4 (glyburide/glibenclamide) and VEGF (bevacizumab) are currently closest to translation based on advances in clinical trials. However, the latter, tested in glioblastoma multiforme, has not demonstrated survival benefit. Research on recombinant proteins and gene therapies for cerebral edema is in its infancy, but early results are encouraging. These newer modalities may facilitate our understanding of the pathobiology underlying cerebral edema.
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Edema Encefálico , Canales Catiónicos TRPM , Edema Encefálico/terapia , Gliburida/farmacología , Humanos , Receptores de Sulfonilureas/genética , Receptores de Sulfonilureas/metabolismo , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismoRESUMEN
The combined antiretroviral therapy era has significantly increased the lifespan of people with HIV (PWH), turning a fatal disease to a chronic one. However, this lower but persistent level of HIV infection increases the susceptibility of HIV-associated neurocognitive disorder (HAND). Therefore, research is currently seeking improved treatment for this complication of HIV. In PWH, low levels of brain derived neurotrophic factor (BDNF) has been associated with worse neurocognitive impairment. Hence, BDNF administration has been gaining relevance as a possible adjunct therapy for HAND. However, systemic administration of BDNF is impractical because of poor pharmacological profile. Therefore, we investigated the neuroprotective effects of BDNF-mimicking 7,8 dihydroxyflavone (DHF), a bioactive high-affinity TrkB agonist, in the memory-involved hippocampus and brain cortex of Tg26 mice, a murine model for HAND. In these brain regions, we observed astrogliosis, increased expression of chemokine HIV-1 coreceptors CXCR4 and CCR5, neuroinflammation, and mitochondrial damage. Hippocampi and cortices of DHF treated mice exhibited a reversal of these pathological changes, suggesting the therapeutic potential of DHF in HAND. Moreover, our data indicates that DHF increases the phosphorylation of TrkB, providing new insights about the role of the TrkB-Akt-NFkB signaling pathway in mediating these pathological hallmarks. These findings guide future research as DHF shows promise as a TrkB agonist treatment for HAND patients in adjunction to the current antiviral therapies.
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Complejo SIDA Demencia/patología , Encéfalo/efectos de los fármacos , Flavonas/farmacología , Glicoproteínas de Membrana/agonistas , Fármacos Neuroprotectores/farmacología , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Gliosis/patología , Ratones , Fosforilación/efectos de los fármacos , Proteínas Tirosina QuinasasRESUMEN
Females have been understudied in pre-clinical and clinical traumatic brain injury (TBI), despite distinct biology and worse clinical outcomes versus males. Sulfonylurea receptor 1 (SUR1) inhibition has shown promising results in predominantly male TBI. A phase II trial is ongoing. We investigated whether SUR1 inhibition effects on contusional TBI differ by sex given that this may inform clinical trial design and/or interpretation. We studied the moderating effects of sex on post-injury brain tissue loss in 142 male and female ATP-binding cassette transporter subfamily C member 8 (Abcc8) wild-type, heterozygote, and knockout mice (12-15 weeks). Monkey fibroblast-like cells and mouse brain endothelium-derived cells were used for in vitro studies. Mice were injured with controlled cortical impact and euthanized 21 days post-injury to assess contusion, brain, and hemisphere volumes (vs. genotype- and sex-matched naïves). Abcc8 knockout mice had smaller contusion volumes (p = 0.012) and larger normalized contralateral (right) hemisphere volumes (nRHV; p = 0.03) after injury versus wild type. This was moderated by sex: Contusions were smaller (p = 0.020), nRHV was higher (p = 0.001), and there was less global atrophy (p = 0.003) in male, but not female, knockout versus wild-type mice after TBI. Less atrophy occurred in males for each copy of Abcc8 lost (p = 0.023-0.002, all outcomes). In vitro, sex-determining region Y (SRY) stimulated Abcc8 promoter activity and increased Abcc8 expression. Loss of Abcc8 strongly protected against post-traumatic cerebral atrophy in male, but not female, mice. This may partly be mediated by SRY on the Y-chromosome. Sex differences may have important implications for ongoing and future trials of SUR1 blockade.
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Lesiones Traumáticas del Encéfalo/patología , Receptores de Sulfonilureas/fisiología , Animales , Atrofia , Lesiones Traumáticas del Encéfalo/etiología , Lesiones Traumáticas del Encéfalo/metabolismo , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores Sexuales , Proteína de la Región Y Determinante del Sexo/fisiologíaRESUMEN
The perivascular astrocyte endfoot is a specialized and diffusion-limited subcellular compartment that fully ensheathes the cerebral vasculature. Despite their ubiquitous presence, a detailed understanding of endfoot physiology remains elusive, in part due to a limited understanding of the proteins that distinguish the endfoot from the greater astrocyte body. Here, we developed a technique to isolate astrocyte endfeet from brain tissue, which was used to study the endfoot proteome in comparison to the astrocyte somata. In our approach, brain microvessels, which retain their endfoot processes, were isolated from mouse brain and dissociated, whereupon endfeet were recovered using an antibody-based column astrocyte isolation kit. Our findings expand the known set of proteins enriched at the endfoot from 10 to 516, which comprised more than 1/5th of the entire detected astrocyte proteome. Numerous critical electron transport chain proteins were expressed only at the endfeet, while enzymes involved in glycogen storage were distributed to the somata, indicating subcellular metabolic compartmentalization. The endfoot proteome also included numerous proteins that, while known to have important contributions to blood-brain barrier function, were not previously known to localize to the endfoot. Our findings highlight the importance of the endfoot and suggest new routes of investigation into endfoot function.
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Astrocitos/metabolismo , Transporte de Electrón/inmunología , Proteoma/metabolismo , Animales , Humanos , Masculino , RatonesRESUMEN
BACKGROUND: Neuropathic pain following peripheral nerve injury (PNI) is linked to neuroinflammation in the spinal cord marked by astrocyte activation and upregulation of interleukin 6 (IL-6), chemokine (C-C motif) ligand 2 (CCL2) and chemokine (C-X-C motif) ligand 1 (CXCL1), with inhibition of each individually being beneficial in pain models. METHODS: Wild type (WT) mice and mice with global or pGfap-cre- or pGFAP-cre/ERT2-driven Abcc8/SUR1 deletion or global Trpm4 deletion underwent unilateral sciatic nerve cuffing. WT mice received prophylactic (starting on post-operative day [pod]-0) or therapeutic (starting on pod-21) administration of the SUR1 antagonist, glibenclamide (10 µg IP) daily. We measured mechanical and thermal sensitivity using von Frey filaments and an automated Hargreaves method. Spinal cord tissues were evaluated for SUR1-TRPM4, IL-6, CCL2 and CXCL1. RESULTS: Sciatic nerve cuffing in WT mice resulted in pain behaviors (mechanical allodynia, thermal hyperalgesia) and newly upregulated SUR1-TRPM4 in dorsal horn astrocytes. Global and pGfap-cre-driven Abcc8 deletion and global Trpm4 deletion prevented development of pain behaviors. In mice with Abcc8 deletion regulated by pGFAP-cre/ERT2, after pain behaviors were established, delayed silencing of Abcc8 by tamoxifen resulted in gradual improvement over the next 14 days. After PNI, leakage of the blood-spinal barrier allowed entry of glibenclamide into the affected dorsal horn. Daily repeated administration of glibenclamide, both prophylactically and after allodynia was established, prevented or reduced allodynia. The salutary effects of glibenclamide on pain behaviors correlated with reduced expression of IL-6, CCL2 and CXCL1 by dorsal horn astrocytes. CONCLUSION: SUR1-TRPM4 may represent a novel non-addicting target for neuropathic pain.
Asunto(s)
Astrocitos/metabolismo , Neuralgia/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Receptores de Sulfonilureas/metabolismo , Animales , Modelos Animales de Enfermedad , Hiperalgesia/metabolismo , Ratones Endogámicos C57BL , Neuralgia/fisiopatología , Nervio Ciático/metabolismo , Médula Espinal/metabolismo , Asta Dorsal de la Médula Espinal/metabolismoRESUMEN
Therapeutic inhibition of critical viral functions is important for curtailing coronavirus disease 2019 (COVID-19). We sought to identify antiviral targets through the genome-wide characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins that are crucial for viral pathogenesis and that cause harmful cytopathogenic effects. All 29 viral proteins were tested in a fission yeast cell-based system using inducible gene expression. Twelve proteins, including eight nonstructural proteins (NSP1, NSP3, NSP4, NSP5, NSP6, NSP13, NSP14, and NSP15) and four accessory proteins (ORF3a, ORF6, ORF7a, and ORF7b), were identified that altered cellular proliferation and integrity and induced cell death. Cell death correlated with the activation of cellular oxidative stress. Of the 12 proteins, ORF3a was chosen for further study in mammalian cells because it plays an important role in viral pathogenesis and its activities are linked to lung tissue damage and a cytokine storm. In human pulmonary and kidney epithelial cells, ORF3a induced cellular oxidative stress associated with apoptosis and necrosis and caused activation of proinflammatory response with production of the cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IFN-ß1, possibly through the activation of nuclear factor kappa B (NF-κB). To further characterize the mechanism, we tested a natural ORF3a Beta variant, Q57H, and a mutant with deletion of the highly conserved residue, ΔG188. Compared with wild-type ORF3a, the ΔG188 variant yielded more robust activation of cellular oxidative stress, cell death, and innate immune response. Since cellular oxidative stress and inflammation contribute to cell death and tissue damage linked to the severity of COVID-19, our findings suggest that ORF3a is a promising, novel therapeutic target against COVID-19. IMPORTANCE The ongoing COVID-19 pandemic caused by SARS-CoV-2 has claimed over 5.5 million lives with more than 300 million people infected worldwide. While vaccines are effective, the emergence of new viral variants could jeopardize vaccine protection. Treatment of COVID-19 by antiviral drugs provides an alternative to battle against the disease. The goal of this study was to identify viral therapeutic targets that can be used in antiviral drug discovery. Utilizing a genome-wide functional analysis in a fission yeast cell-based system, we identified 12 viral candidates, including ORF3a, which cause cellular oxidative stress, inflammation, apoptosis, and necrosis that contribute to cytopathogenicity and COVID-19. Our findings indicate that antiviral agents targeting ORF3a could have a great impact on COVID-19.