Immortalized neurons for the study of hypothalamic function

2011 ◽  
Vol 300 (5) ◽  
pp. R1030-R1052 ◽  
Author(s):  
Prasad S. Dalvi ◽  
Anaies Nazarians-Armavil ◽  
Stephanie Tung ◽  
Denise D. Belsham
Keyword(s):  
Neuronal Cell ◽  
Glucose Sensing ◽  
T Antigen ◽  
Molecular Techniques ◽  
Cell Models ◽  
Hypothalamic Neurons ◽  
In Vivo Models ◽  
Wide Range ◽  
Hypothalamic Function

The hypothalamus is a vital part of the central nervous system: it harbors control systems implicated in regulation of a wide range of homeostatic processes, including energy balance and reproduction. Structurally, the hypothalamus is a complex neuroendocrine tissue composed of a multitude of unique neuronal cell types that express a number of neuromodulators, including hormones, classical neurotransmitters, and specific neuropeptides that play a critical role in mediating hypothalamic function. However, neuropeptide and receptor gene expression, second messenger activation, and electrophysiological and secretory properties of these hypothalamic neurons are not yet fully defined, primarily because the heterogeneity and complex neuronal architecture of the neuroendocrine hypothalamus make such studies challenging to perform in vivo. To circumvent this problem, our research group recently generated embryonic- and adult-derived hypothalamic neuronal cell models by utilizing the novel molecular techniques of ciliary neurotrophic factor-induced neurogenesis and SV40 T antigen transfer to primary hypothalamic neuronal cell cultures. Significant research with these cell lines has demonstrated their value as a potential tool for use in molecular genetic analysis of hypothalamic neuronal function. Insights gained from hypothalamic immortalized cells used in conjunction with in vivo models will enhance our understanding of hypothalamic functions such as neurogenesis, neuronal plasticity, glucose sensing, energy homeostasis, circadian rhythms, and reproduction. This review discusses the generation and use of hypothalamic cell models to study mechanisms underlying the function of individual hypothalamic neurons and to gain a more complete understanding of the overall physiology of the hypothalamus.

scholarly journals From 2D to 3D Cancer Cell Models—The Enigmas of Drug Delivery Research

Nanomaterials ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 2236
Author(s):  
Indra Van Zundert ◽  
Beatrice Fortuni ◽  
Susana Rocha
Keyword(s):  
Drug Delivery ◽  
Cell Culture ◽  
Cancer Cell ◽  
Engineered Nanoparticles ◽  
Cell Models ◽  
In Vivo Models ◽  
Culture Systems ◽  
Cell Culture Systems ◽  
Wide Range

Over the past decades, research has made impressive breakthroughs towards drug delivery systems, resulting in a wide range of multifunctional engineered nanoparticles with biomedical applications such as cancer therapy. Despite these significant advances, well-designed nanoparticles rarely reach the clinical stage. Promising results obtained in standard 2D cell culture systems often turn into disappointing outcomes in in vivo models. Although the overall majority of in vitro nanoparticle research is still performed on 2D monolayer cultures, more and more researchers started acknowledging the importance of using 3D cell culture systems, as better models for mimicking the in vivo tumor physiology. In this review, we provide a comprehensive overview of the 3D cancer cell models currently available. We highlight their potential as a platform for drug delivery studies and pinpoint the challenges associated with their use. We discuss in which way each 3D model mimics the in vivo tumor physiology, how they can or have been used in nanomedicine research and to what extent the results obtained so far affect the progress of nanomedicine development. It is of note that the global scientific output associated with 3D models is limited, showing that the use of these systems in nanomedicine investigation is still highly challenging.

Neurotrophic Properties of Silexan, an Essential Oil from the Flowers of Lavender-Preclinical Evidence for Antidepressant-Like Properties

2020 ◽  
Vol 54 (01) ◽  
pp. 37-46
Author(s):  
Kristina Friedland ◽  
Giacomo Silani ◽  
Anita Schuwald ◽  
Carola Stockburger ◽  
Egon Koch ◽  
...  
Keyword(s):  
Essential Oil ◽  
Hippocampal Neurons ◽  
Day Treatment ◽  
Neuronal Cell ◽  
Antidepressant Activity ◽  
In Vivo Models ◽  
Antidepressant Effects ◽  
Primary Hippocampal Neurons

Abstract Background Silexan, a special essential oil from flowering tops of lavandula angustifolia, is used to treat subsyndromal anxiety disorders. In a recent clinical trial, Silexan also showed antidepressant effects in patients suffering from mixed anxiety-depression (ICD-10 F41.2). Since preclinical data explaining antidepressant properties of Silexan are missing, we decided to investigate if Silexan also shows antidepressant-like effects in vitro as well as in vivo models. Methods We used the forced swimming test (FST) in rats as a simple behavioral test indicative of antidepressant activity in vivo. As environmental events and other risk factors contribute to depression through converging molecular and cellular mechanisms that disrupt neuronal function and morphology—resulting in dysfunction of the circuitry that is essential for mood regulation and cognitive function—we investigated the neurotrophic properties of Silexan in neuronal cell lines and primary hippocampal neurons. Results The antidepressant activity of Silexan (30 mg/kg BW) in the FST was comparable to the tricyclic antidepressant imipramine (20 mg/kg BW) after 9-day treatment. Silexan triggered neurite outgrowth and synaptogenesis in 2 different neuronal cell models and led to a significant increase in synaptogenesis in primary hippocampal neurons. Silexan led to a significant phosphorylation of protein kinase A and subsequent CREB phosphorylation. Conclusion Taken together, Silexan demonstrates antidepressant-like effects in cellular as well as animal models for antidepressant activity. Therefore, our data provides preclinical evidence for the clinical antidepressant effects of Silexan in patients with mixed depression and anxiety.

scholarly journals 'Off-the-shelf’ allogeneic antigen-specific adoptive T-cell therapy for the treatment of multiple EBV-associated malignancies

2021 ◽  
Vol 9 (2) ◽  
pp. e001608
Author(s):  
Debottam Sinha ◽  
Sriganesh Srihari ◽  
Kirrliee Beckett ◽  
Laetitia Le Texier ◽  
Matthew Solomon ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Therapy ◽  
Adoptive T Cell Therapy ◽  
Nuclear Antigen ◽  
T Cell Therapy ◽  
In Vivo Models ◽  
Hla Alleles ◽  
Cell Therapies ◽  
Wide Range

BackgroundEpstein-Barr virus (EBV), an oncogenic human gammaherpesvirus, is associated with a wide range of human malignancies of epithelial and B-cell origin. Recent studies have demonstrated promising safety and clinical efficacy of allogeneic ‘off-the-shelf’ virus-specific T-cell therapies for post-transplant viral complications.MethodsTaking a clue from these studies, we developed a highly efficient EBV-specific T-cell expansion process using a replication-deficient AdE1-LMPpoly vector that specifically targets EBV-encoded nuclear antigen 1 (EBNA1) and latent membrane proteins 1 and 2 (LMP1 and LMP2), expressed in latency II malignancies.ResultsThese allogeneic EBV-specific T cells efficiently recognized human leukocyte antigen (HLA)-matched EBNA1-expressing and/or LMP1 and LMP2-expressing malignant cells and demonstrated therapeutic potential in a number of in vivo models, including EBV lymphomas that emerged spontaneously in humanized mice following EBV infection. Interestingly, we were able to override resistance to T-cell therapy in vivo using a ‘restriction-switching’ approach, through sequential infusion of two different allogeneic T-cell therapies restricted through different HLA alleles. Furthermore, we have shown that inhibition of the programmed cell death protein-1/programmed death-ligand 1 axis in combination with EBV-specific T-cell therapy significantly improved overall survival of tumor-bearing mice when compared with monotherapy.ConclusionThese findings suggest that restriction switching by sequential infusion of allogeneic T-cell therapies that target EBV through distinct HLA alleles may improve clinical response.

Hypothermia and Isoflurane Similarly Inhibit Glutamate Release Evoked by Chemical Anoxia in Rat Cortical Brain Slices

1996 ◽  
Vol 85 (3) ◽  
pp. 600-607. ◽  
Author(s):  
Helge Eilers ◽  
Philip E. Bickler
Keyword(s):  
Minimum Alveolar Concentration ◽  
Glutamate Release ◽  
Brain Slices ◽  
Neuronal Cell ◽  
Release Rates ◽  
Neuroprotective Effects ◽  
In Vivo Models ◽  
Chemical Anoxia

Background Accumulation of the excitatory neurotransmitter glutamate in ischemic brain tissue contributes to neuronal cell death. Volatile anesthetics at clinically relevant concentrations are neuroprotective in in vivo models of brain ischemia and reduce glutamate release in vivo and in vitro, but they appear to have weaker neuroprotective effects than hypothermia. The purpose of this study was to determine whether isoflurane reduces glutamate release in hypoxic brain slices, how large this effect is compared to that of hypothermia, and if it is diminished by hyperthermia. Methods Glutamate released from rat cortical brain slices during chemical anoxia (100 microM NaCN) was measured continuously with a fluorescence assay. The release rate was compared at three temperatures (28 degrees C, 37 degrees C, and 39 degrees C) with and without isoflurane at concentrations equipotent to 1 minimum alveolar concentration. At the same three temperatures, glutamate release rates before and after exposure to isoflurane were compared. Results Isoflurane reduced glutamate release from brain slices during chemical anoxia at 37 degrees C (19.6%, P < 0.01) and at 39 degrees C (25.4%, P < 0.01), but not at 28 degrees C. The reduction in glutamate release with hypothermia was similar to that with isoflurane. Hyperthermia (39 degrees C) caused greater glutamate release under basal and anoxic conditions than normo- and hypothermia. Isoflurane caused a slight increase in basal glutamate release rates, although this effect was smaller than the increase caused by hyperthermia. Conclusions In a brain slice model of cerebral anoxia, 1 minimum alveolar concentration isoflurane decreases glutamate release to a similar extent that hypothermia (28 degrees C) does. The increased glutamate release with hyperthermia (39 degrees C) is not prevented by isoflurane.

scholarly journals Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways

2020 ◽  
Vol 21 (15) ◽  
pp. 5239 ◽  
Author(s):  
Boris Sabirzhanov ◽  
Oleg Makarevich ◽  
James P. Barrett ◽  
Isabel L. Jackson ◽  
Ethan P. Glaser ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Intrinsic Apoptosis ◽  
In Vivo Models ◽  
Neuroprotective Strategy ◽  
Radiation Induced

Radiotherapy for brain tumors induces neuronal DNA damage and may lead to neurodegeneration and cognitive deficits. We investigated the mechanisms of radiation-induced neuronal cell death and the role of miR-711 in the regulation of these pathways. We used in vitro and in vivo models of radiation-induced neuronal cell death. We showed that X-ray exposure in primary cortical neurons induced activation of p53-mediated mechanisms including intrinsic apoptotic pathways with sequential upregulation of BH3-only molecules, mitochondrial release of cytochrome c and AIF-1, as well as senescence pathways including upregulation of p21WAF1/Cip1. These pathways of irradiation-induced neuronal apoptosis may involve miR-711-dependent downregulation of pro-survival genes Akt and Ang-1. Accordingly, we demonstrated that inhibition of miR-711 attenuated degradation of Akt and Ang-1 mRNAs and reduced intrinsic apoptosis after neuronal irradiation; likewise, administration of Ang-1 was neuroprotective. Importantly, irradiation also downregulated two novel miR-711 targets, DNA-repair genes Rad50 and Rad54l2, which may impair DNA damage responses, amplifying the stimulation of apoptotic and senescence pathways and contributing to neurodegeneration. Inhibition of miR-711 rescued Rad50 and Rad54l2 expression after neuronal irradiation, enhancing DNA repair and reducing p53-dependent apoptotic and senescence pathways. Significantly, we showed that brain irradiation in vivo persistently elevated miR-711, downregulated its targets, including pro-survival and DNA-repair molecules, and is associated with markers of neurodegeneration, not only across the cortex and hippocampus but also specifically in neurons isolated from the irradiated brain. Our data suggest that irradiation-induced miR-711 negatively modulates multiple pro-survival and DNA-repair mechanisms that converge to activate neuronal intrinsic apoptosis and senescence. Using miR-711 inhibitors to block the development of these regulated neurodegenerative pathways, thus increasing neuronal survival, may be an effective neuroprotective strategy.

scholarly journals ERK1/2 mediates glucose-regulated POMC gene expression in hypothalamic neurons

2015 ◽  
Vol 54 (2) ◽  
pp. 125-135 ◽  
Author(s):  
Juan Zhang ◽  
Yunting Zhou ◽  
Cheng Chen ◽  
Feiyuan Yu ◽  
Yun Wang ◽  
...  
Keyword(s):  
Specific Inhibitor ◽  
Neuronal Cell ◽  
Glucose Sensing ◽  
Hypothalamic Neurons ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Low Glucose ◽  
Amp Activated Protein Kinase

Hypothalamic glucose-sensing neurons regulate the expression of genes encoding feeding-related neuropetides POMC, AgRP, and NPY – the key components governing metabolic homeostasis. AMP-activated protein kinase (AMPK) is postulated to be the molecular mediator relaying glucose signals to regulate the expression of these neuropeptides. Whether other signaling mediator(s) plays a role is not clear. In this study, we investigated the role of ERK1/2 using primary hypothalamic neurons as the model system. The primary neurons were differentiated from hypothalamic progenitor cells. The differentiated neurons possessed the characteristic neuronal cell morphology and expressed neuronal post-mitotic markers as well as leptin-regulated orexigenic POMC and anorexigenic AgRP/NPY genes. Treatment of cells with glucose dose-dependently increased POMC and decreased AgRP/NPY expression with a concurrent suppression of AMPK phosphorylation. In addition, glucose treatment dose-dependently increased the ERK1/2 phosphorylation. Blockade of ERK1/2 activity with its specific inhibitor PD98059 partially (approximately 50%) abolished glucose-induced POMC expression, but had little effect on AgRP/NPY expression. Conversely, blockade of AMPK activity with its specific inhibitor produced a partial (approximately 50%) reversion of low-glucose-suppressed POMC expression, but almost completely blunted the low-glucose-induced AgRP/NPY expression. The results indicate that ERK1/2 mediated POMC but not AgRP/NPY expression. Confirming the in vitro findings, i.c.v. administration of PD98059 in rats similarly attenuated glucose-induced POMC expression in the hypothalamus, but again had little effect on AgRP/NPY expression. The results are indicative of a novel role of ERK1/2 in glucose-regulated POMC expression and offer new mechanistic insights into hypothalamic glucose sensing.

Microfluidic neural axon diode

TECHNOLOGY ◽  
2016 ◽  
Vol 04 (04) ◽  
pp. 240-248 ◽  
Author(s):  
Sangcheol Na ◽  
Myeongwoo Kang ◽  
Seokyoung Bang ◽  
Daehun Park ◽  
Jinhyun Kim ◽  
...  
Keyword(s):  
Neural Circuit ◽  
Axon Growth ◽  
In Vivo Models ◽  
Starting Point ◽  
Wide Range ◽  
Reproducible Method ◽  
Physical And Chemical ◽  
Biology Research

Neural circuits, groups of neurons connected in directional manner, play a central role in information processing. Advances in neuronal biology research is limited by a lack of appropriate in vitro methods to construct and probe neuronal networks. Here, we describe a microfluidic culture platform that directs the growth of axons using “neural diode” structures to control neural connectivity. This platform is compatible with live cell imaging and can be used to (i) form pre-synaptic and postsynaptic neurons by directional axon growth and (ii) localize physical and chemical treatment to pre- or postsynaptic neuron groups (i.e. virus infection and etc.). The “neural diode” design consist of a microchannel that split into two branches: one is directed straight toward while the other returns back toward the starting point in a closed loop to send the axons back to the origin. We optimized the “neural diode” pattern dimension and design to achieve close to 70% directionality with a single unit of the “diode”. When repeated 3 times, near perfect (98–100% at wide range of cell concentrations) directionality can be achieved. The living neural circuit was characterized using Ca imaging and confirmed their function. The platform also serves as a straightforward, reproducible method to recapitulate a variety of neural circuit in vitro that were previously observable only in brain slice or in vivo models. The microfluidic neural diode may lead to better models for understanding the neural circuit and neurodegenerative diseases.

scholarly journals Toughening mechanisms for the attachment of architectured materials: The mechanics of the tendon enthesis

2021 ◽  
Author(s):  
Mikhail Golman ◽  
Adam C Abraham ◽  
Iden Kurtaliaj ◽  
Brittany P Marshall ◽  
Yizhong Jenny Hu ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Mouse Model ◽  
Energy Absorption ◽  
Protein Denaturation ◽  
Simultaneous Observation ◽  
In Vivo Models ◽  
Trade Offs ◽  
Wide Range ◽  
Architectured Materials

Architectured materials offer tailored mechanical properties but are limited in engineering applications due to challenges in maintaining toughness across their attachments. The enthesis connects tendon and bone, two vastly different architectured materials, and exhibits toughness across a wide range of loadings. Understanding the mechanisms by which this is achieved could inform the development of engineered attachments. Integrating experiments, simulations, and novel imaging that enabled simultaneous observation of mineralized and unmineralized tissues, we identified putative mechanisms of enthesis toughening in a mouse model and then manipulated these mechanisms via in vivo control of mineralization and architecture. Imaging uncovered a fibrous architecture within the enthesis that controls trade-offs between strength and toughness. In vivo models of pathology revealed architectural adaptations that optimize these trade-offs through cross-scale mechanisms including nanoscale protein denaturation, milliscale load-sharing, and macroscale energy absorption. Results suggest strategies for optimizing architecture for tough bimaterial attachments in medicine and engineering.

scholarly journals Regulation of Endosome Sorting by a Specific PP2A Isoform

1998 ◽  
Vol 142 (6) ◽  
pp. 1399-1411 ◽  
Author(s):  
Sean S. Molloy ◽  
Laurel Thomas ◽  
Craig Kamibayashi ◽  
Marc C. Mumby ◽  
Gary Thomas
Keyword(s):  
Protein Localization ◽  
T Antigen ◽  
Regulatory Subunits ◽  
State Dependent ◽  
Early Endosomes ◽  
Wide Range ◽  
Small T Antigen ◽  
Endosomal System

The regulated sorting of proteins within the trans-Golgi network (TGN)/endosomal system is a key determinant of their biological activity in vivo. For example, the endoprotease furin activates of a wide range of proproteins in multiple compartments within the TGN/endosomal system. Phosphorylation of its cytosolic domain by casein kinase II (CKII) promotes the localization of furin to the TGN and early endosomes whereas dephosphorylation is required for efficient transport between these compartments (Jones, B.G., L. Thomas, S.S. Molloy, C.D. Thulin, M.D. Fry, K.A. Walsh, and G. Thomas. 1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:5869–5883). Here we show that phosphorylated furin molecules internalized from the cell surface are retained in a local cycling loop between early endosomes and the plasma membrane. This cycling loop requires the phosphorylation state-dependent furin-sorting protein PACS-1, and mirrors the trafficking pathway described recently for the TGN localization of furin (Wan, L., S.S. Molloy, L. Thomas, G. Liu, Y. Xiang, S.L. Ryback, and G. Thomas. 1998. Cell. 94:205–216). We also demonstrate a novel role for protein phosphatase 2A (PP2A) in regulating protein localization in the TGN/endosomal system. Using baculovirus recombinants expressing individual PP2A subunits, we show that the dephosphorylation of furin in vitro requires heterotrimeric phosphatase containing B family regulatory subunits. The importance of this PP2A isoform in directing the routing of furin from early endosomes to the TGN was established using SV-40 small t antigen as a diagnostic tool in vivo. The role of both CKII and PP2A in controlling multiple sorting steps in the TGN/endosomal system indicates that the distribution of itinerant membrane proteins may be acutely regulated via signal transduction pathways.

scholarly journals Iron-Dependent Mechanism of Neuronal Bdnf Suppression by Cellular Iron Deficiency

2020 ◽  
Vol 4 (Supplement_2) ◽  
pp. 1278-1278
Author(s):  
Phu Tran ◽  
Montana Beeson ◽  
Michael Georgieff
Keyword(s):  
Iron Deficiency ◽  
Neuronal Cell ◽  
Epigenetic Modifications ◽  
Similar Degree ◽  
Bdnf Expression ◽  
In Vivo Models ◽  
Iron Deficient

Abstract Objectives Iron deficiency (ID) during neural development is associated with long-term neurocognitive dysfunction. In rodent models, the cognitive deficit is associated with reduced hippocampal brain-derived neurotrophic factor (Bdnf) expression in adulthood despite early iron treatment. Since a previous study suggested a role of epigenetic modifications at the Bdnf locus, we assessed whether an iron-dependent signaling pathway from ID → HIF1α → JARID1B (Fe-containing histone demethylase) → Bdnf is responsible for Bdnf suppression in iron-deficient neurons. The objective is to determine the effect of ID on the HIF1α/JARID1B/Bdnf pathway in vitro and in vivo. Methods A hippocampal neuronal cell line HT-22 (n = 3/group) was used to assess cellular changes following deferoxamine (10 μM) induced-ID. In parallel, timed pregnant Sprague-Dawley rats were fed a purified iron deficient diet (ID, 4 mg Fe/kg) from gestational day (G)2 to through postnatal day (P)7 to induce a similar degree of neuronal ID. At P7, nursing dams where switched to a purified-iron sufficient diet (IS, 200 mg Fe/kg). Control dams were fed IS diet. Hippocampi (n = 6/group) were collected from P15 ID and IS rats. Enrichment of HIF1α, JARID1B, USF1, histone H3 methylation at the Bdnf promoter in both models was determined using ChIP-qPCR. Results were analyzed using t-test for pairwise comparison and α ≤ 0.05. Results ID increased nuclear HIF1α in HT-22 cells (P = 0.03), suggesting less hydroxylated-HIF1α due to reduced Fe-dependent prolyl hydroxylase (PHD) activity. Increased nuclear HIF1α led to increased binding and transactivation at the VEGF (positive control, P = 0.03)) and JARID1B promoters (P = 0.04), which in turns reduced Bdnf expression in HT-22 cells (P = 0.02). Similar effects were observed in iron-deficient P15 hippocampus. Conclusions This is the first evidence that ID directly regulates long-term neural gene expression through the cellular PHD/HIF1α/JARID1B pathway to induce epigenetic modifications both in vitro and in vivo models. Funding Sources 1R01NS099178.

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