Name: a novel in vitro live-imaging assay of astrocyte-mediated phagocytosis using ph indicator-conjugated synaptosomes
Uploaded: 2018-02-05T13:30:00
Description: Watch this Scientific Journal Video about A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes at JoVE.com

Forfatterne takke Yeon-Joo Jung for hendes eksperimentelle støtte under synaptosome rensning og Jungjoo Park for billeder af synaptosomes med PS eksponering. Derudover takker vi alle medlemmer i Chungs laboratorium for nyttig diskussion. Dette arbejde blev støttet af Danmarks Grundforskningsfond i Korea (NRF) tilskud finansieres af den koreanske regering (MSIP) (NRF-2016M3C7A1905391 og NRF-2016R1C1B3006969) (W.-S. C).

Alle metoder beskrevet her er blevet godkendt af The Korea Advanced Institute of Science og Technology institutionelle Animal Care og brug udvalg (IACUC), KA2016-08.
1. synaptosome rensning
Bemærk: Disse procedurer er tilpasset fra en tidligere udgivne papir19 med flere ændringer til at forbedre udbyttet af renset synaptosomes (figur 1).
<ol><li>Forberede diskontinuert tæthed gradient medier.<ol>
<li>Placer ...

<ol>
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I denne artikel præsenterer vi metoder for en langsigtet live imaging i vitro fagocytose assay ved hjælp af renset glial celler og pH indikator-konjugeret synaptosomes. Vi viser, at sammenlignet med mikroglia, astrocytter besidder forskellige engulfment og nedbrydning kapacitet under fagocytose af synaptosomes. Derudover tyder vores data på, at astrocyte-udskilles faktorer, som indeholder passerelle molekyler som GAS6, proteiner og MEGE8, er afgørende for effektiv PS-afhængige fagocytose af glial celler i h...

Glial celler, som refererer til ikke-overgearet celler i hjernen, er den største celletype i centralnervesystemet (CNS). Tidligere blev glial celler betragtet som blotte støtte celler, der hovedsageligt spiller en passiv rolle i opretholdelse af neuronal overlevelse og basal synaptic egenskaber. Imidlertid har nye beviser afsløret at glial celler spiller en mere aktiv rolle i forskellige aspekter af neurobiologi, såsom opretholdelse af hjernen homøostase, mægle synapse dannelse1,2,3 og synapse eliminering4,5, og modulerende synaptisk plasticitet6,7. Gliaceller i CNS omfatter astrocytter, mikroglia og oligodendrocytes. Blandt disse celler, astrocytter og mikroglia har vist sig at spille fagocyterende roller af oversvømmer synapser4,5, apoptotiske celler8, neurale vragrester9og patogene proteiner, såsom amyloid beta plaques10,11. I udvikle hjernen fjerne astrocytter synapser i den dorsale laterale geniculate kerne (dLGN) gennem MERTK - og MEGF10-afhængige fagocytose4. Ligeledes mikroglia også fjerne C1q-belagt synapser under udviklingsstadier gennem den klassiske supplement cascade5. Interessant, er det blevet foreslået, at defekter i synapse beskæring kan være en af initiativtagerne til flere neurologiske lidelser. For eksempel har været vist, at mutationer i komplement komponent 4 (C4), hvilket øger komplement-medieret synapse beskæring af mikroglia, er stærkt forbundet med forekomsten af skizofreni hos mennesker12. Et nyligt papir har også vist, at den klassiske supplement pathway er hyperactivated i indledningen stadier af AD og inducerer tidlig synapse tab i denne sygdom13.
Sammenlignet med mikroglia-medieret fagocytose, er om astrocyte-medierede fagocytose bidrager til indledningen og progression af forskellige neurologiske lidelser mindre klar. Dog, et nyligt papir tyder på, at faktorer, der ændrer satsen for normale synapse beskæring af astrocytter kan forstyrre hjernen homøostase og bidrage til AD modtagelighed og patologi14. Satsen for synapse beskæring af astrocytter er stærkt kontrolleret af ApoE isomerer, med en beskyttende allel for annonce (ApoE2) stærkt øge hastigheden og en risiko allel for annonce (ApoE4) betydeligt sænke satsen. Derudover akkumuleret Transgene mus udtrykker ApoE4 meget mere synaptic C1q end kontrol eller ApoE2 mus14. Disse data tyder på, at nedsat astrocyte-medieret fagocytose i de tidlige AD hjernen kan fremkalde ophobning af senescent C1q-belagt synapser/synaptic vragrester, der aktiverer komplement-medieret microglial fagocytose, kørsel synaptic degeneration . Astrocytter i ApoE4 luftfartsselskaber nedsat fagocyterende kapacitet kan også bidrage til den ukontrollerede ophobning af amyloid beta plaques i AD-ramte hjerner.
Desuden har det vist at glial celler i alderen Drosophila hjernen mister deres fagocyterende kapacitet på grund af nedsat oversættelse af Draper, en homolog af Megf10 , astrocytter bruger til phagocytosing synapser. Genoprette Draper niveauer reddet glial celler, som effektivt ryddet beskadigede cytoskeletale vragrester i alderen hjernen til en lignende omfang som i den unge hjerne, der angiver, at aldring-induceret fagocyterende kapacitet ændringer i fagocyterende kapacitet astrocytter kan bidrage til forstyrrelser af hjernen homøostase15.
Baseret på disse nye resultater, kan modulerende astrocytter fagocyterende kapacitet være en attraktiv terapeutisk strategi til forebyggelse og behandling af forskellige neurologiske lidelser. I denne forbindelse har der været flere forsøg på at forbedre astrocytter, eksempelvis fagocyterende kapacitet ved overtalelse forsuring af lysosomer med sure nanopartikler16 og overekspression transkriptionsfaktor EB (TFEB), som kan forbedre lysosomet Biogenese17. Trods disse forsøg er det stadig uklart, hvordan astrocytter og microglial celler afviger i deres fagocyterende kinetik og om vi skal øge eller mindske deres fagocyterende kapacitet i forskellige sygdomme.
I dette papir præsenterer vi en roman i vitro assay til påvisning af astrocytter fagocyterende kapacitet i realtid. Dataene viser forskellige kinetik af engulfment og nedbrydning i astrocytter og mikroglia. Astrocyte-aircondition medium (ACM), som indeholder udskilles faktorer fra astrocytter, er afgørende for effektiv fagocytose af både astrocytter og mikroglia. Derudover spiller Megf10, en fagocyterende receptor i astrocytter og en homolog Ced-1 og Draper, vigtige roller i astrocyte-medieret fagocytose8,18.

<table>
	<tbody>
		<tr>
			<td>Synaptosome purification</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>pH indicator conjugation</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>Immunopanning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X Earle&#8217;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 (&#956;-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&#38;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.23</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 &#956;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>Collect IP-ACM</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>Phagocytosis live imaging assay</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

Astrocytter er den største celletype i hjernen og direkte kontakt synapser og blodkar. Selvom microglial celler har været betragtet som den store immunceller og kun fagocytter i hjernen, har nylige undersøgelser vist, at astrocytter også deltage i forskellige fagocyterende processer, såsom udviklingsmæssige synapse eliminering og regnskabsafslutning amyloid beta plaques i Alzheimers sygdom (AD). Trods disse resultater er effektivitet af astrocyte engulfment og forringelse af deres mål uklare sammenlignet med mikroglia. Denne mangel på oplysninger er for det meste på grund af manglende en assay system hvor kinetik af astrocyte - og mikroglia-medieret fagocytose er let sammenlignelige. For at nå dette mål har vi udviklet en langsigtet live imaging i vitro fagocytose assay for at evaluere renset astrocytter og mikroglia fagocyterende kapacitet. I denne analyse er real-time detektering af engulfment og nedbrydning muligt ved hjælp af pH indikator-konjugeret synaptosomes, som udsender lys rød fluorescens i sure organeller, såsom lysosomer. Vores nye assay giver enkel og effektiv påvisning af fagocytose gennem live-imaging. Derudover kan denne in vitro- fagocytose assay bruges som en screening platform til at identificere kemikalier og stoffer, der kan fremme eller hæmme astrocytter fagocyterende kapacitet. Som synaptic beskæring funktionsfejl og patogene protein ophobning har vist sig at forårsage psykiske lidelser eller neurodegenerative sygdomme, bør kemikalier og stoffer, der modulerer gliaceller fagocyterende kapacitet være nyttige i behandling af forskellige neurologiske lidelser.

I dette in vitro- fagocytose assay med langsigtede live-imaging brugte vi synaptosomes fra voksne mus hjernen homogeniseret, som var adskilt i den gradient løsning mellem 23% gradient løsning og 10% gradient løsning af ultracentrifugering ( Figur 3). Efter tilberedning udsat synaptosomes PS i deres ydre membran (figur 4), hvilket tyder på, at de mistede deres funktion og kunne blive anerkendt af PS receptorer i astro...

Watch this Scientific Journal Video about A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes at JoVE.com

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

Denne protokol udgør en in vitro- live imaging fagocytose assay for at måle astrocytter fagocyterende kapacitet. Renset rotte astrocytter og mikroglia bruges sammen med pH indikator-konjugeret synaptosomes. Denne metode kan registrere real-time engulfment og nedbrydning kinetik og giver en passende screening platform til at identificere faktorer modulere astrocyte fagocytose.

En roman In Vitro Live-imaging analyse af Astrocyte-medieret fagocytose bruger pH indikator-konjugeret 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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Neuroscience

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

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