23 results on '"Bhanu P. Tewari"'
Search Results
2. Neonatal Zika virus infection causes transient perineuronal net degradation
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Kaliroi Engel, Ha-Na Lee, Bhanu P. Tewari, Aaron P. Lewkowicz, Derek D. C. Ireland, Mohanraj Manangeeswaran, and Daniela Verthelyi
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Zika virus (ZIKV) ,brain development ,perineuronal net (PNN) ,Wisteria floribunda agglutinin (WFA) ,aggrecan ,brevican ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Perineuronal nets (PNNs) form a specialized extracellular matrix that predominantly surrounds parvalbumin (PV)-expressing GABAergic inhibitory interneurons and help regulate neuronal activity. Their formation early in the postnatal period is regulated by neuronal signaling and glial activation raising concerns that part of the long-term effects ascribed to perinatal viral infections could be mediated by altered PNN formation. Previously, we developed a model of neonatal Zika virus (ZIKV) infection where mice have lifelong neurological sequelae that includes motor disfunction and reduced anxiety coupled with a persistent low-grade expression in proinflammatory markers despite resolving the acute infection. Here, we demonstrate that ZIKV infection to P1 neonatal mice results in a reduction of PNN formation during the acute disease with significant reduction in Wisteria floribunda agglutinin (WFA) staining at the peak of infection [15 days post infection (dpi)] that persisted after the symptoms resolved (30 dpi). At 60 dpi, when there is residual inflammation in the CNS, the number of WFA+ cells and the level of WFA staining as well as levels of aggrecan and brevican in the brains of convalescent mice were not different from those in uninfected controls, however, there was increased frequency of PNNs with an immature phenotype. Over time the impact of the perinatal infection became less evident and there were no clear differences in PNN morphology between the groups at 1 year post infection. Of note, the reduction in PNNs during acute ZIKV infection was not associated with decreased mRNA levels of aggrecan or brevican, but increased levels of degraded aggrecan and brevican indicating increased PNN degradation. These changes were associated with increased expression of matrix metalloproteinase 12 (MMP12) and MMP19, but not MMP9, a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) or ADAMTS5. Together our findings indicate that infection at the time of PNN development interferes with PNN formation, but the nets can reform once the infection and inflammation subside.
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- 2023
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3. A glial perspective on the extracellular matrix and perineuronal net remodeling in the central nervous system
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Bhanu P. Tewari, Lata Chaunsali, Courtney E. Prim, and Harald Sontheimer
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perineuronal nets (PNNs) ,astrocytes ,extracellular matrix (ECM) ,microglia ,PV neurons ,matrix metalloproteinases ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
A structural scaffold embedding brain cells and vasculature is known as extracellular matrix (ECM). The physical appearance of ECM in the central nervous system (CNS) ranges from a diffused, homogeneous, amorphous, and nearly omnipresent matrix to highly organized distinct morphologies such as basement membranes and perineuronal nets (PNNs). ECM changes its composition and organization during development, adulthood, aging, and in several CNS pathologies. This spatiotemporal dynamic nature of the ECM and PNNs brings a unique versatility to their functions spanning from neurogenesis, cell migration and differentiation, axonal growth, and pathfinding cues, etc., in the developing brain, to stabilizing synapses, neuromodulation, and being an active partner of tetrapartite synapses in the adult brain. The malleability of ECM and PNNs is governed by both intrinsic and extrinsic factors. Glial cells are among the major extrinsic factors that facilitate the remodeling of ECM and PNN, thereby acting as key regulators of diverse functions of ECM and PNN in health and diseases. In this review, we discuss recent advances in our understanding of PNNs and how glial cells are central to ECM and PNN remodeling in normal and pathological states of the CNS.
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- 2022
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4. Perineuronal nets decrease membrane capacitance of peritumoral fast spiking interneurons in a model of epilepsy
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Bhanu P. Tewari, Lata Chaunsali, Susan L. Campbell, Dipan C. Patel, Adam E. Goode, and Harald Sontheimer
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Science - Abstract
Brain tumours are associated with epilepsy. Here the authors show, using a mouse model, that the degradation of perineuronal nets around fast spiking interneurons near the tumour contribute to seizures by increasing their membrane capacitance and firing.
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- 2018
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5. Dysregulation of Ambient Glutamate and Glutamate Receptors in Epilepsy: An Astrocytic Perspective
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Oscar B. Alcoreza, Dipan C. Patel, Bhanu P. Tewari, and Harald Sontheimer
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glutamate homeostasis ,System xc- ,epilepsy ,astroglia ,metabotrophic glutamate receptor ,NMDAR ,Neurology. Diseases of the nervous system ,RC346-429 - Abstract
Given the important functions that glutamate serves in excitatory neurotransmission, understanding the regulation of glutamate in physiological and pathological states is critical to devising novel therapies to treat epilepsy. Exclusive expression of pyruvate carboxylase and glutamine synthetase in astrocytes positions astrocytes as essential regulators of glutamate in the central nervous system (CNS). Additionally, astrocytes can significantly alter the volume of the extracellular space (ECS) in the CNS due to their expression of the bi-directional water channel, aquaporin-4, which are enriched at perivascular endfeet. Rapid ECS shrinkage has been observed following epileptiform activity and can inherently concentrate ions and neurotransmitters including glutamate. This review highlights our emerging knowledge on the various potential contributions of astrocytes to epilepsy, particularly supporting the notion that astrocytes may be involved in seizure initiation via failure of homeostatic responses that lead to increased ambient glutamate. We also review the mechanisms whereby ambient glutamate can influence neuronal excitability, including via generation of the glutamate receptor subunit GluN2B-mediated slow inward currents, as well as indirectly affect neuronal excitability via actions on metabotropic glutamate receptors that can potentiate GluN2B currents and influence neuronal glutamate release probabilities. Additionally, we discuss evidence for upregulation of System xc-, a cystine/glutamate antiporter expressed on astrocytes, in epileptic tissue and changes in expression patterns of glutamate receptors.
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- 2021
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6. Glioma‐induced peritumoral hyperexcitability in a pediatric glioma model
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Lata Chaunsali, Bhanu P. Tewari, Allison Gallucci, Emily G. Thompson, Andrew Savoia, Noah Feld, and Susan L. Campbell
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development ,glioma ,hyperexcitability ,pediatric ,Physiology ,QP1-981 - Abstract
Abstract Epileptic seizures are among the most common presenting symptom in patients with glioma. The etiology of glioma‐related seizures is complex and not completely understood. Studies using adult glioma patient tissue and adult glioma mouse models, show that neurons adjacent to the tumor mass, peritumoral neurons, are hyperexcitable and contribute to seizures. Although it is established that there are phenotypic and genotypic distinctions in gliomas from adult and pediatric patients, it is unknown whether these established differences in pediatric glioma biology and the microenvironment in which these glioma cells harbor, the developing brain, differentially impacts surrounding neurons. In the present study, we examine the effect of patient‐derived pediatric glioma cells on the function of peritumoral neurons using two pediatric glioma models. Pediatric glioma cells were intracranially injected into the cerebrum of postnatal days 2 and 3 (p2/3) mouse pups for 7 days. Electrophysiological recordings showed that cortical layer 2/3 peritumoral neurons exhibited significant differences in their intrinsic properties compared to those of sham control neurons. Peritumoral neurons fired significantly more action potentials in response to smaller current injection and exhibited a depolarization block in response to higher current injection. The threshold for eliciting an action potential and pharmacologically induced epileptiform activity was lower in peritumoral neurons compared to sham. Our findings suggest that pediatric glioma cells increase excitability in the developing peritumoral neurons by exhibiting early onset of depolarization block, which was not previously observed in adult glioma peritumoral neurons.
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- 2020
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7. Infection-induced epilepsy is caused by increased expression of chondroitin sulfate proteoglycans in hippocampus and amygdala
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Dipan C. Patel, Nathaniel Swift, Bhanu P. Tewari, Jack L. Browning, Courtney Prim, Lata Chaunsali, Ian Kimbrough, Michelle L. Olsen, and Harald Sontheimer
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Article - Abstract
Alterations in the extracellular matrix (ECM) are common in epilepsy, yet whether they are cause or consequence of disease is unknow. Using Theiler’s virus infection model of acquired epilepsy we findde novoexpression of chondroitin sulfate proteoglycans (CSPGs), a major ECM component, in dentate gyrus (DG) and amygdala exclusively in mice with seizures. Preventing synthesis of CSPGs specifically in DG and amygdala by deletion of major CSPG aggrecan reduced seizure burden. Patch-clamp recordings from dentate granule cells (DGCs) revealed enhanced intrinsic and synaptic excitability in seizing mice that was normalized by aggrecan deletion.In situexperiments suggest that DGCs hyperexcitability results from negatively charged CSPGs increasing stationary cations (K+, Ca2+) on the membrane thereby depolarizing neurons, increasing their intrinsic and synaptic excitability. We show similar changes in CSPGs in pilocarpine-induced epilepsy suggesting enhanced CSPGs in the DG and amygdala may be a common ictogenic factor and novel therapeutic potential.
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- 2023
8. Development of astrocyte morphology and function in mouse visual thalamus
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Lata Chaunsali, Michael A. Fox, Bhanu P Tewari, Ubadah Sabbagh, Gabriela L. Carrillo, Natalie A Huebschman, and Rachana D. Somaiya
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genetic structures ,General Neuroscience ,Transgene ,Thalamus ,Glutamate receptor ,Geniculate Bodies ,Sensory system ,In situ hybridization ,Biology ,Axons ,Article ,eye diseases ,Mice ,medicine.anatomical_structure ,Astrocytes ,Synapses ,medicine ,Animals ,Humans ,Immunohistochemistry ,Visual Pathways ,Neuroscience ,Immunostaining ,Astrocyte - Abstract
The rodent visual thalamus has served as a powerful model to elucidate the cellular and molecular mechanisms that underlie sensory circuit formation and function. Despite significant advances in our understanding of the role of axon-target interactions and neural activity in orchestrating circuit formation in visual thalamus, the role of non-neuronal cells, such as astrocytes, is less clear. In fact, we know little about the transcriptional identity and development of astrocytes in mouse visual thalamus. To address this gap in knowledge, we studied the expression of canonical astrocyte molecules in visual thalamus using immunostaining, in situ hybridization, and reporter lines. While our data suggests some level of heterogeneity of astrocytes in different nuclei of the visual thalamus, the majority of thalamic astrocytes appeared to be labelled in Aldh1l1-EGFP mice. This led us to use this transgenic line to characterize the neonatal and postnatal development of these cells in visual thalamus. Our data show that not only have the entire cohort of astrocytes migrated into visual thalamus by eye-opening but they also have acquired their adult-like morphology, even while retinogeniculate synapses are still maturing. Furthermore, ultrastructural, immunohistochemical, and functional approaches revealed that by eye-opening, thalamic astrocytes ensheath retinogeniculate synapses and are capable of efficient uptake of glutamate. Taken together, our results reveal that the morphological, anatomical, and functional development of astrocytes in visual thalamus occurs prior to eye-opening and the emergence of experience-dependent visual activity. This article is protected by copyright. All rights reserved.
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- 2021
9. Pericyte Progenitor Coupling to the Emerging Endothelium during Vasculogenesis via Connexin43
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Laura Beth Payne, Bhanu P. Tewari, Logan Dunkenberger, Samantha Bond, Alyssa Savelli, Jordan Darden, Huaning Zhao, Caroline Willi, Ronak Kanodia, Rosalie Gude, Michael D. Powell, Kenneth J. Oestreich, Harald Sontheimer, Sophie Dal-Pra, and John C. Chappell
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Mice ,Connexin 43 ,cardiovascular system ,Animals ,Endothelial Cells ,Cell Differentiation ,Cardiology and Cardiovascular Medicine ,Pericytes ,Article ,Connexins - Abstract
Background: Vascular pericytes stabilize blood vessels and contribute to their maturation, while playing other key roles in microvascular function. Nevertheless, relatively little is known about involvement of their precursors in the earliest stages of vascular development, specifically during vasculogenesis. Methods: We combined high-power, time-lapse imaging with transcriptional profiling of emerging pericytes and endothelial cells in reporter mouse and cell lines. We also analyzed conditional transgenic animals deficient in Cx43/Gja1 (connexin 43/gap junction alpha-1) expression within Ng2+ cells. Results: A subset of Ng2-DsRed+ cells, likely pericyte/mural cell precursors, arose alongside endothelial cell differentiation and organization and physically engaged vasculogenic endothelium in vivo and in vitro. We found no overlap between this population of differentiating pericyte/mural progenitors and other lineages including hemangiogenic and neuronal/glial cell types. We also observed cell-cell coupling and identified Cx43-based gap junctions contributing to pericyte–endothelial cell precursor communication during vascular assembly. Genetic loss of Cx43/Gja1 in Ng2+ pericyte progenitors compromised embryonic blood vessel formation in a subset of animals, while surviving mutants displayed little-to-no vessel abnormalities, suggesting a resilience to Cx43/Gja1 loss in Ng2+ cells or potential compensation by additional connexin isoforms. Conclusions: Together, our data suggest that a distinct pericyte lineage emerges alongside vasculogenesis and directly communicates with the nascent endothelium via Cx43 during early vessel formation. Cx43/Gja1 loss in pericyte/mural cell progenitors can induce embryonic vessel dysmorphogenesis, but alternate connexin isoforms may be able to compensate. These data provide insight that may reshape the current framework of vascular development and may also inform tissue revascularization/vascularization strategies.
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- 2022
10. Author response for 'Development of astrocyte morphology and function in mouse visual thalamus'
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null Rachana D. Somaiya, null Natalie A. Huebschman, null Lata Chaunsali, null Ubadah Sabbagh, null Gabriela L. Carrillo, null Bhanu P. Tewari, and null Michael A. Fox
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- 2021
11. Author response for 'Development of astrocyte morphology and function in mouse visual thalamus'
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Lata Chaunsali, Ubadah Sabbagh, Gabriela L. Carrillo, Michael A. Fox, Bhanu P. Tewari, Rachana D. Somaiya, and Natalie A Huebschman
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medicine.anatomical_structure ,Thalamus ,medicine ,Morphology (biology) ,Biology ,Neuroscience ,Function (biology) ,Astrocyte - Published
- 2021
12. Development and implementation of a scalable and versatile test for COVID-19 diagnostics in rural communities
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Robyn A. Umans, N. Bissell, Harald Sontheimer, F. M. Michel, Dipan C. Patel, Carmen Muñoz-Ballester, Carla V. Finkielstein, Oscar B. Alcoreza, Michael J. Friedlander, T. Maynard, P. Bordwine, Bhanu P. Tewari, Allison N. Tegge, A. Ceci, Daniel Martinez-Martinez, Joelle Martin, and K. L. Brown
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0301 basic medicine ,Emergency Use Authorization ,medicine.medical_specialty ,Public land ,Computer science ,Science ,Supply chain ,General Physics and Astronomy ,Real-Time Polymerase Chain Reaction ,Sensitivity and Specificity ,General Biochemistry, Genetics and Molecular Biology ,Article ,Specimen Handling ,Genomic analysis ,03 medical and health sciences ,0302 clinical medicine ,Limit of Detection ,Nasopharynx ,Pandemic ,medicine ,Humans ,Pandemics ,Health policy ,Multidisciplinary ,SARS-CoV-2 ,Public health ,COVID-19 ,General Chemistry ,Equipment Design ,Gene expression profiling ,Test (assessment) ,Identification (information) ,030104 developmental biology ,Risk analysis (engineering) ,COVID-19 Nucleic Acid Testing ,Communicable Disease Control ,Printing, Three-Dimensional ,RNA, Viral ,Reagent Kits, Diagnostic ,Rural Health Services ,Rural area ,030217 neurology & neurosurgery - Abstract
Rapid and widespread testing of severe acute respiratory coronavirus 2 (SARS-CoV-2) is essential for an effective public health response aimed at containing and mitigating the coronavirus disease 2019 (COVID-19) pandemic. Successful health policy implementation relies on early identification of infected individuals and extensive contact tracing. However, rural communities, where resources for testing are sparse or simply absent, face distinctive challenges to achieving this success. Accordingly, we report the development of an academic, public land grant University laboratory-based detection assay for the identification of SARS-CoV-2 in samples from various clinical specimens that can be readily deployed in areas where access to testing is limited. The test, which is a quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based procedure, was validated on samples provided by the state laboratory and submitted for FDA Emergency Use Authorization. Our test exhibits comparable sensitivity and exceeds specificity and inclusivity values compared to other molecular assays. Additionally, this test can be re-configured to meet supply chain shortages, modified for scale up demands, and is amenable to several clinical specimens. Test development also involved 3D engineering critical supplies and formulating a stable collection media that allowed samples to be transported for hours over a dispersed rural region without the need for a cold-chain. These two elements that were critical when shortages impacted testing and when personnel needed to reach areas that were geographically isolated from the testing center. Overall, using a robust, easy-to-adapt methodology, we show that an academic laboratory can supplement COVID-19 testing needs and help local health departments assess and manage outbreaks. This additional testing capacity is particularly germane for smaller cities and rural regions that would otherwise be unable to meet the testing demand., Here, the authors report the development of a versatile academic, SARSCoV-2 RT-qPCR molecular diagnostic test that uses 3D printed technology for sample collection, is implemented in rural setting in the US state of Virginia and validated in its population.
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- 2021
13. Frontiers in Neurology
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Oscar B. Alcoreza, Dipan C. Patel, Bhanu P. Tewari, Harald Sontheimer, Fralin Biomedical Research Institute, and Virginia Tech Carilion School of Medicine
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astroglia ,Chemistry ,Mini Review ,Central nervous system ,Glutamate receptor ,Neurotransmission ,glutamate homeostasis ,System x(c)(-) ,lcsh:RC346-429 ,NMDAR ,medicine.anatomical_structure ,Neurology ,Glutamate homeostasis ,Metabotropic glutamate receptor ,Glutamine synthetase ,metabotrophic glutamate receptor ,medicine ,Excitatory postsynaptic potential ,NMDA receptor ,epilepsy ,Neurology (clinical) ,System xc ,Neuroscience ,lcsh:Neurology. Diseases of the nervous system - Abstract
Given the important functions that glutamate serves in excitatory neurotransmission, understanding the regulation of glutamate in physiological and pathological states is critical to devising novel therapies to treat epilepsy. Exclusive expression of pyruvate carboxylase and glutamine synthetase in astrocytes positions astrocytes as essential regulators of glutamate in the central nervous system (CNS). Additionally, astrocytes can significantly alter the volume of the extracellular space (ECS) in the CNS due to their expression of the bi-directional water channel, aquaporin-4, which are enriched at perivascular endfeet. Rapid ECS shrinkage has been observed following epileptiform activity and can inherently concentrate ions and neurotransmitters including glutamate. This review highlights our emerging knowledge on the various potential contributions of astrocytes to epilepsy, particularly supporting the notion that astrocytes may be involved in seizure initiation via failure of homeostatic responses that lead to increased ambient glutamate. We also review the mechanisms whereby ambient glutamate can influence neuronal excitability, including via generation of the glutamate receptor subunit GluN2B-mediated slow inward currents, as well as indirectly affect neuronal excitability via actions on metabotropic glutamate receptors that can potentiate GluN2B currents and influence neuronal glutamate release probabilities. Additionally, we discuss evidence for upregulation of System xc-, a cystine/glutamate antiporter expressed on astrocytes, in epileptic tissue and changes in expression patterns of glutamate receptors. NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [1R01CA227149-01A1, 1R01NS036692-01A1]; DODUnited States Department of Defense [W81XWH-18-1-0521] This work was supported by NIH grants 1R01CA227149-01A1 and 1R01NS036692-01A1, and DOD grant W81XWH-18-1-0521.
- Published
- 2021
14. Vasculogenic Pericytes Directly Couple to the Emerging Endothelium During Vessel Formation
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Jordan Darden, Michael Powell, Bhanu P. Tewari, Sophie Dal-Pra, Samantha Bond, Laura Beth Payne, Logan Dunkenberger, John C. Chappell, Huaning Zhao, Harald Sontheimer, Kenneth J Oestreich, and Alyssa Savelli
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Cell type ,Vasculogenesis ,medicine.anatomical_structure ,Endothelium ,Cell culture ,In vivo ,cardiovascular system ,Gap junction ,medicine ,Biology ,Embryonic stem cell ,Function (biology) ,Cell biology - Abstract
Vascular pericytes (PCs) stabilize blood vessels and contribute to their maturation, while also playing key roles in microvascular function. Nevertheless, relatively little is known about their involvement in the earliest stages of vascular development, specifically during vasculogenesis. We combined high-power, time-lapse imaging with transcriptional profiling of PCs and endothelial cells (ECs) in reporter mouse and cell lines. PCs emerged alongside EC differentiation, and physically engaged the vasculogenic endothelium in vivo and in vitro. We observed PC-EC coupling and identified Connexin43 (Cx43)-based gap junctions as key elements in PC-EC communication during vessel assembly. We found no overlap between differentiating PCs and other lineages including hemangiogenic and neuronal/glial cell types. Together, our data suggest that a distinct PC lineage emerges prior to vasculogenesis and participates in early vessel formation through direct communication with nascent endothelium via Cx43 – providing insight that reshapes the current framework of vascular development and informs tissue vascularization strategies.
- Published
- 2021
15. Pericytes Directly Communicate with Emerging Endothelial Cells During Vasculogenesis
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Jordan Darden, Harald Sontheimer, John C. Chappell, Huaning Zhao, Alyssa Savelli, Michael Powell, Samantha Bond, Sophie Dal-Pra, Laura Beth Payne, Kenneth J Oestreich, Bhanu P. Tewari, and Logan Dunkenberger
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Vasculogenesis ,medicine.anatomical_structure ,Live cell imaging ,Mechanism (biology) ,Cell ,medicine ,Gap junction ,Mouse Embryonic Stem Cell ,Biology ,Microinjection ,Embryonic stem cell ,Cell biology - Abstract
SummaryPericytes (PCs), cells that extend along capillaries to contribute stability and other critical functions to established vasculature, are attracting attention from various fields involving vascular-related pathologies. Here, we demonstrate primary evidence of PC communication with endothelial cells (ECs) prior to tube coalescence. Observations of apparent PCs during early embryogenesis urged development of a mouse embryonic stem cell line (DR-ESCs), enabling unique dual-reporter investigations into earliest PC-EC interactions. Live imaging of differentiating DR-ESCs corroborated emergence of a PC lineage, which preceded EC differentiation, and further revealed highly dynamic PC-EC interactions during coordinated vessel formation. We show direct PC-EC communication via cell microinjection and dye-transfer, and RNA-seq analysis indicates a PC-EC coupling mechanism via gap junction Connexin43 (Cx43), exclusively up-regulated throughout DR-ESC differentiation. High resolution imaging of embryonic and postnatal mouse vasculature substantiates Cx43 plaques at PC-EC borders. These findings indicate a new role for PCs during vasculogenesis via Cx43-mediated communication with ECs.Competing Interest StatementThe authors have declared no competing interest.View Full Text
- Published
- 2020
16. Sulfasalazine decreases mouse cortical hyperexcitability
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Bhanu P. Tewari, Susan Campbell, Andrew Savoia, Oscar B. Alcoreza, Harald Sontheimer, and Allison Bouslog
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0301 basic medicine ,Topiramate ,Patch-Clamp Techniques ,seizure ,Pharmacology ,Inhibitory postsynaptic potential ,03 medical and health sciences ,Epilepsy ,Mice ,0302 clinical medicine ,medicine ,Animals ,Patch clamp ,antiepileptic drugs ,business.industry ,Glutamate receptor ,Brain ,Excitatory Postsynaptic Potentials ,system xc‐ ,Bicuculline ,medicine.disease ,3. Good health ,Cortex (botany) ,Mice, Inbred C57BL ,Sulfasalazine ,Disease Models, Animal ,030104 developmental biology ,Neurology ,sulfasalazine ,Excitatory postsynaptic potential ,Full‐length Original Research ,epilepsy ,Anticonvulsants ,Neurology (clinical) ,business ,030217 neurology & neurosurgery ,medicine.drug - Abstract
Objective: Currently prescribed antiepileptic drugs (AEDs) are ineffective in treating approximately 30% of epilepsy patients. Sulfasalazine (SAS) is an US Food and Drug Administration (FDA)–approved drug for the treatment of Crohn disease that has been shown to inhibit the cystine/glutamate antiporter system xc‐ (SXC) and decrease tumor‐associated seizures. This study evaluates the effect of SAS on distinct pharmacologically induced network excitability and determines whether it can further decrease hyperexcitability when administered with currently prescribed AEDs. Methods: Using in vitro cortical mouse brain slices, whole‐cell patch‐clamp recordings were made from layer 2/3 pyramidal neurons. Epileptiform activity was induced with bicuculline (bic), 4‐aminopyridine (4‐AP) and magnesium‐free (Mg2+‐free) solution to determine the effect of SAS on epileptiform events. In addition, voltagesensitive dye (VSD) recordings were performed to characterize the effect of SAS on the spatiotemporal spread of hyperexcitable network activity and compared to currently prescribed AEDs. Results: SAS decreased evoked excitatory postsynaptic currents (eEPSCs) and increased the decay kinetics of evoked inhibitory postsynaptic currents (eIPSCs) in layer 2/3 pyramidal neurons. Although application of SAS to bic and Mg2+‐free–induced epileptiform activity caused a decrease in the duration of epileptiform events, SAS completely blocked 4‐AP–induced epileptiform events. In VSD recordings, SAS decreased VSD optical signals induced by 4‐AP. Co‐application of SAS with the AED topiramate (TPM) caused a significantly further decrease in the spatiotemporal spread of VSD optical signals. Significance: Taken together this study provides evidence that inhibition of SXC by SAS can decrease network hyperexcitability induced by three distinct pharmacologic agents in the superficial layers of the cortex. Furthermore, SAS provided additional suppression of 4‐AP–induced network activity when administered with the currently prescribed AED TPM. These findings may serve as a foundation to assess the potential for SAS or other compounds that selectively target SXC as an adjuvant treatment for epilepsy. Foundation for the National Institutes of Health, Grant/Award Number: 5R01‐NS036692, R01‐NS052634 and R01CA227149
- Published
- 2019
17. Protocol to quantitatively assess the structural integrity of Perineuronal Nets ex vivo
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Harald Sontheimer and Bhanu P. Tewari
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biology ,Chemistry ,Strategy and Management ,Mechanical Engineering ,Perineuronal net ,Central nervous system ,Metals and Alloys ,Industrial and Manufacturing Engineering ,chemistry.chemical_compound ,medicine.anatomical_structure ,Cerebral cortex ,Chondroitin sulfate proteoglycan ,Neuroplasticity ,medicine ,biology.protein ,Methods Article ,NeuN ,Neuroscience ,Parvalbumin ,Ex vivo - Abstract
Perineuronal nets (PNNs) are extracellular matrix assemblies of highly negatively charged proteoglycans that wrap around fast-spiking parvalbumin (PV) expressing interneurons in the cerebral cortex. PNNs play important roles in neuronal plasticity and modulate biophysical properties of the enclosed interneurons. Various central nervous system diseases including schizophrenia, Alzheimer disease and epilepsy present with qualitative alteration in PNNs, however prior studies failed to quantitatively assess such changes at single PNN level and correlate them with functional changes in disease. We describe a method to quantify the structural integrity of PNNs using high magnification image analysis of Wisteria Floribunda Agglutinin (WFA)-labeled PNNs in combination with cell-type-specific marker such as PV and NeuN. A polyline intensity profile of WFA along the entire perimeter of cell shows alternate segments with and without WFA labeling, indicating the intact chondroitin sulfate proteoglycan (CSPG) and holes of PNN respectively. This line intensity profile defines CSPG peaks, where intact PNN is present, and CSPG valleys (holes) where the PNN is missing. The average number of peaks reflect the integrity of the lattice assembly of PNN. The average size of PNN holes can be readily computed using image analysis software. Furthermore, degradation of PNNs using a bacterial-derived enzyme, Chondroitinase ABC (ChABC), allows to experimentally manipulate PNNs in situ brain slices during which biophysical properties can be assessed by patch-clamp recordings. We describe optimized experimental parameters to degrade PNNs in brain slices before as well as during recordings to study the possible change in function in real time. Our protocols provide effective and appropriate methods to modulate and quantify the PNN's experimental manipulations.
- Published
- 2019
18. Neuron-glia interactions in the pathophysiology of epilepsy
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Dipan C. Patel, Bhanu P. Tewari, Lata Chaunsali, and Harald Sontheimer
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0301 basic medicine ,Neurological disorder ,Neurotransmission ,Inhibitory postsynaptic potential ,Synaptic Transmission ,Article ,03 medical and health sciences ,Epilepsy ,0302 clinical medicine ,medicine ,Premovement neuronal activity ,Animals ,Humans ,Neurons ,Microglia ,business.industry ,General Neuroscience ,medicine.disease ,030104 developmental biology ,medicine.anatomical_structure ,nervous system ,Excitatory postsynaptic potential ,Neuron ,business ,Neuroscience ,Neuroglia ,030217 neurology & neurosurgery - Abstract
Epilepsy is a neurological disorder afflicting ~65 million people worldwide. It is caused by aberrant synchronized firing of populations of neurons primarily due to imbalance between excitatory and inhibitory neurotransmission. Hence, the historical focus of epilepsy research has been neurocentric. However, the past two decades have enjoyed an explosion of research into the role of glia in supporting and modulating neuronal activity, providing compelling evidence of glial involvement in the pathophysiology of epilepsy. The mechanisms by which glia, particularly astrocytes and microglia, may contribute to epilepsy and consequently could be harnessed therapeutically are discussed in this Review.
- Published
- 2019
19. Perineuronal nets decrease membrane capacitance of peritumoral fast spiking interneurons in a model of epilepsy
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Adam E. Goode, Susan Campbell, Harald Sontheimer, Lata Chaunsali, Bhanu P. Tewari, and Dipan C. Patel
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0301 basic medicine ,Male ,Science ,General Physics and Astronomy ,Action Potentials ,Mice, Nude ,Mice, SCID ,Electric Capacitance ,Article ,General Biochemistry, Genetics and Molecular Biology ,Biophysical Phenomena ,03 medical and health sciences ,Epilepsy ,0302 clinical medicine ,Interneurons ,medicine ,Animals ,Gliosis ,lcsh:Science ,Membrane potential ,Multidisciplinary ,Extramural ,Chemistry ,Brain Neoplasms ,musculoskeletal, neural, and ocular physiology ,Perineuronal net ,Myelin sheaths ,Cell Membrane ,Proteolytic enzymes ,General Chemistry ,Glioma ,medicine.disease ,Current Literature in Basic Science ,Extracellular Matrix ,Disease Models, Animal ,030104 developmental biology ,nervous system ,lcsh:Q ,Gabaergic inhibition ,Female ,Neuroscience ,030217 neurology & neurosurgery ,Peptide Hydrolases - Abstract
Brain tumor patients commonly present with epileptic seizures. We show that tumor-associated seizures are the consequence of impaired GABAergic inhibition due to an overall loss of peritumoral fast spiking interneurons (FSNs) concomitant with a significantly reduced firing rate of those that remain. The reduced firing is due to the degradation of perineuronal nets (PNNs) that surround FSNs. We show that PNNs decrease specific membrane capacitance of FSNs permitting them to fire action potentials at supra-physiological frequencies. Tumor-released proteolytic enzymes degrade PNNs, resulting in increased membrane capacitance, reduced firing, and hence decreased GABA release. These studies uncovered a hitherto unknown role of PNNs as an electrostatic insulator that reduces specific membrane capacitance, functionally akin to myelin sheaths around axons, thereby permitting FSNs to exceed physiological firing rates. Disruption of PNNs may similarly account for excitation-inhibition imbalances in other forms of epilepsy and PNN protection through proteolytic inhibition may provide therapeutic benefits., Brain tumours are associated with epilepsy. Here the authors show, using a mouse model, that the degradation of perineuronal nets around fast spiking interneurons near the tumour contribute to seizures by increasing their membrane capacitance and firing.
- Published
- 2017
20. AMPA receptor activation causes preferential mitochondrial Ca2+ load and oxidative stress in motor neurons
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Dinesh C. Joshi, Preeti G. Joshi, Mahendra Singh, Bhanu P. Tewari, and Nanda B. Joshi
- Subjects
Voltage-dependent calcium channel ,General Neuroscience ,MPTP ,Glutamate receptor ,Excitotoxicity ,AMPA receptor ,Biology ,Mitochondrion ,medicine.disease_cause ,Cell biology ,chemistry.chemical_compound ,nervous system ,chemistry ,Mitochondrial permeability transition pore ,medicine ,Neurology (clinical) ,Molecular Biology ,Neuroscience ,Developmental Biology ,Calcium signaling - Abstract
It is well established that motor neurons are highly vulnerable to glutamate induced excitotoxicity. The selective vulnerability of these neurons has been attributed to AMPA receptor mediated excessive rise in cytosolic calcium and consequent mitochondrial Ca(2+) loading. Earlier we have reported that in motor neurons a generic rise in [Ca(2+)]i does not always lead to mitochondrial Ca(2+) loading and membrane depolarization but it occurs upon AMPA receptor activation. The mechanism of such specific mitochondrial involvement upon AMPA receptor activation is not known. The present study examines the mitochondrial Ca(2+) regulation and oxidative stress in spinal cord neurons upon AMPA subtype of glutamate receptor activation. Stimulating the spinal neurons with AMPA exhibited a sharp rise in [Ca(2+)]m in both motor and other spinal neurons that was sustained up to the end of recording time of 30min. The rise in [Ca(2+)]m was substantially higher in motor neurons than in other spinal neurons which could be due to the differential mitochondrial homeostasis in two types of neurons. To examine this possibility, we measured AMPA induced [Ca(2+)]m loading in the presence of mitochondrial inhibitors. In both cell types the AMPA induced [Ca(2+)]m loading was blocked by mitochondrial calcium uniporter blocker ruthenium red. In motor neurons it was also inhibited substantially by CGP37157 and cyclosporine-A, the blockers of Na(+)/Ca(2+) exchanger and mitochondrial permeability transition pore (MPTP) respectively, whereas no effect of these agents was observed in other spinal neurons. Thus in motor neurons the Ca(2+) sequestration by mitochondria occurs through mitochondrial calcium uniporter as well as due to reversal of Na(+)/Ca(2+) exchanger, in contrast the latter pathway does not contribute in other spinal neurons. The ROS formation was inhibited by nitric oxide synthase (NOS) inhibitor L-NAME in both types of neurons, however the mitochondrial complex-I inhibitor rotenone suppressed the ROS formation only in motor neurons. It appears that activation of cytoplasmic nNOS leads to ROS formation in both types of spinal neurons but mitochondria is the major source of ROS in motor neurons. Spinal neurons exhibited a significant time dependent fall in glutathione (GSH) level. The GSH level in motor neurons did not recover even at 24h after AMPA exposure, whereas the other spinal neurons exhibited a tendency to maintain the GSH after a certain level suggesting that the oxidative stress is arrested in other spinal neurons but it continues to increase in motor neurons. Thus our results demonstrate that upon AMPA receptor stimulation the motor neurons employ some additional pathways for regulation of mitochondrial calcium and oxidative stress as compared to other spinal neurons. It is suggested that such differential signaling mechanisms in motor neurons could be crucial for their selective vulnerability to excitotoxicity.
- Published
- 2015
21. Depalmitoylation preferentially downregulates AMPA induced Ca2+ signaling and neurotoxicity in motor neurons
- Author
-
Karthik Krishnamurthy, Bhupesh Mehta, Nanda B. Joshi, Bhanu P. Tewari, Mahendra Singh, and Preeti G. Joshi
- Subjects
Cell Survival ,Lipoylation ,Palmitates ,Excitotoxicity ,AMPA receptor ,medicine.disease_cause ,Rats, Sprague-Dawley ,Palmitoylation ,medicine ,Animals ,Hypoglycemic Agents ,Calcium Signaling ,Receptors, AMPA ,Amyotrophic lateral sclerosis ,alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid ,Molecular Biology ,Cells, Cultured ,Calcium signaling ,Motor Neurons ,Chemistry ,musculoskeletal, neural, and ocular physiology ,General Neuroscience ,Sodium ,Intracellular Signaling Peptides and Proteins ,Neurotoxicity ,Membrane Proteins ,Motor neuron ,Embryo, Mammalian ,Spinal cord ,medicine.disease ,Rats ,Cell biology ,medicine.anatomical_structure ,Spinal Cord ,nervous system ,Calcium ,Neurology (clinical) ,Disks Large Homolog 4 Protein ,Microtubule-Associated Proteins ,Developmental Biology - Abstract
Excessive activation of AMPA receptor has been implicated in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). However, it is not clear why motor neurons are preferentially sensitive to AMPA receptor mediated excessive [Ca2+]i rise and excitotoxicity. In the present study we examined whether palmitoylation regulates Ca2+ permeability of AMPA receptor and excitotoxicity in cultured spinal cord neurons. We adapted chronic 2-bromopalmitate (2-BrP) treatment to achieve depalmitoylation and examined its effect on the cytotoxicity in spinal cord neurons exposed to AMPA. The change in AMPA induced signaling and cytotoxicity in motor neurons and other spinal neurons under identical conditions of exposure to AMPA was studied. 2-BrP treatment inhibited AMPA induced rise in [Ca2+]i and cytotoxicity in both types of neurons but the degree of inhibition was significantly higher in motor neurons as compared to other spinal neurons. The AMPA induced [Na+]i rise was moderately affected in both type of neurons on depalmitoylation. Depalmitoylation reduced the expression levels of AMPA receptor subunits (GluR1 and GluR2) and also PSD-95 but stargazin levels remained unaffected. Our results demonstrate that 2-BrP attenuates AMPA receptor activated Ca2+ signaling and cytotoxicity preferentially in motor neurons and suggest that AMPA receptor modulation by depalmitoylation could play a significant role in preventing motor neuron degeneration.
- Published
- 2013
22. AMPA receptor activation causes preferential mitochondrial Ca²⁺ load and oxidative stress in motor neurons
- Author
-
Dinesh C, Joshi, Bhanu P, Tewari, Mahendra, Singh, Preeti G, Joshi, and Nanda B, Joshi
- Subjects
Motor Neurons ,Analysis of Variance ,Embryo, Mammalian ,Glutathione ,Mitochondria ,Potassium Chloride ,Rats ,Rats, Sprague-Dawley ,Oxidative Stress ,Spinal Cord ,Excitatory Amino Acid Agonists ,Animals ,Calcium ,Receptors, AMPA ,Reactive Oxygen Species ,alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid ,Cells, Cultured - Abstract
It is well established that motor neurons are highly vulnerable to glutamate induced excitotoxicity. The selective vulnerability of these neurons has been attributed to AMPA receptor mediated excessive rise in cytosolic calcium and consequent mitochondrial Ca(2+) loading. Earlier we have reported that in motor neurons a generic rise in [Ca(2+)]i does not always lead to mitochondrial Ca(2+) loading and membrane depolarization but it occurs upon AMPA receptor activation. The mechanism of such specific mitochondrial involvement upon AMPA receptor activation is not known. The present study examines the mitochondrial Ca(2+) regulation and oxidative stress in spinal cord neurons upon AMPA subtype of glutamate receptor activation. Stimulating the spinal neurons with AMPA exhibited a sharp rise in [Ca(2+)]m in both motor and other spinal neurons that was sustained up to the end of recording time of 30min. The rise in [Ca(2+)]m was substantially higher in motor neurons than in other spinal neurons which could be due to the differential mitochondrial homeostasis in two types of neurons. To examine this possibility, we measured AMPA induced [Ca(2+)]m loading in the presence of mitochondrial inhibitors. In both cell types the AMPA induced [Ca(2+)]m loading was blocked by mitochondrial calcium uniporter blocker ruthenium red. In motor neurons it was also inhibited substantially by CGP37157 and cyclosporine-A, the blockers of Na(+)/Ca(2+) exchanger and mitochondrial permeability transition pore (MPTP) respectively, whereas no effect of these agents was observed in other spinal neurons. Thus in motor neurons the Ca(2+) sequestration by mitochondria occurs through mitochondrial calcium uniporter as well as due to reversal of Na(+)/Ca(2+) exchanger, in contrast the latter pathway does not contribute in other spinal neurons. The ROS formation was inhibited by nitric oxide synthase (NOS) inhibitor L-NAME in both types of neurons, however the mitochondrial complex-I inhibitor rotenone suppressed the ROS formation only in motor neurons. It appears that activation of cytoplasmic nNOS leads to ROS formation in both types of spinal neurons but mitochondria is the major source of ROS in motor neurons. Spinal neurons exhibited a significant time dependent fall in glutathione (GSH) level. The GSH level in motor neurons did not recover even at 24h after AMPA exposure, whereas the other spinal neurons exhibited a tendency to maintain the GSH after a certain level suggesting that the oxidative stress is arrested in other spinal neurons but it continues to increase in motor neurons. Thus our results demonstrate that upon AMPA receptor stimulation the motor neurons employ some additional pathways for regulation of mitochondrial calcium and oxidative stress as compared to other spinal neurons. It is suggested that such differential signaling mechanisms in motor neurons could be crucial for their selective vulnerability to excitotoxicity.
- Published
- 2014
23. Aging-Related Changes in Expression and Function of Glutamate Transporters in Rat Spinal Cord Astrocytes
- Author
-
Shiksha Sharan, Bhanu Prakash Tewari, and Preeti G. Joshi
- Subjects
astrocytes ,excitotoxicity ,aging ,glutamate ,transporter ,spinal cord ,Neurology. Diseases of the nervous system ,RC346-429 - Abstract
Astrocytes make up the predominant cell population among glial cells in the mammalian brain, and they play a vital role in ensuring its optimal functioning. They promote neuronal health and survival and protect neurons from glutamate-induced excitotoxicity. In the spinal cord’s dorsal horn (DH) and ventral horn (VH) regions, astrocytes serve crucial roles. Notably, VH motor neurons exhibit a heightened sensitivity to glutamate-induced damage. It is posited that this selective sensitivity could be related to their localized presence within the VH, where astrocytes possess a distinct set of mechanisms for managing glutamate. As organisms age, the risk of damage from glutamate increases, indicating a potential decline in the efficiency of astrocytic glutamate regulation. Our research involved an analysis of astrocytic structure, glutamate transporter levels, and glutamate uptake capabilities within the DH and VH through immunohistochemical methods, protein analysis via Western blot, and patch-clamp studies in electrophysiology. The investigations revealed a decrease in both the number and coverage of astroglia in the spinal cord, more so within the VH as aging progressed. Notably, levels of the excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2) also decreased with age, particularly within the VH. Patch-clamp analyses of astrocytes from both spinal regions confirmed a significant reduction in glutamate uptake activity as age advanced, indicating an age-related impairment in glutamate processing. The findings indicate aging leads to distinct changes in DH and VH astrocytes, impairing their glutamate management abilities, which could contribute significantly to the development of late-onset neurodegenerative conditions.
- Published
- 2023
- Full Text
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