Visualisere cytoskeletale vækst kegle sammenbrud og tidlige Amyloid β effekter i kulturperler mus neuroner
Summary
Her præsenteres en protokol til at undersøge de tidlige effekter af amyloid-β (Aβ) i hjernen. Dette viser, at Aβ fremkalder clathrin-medieret endocytose og sammenbrud af cytoskeletale vækst kegler. Protokollen er nyttige i at studere tidlige effekter af Aβ på cytoskeletale vækst kegler og kan lette forebyggelse af Alzheimers sygdom.
Abstract
Amyloid-β (Aβ) forårsager hukommelse funktionshæmninger i Alzheimers sygdom (AD). Selvom terapi har vist sig at reducere Aβ i hjernen hos AD patienter, forbedrer disse ikke hukommelsesfunktioner. Da Aβ aggregater i hjernen før fremkomsten af hukommelse svækkelser, kan målretning Aβ være ineffektivt til behandling af annonce patienter, der allerede udviser hukommelse underskud. Derfor bør downstream signalering på grund af Aβ deposition blokeres før AD udvikling. Aβ inducerer cytoskeletale degeneration, fører til forstyrrelser af neuronal netværk og hukommelse funktionshæmninger. Selvom der er mange undersøgelser om mekanismerne af Aβ toksicitet, kilden til Aβ toksicitet er fortsat ukendt. For at hjælpe med at identificere kilden, foreslår vi en roman protocol, der bruger mikroskopi, gen Transfektion og levende celle imaging for at undersøge tidlige ændringer forårsaget af Aβ i cytoskeletale vækst kogler af kulturperler neuroner. Denne protokol afslørede, at Aβ induceret clathrin-medieret endocytose i cytoskeletale vækst kegler efterfulgt af vækst kegle sammenbrud, demonstrerer, at hæmning af endocytose forhindrer Aβ toksicitet. Denne protokol vil være nyttige i at studere de tidlige effekter af Aβ og kan føre til mere effektive og forebyggende AD behandling.
Introduction
Amyloid-β (Aβ) indlån findes i hjernen hos patienter med Alzheimers sygdom (AD) og betragtes som en afgørende årsag til annonce1 der forstyrrer neuronal netværk, fører til hukommelse nedskrivninger2,3,4. Mange kliniske lægemiddelkandidater har vist sig at effektivt forhindre amyloid-β (Aβ) produktion eller fjerne Aβ-aflejringer. Dog lykkedes ingen forbedring hukommelsesfunktion i AD patienter5. Aβ er allerede deponeret i hjernen før igangsættelsen af hukommelse funktionshæmninger6; Derfor kan faldende Aβ niveauer i hjernen hos patienter udstillende hukommelse funktionsnedsættelser være ineffektive. Aβ deposition er til stede i prækliniske AD patienter; men disse patienter sjældent præsentere med neuronal degeneration og hukommelse underskud6. Der er en tidsforskydning mellem Aβ deposition og hukommelse funktionshæmninger. Derfor en kritisk strategi for forebyggelse af AD blokering Aβ toksicitet signalering i de tidlige stadier af Annoncen, før udviklingen af hukommelse underskud. Aβ deposition inducerer axon degeneration7,8,9,10,11,12,13, hvilket kan føre til en afbrydelse af neurale netværk og permanent svækkelse af hukommelsesfunktion. Mange studier har undersøgt mekanismerne i Aβ toksicitet; for eksempel, har de degenererede axoner af annonce mus hjerner vist sig at har øget autophagy14. Calcineurin aktivering er blevet rapporteret som en mulig mekanisme af Aβ-induceret cytoskeletale degeneration15; men den direkte udløsende faktor for cytoskeletale degeneration forbliver ukendt.
Denne undersøgelse fokuserer på sammenbruddet af cytoskeletale endelser kaldet vækst kegler. Sammenbruddet af cytoskeletale vækst kegler kan være forårsaget af cytoskeletale vækst afskrækningsmidler, såsom semaphorin 3A og ephrin-A516,17,18,19,20. Sammenbrud-lignende dystrofe cytoskeletale endelser er blevet observeret i hjernen hos AD patienter21,22. Derudover kan manglende vækst kegle funktion provokere cytoskeletale degeneration23. Men det er uvist om Aβ inducerer vækst kegle sammenbrud. Derfor, denne undersøgelse præsenterer en roman protokol for at observere de tidlige effekter af Aβ i kulturperler neuroner og undersøge Aβ-induceret vækst kegle sammenbrud.
Protocol
Representative Results
Discussion
Den protokol, der er beskrevet i denne undersøgelse aktiveret observation af tidlige fænomener i cytoskeletale vækst kegler efter Aβ1-42 behandling. Aβ1-42 induceret endocytose i cytoskeletale vækst kegler indenfor 20 min, og vækst kegle sammenbrud blev observeret i 1 h af behandling. Denne endocytose var sandsynligvis medieret af clathrin. Ved hjælp af denne protokol, blev hæmning af clathrin-medieret endocytose bekræftet for at forhindre Aβ1-42-induceret vækst kegle sammenbrud og cytoskeletale degeneration …
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev delvist støttet af forskningstilskud fra JSP’ER (KAKENHI 18K 07389), Japan, Takeda Science Foundation, Japan og Kobayashi Pharmaceutical Co, Ltd, Japan.
Materials
| ddY mice | SLC | ||
| Eight-well culture slide | Falcon | 354108 | |
| poly D lysine | Wako | 168-19041 | |
| Culture medium, Neurobasal medium | Gibco | 21103-049 | |
| house serum | Gibco | 26050-088 | |
| glucose | Wako | 049-31165 | |
| L-glutamine | Wako | 074-00522 | |
| 0.05% trypsin | Gibco | 25300-054 | |
| DNase I | Worthington | DP | |
| soybean trypsin inhibitor | Gibco | 17075-029 | |
| Filter with 70 µm mesh size, cell strainer | Falcon | 352350 | |
| B-27 supplement | Gibco | 17504-044 | |
| CO2 incubator | Astec | SCA-165DS | |
| Amyloid β1-42 | Sigma-Aldrich | A9810 | |
| paraformaldehyde | Wako | 162-16065 | |
| sucrose | Wako | 196-00015 | |
| Aqueous mounting medium, Aqua-Poly/Mount | polysciences | 18606-20 | |
| Inverted microscope A | Carl Zeiss | Axio Observer Z1 | Connected with AxioCam MRm, Heating Unit XL S, CO2 Module S1, and TempModule S1 |
| Objective Plan-Apochromat 20x | Carl Zeiss | 420650-9901 | |
| Objective Plan-Apochromat 63x | Carl Zeiss | 440762-9904 | |
| Objective, CFI Plan Apo Lambda 40X | Nikon | ||
| anti-MAP2 IgG | Abcam | ab32454 | |
| anti-tau-1 IgG | Chemicon | MAB3420 | |
| anti-amyloid β antibody | IBL | 10379 | clone 11A1 |
| normal goat serum | Wako | 143-06561 | |
| bovine serum albumin | Wako | 010-25783 | |
| t-octylphenoxypolyethoxyethanol | Wako | 169-21105 | |
| goat anti-mouse IgG conjugated with AlexaFluor 594 | Invitrogen | A11032 | |
| goat anti-rabbit IgG conjugated with AlexaFluor 488 | Invitrogen | A11029 | |
| hot plate | NISSIN | NHP-M30N | |
| cover glass | Fisher Scientific | 12-545-85 | |
| 35 mm dish | IWAKI | 1000-035 | |
| Silicone RTV | Shin-Etsu | KE42T | |
| hand punch | Roper Whitney | No. XX | |
| Fluorescence membrane probe, FM1-43FX | Invitrogen | F35355 | |
| Ca2+– and Mg2+-free Hanks' balanced salt solution | Gibco | 14175-095 | |
| Transfection solution, Nucleofector solution | Lonza | VPG-1001 | |
| Electroporator, Nucleofector I | Amaxa | ||
| Inverted microscope B | Keyence | BZ-X710 | |
| Image software, ImageJ | NIH | https://imagej.nih.gov/ij/ |
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