Die Autoren danken Yeon-Joo Jung für ihre experimentelle Unterstützung während Synaptosome Reinigung und Jungjoo Park für Bilder von Synaptosomes mit PS-Exposition. Darüber hinaus danken wir allen Mitgliedern in chungs Labor für hilfreiche Diskussion. Diese Arbeit wurde unterstützt durch die National Research Foundation of Korea (NRF) Zuschuss finanziert von der koreanischen Regierung (MSIP) (NRF-2016M3C7A1905391 und NRF-2016R1C1B3006969) (W.-S. (C).

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In diesem Artikel präsentieren wir Methoden für einen langfristigen live-Imaging in Vitro Phagozytose-Assay mit gereinigtem Gliazellen und pH-Indikator-konjugiert Synaptosomes. Wir zeigen, dass im Vergleich zu Mikroglia, Astrozyten besitzen unterschiedlichen Engulfment und Abbau Kapazität während der Phagozytose von Synaptosomes. Darüber hinaus unsere Daten deuten darauf hin, dass Astrozyten abgesondert Faktoren, die Überbrückung Moleküle wie GAS6, Proteine und MEGE8 enthalten, entscheidend für effizien...

Gliazellen, die nicht leicht erregbaren Zellen im Gehirn beziehen, sind die wichtigen Zelltyp in das zentrale Nervensystem (ZNS). Zuvor galten Gliazellen als bloße Stützzellen, die vor allem passive Rollen bei der Aufrechterhaltung neuronaler überleben und basalen synaptischen Eigenschaften spielen. Anzeichen dafür ergab jedoch, dass Gliazellen in verschiedene Aspekte der Neurobiologie, wie Gehirn Homöostase, Vermittlung von Synapse Bildung1,2,3 und Synapse eine aktivere Rolle spielen Beseitigung4,5, und modulierenden synaptische Plastizität6,7. Gliazellen des ZNS gehören Oligodendrozyten, Astrozyten und Mikroglia. Zwischen diesen Zellen, Astrozyten und Mikroglia haben gezeigt, dass phagocytic Rollen zu spielen, indem Sie verschlingen Synapsen4,5, apoptotischen Zellen8, neuronale Trümmer9und Pathogene Proteine wie Amyloid beta Plaketten10,11. In das sich entwickelnde Gehirn beseitigen Astrozyten Synapsen im dorsalen seitlichen gekniet Nucleus (CGN) durch MERTK und MEGF10-abhängige Phagozytose4. In ähnlicher Weise Mikroglia auch beseitigen C1q-beschichtete Synapsen während Entwicklungsstadien durch die klassische Ergänzung Kaskade5. Interessanterweise wurde vermutet, dass Mängel in Synapse beschneiden einer der Initiatoren von verschiedenen neurologischen Störungen sein können. Zum Beispiel hat sich gezeigt, dass Mutationen in Ergänzung Komponente 4 (C4), die Ergänzung-vermittelten Synapse Beschneiden von Mikroglia zunimmt, eng mit der Prävalenz von Schizophrenie in Menschen12 sind. Eine neuere Arbeit hat auch gezeigt, dass die klassische Ergänzung Bahn Überaktivierung in der Einleitung von AD und frühem Verlust der Synapse in dieser Krankheit13induziert.
Verglichen mit Mikroglia-vermittelten Phagozytose, ob Astrozyten vermittelt Phagozytose, die Initiierung und Progression von verschiedenen neurologischen Erkrankungen beiträgt, ist weniger klar. Eine neuere Arbeit legt jedoch nahe, dass Faktoren, die die Rate der normalen Synapse Beschneidung von Astrozyten verändern können Gehirn Homöostase zu stören und zu AD Anfälligkeit und Pathologie14beitragen. Die Rate der Synapse Beschneidung von Astrozyten steuert kraftvoll ApoE -Isomere, mit einer schützenden Allel für AD (ApoE2) stark erhöhen die Rate und eine Risiko-Allel für AD (ApoE4) die Rate deutlich zu senken. Darüber hinaus angesammelt transgene Mäuse, die mit dem Ausdruck ApoE4 viel mehr synaptische C1q als Kontrolle oder ApoE2 Mäuse14. Diese Daten deuten darauf hin, dass Sehbehinderte Astrozyten vermittelt Phagozytose in den frühen AD Gehirn kann induzieren die Anhäufung der alternde C1q-beschichtete Synapsen/synaptische Trümmer, die Ergänzung-vermittelten Mikroglia Phagozytose, fahren synaptischen Degeneration aktiviert . Die beeinträchtigte phagocytic Kapazität der Astrozyten in ApoE4 Trägern kann auch die unkontrollierte Anhäufung von Beta Amyloid-Plaques im Gehirn AD betroffen beitragen.
Darüber hinaus hat gezeigt, dass Gliazellen im Gehirn im Alter von Drosophila ihre phagocytic Kapazität aufgrund der verringerten Übersetzung von Draper, ein Homolog des Megf10 , die Astrozyten verwenden verlieren für phagocytosing Synapsen. Wiederherstellen von Draper Ebenen gerettet phagocytic Kapazität von Gliazellen, die effizient beschädigte axonalen Schutt im alten Gehirn in ähnlichem Ausmaß wie im jungen Gehirn darauf hinweist, dass Altern ausgeglichen-induzierte Veränderungen in der phagocytic Kapazität von Astrozyten können zu Störungen des Gehirns Homöostase15beitragen.
Möglicherweise modulieren die phagocytic Kapazität der Astrozyten basierend auf diese neuen Erkenntnisse, eine attraktive therapeutische Strategie zur Vorbeugung und Behandlung von verschiedenen neurologischen Erkrankungen. In diesem Zusammenhang gab es mehrere Versuche, phagocytic Astrozyten, beispielsweise verstärkt durch Induktion Versauerung der Lysosomen mit sauren Nanopartikel16 und überexprimierenden Transkriptionsfaktor EB (TFEB), die erhöhen können Lysosomen Biogenese17. Trotz dieser Bemühungen ist noch unklar, wie Astrozyten und Mikroglia-Zellen unterscheiden sich in ihrer phagocytic Kinetik und ob wir erhöhen oder verringern Sie ihre phagocytic Kapazitäten bei verschiedenen Erkrankungen sollten.
In diesem Beitrag präsentieren wir eine neuartige in-vitro- Test zur Erkennung von phagocytic Kapazität der Astrozyten in Echtzeit. Die Daten zeigen unterschiedliche Kinetik der Engulfment und Abbau in Astrozyten und Mikroglia. Astrozyten konditioniertes Medium (ACM), die von Astrozyten sezernierten Faktoren enthält, ist essentiell für effektive Phagozytose von Astrozyten und Mikroglia. Darüber hinaus spielt Megf10, einen phagocytic Rezeptor in Astrozyten und ein Homolog von Ced-1 und Draper, wichtige Rollen in Astrozyten vermittelt Phagozytose8,18.

<table><tbody><tr><td>&lt;strong&gt;Synaptosome purification&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Percoll</td><td>GE healthcare life sciences</td><td>17-0891-01</td><td></td></tr><tr><td>Quick Start Bradford Protein Assay Kit 2</td><td>BIO-RAD</td><td>5000202</td><td></td></tr><tr><td>&lt;strong&gt;pH indicator conjugation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dimethyl sulfoxide(DMSO)</td><td>LPS solution</td><td>DMSO100</td><td></td></tr><tr><td>pHrodo red, succinimidyl ester</td><td>Molecula probes</td><td>P36600</td><td></td></tr><tr><td>&lt;strong&gt;Immunopanning&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10X Earle&amp;rsquo;s balanced salt solution (EBSS)</td><td>Sigma</td><td>E7510</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Bovogen</td><td>BSA025</td><td></td></tr><tr><td>Deoxyrebonuclease 1 (DNase)</td><td>Worthington</td><td>Is002007</td><td></td></tr><tr><td>(DMEM)</td><td>Gibco</td><td>11960-044</td><td></td></tr><tr><td>(dPBS)</td><td>Welgene</td><td>LB001-02</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>Griffonia Simplicifolia Lectin(BSL-1)</td><td>Vector Labs</td><td>L-1100</td><td></td></tr><tr><td>Goat anti-mouse IgG+IgM(H+L)</td><td>Jackson ImmunoResearch</td><td>115-005-044</td><td></td></tr><tr><td>Goat anti-mouse IgM (&amp;mu;-chain)</td><td>Jackson ImmunoResearch</td><td>115-005-020</td><td></td></tr><tr><td>Heparin-binding epidermal growth factor</td><td>Sigma</td><td>E4643</td><td></td></tr><tr><td>Human HepaCAM antibody</td><td>R&amp;amp;D systems</td><td>MAB4108</td><td></td></tr><tr><td>Integrin beta 5 monoclonal antibody (KN52)</td><td>eBioscience</td><td>14-0497-82</td><td></td></tr><tr><td>L-cysteine</td><td>Sigma</td><td>C7880</td><td></td></tr><tr><td>L-glutamate</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>N-acetly-L-cyteine (NAC)</td><td>Sigma</td><td>A8199</td><td></td></tr><tr><td>Neurobasal media</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>O4 hybridoma supernatant(mouse IgM)</td><td>Bansal et al.&lt;sup class=&quot;xref&quot;&gt;23&lt;/sup&gt;</td><td></td><td></td></tr><tr><td>Papain</td><td>Worthington</td><td>Is003126</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Pluristrainer 20 &amp;mu;m</td><td>PluriSelect</td><td>43-50020-03</td><td></td></tr><tr><td>Poly-D-lysine</td><td>Sigma</td><td>P6407</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>Putrescine dihydrochloride</td><td>Sigma</td><td>P5780</td><td></td></tr><tr><td>Purified rat anti-mouse CD45</td><td>BD Pharmingen</td><td>550539</td><td></td></tr><tr><td>Purified mouse anti-rat CD45</td><td>BD Pharmingen</td><td>554875</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Gibco</td><td>11360-070</td><td></td></tr><tr><td>Sodium selenite</td><td>Sigma</td><td>S5261</td><td></td></tr><tr><td>Transferrin</td><td>Sigma</td><td>T1147</td><td></td></tr><tr><td>Trypsin</td><td>Sigma</td><td>T9935</td><td></td></tr><tr><td>Trypsin inhibitor</td><td>Worthington</td><td>LS003086</td><td></td></tr><tr><td>Ultra-clear tube (Tube, Thinwall, Ultra-Clear)</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>&lt;strong&gt;Collect IP-ACM&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Macrosep Advance Centrifugal Devices with Omega Membrane (10k)</td><td>PALL</td><td>MAP010C37</td><td></td></tr><tr><td>Macrosep Advance Centrifugal Devices with Omega Membrane (30k)</td><td>PALL</td><td>MAP030C37</td><td></td></tr><tr><td>&lt;strong&gt;Phagocytosis live imaging assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Juli stage</td><td>NanoEntek</td><td></td><td></td></tr><tr><td>Time Series Analyzer V3 plugins</td><td>https://imagej.nih.gov/ij/plugins/time-series.html</td><td></td><td></td></tr></tbody></table>

a novel in vitro live-imaging assay of astrocyte-mediated phagocytosis using ph indicator-conjugated synaptosomes

Astrozyten sind die wichtigen Zelltyp im Gehirn und Synapsen und Blutgefäße direkt zu kontaktieren. Obwohl Mikroglia-Zellen die wichtigsten Immunzellen und nur Fresszellen im Gehirn betrachtet, haben neuere Studien gezeigt, dass Astrozyten auch Teilnahme an verschiedenen phagocytic Prozesse, z. B. Entwicklungsstörungen Synapse Beseitigung und Clearance von Beta Amyloid-Plaques bei Alzheimer-Krankheit (AD). Trotz dieser Erkenntnisse ist die Effizienz der Astrozyten Engulfment und Abbau ihrer Ziele unklar im Vergleich mit der Mikroglia. Dieser Mangel an Informationen ist meist aufgrund des Fehlens von einem Testsystem, in denen die Kinetik der Astrozyten und Mikroglia vermittelt Phagozytose sind leicht vergleichbar. Um dieses Ziel zu erreichen, entwickelten wir einen langfristige live-Imaging in Vitro Phagozytose Assay zur Bewertung der phagocytic Kapazität der gereinigten Astrozyten und Mikroglia. In diesem Test ist Echtzeit-Erkennung von Engulfment und Abbau möglich mit pH-Indikator-konjugiert Synaptosomes, die leuchtend rote Fluoreszenz in sauren Organellen, wie Lysosomen abgeben. Unsere neuartige Assay bietet einfache und effektive Erkennung von Phagozytose durch live-Imaging. Darüber hinaus kann dieser in-vitro- Phagozytose Assay als Screening-Plattform verwendet werden, um zu identifizieren, Chemikalien und Substanzen, die zu verbessern oder hemmen die phagocytic Kapazität der Astrozyten. Wie synaptic Pruning Fehlfunktion und Pathogene Protein Akkumulation gezeigt wurden, psychischen Störungen oder neurodegenerativen Erkrankungen verursachen, sollte Chemikalien und Substanzen, die die phagocytic Kapazität von Gliazellen modulieren hilfreich bei der Behandlung von verschiedenen neurologische Erkrankungen.

In dieser in Vitro Phagozytose Assay mit langfristigen live-Imaging verwendeten wir Synaptosomes von Erwachsenen Maus Gehirn Homogenates, die in der gradient Lösung zwischen 23 % Steigung und 10 % Steigung Lösung, durch Ultrazentrifugation getrennt waren ( ( Abbildung 3). Nach der Zubereitung ausgesetzt Synaptosomes PS in der äußeren Membran (Abbildung 4), was darauf hindeutet, dass sie ihre Funktion verloren und dur...

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A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Dieses Protokoll stellt einen in-vitro- live-Imaging Phagozytose Assay phagocytic Kapazität von Astrozyten zu messen. Gereinigte Ratte Astrozyten und Mikroglia sind zusammen mit pH-Indikator-konjugiert Synaptosomes verwendet. Diese Methode kann in Echtzeit Engulfment und Abbau Kinetik erkennen und bietet eine geeignete Screening-Plattform für Astrozyten Phagozytose modulierende Faktoren zu identifizieren.

Ein neuartiges In vitro- Live-Imaging Assay Astrozyten vermittelt Phagozytose mittels pH Indikator konjugiert Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

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A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

12.1K Aufrufe.  Korea Advanced Institute of Science and Technology. Dieses Protokoll stellt einen in-vitro- live-Imaging Phagozytose Assay phagocytic Kapazität von Astrozyten zu messen. Gereinigte Ratte Astrozyten und Mikroglia sind zusammen mit pH-Indikator-konjugiert Synaptosomes verwendet. Diese Methode kann in Echtzeit Engulfment und Abbau Kinetik erkennen und bietet eine geeignete Screening-Plattform für Astrozyten Phagozytose modulierende Faktoren zu identifizieren.

Serie: A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

Ein Engulfment Assay: Ein Protokoll, um Interaktionen zwischen Fresszellen und Neuronen CNS bewerten

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

Forschung

Neurowissenschaften

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Dreidimensionale Gewebezüchtungen ausgerichtet Astrozyten Netzwerke Nervensystem Regeneration zu rekapitulieren Entwicklungsmechanismen

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Biotechnik

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Die Beurteilung Retinal Microglial Phagozytische Funktion<em&gt; In Vivo</em&gt; Mit einem Durchfluss-Zytometrie basierten Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Immunologie und Infektion

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

Überwachung der Astrozyten Reaktivität und Proliferation in Vitro unter ischämischen-ähnlichen Bedingungen

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

Visualisierung und Analyse des intrazellulären Transports von Organellen und anderen Ladungen in Astrozyten

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

In vitro Quantitative Imaging Assay für Phagozytose toter Neuroblastomzellen durch iPSC-Makrophagen

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Humane Mikroglia-ähnliche Zellen: Differenzierung aus induzierten pluripotenten Stammzellen und In-vitro-Lebendzell-Phagozytose-Assay mit humanen Synaptosomen

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

Durchflusszytometrie-basierte Assays für das mikrogliale Engulfment von synaptischen Materialien

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry.
In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo&#160;Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

Autor im Rampenlicht: Förderung des Verständnisses durch technologische Innovationen in der Psychoneuroimmunologie

Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo&#160;Red-labeled crude synaptosomes using flow cytometry.

Quantifizierung des mikroglialen Engulfments von synaptischem Material mittels Durchflusszytometrie

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this ...

Ein neuartiges In vitro- Live-Imaging Assay Astrozyten vermittelt Phagozytose mittels pH Indikator konjugiert Synaptosomes

Zusammenfassung

Weitere Videos entdecken

Ein Engulfment Assay: Ein Protokoll, um Interaktionen zwischen Fresszellen und Neuronen CNS bewerten

Dreidimensionale Gewebezüchtungen ausgerichtet Astrozyten Netzwerke Nervensystem Regeneration zu rekapitulieren Entwicklungsmechanismen

Die Beurteilung Retinal Microglial Phagozytische Funktion

Überwachung der Astrozyten Reaktivität und Proliferation in Vitro unter ischämischen-ähnlichen Bedingungen

Visualisierung und Analyse des intrazellulären Transports von Organellen und anderen Ladungen in Astrozyten

In vitro Quantitative Imaging Assay für Phagozytose toter Neuroblastomzellen durch iPSC-Makrophagen

Humane Mikroglia-ähnliche Zellen: Differenzierung aus induzierten pluripotenten Stammzellen und In-vitro-Lebendzell-Phagozytose-Assay mit humanen Synaptosomen

Quantifizierung des mikroglialen Engulfments von synaptischem Material mittels Durchflusszytometrie

Quantifizierung des mikroglialen Engulfments von synaptischem Material mittels Durchflusszytometrie

Quantifizierung des mikroglialen Engulfments von synaptischem Material mittels Durchflusszytometrie