Aktivering Autophagy ved aerob træning i mus
Summary
Autophagy aktivering er gavnlig til forebyggelse af en række sygdomme. En af de fysiologiske fremgangsmåder til at inducere autofagi in vivo er fysisk træning. Her viser vi, hvordan du aktiverer autophagy af aerob træning og måle autofagi niveauer i mus.
Abstract
Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.
Introduction
Autophagy er en evolutionært bevaret nedbrydningsvej, som induceres som respons på forskellige stressbetingelser såsom sult og hypoxi 1, 2. Under autophagy, dobbelt-membran vesikler, kaldet autophagosomes, indarbejde unødvendige eller beskadigede subcellulære komponenter og transportere dem i lysosomer for nedbrydning 3. Basal autophagy er afgørende for cellefunktion og organisme udvikling, og nedsat basal autofagi er impliceret i mange lidelser, herunder neurodegeneration, tumordannelse og type 2 diabetes 4, 5, 6.
Den bedst kendte fysiologiske autophagy inducer er sult. Men det har to store begrænsninger. Først sult tager lang tid til effektivt at fremkalde autophagy hos dyr, fx 48 timers mad begrænsning i musi de fleste organer. Sekund, sult inducerer næppe hjerne autofagi, på grund af en relativt stabil forsyning af næringsstoffer i hjernen. Faktisk er det også vanskeligt at påvise autophagy induktion med små molekyler induktorer, som mange lægemidler ikke kan passere blod-hjerne barrieren. Således bedre til at analysere funktionen af autofagi aktivering i sygdom patogenese, vi for nylig opdaget, at motion er en mere potent fysiologisk metode til at inducere autofagi i en kort periode 7, 8, 9. Sammenlignet med sult, er autophagy effektivt fremkaldt af løbebånd køre så hurtigt som 30 min. , Motion er således en praktisk og potent fysiologisk tilgang til at studere den mekanisme af autofagi ved mediering sundhedsmæssige fordele og forebygge sygdomme.
Der er flere proteinmarkører til påvisning af autophagy aktivitet, herunder LC3 og P62. LC3 (mikrotubulus-associeret protein 1A / 1B-light cHain 3) er et cytosolisk protein (LC3-I-form), der er konjugeret til PE (phosphatidylethanolamin) ved autophagy induktion. PE-lipiderede LC3 (LC3-II form) rekrutteres på autophagosomal membraner og kan bruges til at visualisere autophagosomes når mærket med GFP. Dens translokation fra cytosolen til punktformet strukturer af autophagosomes under mikroskopi er en indikation af autofagi induktion. P62 er en last receptor for Autophagy substrater (såsom ubiquitinering proteiner), og er indarbejdet i autophagosomes samt. Da proteinet nedbrydes i lysosomer sammen med autophagosomes, kan dets niveau anvendes til at måle autofagi flux. Her viser vi, hvordan man bruger disse markører til at kvantificere autofagi i forskellige musevæv fremkaldt af aerobe motion, herunder tvungen motion (løbebånd) og frivillig motion (løb hjul). De samme fremgangsmåder kan også anvendes til in vivo måling af autophagy efter behandling af andre induktorer.
Protocol
Representative Results
Discussion
Autophagy er en katabolisk proces, der giver energi og reducerer cytotoksicitet ved lysosomal nedbrydning af cytoplasma komponenter eller beskadigede organeller. Studere autophagy er vigtigt at forstå reguleringen af cellulære homeostase og mekanismerne i stressrespons. Nye modeller og metoder dukker på forskningsområdet 15, for at studere, hvordan nedsat autofagi bidrager til en lang række patologiske processer 16, 17.
<p …Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We thank the Northwestern University Mouse Histology and Phenotyping Laboratoryfor technical support and assistance, and Noboru Mizushima (University of Tokyo) for providing GFP-LC3 transgenic mice. A. R. and C. H. were supported by the startup funds from Northwestern University and the grant from National Institutes of Health (DK094980).
Materials
| Treadmill | Columbus Instruments | 150-RM Exer 3/6 | |
| Mouse running wheel | Super Pet | 100079365 | diameter 11.4 cm |
| Odometer | Bell | DASHBOARD 100 | |
| Syringe pump | KD Scientific | KDS100 | |
| Fluorescence microscope | Nikon | Model: inverted microscope ECLIPSE | |
| Cryostat | Leica | CM 1850UV | |
| Homogenizer | IKA | 003737001 / Model: T10 Basic S1 | |
| Chloroquine | CAYMAN CHEMICAL COMPANY | 14194 | |
| Parafolmaldehye | SIGMA-ALDRICH | P6148 | Personal protection equipment required. This product may release formaldehyde gas, a chemical known to cause cancer |
| Mounting media | Vector Laboratories | H-1200 | |
| p62 antibody | BD Biosciences | 610833 | |
| LC3 antibody | Novus Biologicals | NB100-2220 | |
| 2X Laemmli Sample Buffer | Bio-Rad Laboratories | 161-0737 | |
| ImageJ | NIH |
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