脊髄損傷におけるシャペロン介在性オートファジーの検討

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脊髄損傷におけるシャペロン介在性オートファジー

の検討

著者

半田恭一

学位授与機関

Tohoku University

学位授与番号

11301甲第19129号

URL

http://hdl.handle.net/10097/00129224

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3 7 α 8 12 13 21 25 31 32 33 38

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α β β

chaperone-mediated

autophagy: CMA 3 CMA

CMA

CMA lysosome-associated membrane protein type 2A LAMP2A

CMA

C57BL/6J 10

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n = 5 LAMP2A CMA heat shock cognate 70 kDa protein HSC70

lysosome-associated membrane protein type 1 LAMP1

sham LAMP2A HSC70 β 4 24 3 7 21 sham LAMP2A HSC70 n = 5 LAMP2A β β LAMP2A 3 sham LAMP2A LAMP2A β LAMP2A HSC70 LAMP1 β LAMP2A β 4 3 7 24 3 7 LAMP2A β sham p < 0.05 3 LAMP2A β p < 0.05 HSC70

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HSC70 β LAMP2A 4 3 21 3 LAMP2A sham p < 0.05 HSC70 3 β LAMP2A sham β β β β β LAMP2A sham LAMP2A CMA CMA β CMA

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CMA: chaperone-mediated autophagy DAB: diaminobenzidine tetrahydrochloride FBS: fetal bovine serum

HO-1: hemo oxygenase-1

HSC70: heat shock cognate 70 kDa protein HSP70: heat shock protein 70

LAMP1: lysosome-associated membrane protein 1 LAMP2A: lysosome-associated membrane protein 2A LC3: light chain 3

mTOR: mammalian target of rapamycin OCT: optimal-cutting temperature PBS: phosphate buffered saline

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α

100 3.6 195 1)

National Spinal Cord Injury Statistical Center

100 54 30 2,3) 10 5000 4) β β β 5,6) apoptosis caspase caspase 8)

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apoptosis

9) _β

β β

9,10) _{mammalian target of rapamycin mTOR}

11,12) β 13,14) chaperone-mediated autophagy: CMA 3 15) β 16) β

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17) _CMA

CMA 5 KFERQ

heat shock protein 70 HSP70 heat shock cognate 70 kDa protein HSC70

lysosome-associated membrane protein 2A

LAMP2A 18)

1 15,19) _LAMP2A _CMA

LAMP2A CMA 20,21) _CMA

22) _CMA β 23-25) CMA × β CMA DNA β 21) CMA

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26-31) CMA × -CMA -32) CMA Htt 27,33) CMA 34,35) CMA 36,37) CMA 2 CMA CMA LAMP2A

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CMA LAMP2A CMA

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2016 -182 10 12 C57BL/6J 70 4 24 3 7 21 5 sham 5 3 2 sham 2 3 3 sham 3 1 4 5 24 5% 3% 37

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α 15 mm

9 - 11 10

Feather disposable scalpel No. 11, Feather Safety

Razor 3 38,39) 4 × 4 24 3 7 21 sham sham ± 4 Phosphate

Buffered Saline PBS 2.9 mM NaH2PO4 9.0mM Na2HPO4 138 mM NaCl pH 7.2

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Optimal-Cutting Temperature OCT compound Thermo Scientific

Ltd. 15 µm

1500 µm 3000 µm 250 µm

4 24 3 7 21 sham

lysosome-associated membrane protein 1 LAMP1 LAMP2A HSC70

β LAMP1

PBS 3 5

Fetal Bovine Serum FBS 2

LAMP1 1: 100; H4A3, Abcam LAMP2A 1: 250; 51-2200, Thermo Fisher HSC70 1: 200; ab19136,

Abcam 4 Alexa Fluor 488

IgG 1: 500; A11029, Invitrogen Alexa Fluor 594 IgG 1: 500; A11012, Invitrogen Alexa Fluor 594 IgG 1: 500;

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A11007, Invitrogen 1

Vectashield containing DAPI to label the nuclei H-1200, Vector Laboratories BX51, Olympus LAMP2A HSC70 β LAMP2A HSC70 4 ± ± 16 β 4 DAPI β β 250 µm 3 3 β LAMP2A β

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sham 10 LAMP2A HSC70

Lysosome isolation kit

ab2340471: abcam 4 40)

200 µl Lysosome isolation buffer ±

± 30 500 µl Lysosome enrichment buffer 500 ×g 4 °C 10 3 Lysosome gradient

Lysosome gradient Lysosome enrichment buffer

5 17 20 23 27 30% Optima XPN-100, Beckman 145,000 ×g 4 °C 120 10% 2 PBS 18,000 ×g 4 °C 30 100 µl PBS -80 10% SDS-PAGE

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Polyvinylidene difluoride membrane Bio-Rad 12 V 40

5 1

LAMP2A 1: 1000; 51-2200, Thermo Fisher HSC70 1: 200; ab19136, Abcam 4 2500

HRP IgG P0448, Dako 1000 HRP

IgG P0450, Dako 1 ECL Prime Western Blotting Detection System RPN2232, Amersham Biosciences

Chemi Doc MP system Bio-Rad Laboratories Image Lab version 6.0 Bio-Rad Laboratories

LAMP2A β

LAMP2A 3

LAMP2A NeuN β GFAP

Olig2

LAMP2A 1: 250; 51-2200, Thermo Fisher NeuN 1:100; Chemicon GFAP 1:50; DAKO Olig2 1:100;

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Santa Cruz Biotechnology Alexa Fluor 594

IgG 1: 500; A11012, Invitrogen Alexa Fluor 488 IgG 1:500; Molecular Probes Alexa Fluor 488 IgG 1:500; Molecular Probes CMA 3 sham ± 25% 4 12

30 OCT compound Thermo Scientific

Ltd. 6 µm

LAMP2A

pre-embedding 10%

PBS LAMP2A 1: 500;

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5nm ± HRP IgG P0448, Dako 3 PBS 0.5% 1 PBS DAB 30 Graham-Karnovsky 5 PBS 1% 30 PBS JEM-1400, JOEL LAMP2A HSC70 β

Mann-Whitney U test LAMP2A HSC70 β

LAMP2A HSC70 sham

one-way ANOVA post-hoc test Tukey-Kramer

± JMP

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× 1 1 8 LAMP2A Sham LAMP2A β 3 LAMP2A β 5 LAMP2A β LAMP2A β 500 µm 5B

LAMP2A LAMP1 LAMP2A

LAMP1 β 6 HSC70 LAMP1 β

7

LAMP2A HSC70

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7 8 LAMP2A β sham 36 1 4 50 9 24 84 25 3 280 54 7 108 28 21 48 7 LAMP2A β sham 44 5 4 47 6 24 44 6 3 123 19 7 52 25 21 40 6 24 3 7 LAMP2A β sham P < 0.05 9 3 LAMP2A β P < 0.05 9 HSC70 β 10 LAMP2A β sham 93 10 4 92 11 24 101 12 3 104 10 7 98 14 21 83 8 LAMP2A β sham 95 13 4 95 15 24 100 20 3 102 13 7 95 17 21 88 8 HSC70 β β 11 HSC70 β 11

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LAMP2A HSC70 LAMP2A sham 1.00 0.12 4 1.34 0.20 24 1.49 0.16 3 2.12 0.41 7 1.66 0.58 21 1.20 0.16 LAMP2A 4 3 21 12 3 LAMP2A sham P < 0.05 12 HSC70 sham 1.00 0.18 4 1.03 0.14 24 0.92 0.17 3 0.84 0.12 7 0.87 0.16 21 0.81 0.10 HSC70 13 LAMP2A β 3 NeuN β LAMP2A β LAMP2A 14A B

GFAP β 14C D Olig2 β 14E F LAMP2A

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Sham β β β β 15A D E 3 β 15B C β 15F I 3 β LAMP2A 15K L O R sham 15J M N

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LAMP2A LAMP2A β 24 3 7 HSC70 LAMP2A LAMP2A β β LAMP2A β CMA CMA CMA β CMA 26,27,29-31) CMA

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27,34,35) _CMA β β CMA 35) _CMA _β LAMP2A β LAMP2A β LAMP2A CMA β CMA 24 3 41-44)

beclin 1 light chain 3

LC3 3

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11,46) CMA 48 CMA 35) _CMA 3 34) CMA LAMP2A 24 3 LAMP2A LAMP2A CMA CMA β CMA CMA β 34,35) β CMA 27) _LAMP2A _β CMA β

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CMA × CMA 26) 41) LAMP2A CMA β HSC70 CMA HSC70 LAMP2A 47,48) LAMP2A HSC70 34) _HSC70 _HSC70 18) _HSC70

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CMA β 49,50) CMA CMA 27,34,35) CMA 36,37) CMA β 35) _A CMA β 51) _LAMP2 52) CMA CMA CMA CMA

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CMA CMA × × CMA LAMP2 CMA LAMP2A β LAMP2A β × β β CMA

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LAMP2A

CMA CMA

β

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1) S. B. Jazayeri, S. Beygi, F. Shokraneh, E. M. Hagen, and V.

Rahimi-Movaghar: Incidence of traumatic spinal cord injury worldwide: a systematic review. Eur. Spine J. 2015;24:905-918

2) S. Selvarajah, E. R. Hammond, A. H. Haider, C. J. Abularrage, D. Becker, N. Dhiman, O. Hyder, D. Gupta, J. H. Black, and E. B. Schneider: The burden of acute traumatic spinal cord injury among adults in the United States: An update. J. Neurotrauma. 2014;31:228-238

3) _{Shannon D. Sullivan, M. S. Nash, and Eshetu Tefera: Prevalence and}

Etiology of Hypogonadism in Young Men with Chronic Spinal Cord Injury: A Cross-Sectional Analysis from Two University-Based Rehabilitation Centers Shannon. PM R. 2017;9:751-760

4) T. I. H shingu, M Ohama: A nationwide epidemiological survery of spinal cord injuries in Japan from January 1990 to December 1992. Paraplegia. 1995;33:183-188

5) M. E. Schwab and D. Bartholdi: Degeneration and regeneration of axons in the lesioned spinal cord. Physiol. Rev. 1996;76:319-370

6) _{H. Kanno, H. Ozawa, A. Sekiguchi, and E. Itoi: The role of autophagy in} spinal cord injury. Autophagy. 2009;5:390-392

7) C. it lin E. Hill, M. S, and and J. acqueline C. B. Bea t t ie: Degeneration and Sprouting of Identified Descending Supraspinal Axons after Contusive Spinal Cord Injury in the Rat. Exp. Neurol. 2001;171:153-169

8) H. Ozawa, R. W. Keane, and A. E. MarCillo: Therapeutic Strategies Targeting Caspase Inhibition Following Spinal Cord Injury in Rats. 2002;177:306-313

9) H. Kanno, H. Ozawa, Y. Dohi, A. Sekiguchi, K. Igarashi, and E. Itoi: Genetic Ablation of Transcription Repressor Bach1 Reduces Neural Tissue Damage and Improves Locomotor Function after Spinal Cord Injury in Mice. J. Neurotrauma. 2009;26:31-39

10) _{H. Kanno, H. Ozawa, A. Sekiguchi, S. Yamaya, and E. Itoi: Induction of} autophagy and autophagic cell death in damaged neural tissue after acute spinal cord injury in mice. Spine (Phila. Pa. 1976). 2011;36:1427-1434 11) _{A. Sekiguchi, H. Kanno, H. Ozawa, S. Yamaya, and E. Itoi: Rapamycin}

(35)

Impairment after Spinal Cord Injury in Mice. J. Neurotrauma. 2012;29:946-956

12) S. Tateda, H. Kanno, H. Ozawa, A. Sekiguchi, K. Yahata, S. Yamaya, and E. Itoi: Rapamycin Suppresses Microglial Activation and Reduces the

Development of Neuropathic Pain After Spinal Cord Injury. J. Orthop. Res. 2017;93-103

13) _{N. Mizushima and M. Komatsu: Autophagy: Renovation of cells and tissues.} Cell. 2011;147:728-741

14) I. Dikic and Z. Elazar: Mechanism and medical implications of mammalian autophagy. Nat. Rev. Mol. Cell Biol. 2018;19:349-364

15) A. M. Cuervo: Autophagy: Many paths to the same end. Mol. Cell. Biochem. 2004;263:55-72

16) _{Z. Yang and D. J. Klionsky: Eaten alive : a history of macroautophagy. Nat.} Cell Biol. 2010;12:814-822

17) D. J. Klionsky: The molecular machinery of autophagy : unanswered questions. 2005;

18) F. A. Agarraberes and J. F. Dice: A molecular chaperone complex at the lysosomal membrane is required for protein translocation. J. Cell Sci. 2001;114:2491-2499

19) B. Patel and A. M. Cuervo: Methods to study chaperone-mediated autophagy. Methods. 2015;75:133-140

20) _{U. Bandyopadhyay, S. Kaushik, L. Varticovski, and A. M. Cuervo: The} Chaperone-Mediated Autophagy Receptor Organizes in Dynamic Protein Complexes at the Lysosomal Membrane. Mol. Cell. Biol. 2008;28:5747-5763 21) _{S. Kaushik and A. M. Cuervo: The coming of age of chaperone-mediated}

autophagy. Nat Rev Mol Cell Biol. 2018;19:365-381

22) K. S. and C. A.M.: Chaperone-mediated autophagy: A unique way to enter the lysosome world. Trends Cell Biol. 2012;22:407-417

23) A. N. A. M. Cuervo, E. Knecht, S. R. Terlecky, and J. F. DIce: Activation of a selective pathway of lysosomal proteolysis in rat liver by prolonged

starvation. Am. J. Physiol. 1995;269:C1200-1208

24) M. E. Hubbi, H. Hu, Kshitiz, I. Ahmed, A. Levchenko, and G. L. Semenza: Chaperone-mediated Autophagy Targets Hypoxia-inducible Factor-1β(HIF- 1β) for Lysosomal Degradation. J. Biol. Chem. 2013;288:10703-10714

(36)

Chaperone-mediated Autophagy during Oxidative Stress. Mol. Biol. Cell. 2004;15:4829-4840

26) A. di Domenico, G. Carola, C. Calatayud, M. Pons-Espinal, J. P. Muñoz, Y. Richaud-Patin, I. Fernandez-Carasa, M. Gut, A. Faella, J. Parameswaran, J. Soriano, I. Ferrer, E. Tolosa, A. Zorzano, A. M. Cuervo, A. Raya, and A. Consiglio: Patient-Specific iPSC-Derived Astrocytes Contribute to

Non-Cell-Autonomous Neurodegeneration in Parkinson’s Disease. Stem Cell Reports. 2019;12:213-229

27) H. Koga, M. Martinez-vicente, E. Arias, S. Kaushik, D. Sulzer, and A. M. Cuervo: Constitutive Upregulation of Chaperone-Mediated Autophagy in Huntington’s Disease. J. Neurosci. 2011;31:18492-18505

28) M. Kon, R. Kiffin, H. Koga, J. Chapochnick, F. Macian, L. Varticovski, and A. M. Cuervo: Chaperone-Mediated Autophagy Is Required for Tumor

Growth. Sci Transl Med. 2011;3:109ra117

29) S. Sooparb, S. R. Price, J. Shaoguang, and H. A. Franch: Suppression of chaperone-mediated autophagy in the renal cortex during acute diabetes mellitus. Kidney Int. 2004;65:2135-2144

30) B. Venugopal, N. T. Mesires, J. C. Kennedy, C. Curcio-morelli, J. M. Laplante, J. F. Dice, and S. A. Slaugenhaupt: Chaperone-Mediated Autophagy is Defective in Mucolipidosis Type IV. Jounal Cell. Physiol. 2009;219:344-353

31) _{I. Nishino, J. Fu, K. Tanji, T. Yamada, S. Shimojo, T. Koori, M. Mora, J. E.} Riggs, S. J. Oh, Y. Koga, C. M. Sue, A. Yamamoto, N. Murakami, S. Shanske, E. Byrne, E. Bonilla, I. Nonaka, S. Dimauro, and M. Hirano: Primary

LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease). Nature. 2000;406:906-910

32) A. M. Cuervo, L. Stefanis, R. Fredenburg, P. T. Lansbury, and D. Sulzer: Impaired Degradation of Mutant α-Synuclein by Chaperone-Mediated Autophagy. Sceince. 2004;305:1292-1295

33) Y. Wang, M. Martinez-Vicente, U. Krüger, S. Kaushik, E. Wong, E. M. Mandelkow, A. M. Cuervo, and E. Mandelkow: Tau fragmentation,

aggregation and clearance: The dual role of lysosomal processing. Hum. Mol. Genet. 2009;18:4153-4170

(37)

Neurotrauma. 2015;32:1449-1457

35) E. Dohi, S. Tanaka, T. Seki, T. Miyagi, I. Hide, T. Takahashi, M. Matsumoto, and N. Sakai: Hypoxic stress activates chaperone-mediated autophagy and modulates neuronal cell survival. Neurochem. Int.

2012;60:431-442

36) S. Brun, N. Schall, S. R. Bonam, K. Bigaut, A. G. Mensah-Nyagan, J. de Sèze, and S. Muller: An autophagy-targeting peptide to treat chronic inflammatory demyelinating polyneuropathies. J. Autoimmun. 2018;92:114-125

37) _{S. Muller, S. Brun, F. René, J. De Sèze, J. Loef, and H. Jeltsch-david:} Autophagy in neuroinflammatory diseases. Autoimmun. Rev.

2017;16:856-874

38) _{H. Dong, A. Fazzaro, C. Xiang, S. J. Korsmeyer, M. F. Jacquin, and J. W.} McDonald: Enhanced oligodendrocyte survival after spinal cord injury in bax-deficient mice and mice with delayed Wallerian degeneration. J. Neurosci. 2003;23:8682-8691

39) N. Kalderon and Z. Fuks: Structural recovery in lesioned adult mammalian spinal cord by x-irradiation of the lesion site. Proc. Natl. Acad. Sci. U. S. A. 1996;93:11179-11184

40) M. Yang, P. Chen, J. Liu, S. Zhu, and G. Kroemer: Clockophagy is a novel selective autophagy process favoring ferroptosis. Sci. Adv. 2019;5:eaaw2238 41) _{C. M. Smith, J. A. Mayer, and I. D. Duncan: Autophagy promotes}

oligodendrocyte survival and function following dysmyelination in a long-lived myelin mutant. J. Neurosci. 2013;33:8088-8100

42) _{J. Lu, K. W. S. Ashwell, and P. Waite: Advances in secondary spinal cord} injury: Role of apoptosis. Spine (Phila. Pa. 1976). 2000;25:1859-1866

43) B. A. Citron, P. M. Arnold, C. Sebastian, F. Qin, S. Malladi, S. Ameenuddin, M. E. Landis, and B. W. Festoff: Rapid Upregulation of Caspase-3 in Rat Spinal Cord after Injury: mRNA, Protein, and Cellular Localization Correlates with Apoptotic Cell Death. Exp. Neurol. 2000;166:213-226 44) _{C. H. I. Yong, M. Arnold, N. Zoubine, and A. Citron: Apoptosis in cellular}

compartments of rat spinal cord after severe contusion Injury. J. Neurotrauma. 1998;15:459-472

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2009;33:143-148

46) H. Kanno, H. Ozawa, A. Sekiguchi, S. Yamaya, S. Tateda, K. Yahata, and E. Itoi: The role of mTOR signaling pathway in spinal cord injury. Cell Cycle. 2012;11:3175-3179

47) A. M. Cuervo, J. F. Dice, and E. Knecht: A population of rat liver lysosomes responsible for the selective uptake and degradation of cytosolic proteins. J. Biol. Chem. 1997;272:5606-5615

48) S. Kaushik and A. M. Cuervo: Methods to Monitor Chaperone-Mediated Autophagy. Methods Enzym. 2009;452:297-324

49) _{S. Lindquist and E. A. Craig: THE HEAT-SHOCK PROTEINS. Annu. Rev.} Gent. 1988;22:631-677

50) B. Bukau, J. Weissman, and A. Horwich: Molecular Chaperones and Protein Quality Control. Cell. 2006;125:443-451

51) J. Anguiano, T. P. Garner, M. Mahalingam, B. C. Das, E. Gavathiotis, and A. M. Cuervo: Chemical modulation of chaperone-mediated autophagy by retinoic acid derivatives. Nat. Chem. Biol. 2013;9:374-382

52) M. Rothaug, S. Stroobants, M. Schweizer, J. Peters, F. Zunke, M. Allerding, R. D. Hooge, P. Saftig, and J. Blanz: LAMP-2 deficiency leads to hippocampal dysfunction but normal clearance of neuronal substrates of

chaperone-mediated autophagy in a mouse model for Danon disease. 2015;1-17

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1. CMA β β KFERQ HSC70 / LAMP2A Lys-HSC70 2. CMA CMA β β β β 3. 10 4. ± 500 g, 10 min 145,000 g, 120 min

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10% 2 PBS 18,000 g, 30 min 5. sham LAMP2A A, B A B sham LAMP2A β R L LAMP2A β C LAMP2A β Scale bars: 500 µm A, B , 10 µm C 6. LAMP1 LAMP2A LAMP2A LAMP1 β Scale bars: 100 µm A , 50 µm B

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7. HSC70 LAMP1 HSC70 LAMP1 β Scale bars: 100 µm A , 50 µm B 8. LAMP2A β A LAMP2A β 24 3 7 Scale bars: 100 µm , 10 µm B A 9. 250 µm 3 LAMP2A β 24 , 3, 7 L LAMP2A β sham P < 0.05, n = 5 3 L LAMP2A β R †_{P < 0.05,} _{n = 5}

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10. HSC70 β A HSC70 β Scale bar: 100 µm , 10 µm B A 11. 250 µm 3 HSC70 β HSC70 β L R HSC70 β n = 5 12. LAMP2A LAMP2A 4 3 21 3 LAMP2A sham P < 0.05, n = 5

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13. HSC70 HSC70 n = 5 14. LAMP2A β A, B 3 NeuN β LAMP2A C, D GFAP β LAMP2A E, F Olig2 β LAMP2A ) Scale bars: 20 µm A, C, E , 10 µm B, D, F G A 15. sham A-I Sham β A, nc D, β E, 3 β B, C, nc β B, C, F-I, J-R 3 β

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LAMP2A K, L, O-R,

sham J, M, N,

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HSC70

LAMP2A

Lys-HSC90 Lys-HSC70 GFAP P KFERQ KFERQ HSC70 1. CMA H SC 7 0 _P KF ER Q

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CMA

?

2. CMA

• 

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. 1 6

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4. 33

145,000 ×g 120 min

18,000 ×g 30 min 500 ×g 10 min

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5. sham LAMP2A

A

L

R

DAPI

LAMP2A

L

R

B

DAPI

LAMP2A

DAPI

LAMP2A

Sham

Hemisection

DAPI

LAMP2A

DAPI

LAMP2A

DAPI

LAMP2A

C

L

R

L

R

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. LAMP1 LAMP2A

A

B

DAPI LAMP1 DAPI

LAMP1 LAMP2A LAMP2A DAPI LAMP1 LAMP2A LAMP2A LAMP1 DAPI

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7. HSC70 LAMP1 DAPI LAMP1 HSC70 HSC70 LAMP1 DAPI DAPI LAMP1 HSC70 HSC70 LAMP1 DAPI

A

B

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8. LAMP2A Sham 4 h 24 h 3 d 7 d 21 d Ventral

DAPI LAMP2A DAPI LAMP2A DAPI LAMP2A DAPI LAMP2A

DAPI LAMP2A DAPI LAMP2A

DAPI LAMP2A DAPI LAMP2A DAPI LAMP2A DAPI LAMP2A DAPI LAMP2A DAPI LAMP2A DAPI LAMP2A DAPI LAMP2A Dorsal A B Left _Right

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. 250 µm 3 LAMP2A 0 100 200 300 400 Sham L R 4h 24h 3d 7d 21d

†

L R L R L R L R L R The number of L A MP2 A -p o s iti v e c e ll s

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10. HSC70 Dorsal Ventral DAPI HSC70 DAPI HSC70 DAPI HSC70 DAPI HSC70 DAPI HSC70 DAPI HSC70 DAPI HSC70 DAPI HSC70 DAPI HSC70 DAPI HSC70 DAPI HSC70 DAPI HSC70 DAPI HSC70 DAPI HSC70 DAPI HSC70 DAPI HSC70 DAPI HSC70 DAPI HSC70 A B Left _Right Sham 4 h 24 h 3 d 7 d 21 d

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. 250 µm 3 HSC70 The number of H SC 7 0 -p o s iti v e c e ll s 0 50 100 150 1 2 3 4 5 6 Sham L R 4h 24h 3d 7d 21d L R L R L R L R L R

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12. 1 9 LAMP2A Sham 4h 24h 3d 7d 21d LAMP2A β-tubulin 0 1 2 3 1 2 3 4 5 6 Sham 4h 24h 3d 7d 21d Pr o te in e x p re s s io n (A rb itr a ry u n it)

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13. 1 9 HSC70 HSC70 β-tubulin Pr o te in e x p re s s io n (A rb itr a ry u n it) 0 0.5 1 1.5 sham 4h 1d 3d 7d 21d Sham 4h 24h 3d 7d 21d Sham 4h 24h 3d 7d 21d

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14. LAMP2A

Dorsal

Ventral

NeuN LAMP2A NeuN

LAMP2A

A NeuN LAMP2A NeuN

LAMP2A

C GFAP LAMP2A GFAP

LAMP2A

GFAP LAMP2A GFAP

LAMP2A

E Olig2 LAMP2A Olig2

LAMP2A

Olig2 LAMP2A Olig2

LAMP2A B D F G Right Left

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15. sham

EM

Im

m

u

n

e

EM

A

nc nc nc

Sham

Hemisection

nc nc nc

脊髄損傷におけるシャペロン介在性オートファジーの検討