Amyloid -peptide ࡟ࡼࡿᑠ⬊యࢫࢺࣞࢫㄏⓎᾏ㤿⚄⤒⣽⬊

Amyloid -peptide ࡟ࡼࡿᑠ⬊యࢫࢺࣞࢫㄏⓎᾏ㤿⚄⤒⣽⬊

Ṛቑᙉᶵᵓ࡜ S -allyl- L -cysteine ࡢ⚄⤒ಖㆤస⏝࡟㛵ࡍࡿ◊✲

௒ ஭ ᚭ

2014

1

┠ḟ

➨ 1 ❶ ㅖゝ

2

➨ 2 ❶ ᇵ㣴ᾏ㤿ษ∦࡟࠾ࡅࡿ A

ቑᙉᶵᵓ

8

2-1 ᗎㄽ

9

2-2 ᪉ἲ

11

2-3 ⤖ᯝ

15

2-4 ⪃ᐹ

24

2-5 ᑠᣓ

28

➨ 3 ❶ ᑠ⬊యࢫࢺࣞࢫㄏⓎᾏ㤿⚄⤒⣽⬊Ṛ࡟࠾ࡅࡿ calpain ࡟ᑐࡍࡿ SAC ࡢ స⏝

30

2-1 ᗎㄽ

31

3-2 ᪉ἲ

33

3-3 ⤖ᯝ

35

3-4 ⪃ᐹ

41

3-5 ᑠᣓ

45

➨ 4 ❶ ⥲ᣓ

47

ཧ⪃ᩥ⊩

50

ㅰ㎡

60

2

➨ 1 ❶ ㅖゝ

3

࢔ࣝࢶࣁ࢖࣐࣮⑓ (AD)ࡣࠊㄆ▱⑕ࡢ 1 ࡘ࡟ศ㢮ࡉࢀࡿ⚄⤒ኚᛶ⑌ᝈ࡛ࠊ⌧ᅾ ࡢᡃࡀᅜ࡟࠾ࡅࡿㄆ▱⑕ࡢ௦⾲ⓗ࡞⑌ᝈ࡛࠶ࡿࠋ᭱኱ࡢ༴㝤ᅉᏊࡀࠕຍ㱋࡛ࠖ

࠶ࡿ AD ࡣࠊ㉸㧗㱋໬♫఍ࢆ㏄࠼࡚࠸ࡿᡃࡀᅜ࡟࠾࠸࡚ࠊᝈ⪅ᩘࡀቑຍࡢ୍㏵

ࢆࡓ࡝ࡾࠊཌ⏕ປാ┬ࡢᝈ⪅ㄪᰝࡢᴫἣ࡟ࡼࡿ࡜ࠊᖹᡂ 20 ᖺᗘࡣ 24 ୓ே࡜᥎ ᐃࡉࢀࡓᝈ⪅ᩘ (1)ࡀࠊᖹᡂ 23 ᖺᗘ࡟ࡣ 36 ୓ே࡜ 1.5 ಸ࡟ቑຍࡋ࡚࠾ࡾ (2)ࠊ

♫఍ⓗ࡟ࡶ኱ࡁ࡞ၥ㢟࡜࡞ࡗ࡚࠸ࡿࠋAD ࡢ⑓⌮Ꮫⓗ≉ᚩ࡜ࡋ࡚ࡣࠊ᪩ᮇ⑓ኚ

࡜ࡋ࡚⪁ேᩬࡀㄆࡵࡽࢀࠊࡑࡢᚋࠊ⚄⤒ཎ⥺⥔ኚ໬ࡀ⏕ࡌࠊ᭱⤊ⓗ࡟ࡣ⚄⤒⣽

⬊ࡢኚᛶ࣭⬺ⴠ࡞࡝ࢆ⏕ࡎࡿࡇ࡜ࡀ᫂࠿࡜࡞ࡗ࡚࠸ࡿ (3)ࠋ⪁ேᩬࡣ୺࡜ࡋ࡚

⚄⤒⣽⬊እ࡟ỿ╔ࡍࡿ amyloid -peptide (A )࡟ࡼࡾᙧᡂࡉࢀࠊ⚄⤒ཎ⥺⥔ኚ໬

ࡣ⚄⤒⣽⬊ෆ࡟⵳✚ࡍࡿ⥺⥔≧ࡢࢱࣥࣃࢡ㉁ࡼࡾ࡞ࡾࠊ tau ࢱࣥࣃࢡ㉁ࡀ㐣๫࡟

ࣜࣥ㓟໬ࡉࢀ࡚ᙧᡂࡉࢀࡿࠋ AD ࡢཎᅉ࡟ࡘ࠸࡚ࡣㅖㄝ࠶ࡿࡶࡢࡢࠊᐙ᪘ᛶ AD ࡢཎᅉ㑇ఏᏊ࡜ࡋ࡚ྠᐃࡉࢀࡓ amyloid precursor protein (APP)ࠊ presenilin 1 (PS1)ࠊ presenilin 2 (PS2)࡞࡝ࡢኚ␗ࡢࡍ࡭࡚ࡀ A ࡢ⏘⏕㔞ࢆቑຍࡉࡏࡿࡇ࡜ (4)ࠊ⚄⤒

ཎ⥺⥔ኚ໬ࡀ AD ࡔࡅ࡛ࡣ࡞ࡃࠊከࡃࡢ⚄⤒ኚᛶ⑌ᝈ࡛ㄆࡵࡽࢀࡿࡢ࡟ᑐࡋ࡚ࠊ AD ࡟࠾࠸࡚ࡣ A

A ࡀ AD Ⓨ⑕࡟㔜せ࡞ᙺ๭ࢆ₇ࡎࡿ࡜࠸࠺ -࢔࣑ࣟ࢖ࢻ௬ㄝࡀᗈࡃཷࡅධࢀࡽ

ࢀ࡚࠸ࡿࠋࡋ࠿ࡋࠊ㏆ᖺࡢ⏬ീデ᩿ᢏ⾡ࡢ㐍Ṍ࡟ࡼࡾࠊ A ࡢ⵳✚ࢆྍど໬ࡍࡿ

ࡇ࡜ࡀྍ⬟࡟࡞ࡿ࡜ (6)ࠊA

࿌ࡉࢀࡿࡼ࠺࡟࡞ࡿ (7)࡜࡜ࡶ࡟ࠊኚ␗ APP ࡢࢺࣛࣥࢫࢪ࢙ࢽࢵࢡ࣐࢘ࢫ⬻࡛

ࡣ A ࡢỿ╔ࡀከᩘほᐹࡉࢀࡿࡀࠊ⚄⤒⣽⬊Ṛࡀㄆࡵࡽࢀ࡞࠸ࡇ࡜ࡶሗ࿌ࡉࢀ࡚

࠸ࡿ (8)ࠋࡇࢀࡽࡢࡇ࡜࠿ࡽࠊA ࡢ⵳✚ࡔࡅ࡛ࡣ AD Ⓨ⑕ࡣㄝ࡛᫂ࡁࡎࠊ㛗ᮇ 㛫࡟ࢃࡓࡿ A

AD

ࢆⓎ⑕ࡍࡿ࡜࠸࠺᪂ࡓ࡞ㄝࡀᥦၐࡉࢀࡿࡼ࠺࡟࡞ࡾࠊ A

せᅉࡢ᳨ドࡀ┒ࢇ࡟⾜ࢃࢀ࡚࠸ࡿࠋᾏ㤿ࡣࠊ኱⬻⓶㉁ഃ㢌ⴥࡢෆഃ㠃࡟఩⨨ࡋࠊ グ᠈ࠊᏛ⩦࡟㔜せ࡞⬻㒊఩࡛ࠊAD ࡟࠾࠸࡚㞀ᐖࢆཷࡅࡸࡍࡃࠊᾏ㤿ࡢ⚄⤒⣽

⬊Ṛࡀㄆ▱㞀ᐖⓎ⑕࡟㛵୚ࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠾ࡾ (9)ࠊ AD ࡢண㜵ἲࡸ἞⒪ἲ

4

࡟㛵ࡍࡿᐇ㦂ⓗ◊✲࡟ከ⏝ࡉࢀ࡚࠸ࡿࠋᙜ◊✲ᐊ࡛ࡣࠊࣛࢵࢺᾏ㤿ษ∦ᇵ㣴⣔

࡟࠾࠸࡚ࠊAD Ⓨ⑕࡟㛵㐃ࡍࡿ࡜⪃࠼ࡽࢀࡿࢢࣝࢱ࣑ࣥ㓟ཷᐜయࡢࢧࣈࢱ࢖ࣉ

࡛࠶ࡿ

-methyl-

-aspartate (NMDA)ཷᐜయࡢ␗ᖖ࡞άᛶ໬࡟ࡼࡾㄏⓎࡉࢀࡿ⚄

⤒⣽⬊Ṛࢆࠊ A ࡀቑᙉࡍࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋ࡚࠾ࡾࠊ⯆ዧẘᛶࡀࡑࡢせᅉࡢ୍

ࡘ࡛࠶ࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓ (10)ࠋ

㏆ᖺࠊAD Ⓨ⑕࡟ᑠ⬊యࡢᶵ⬟୙඲࡟ࡼࡗ࡚⏕ࡌࡿᑠ⬊యࢫࢺࣞࢫࡀ㛵୚ࡋ

࡚࠸ࡿࡇ࡜ࡀ♧ࡉࢀ (11)ࠊA ࡢẘᛶ࡜ࡢ㛵㐃ᛶࡀὀ┠ࡉࢀ࡚࠸ࡿࠋᑠ⬊యࡣࠊ

⭷ࢱࣥࣃࢡ㉁ࡸศἪࢱࣥࣃࢡ㉁࡞࡝ࡢྜᡂ࡜ಟ㣭ࠊ⣽⬊ෆ Ca

ࢫࢺ࢔࡜ࡋ࡚ࡢ ᶵ⬟ࡸ⬡㉁௦ㅰ࡞࡝ࢆᢸ࠺㔜せ࡞⣽⬊ෆᑠჾᐁ࡛࠶ࡿࠋࡇࡢᑠ⬊యᶵ⬟ࡀឤᰁࠊ

⅖⑕ࠊ㑇ఏᏊኚ␗ࠊຍ㱋ࠊ⹫⾑➼ࡢ่⃭࡟ࡼࡾጉࡆࡽࢀࠊᵓ㐀␗ᖖࢱࣥࣃࢡ㉁

ࡀ⵳✚ࡍࡿ࡜ᑠ⬊యࢫࢺࣞࢫ࡜࿧ࡤࢀࡿ≧ែࡀ⏕ࡌࡿ(12)ࠋᑠ⬊యࢫࢺࣞࢫࡀ

Ⓨ⏕ࡍࡿ࡜ࠊinositol-requiring enzyme (IRE1)ࠊPKR-like endoplasmic reticulum kinase (PERK)ࠊactivating transcription factor 6 (ATF6)࡞࡝ࡢࢫࢺࣞࢫࢭࣥࢧ࣮࡟

ࡼࡾ unfolded protein response (UPR)࡜࿧ࡤࢀࡿᶵᵓࡀ㥑ືࡉࢀࠊᵓ㐀␗ᖖࢱࣥࣃ

ࢡ㉁ࡢᢡࡾࡓࡓࡳࢆಁ㐍ࡍࡿ glucose-regulated protein (GRP)94ࠊ GRP78 ࡞࡝ࡢศ Ꮚࢩࣕ࣌ࣟࣥࢆㄏᑟࡍࡿࡇ࡜࡛ࠊࢱࣥࣃࢡ㉁ࡢྜᡂࡸศゎࢆไᚚࡋࠊࢫࢺࣞࢫ

ࢆᅇ㑊ࡍࡿ (12)ࠋࡋ࠿ࡋࠊᑠ⬊యࢫࢺࣞࢫࢆᘬࡁ㉳ࡇࡍ่⃭ࡀ㐣๫࡞ሙྜࡸࢫ ࢺࣞࢫࡀ㛗᫬㛫ᣢ⥆ࡋᅇ㑊࡛ࡁ࡞࠿ࡗࡓሙྜ࡟ࡣࠊ࢔࣏ࢺ࣮ࢩࢫ⤒㊰ࢆάᛶ໬

ࡋ࡚᰾ࡢ᩿∦໬ࡸจ㞟ࢆ㉳ࡇࡋࠊ⣽⬊ṚࢆㄏⓎࡍࡿࡇ࡜ࡀ᫂࠿࡜࡞ࡗ࡚࠸ࡿ

(11)ࠋ AD ࡢ༴㝤ᅉᏊ࡛࠶ࡿຍ㱋ࡸ⚄⤒⅖⑕ࡣࠊ UPR ࢆపୗࡉࡏࠊᵓ㐀␗ᖖࢱࣥ

ࣃࢡ㉁ࡢ⵳✚࡟ࡼࡾࠊᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛࢆᘬࡁ㉳ࡇࡍࡇ࡜ (13, 14)ࠊ

࠾ࡼࡧ A

caspase-12 ࡢάᛶ໬ࢆ௓ࡋ࡚⣽⬊ṚࢆㄏⓎࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (15)ࠋᙜ◊✲ᐊ࡟࠾

࠸࡚ࡶࠊึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊࡟࠾࠸࡚ࠊA ㄏⓎ⣽⬊Ṛ࡟ caspase-12 ࡢάᛶ໬

ࢆ௓ࡋࡓᑠ⬊యࢫࢺࣞࢫࡀ㛵୚ࡋ࡚࠸ࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿ (16)ࠋࡲࡓࠊ㛗ᮇ

㛫 (6~7 㐌㛫)ᇵ㣴ࡋࡓࣛࢵࢺᾏ㤿ษ∦ᇵ㣴⣔࡟࠾࠸࡚ࠊ A

caspase-12 ࡢάᛶ

5

໬ࢆ௓ࡋ࡚⣽⬊ṚࢆㄏⓎࡍࡿࡇ࡜ࢆぢฟࡋ࡚࠸ࡿ (17)ࠋࡇࡢࡼ࠺࡟ࠊ A

యࢫࢺࣞࢫ࡟㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀᙉࡃ♧၀ࡉࢀࡿࡀࠊຍ㱋࡞࡝࡟ࡼࡾㄏⓎࡉࢀ

ࡿᑠ⬊యࢫࢺࣞࢫ࡟ࡼࡿ⣽⬊ẘᛶ࡟ᑐࡋ࡚ࠊ A ࡀ࡝ࡢࡼ࠺࡞ᙳ㡪ࢆཬࡰࡍࡢ࠿

࡟ࡘ࠸࡚ࡣࠊ୙᫂ࡢࡲࡲ࡛࠶ࡿࠋ

AD ࡛ࡣⓎ⑕ࡢึᮇ࠿ࡽ࣐࢖ࢿࣝࢺࡢᇶᗏ᰾ࡸᾏ㤿࡟࠾࠸࡚ࢥࣜࣥసືᛶ⚄

⤒ࡀኚᛶ࣭⬺ⴠࡋࠊࡇࢀࡽ࡟ࡼࡾグ᠈㞀ᐖࢆⓎ⑕ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀࡓࡇ

࡜࠿ࡽ (18)ࠊ࢔ࢭࢳࣝࢥࣜࣥ㔞ࢆ⥔ᣢࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋ࡚୰ᯡᣦྥᛶࡢ࢔ࢭ ࢳࣝࢥ࢚ࣜࣥࢫࢸ࣮ࣛࢮ㜼ᐖ⸆ࡀ㛤Ⓨࡉࢀࠊdonepezil (19)ࠊgalantamine (20)ࠊ

rivastigmine (21)ࡀୖᕷ࡟⮳ࡗ࡚࠸ࡿࠋࡲࡓࠊAD ࡢ㐍⾜ᮇ࡛ࡣ NMDA ཷᐜయࡢ

␗ᖖ⯆ዧࡀㄆࡵࡽࢀࠊ⚄⤒ఏ㐩࡟࠾ࡅࡿࣀ࢖ࢬࡀቑ኱ࡍࡿࡇ࡜ࠊ࠾ࡼࡧ NMDA

ཷᐜయᣕᢠ⸆࡛࠶ࡿ memantine ࡀࠊࡑࡢ␗ᖖ≧ែࢆᢚไࡋㄆ▱ᶵ⬟ࢆᨵၿࡍࡿ

ࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀ (22)ࠊ἞⒪⸆࡜ࡋ࡚ୖᕷࡉࢀࡓࠋࡋ࠿ࡋࠊࡇࢀࡽࡢ⸆≀ࡣࠊ

࠸ࡎࢀࡶ⑓ែࡢ㐍⾜ࢆ⦆ᚎ࡟ࡍࡿᑐ⑕⒪ἲ⸆࡛࠶ࡾࠊ᰿ᮏ἞⒪ࡣ୙ྍ⬟࡛࠶ࡿࠋ

ࡲࡓࠊ↓ຠ࡞⑕౛ࡸ๪స⏝ࡢࡓࡵ࡟౑⏝࡛ࡁ࡞࠸⑕౛ࡶሗ࿌ࡉࢀ࡚࠾ࡾ (22, 23)ࠊ⌧ᅾࡶ᪂ࡓ࡞἞⒪⸆ࡢ㛤Ⓨࡀᚅࡓࢀ࡚࠸ࡿࠋ

ࢽࣥࢽࢡࡣ⁠㣴ᙉኊࠊ⑂ປᅇ᚟࡞࡝ࡢᵝࠎ࡞೺ᗣຠᯝࢆᮇᚅࡋ࡚ࠊẸ㛫⸆࡜

ࡋ࡚ᗈࡃ⏝࠸ࡽࢀ࡚࠸ࡿࠋ⏕ࢽࣥࢽࢡࢆ࢚ࢱࣀ࣮ࣝỈ⁐ᾮ୰࡛⇍ᡂࡉࡏࡓ⇍ᡂ ࢽࣥࢽࢡ࢚࢟ࢫ୰࡟ྵࡲࢀࡿỈ⁐ᛶపศᏊ

ࢳ࢜࢔ࣜࣝ໬ྜ≀࡛࠶ࡿ

-allyl-

-cysteine (SAC) (Fig. 1)ࡣࠊᢠ㓟໬స⏝ (24-26)ࠊᢠࡀ

ࢇస⏝(27)ࠊᢠ⫢ẘᛶస⏝ (28)ࠊ⚄⤒ᰤ㣴

ᅉᏊస⏝ (29)࡞࡝ᵝࠎ࡞⏕⌮άᛶࢆᣢࡘࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋ SAC ࡣ㧗࠸Ỉ

⁐ᛶ࡜Ᏻᐃᛶࢆ᭷ࡋࠊ⤒ཱྀᦤྲྀ࡟ࡼࡾᾘ໬⟶࠿ࡽ྾཰ࡉࢀࡿࡀࠊ㔜⠜࡞๪స⏝

ࢆ♧ࡉ࡞࠸ࡇ࡜ࡀࡍ࡛࡟᫂ࡽ࠿࡜࡞ࡗ࡚࠸ࡿ (30)ࠋࡉࡽ࡟ࠊ SAC ࡢ⤒ཱྀᢞ୚࡟

ࡼࡾ⪁໬ಁ㐍࣐࢘ࢫ࡛ㄆࡵࡽࢀࡿຍ㱋࡟క࠺Ꮫ⩦㞀ᐖࢆᨵၿࡍࡿࡇ࡜ (31)ࡸࠊ

ࣛࢵࢺᾏ㤿ෆ࡬ࡢ A

ࡢ⭡⭍ෆᢞ

Fig. 1. Chemical structure of S-allyl-^L- cysteine㻌

6

୚ࡀᢚไࡍࡿࡇ࡜ (32)ࡀሗ࿌ࡉࢀ࡚࠾ࡾࠊᮎᲈᢞ୚࡟ࡼࡾ୰ᯡ⚄⤒࡬⛣⾜ࡋࠊ

୰ᯡಖㆤస⏝ࢆ᭷ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀ࡚࠸ࡿࠋᙜ◊✲ᐊࡣࠊ SAC ࡢ⸆⌮స⏝࡟

ὀ┠ࡋ࡚࠾ࡾࠊ SAC ࡀ PC12 ⣽⬊࡟࠾࠸࡚ A ㄏⓎ⣽⬊Ṛࢆᢚไࡍࡿࡇ࡜ (33)ࠊ

ึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊࡟࠾࠸࡚ A ࡟ࡼࡾㄏⓎࡉࢀࡓᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊

Ṛࡸᑠ⬊యࢫࢺࣞࢫㄏᑟ⸆࡛࠶ࡿ tunicamycin (TM)ㄏⓎ⣽⬊Ṛࢆ㢧ⴭ࡟ᢚไࡍ

ࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿ (16)ࠋࡇࡢࡼ࠺࡟ SAC ࡣࠊᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛ

࡟ᑐࡋ࡚ᢚไస⏝ࢆ♧ࡍࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀࡓࡶࡢࡢࠊ SAC ࡀᾏ㤿⚄⤒⣽⬊࡟

࠾࠸࡚ࠊ࡝ࡢࡼ࠺࡞࣓࢝ࢽࢬ࣒࡛ᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛᢚไస⏝ࢆ♧ࡍࡢ

࠿࡟ࡘ࠸࡚ࡣ୙᫂ࡢࡲࡲ࡛࠶ࡿࠋ

ࡑࡇ࡛ࠊᮏ◊✲࡛ࡣࠊ A

ᾏ㤿≉᭷ࡢ⚄⤒ᅇ㊰࠾ࡼࡧࡑࡢᶵ⬟ࡀಖᣢࡉࢀ࡚࠸ࡿษ∦ᇵ㣴⣔࡟࠾࠸࡚ࠊ A ࡀᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛ࡟ཬࡰࡍᙳ㡪࡟ࡘ࠸࡚⢭ᰝࡋࡓࠋࡲࡓࠊSAC ࡢ⚄

⤒ಖㆤస⏝ࡢᶵᗎ࡟ࡘ࠸࡚ヲ⣽࡟᳨ウࡋࡓࠋ

7

ᮏㄽᩥࡣ௨ୗࡢㄽᩥࢆᇶ♏࡜ࡍࡿࡶࡢ࡛࠶ࡿࠋ

1. Toru Imai, Yasuhiro Kosuge, Kaori Endo-Umeda, Hiroko Miyagishi, Kumiko Ishige, Makoto Makishima, Yoshihisa Ito.

Protective effect of

-allyl-

-cysteine against endoplasmic reticulum stress-induced neuronal death is mediated by inhibition of calpain.

Amino Acids, 46, 385-393, 2014.

2. Toru Imai, Yasuhiro Kosuge, Kumiko Ishige, Yoshihisa Ito.

Amyloid -protein potentiates tunicamycin-induced neuronal death in organotypic hippocampal slice cultures.

Neuroscience, 147, 639-651, 2007.

8

➨ 2 ❶

ᇵ㣴ᾏ㤿ษ∦࡟࠾ࡅࡿ A ࡟ࡼࡿ

ᑠ⬊యࢫࢺࣞࢫㄏⓎᾏ㤿⚄⤒⣽⬊Ṛቑᙉᶵᵓ

9

2-1 ᗎ ᗎㄽ

A

APP ࢆ⣽⬊እ࡛ษ᩿ࡍࡿ -ࢭࢡࣞࢱ࣮ࢮ࡜⭷

㈏㏻㡿ᇦࡢ⣽⬊㉁ഃ࡛ษ᩿ࡍࡿ -ࢭࢡࣞࢱ࣮ࢮ࡟ࡼࡗ࡚ẁ㝵ⓗ࡟ษ᩿ࡉࢀ࡚⏕

ᡂࡍࡿࠋ -ࢭࢡࣞࢱ࣮ࢮࡣࠊAPP ࢆ⭷㈏㏻㒊఩ෆ࡟࠾࠸࡚ࠊ40 ࡶࡋࡃࡣ 42 ␒

┠ࡢ࢔࣑ࣀ㓟࡛ษ᩿ࡋࠊ A

ࡲࡓࡣ A

ࢆ⏘⏕ࡉࡏࡿ(34)ࠋࡲࡓࠊࡇࡢ A

ࡲࡓࡣ A

ࡢ 25~35 ␒┠࡟࠶ࡿ 11 ṧᇶࡀࠊ⣽⬊ẘᛶࢆⓎ᥹ࡍࡿࡢ࡟ᚲせ࡞ࢥ

࢔㡿ᇦ࡜ࡋ࡚▱ࡽࢀ࡚࠸ࡿ (35)ࠋ A

(36, 37)ࡸ NMDA ཷᐜయάᛶ໬ (22)ࠊ c-Jun N-terminal kinase (JNK)ࡢάᛶ໬ (38)

࡞࡝ࢆ௓ࡋ࡚⚄⤒⣽⬊ṚࢆㄏⓎࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡋ࠿ࡋࠊ๓㏙ࡋࡓ

ࡼ࠺࡟ AD ࡢⓎ⑕࣓࢝ࢽࢬ࣒ࡣࠊA

࡞せᅉࡀ A

AD ࢆᘬࡁ㉳ࡇࡍ࡜⪃࠼ࡽࢀࡿࡼ࠺࡟࡞ࡾࡘࡘ࠶ࡿࠋ஦

ᐇࠊA ࡣ㓟໬ⓗࢫࢺࣞࢫ࡜㛵㐃ࡋࠊp53 ࡢ࢔࣏ࢺ࣮ࢩࢫ⤒㊰ࢆ௓ࡋࡓ⣽⬊Ṛࢆ

ቑᙉࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (39)ࠋࡲࡓࠊᙜ◊✲ᐊ࡟࠾࠸࡚ࡶࠊ3 㐌㛫ࡢᇵ 㣴࡟ࡼࡾᡂ⇍ࡋࡓᇵ㣴ᾏ㤿ษ∦࡟ᑐࡋ࡚ࡣẘᛶࢆ♧ࡉ࡞࠸⃰ᗘࡢ A

(6~7 㐌㛫)ᇵ㣴ࡋࡓᇵ㣴ᾏ㤿ษ∦࡟࠾࠸࡚ࡣࠊcaspase-12 ࡢάᛶ໬ࢆ௓ࡋ࡚⣽⬊

ṚࢆㄏⓎࡍࡿࡇ࡜ࢆぢฟࡋ࡚࠸ࡿ (17)ࠋࡋࡓࡀࡗ࡚ࠊAD ࡢ⑓ᅉゎ᫂ࡢࡓࡵ࡟

ࡣࠊA ༢⊂࡟ࡼࡿ⣽⬊ẘᛶࡢ◊✲ࡔࡅ࡛ࡣ࡞ࡃࠊAD Ⓨ⑕࡟㛵୚ࡍࡿせᅉࢆࡩ

ࡲ࠼࡚ࠊA ࡢ⣽⬊ṚㄏⓎ࣓࢝ࢽࢬ࣒ࢆ᳨ウࡍࡿᚲせࡀ࠶ࡿࠋ

๓㏙ࡋࡓᑠ⬊యࢫࢺࣞࢫࡀᘬࡁ㉳ࡇࡍ࢔࣏ࢺ࣮ࢩࢫ⤒㊰࡜ࡋ࡚ࠊJNK ⤒㊰

(38, 40)ࠊC/EBP-homologous protein (CHOP)⤒㊰ (41, 42)ࠊᑠ⬊య㛵㐃 caspase ⤒

㊰ (15) ࡀ ሗ ࿌ ࡉ ࢀ ࡚ ࠸ ࡿ ࠋ JNK ⤒ ㊰ ࡣ ᑠ ⬊ య ࢫ ࢺ ࣞ ࢫ ࡟ ࡼ ࡾ apoptosis signal-regulating kinase 1 ࡀ IRE1 ࡜」ྜయࢆᙧᡂࡍࡿࡇ࡜࡟ࡼࡾࠊ JNK ࢆࣜࣥ㓟

໬ࡋࠊ࢔࣏ࢺ࣮ࢩࢫࢆㄏᑟࡍࡿ (38, 40)ࠋࡲࡓࠊCHOP ࡣ C/EBP ࣇ࢓࣑࣮ࣜࡢ

㌿෗ᅉᏊ࡜ࡋ࡚ᶵ⬟ࡋࠊᑠ⬊యࢫࢺࣞࢫ࡟ࡼࡾ ATF6 ࠾ࡼࡧ PERK ࡢ⤒㊰࡟ࡼ

ࡗ࡚㌿෗ㄏᑟࡉࢀࠊBax ࡢ࣑ࢺࢥࣥࢻࣜ࢔⛣⾜ࢆ௓ࡋ࡚࢔࣏ࢺ࣮ࢩࢫࢆㄏᑟࡍ

ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (41) ࠋ୍᪉ࠊᑠ⬊యࢫࢺࣞࢫ≉␗ⓗ࡟άᛶ໬ࡍࡿ

10

caspase ⤒ ㊰ ࡜ ࡋ ࡚ ࠊ caspase-12 ࡀ 㱎 ṑ 㢮 ࡟ ࠾ ࠸ ࡚ ྠᐃ ࡉ ࢀ ࡚ ࠸ ࡿ (15) ࠋ Caspase-12 ࡣࠊ௚ࡢ caspase ࣇ࢓࣑࣮ࣜ࡜ྠᵝ࡟๓㥑య࡛࠶ࡿ pro-caspase-12 (60 kDa)ࡀྜᡂࡉࢀࠊᑠ⬊య⭷ࡢ⣽⬊㉁ഃ࡟ᒁᅾࡋࠊᑠ⬊యࢫࢺࣞࢫ㈇Ⲵ᫬࡟ษ᩿

ࡉࢀ࡚άᛶ໬యࡢ caspase-12 (42 kDa)࡜ࡋ࡚స⏝ࡋ⣽⬊Ṛࢆᘬࡁ㉳ࡇࡍ (15)ࠋ

Caspase-12 ࡢάᛶ໬ᶵᵓ࡟ࡘ࠸࡚ࡣࠊ⣽⬊ෆ Ca

⃰ᗘࡢୖ᪼࡟ࡼࡾάᛶ໬ࡉࢀ

ࡿ Ca

౫Ꮡᛶࢩࢫࢸ࢖ࣥࣉࣟࢸ࢔࣮ࢮ࡛࠶ࡿ calpain (43)࠾ࡼࡧ IRE1 ࡜ tumor necrosis factor receptor-associated factor 2 ࡢ」ྜయࡀ㛵୚ࡋ࡚࠸ࡿ࡜࠸࠺ሗ࿌ࡀ

࠶ ࡿ (44, 45) ࠋ ά ᛶ ໬ ࡉ ࢀ ࡓ caspase-12 ࡣ ࠊ cytochrome c ࡸ apoptotic protease-activating factor 1 ࡜ࡣ㛵ಀࡏࡎࠊ caspase-9ࠊ caspase-3 ࢆάᛶ໬ࡍࡿ⤒㊰

ࡀ♧ࡉࢀ࡚࠾ࡾࠊᚑ᮶ࡲ࡛▱ࡽࢀ࡚࠸ࡓ࣑ࢺࢥࣥࢻࣜ࢔⤒㊰ࡢάᛶ໬࡟ࡼࡿ⣽

⬊Ṛᐇ⾜࢝ࢫࢣ࣮ࢻ࡜␗࡞ࡿ⤒㊰ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (44, 46, 47)ࠋࡋ࠿ࡋࠊA ࡀࠊࡇࡢࡼ࠺࡞ᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛ࡟ᑐࡋ࡚ࠊ࡝ࡢࡼ࠺࡞ᙳ㡪ࢆཬࡰࡍ ࡢ࠿ࡣ୙࡛᫂࠶ࡿࠋ

ᾏ㤿ࡣ㛫⬻ࡢ⫼ഃࢆ㉮ࡿ୕᪥᭶ᵝᵓ㐀ࢆ࡞ࡋࠊ㗹య⣽⬊ࡀ༨ࡵࡿ࢔ࣥࣔࣥゅ (cornu ammonis : CA)࡜㢛⢏⣽⬊ࡀ༨ࡵࡿṑ≧ᅇ (dentate gyrus : DG)࡟ࡼࡾᵓᡂ ࡉࢀࡿࠋࡉࡽ࡟ࠊᾏ㤿ࡢ㗹య⣽⬊ࡣࠊ⣽⬊ࡢ኱ࡁࡉ࡟ࡼࡾᑠ⣽⬊ᛶࡢ CA1 ࡜኱

⣽⬊ᛶࡢ CA3 㡿ᇦ࡟ศ㢮ࡉࢀࡿࠋᾏ㤿ษ∦ᇵ㣴⣔ࡣ⣽⬊ࡢ఩⨨㛵ಀࡸ⚄⤒ᅇ㊰

ࡀ

࡜ྠࡌ≧ែࢆಖᣢࡋ࡚࠾ࡾࠊ㛗ᮇ࡟ࢃࡓࡿ⸆≀ࡢฎ⨨ࡀྍ⬟࡞࡝ࡢ฼

Ⅼ࠿ࡽࠊ⚄⤒⣽⬊Ṛࡢ࣓࢝ࢽࢬ࣒ࡢゎ᫂ࡢࡓࡵ࡟✚ᴟⓗ࡟⏝࠸ࡽࢀ࡚࠸ࡿ (48, 49)ࠋ

ࡑࡇ࡛ࠊᮏ❶࡛ࡣࠊA ࡢ⣽⬊ṚㄏⓎ࣓࢝ࢽࢬ࣒ࢆ᫂ࡽ࠿࡟ࡍࡿ┠ⓗ࡛ࠊᾏ㤿 ษ∦ᇵ㣴⣔࡟࠾࠸࡚ࠊ A

ウࡋࡓࠋࡲࡓࠊ SAC ࡢᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛ࡟ᑐࡍࡿಖㆤస⏝࡟ࡘ࠸࡚ࡶ

ేࡏ᳨࡚ウࡋࡓࠋ

11

2-2 ᐇ㦂᪉ἲ

1.

౑⏝⸆≀

ᮏ◊✲࡛౑⏝ࡋࡓ⸆ရࡣ௨ୗࡢ఍♫࠿ࡽ㉎ධࡋࡓࡶࡢࢆ⏝࠸ࡓࠋ A

ࡣᰴᘧ

఍♫࣌ࣉࢳࢻ◊✲ᡤࡼࡾࠊanti-caspase-12 monoclonal antibodyࠊHanks’ balanced salts (HBSS)ࠊprotease inhibitor cocktailࠊtunicamycinࠊanti-caspase-12 polyclonal antibody ࡣ Sigma-Aldrich ࡼࡾࠊ sodium hydrogen carbonateࠊ D (+)-glucoseࠊ bovine serum albumin (BSA)ࡣ࿴ග⣧⸆ᕤᴗᰴᘧ఍♫ࡼࡾࠊMinimum Essential Medium

(MEM)ࡣ Invitrogen ࡼࡾࠊ⤖ᬗ࣌ࢽࢩࣜࣥ Gࠊ◲㓟ࢫࢺࣞࣉࢺ࣐࢖ࢩࣥࡣ᫂἞

〇Ⳬᰴᘧ఍♫ࡼࡾࠊ࣐࢘⾑Ύࡣ TRACE ࡼࡾࠊpropidium iodide (PI)ࡣ Molecular Probes ࡼࡾࠊ caspase-12 inhibitor ࡣ Biovision ࡼࡾࠊHEPESࠊ BAPTA-AM ࡣྠோ

໬Ꮫ◊✲ᡤࡼࡾࠊ Tween 20 ࡣ National Diagnostics ࡼࡾࠊ anti- -spectrin monoclonal antibody ࡣ CHEMICOM ࡼࡾࠊ anti-caspase-3 monoclonal antibody ࡣ Cell Signaling Technology, Inc ࡼ ࡾ ࠊ anti-KDEL monoclonal antibody ࡣ stressgen ࡼ ࡾ ࠊ anti-GADD153/CHOP polyclonal antibodyࠊ anti-rat-IgG-horseradish peroxidase (HRP)ࠊ anti-rabbit-IgG-horseradish peroxidase ࠊ anti-mouse-IgG-HRP ࡣ Santa Cruz Biotechnology, Inc ࡼࡾࠊECL ࡣ Amersham Biosciences ࡼࡾ㉎ධࡋࡓࠋࡲࡓࠊࡑ ࡢ௚ࡢ⸆ရࡣヨ⸆≉⣭ࢆ౑⏝ࡋࡓࠋ

2.

ᇵ㣴ᾏ㤿ษ∦ࡢㄪ〇

ᇵ㣴ᾏ㤿ษ∦ࡢㄪ〇ࡣ Ito ࡽࡢሗ࿌ (10)࡟ᚑࡗࡓࠋື≀ᐇ㦂ࡣࠊ᪥ᮏ኱Ꮫ⸆

Ꮫ㒊ື≀ᐇ㦂ᣦ㔪࡟ᇶ࡙࠸࡚⾜ࡗࡓࠋWistar ⣔㞤㞝ࣛࢵࢺࡣࠊ୕༠ࣛ࣎ࢧ࣮ࣅ

ࢫᰴᘧ఍♫ࡼࡾ㉎ධࡋࠊ᪥ᮏ኱Ꮫ⸆Ꮫ㒊ᐇ㦂ື≀ࢭࣥࢱ࣮࡛㣫⫱࣭஺㓄ࡋࠊዷ ፎࣛࢵࢺࢆᚓࡓࠋᐇ㦂࡟ࡣዷፎࣛࢵࢺࡢฟ⏘࡟ࡼࡾᚓࡽࢀࡓ 6~7 ᪥㱋ࡢ᪂⏕ࣛ

ࢵࢺࢆ⏝࠸ࡓࠋ⏕ᚋ 6~7 ᪥㱋ࡢ Wistar ⣔ࣛࢵࢺ࠿ࡽ㯞㓉ୗࠊ↓⳦ⓗ࡟ᾏ㤿ࢆ᦬

ฟࡋࠊMcIlwain ᆺ tissue chopper (The Mickle Laboratory Engineering)ࢆ⏝࠸࡚ࠊ

ཌࡉ 400 m ࡟ⷧษࡋࠊ6-well culture plate (IWAKI) ࡟ᤄධࡋࡓ Millicell-CM

12

(Millipore)ࡢ⭷ୖ࡛ᇵ㣴ࡋࡓࠋ࣓ࢹ࢕࣒࢘ࡣ MEM 50%ࠊHBSS 25%ࠊ࣐࢘⾑Ύ

25%࡜࡞ࡿࡼ࠺࡟ΰྜࡋࠊࡇࡇ࡟࣌ࢽࢩࣜࣥ࡜ࢫࢺࣞࣉࢺ࣐࢖ࢩࣥࢆࡑࢀࡒࢀ

100 units/mLࠊ 0.1 mg/mL ࡜࡞ࡿࡼ࠺࡟ῧຍࡋࡓࡶࡢࢆ⏝࠸ࡓࠋᾏ㤿ษ∦ࡣࠊ 5%

CO

/ 95% airࠊ32

Cࠊຍ‵᮲௳ୗࡢ࢖࣮ࣥ࢟ࣗ࣋ࢱ࣮ෆ୍࡛ᐃᮇ㛫ᇵ㣴ࡋࡓࠋ

ᇵ㣴ᮇ㛫୰ࡣ࣓ࢹ࢕࣒࢘ࡢ඲㔞஺᥮ࢆ 2~3 ᪥ẖ࡟⾜ࡗࡓࠋ

3.

ᇵ ᇵ㣴ᾏ㤿ษ∦࡟ᑐࡍࡿ⸆≀ࡢᭀ㟢

⸆≀ࡢᭀ㟢┤๓࡟ᇵ㣴⏝ࡢ࣓ࢹ࢕࣒࢘ࢆ↓⾑Ύ࣓ࢹ࢕࣒࢘࡟⨨᥮ࡋࡓࠋᭀ㟢

ࡣ Bruce ࡽࡢሗ࿌ (36)࡟ᚑ࠸ࠊ⸆≀ࢆ⁐ゎࡋࡓ↓⾑Ύ࣓ࢹ࢕࣒࢘ 100 L ࢆษ∦

࡟┤᥋⁲ୗࡋࡓࠋ

4.

⣽⬊Ṛࡢホ౯

⣽⬊Ṛࡢホ౯࡟ࡣࠊPI ᰁⰍࡲࡓࡣ Lactate dehydrogenase (LDH) release ἲࢆ⏝

࠸ࡓࠋ

ᰁⰍ

୍ᐃ᫬㛫ࡢ⸆≀ᭀ㟢ᚋࠊ࣓ࢹ࢕࣒࢘ࢆ PI (50 g/mL)ࢆྵࡴ Locke ᾮ [154.0 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl

, 3.6 mM NaHCO

, 5.0 mM HEPES-NaOH, pH

7.4]࡟⨨᥮ࡋࠊ 30 ศ㛫ࡢ࢖࣮ࣥ࢟ࣗ࣋ࢺᚋࠊಽ❧ᘧ఩┦ᕪ㢧ᚤ㙾ࢆ⏝࠸ࠊ 340 nm

ࡢບ㉳ගࢆ↷ᑕࡋ࡚⺯ගീࢆほᐹࡋࠊ෗┿᧜ᙳࢆ⾜ࡗࡓࠋ PI ࡢ⺯ගᙉᗘࡣ Sion image software ࢆ⏝࠸࡚ゎᯒࡋࡓࠋᐃ㔞ἲࡣ Abdel ࡽࡢሗ࿌ (50)࡟ᚑ࠸ࠊ⺯ගࡀ

᭱኱࡛ࣉࣛࢺ࣮࡟࡞ࡿ TM 320 g/mL ࡛ฎ⨨ࡋࡓษ∦ࡢ PI ⺯ගᙉᗘࢆ᭱኱⺯ග ᙉᗘ (

max)ࠊྛ⸆≀ฎ⨨࡟ࡼࡿ⺯ගᙉᗘࢆ (

i)ࠊࣂࢵࢡࢢࣛ࢘ࣥࢻࢆ (

)࡜

ࡋࠊ௨ୗࡢᘧ࡟ࡼࡾ⣽⬊Ṛࡢ๭ྜࢆ⟬ฟࡋࡓࠋ

Cell death (%) = (

i –

) / (

max –

) × 100

13 (2) LDH release

ἲ ἲ

࣓ࢹ࢕࣒࢘୰࡬ࡢ LDH ࡢ㐟㞳㔞ࡢ ᐃࡣࠊLDH-Cytotoxic Test Wako (࿴ග⣧

⸆ᕤᴗᰴᘧ఍♫)ࢆ⏝࠸࡚⾜ࡗࡓࠋ୍ᐃ᫬㛫ࡢ⸆≀ᭀ㟢ᚋࠊ well ୰ࡢ࣓ࢹ࢕࣒࢘

50 L ࢆ 96-well culture plate ࡟⛣ࡋࠊⓎⰍヨ⸆ࢆ 50 L ࡎࡘຍ࠼ࠊᐊ ࡛ 45 ศ 㛫཯ᛂࡉࡏࡓᚋࠊ཯ᛂ೵Ṇᾮࢆ 100 L ࡎࡘຍ࠼ࡓࠋ཯ᛂ೵Ṇ࠿ࡽ 10 ศᚋ࡟࣐

࢖ࢡࣟࣉ࣮ࣞࢺ࣮ࣜࢲ࣮ (BIO-RAD)ࢆ⏝࠸࡚ 570 nm ࡢ྾ගᗘࢆ ᐃࡋࡓࠋ

5. Nissl

ᰁⰍ

ᇵ㣴ᾏ㤿ษ∦࡟ᑐࡋ࡚ࠊtoluidine blue (1 mg/mL)ࢆ 15 ศ㛫ฎ⨨ࡋࠊ⢭〇Ỉ࡜

࢚ࢱࣀ࣮࡛ࣝὙίࡋࡓࠋࡉࡽ࡟ࠊtoluidine blue (1 mg/mL)ࢆ 15 ศ㛫ฎ⨨ࡋࠊ࢚

ࢱࣀ࣮࡛ࣝὙίࡋࡓࠋᰁⰍࡋࡓษ∦ࢆಽ❧ᘧ఩┦ᕪ㢧ᚤ㙾ࢆ⏝࠸࡚ࠊ෗┿᧜ᙳ

ࢆ⾜ࡗࡓࠋ

6.

ࢱࣥࣃࢡ㉁Ⓨ⌧ࡢホ౯

ࢱࣥࣃࢡ㉁Ⓨ⌧ࡢホ౯࡟ࡣࠊ Western blot ἲࢆ⏝࠸ࡓࠋ୍ᐃ᫬㛫ࡢ⸆≀ᭀ㟢ᚋ

࡟࣓ࢹ࢕࣒࢘ࢆ㝖ཤࡋࠊphosphate buffered saline (PBS) [137 mM NaCl, 2.7 mM KCl, 1.5 mM Na

HPO

, 8.1 mM KH

PO

]࡛Ὑίࡋࡓᚋࠊlysis buffer [25 mM HEPES-NaOH, pH 7.4, 10 mM EGTA, 10 mM EDTA, 50 mM NaCl, 1% TritonX-100, 0.5% SDS, 1% sodium deoxycholate, 20 mM 2-mercaptoethanol, Complete protease inhibitor cocktail (Roche Diagnostics), phosphatase inhibitor cocktail (Sigma-Aldrich)]୰࡟ᅇ཰ࡋࡓࠋࢱࣥࣃࢡᐃ㔞ࢆ⾜࠸ࠊ୍ᐃࡢ⃰ᗘ࡟࡞ࡿࡼ࠺࡟

sample buffer [125 mM Tris-HCl (pH 6.0), 3% SDS, 10% glycerol, 5% 2-mercapto

ethanol, 0.2% bromophenol blue]ࢆຍ࠼࡚ SDS ໬ࡋࡓࡶࡢࢆࢧࣥࣉࣝ࡜ࡋࠊ㟁Ẽ

ὋືࡋࡓࠋὋື⤊஢ᚋ࡟ Immobilon™-P Transfer Membrane (Millipore)࡟㌿෗ࡋࠊ

membrane ࢆ blocking buffer [5% skim milk in Tris buffered saline (TBS) [20 mM

Tris-HCl (pH 7.6), 137 mM NaCl] ]୰࡟⛣ࡋࠊ✜ࡸ࠿࡟ᨩᢾࡋ࡞ࡀࡽᐊ ࡛ 1 ᫬㛫

14

ࣈࣟࢵ࢟ࣥࢢࡋࡓࠋࣈࣟࢵ࢟ࣥࢢ⤊஢ᚋࠊ0.05% Tween 20 ࢆྵࡴ Tris buffered

saline (TTBS)࡛Ὑίࡋࠊ┠ⓗࢱࣥࣃࢡ㉁ࡢ୍ḟᢠయࢆ 4

C ୍࡛ᬌ཯ᛂࡉࡏࡓࠋ

TTBS ࡛ὙίᚋࠊHRP ࡛ᶆ㆑ࡉࢀࡓ஧ḟᢠయࢆᐊ ࡛✜ࡸ࠿࡟᧠ᢾࡋ࡞ࡀࡽ 1

᫬㛫཯ᛂࡉࡏࠊὙίᚋ࡟ ECL ࡟ࡼࡾⓎⰍࡉࡏࡓࠋෆ㒊ᶆ‽࡜ࡋ࡚ -actin ࢆ⏝࠸

ࡓࠋࣂࣥࢻࡣ Scion image software ࢆ⏝࠸࡚ゎᯒࡋࡓࠋࣂࣥࢻࡢᐃ㔞ࡣࠊ -actin ࡢࣂࣥࢻᙉᗘ࡟ᑐࡍࡿྛࢱࣥࣃࢡ㉁ࡢࣂࣥࢻᙉᗘࡢ๭ྜ࡛⾲ࡋࡓࠋ

7.

⤫ ⤫ィᏛⓗฎ⌮

ࡍ࡭࡚ࡢ್ࡣᖹᆒ್ ± ᶆ‽ㄗᕪ࡛♧ࡋࡓࠋ᭷ពᕪ᳨ᐃࡣ୍ඖ㓄⨨ศᩓศᯒἲ

(one-way ANOVA)࡛ゎᯒᚋࠊTukey

s multiple test ࢆ⏝࠸ࡓࠋ

15

2-3 ⤖ᯝ

1. A25-35

ࠊTM ࠾ࡼࡧࡑࡢే⏝ࡀᇵ㣴ᾏ㤿ษ∦ࡢ⏕Ꮡ࡟ཬࡰࡍᙳ㡪

ᇵ㣴ᾏ㤿ษ∦ࡢ⏕Ꮡ࡟ཬࡰࡍ A ࠾ࡼࡧ TM ࡢᙳ㡪࡟ࡘ࠸᳨࡚ウࡋࡓࠋᐇ㦂࡟

ࡣࠊA

࠾ࡼࡧ A

ࡢࢥ࢔㓄ิ㒊ศ࡛ࠊA

ࠊA

࡜࡯ࡰྠ➼ࡢ⣽⬊ẘ ᛶࢆ♧ࡍࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿ A

ࢆ⏝࠸ࡓࠋᇵ㣴 3 㐌㛫ᚋࡢᡂ⇍ࣛࢵ

ࢺᾏ㤿ษ∦࡟ࠊ2-2 ࡛♧ࡋࡓ᪉ἲ࡛ A

25 Mࠊ50 M ࢆ 48 ᫬㛫༢⊂ᭀ㟢ࡋ ࡓ࡜ࡇࢁࠊṚ⣽⬊ࢆ♧ࡍ PI 㝧ᛶ⣽⬊ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ (Fig. 2A, B)ࠋ୍᪉ࠊ

TM 20ࠊ40ࠊ80 g/mL ࢆ༢⊂࡛ 48 ᫬㛫ᭀ㟢ࡋࡓ࡜ࡇࢁࠊ⃰ᗘ౫Ꮡⓗ࡞ PI 㝧ᛶ

⣽⬊ࡢቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2A, B)ࠋTM ࡣࠊ20 g/mL ࡢᭀ㟢࡟ࡼࡾࠊDG 㡿 ᇦ࡟࠾࠸࡚ࡢࡳ PI 㝧ᛶ⣽⬊ࢆቑຍࡉࡏࡓࡀࠊ 40 g/mL ࡛ࡣࠊ DG 㡿ᇦࡔࡅ࡛ࡣ

࡞ࡃ CA1 㡿ᇦ࡟࠾࠸࡚ࡶ PI 㝧ᛶ⣽⬊ࢆቑຍࡉࡏࠊ80 g/mL ࡢᭀ㟢࡟ࡼࡾࠊᾏ 㤿ࡢ඲㡿ᇦ࡛ PI 㝧ᛶ⣽⬊ࢆቑຍࡉࡏࡓ (Fig. 2D)ࠋḟ࡟ࠊ⣽⬊ṚࢆㄏⓎࡋ࡞࠿

ࡗࡓ A

25 M ࡀ TM ㄏⓎ⣽⬊Ṛ࡟ཬࡰࡍᙳ㡪࡟ࡘ࠸᳨࡚ウࡋࡓࠋ A

25

M ࡜ TM 20 g/mL ࡢే⏝ᭀ㟢ࡣࠊ TM 20 g/mL ༢⊂࡟ẚ࡭ PI 㝧ᛶ⣽⬊ࢆቑຍ ࡉࡏࡓ (Fig. 2A, B)ࠋࡉࡽ࡟ࠊA

25 M ࡜ TM 40 g/mL ࡢే⏝ᭀ㟢࡟࠾࠸

࡚ࡶࠊ TM 40 g/mL ༢⊂࡟ẚ࡭ PI 㝧ᛶ⣽⬊ࡢ㢧ⴭ࡞ቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2A,

B)ࠋᇵ㣴ᾏ㤿ษ∦࠿ࡽࡢ LDH ࡢ㐟㞳㔞ࡢኚ໬࡟ࡘ࠸࡚ࡶ᳨ウࡋࡓ࡜ࡇࢁࠊ

A

25 M ࡢ༢⊂ᭀ㟢࡛ࡣࠊLDH 㐟㞳㔞࡟ኚ໬ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࡀࠊTM

ᭀ㟢࡟ࡼࡾ⃰ᗘ౫Ꮡⓗ࡞ LDH 㐟㞳㔞ࡢቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2C)ࠋࡲࡓࠊ

A

25 M ࡜ TM 20 g/mL ࠾ࡼࡧ TM 40 g/mL ࡢే⏝ᭀ㟢ࡣࠊ TM ༢⊂ᭀ㟢

࡜ẚ㍑ࡋ᭷ព࡞ LDH 㐟㞳㔞ࡢቑຍࡀㄆࡵࡽࢀࡓ (Fig. 2C)ࠋࡑࡇ࡛ࠊA

࡟

ࡼࡿ TM ㄏⓎ⣽⬊Ṛቑᙉస⏝࡟ࡘ࠸࡚ࠊ㡿ᇦู࡟ゎᯒࢆ⾜ࡗࡓ࡜ࡇࢁࠊTM 20 g/mL ࡜ࡢే⏝ᭀ㟢ࡢሙྜ࡟ࡣࠊTM ༢⊂࡜ẚ㍑ࡋ࡚ࠊCA1ࠊ DG 㡿ᇦ࡟࠾࠸࡚

㢧ⴭ࡞ PI 㝧ᛶ⣽⬊ࡢቑຍࡀㄆࡵࡽࢀࡓࡀࠊ CA3 㡿ᇦ࡟ࡣ᭷ព࡞ኚ໬ࡀㄆࡵࡽࢀ

࡞࠿ࡗࡓࠋ୍᪉ࠊTM 40 g/mL ࡜ࡢే⏝ᭀ㟢ࡢሙྜࡣࠊTM ༢⊂࡜ẚ㍑ࡋ࡚ࠊ

CA3 㡿ᇦ࡟࠾࠸࡚㢧ⴭ࡞ PI 㝧ᛶ⣽⬊ࡢቑຍࡀ㉳ࡇࡾࠊCA1ࠊDG 㡿ᇦ࡛ࡣ᭷ព

16

࡞ኚ໬ࡀㄆࡵࡽࢀ࡞࠿ࡗࡓ (Fig. 2D)ࠋ

TM 20g/mL TM 40 g/mL TM 80 g/mL

+A25 M +A25 M

control A25M A50M

(A)

(B) (C)

(D)

None TM None

20 g/mL

TM 20 g/mL TM

40 g/mL

TM 40 g/mL A(-)

A(+)

A(-) A(+)

*

* * **

*

*

**

CA1 CA3 DG

LDH release (% of control) Fluorescence (% of maximum cell death) Fluorescence (% of maximum cell death)

#

#

+ +

+

$

CA1

CA3 DG

Nissl staining

Fig. 2. Effects of A25-35, TM and A25-35 in combination with TM (A+TM) on PI fluorescence and LDH release in rat organotypic hippocampal slice cultures (OHCs). The OHCs were prepared as described in Materials and Methods. (A) Representative photomicrographs of neuronal death assessed by PI fluorescence in OHCs treated with A25-35, TM or A+TM. Aliquots (100 L) containing A25-35, TM or A+TM were applied directly to the top of each slice. PI fluorescence was measured 48 h after addition of each drug solution. Nissl-stained sections of 3-week-old OHC. (B) Quantitative analysis of A25-35-, TM- and A+TM-induced neuronal death in OHCs. (C) After exposure, the amount of LDH released into the medium was assayed as described in Materials and Methods. (D) Quantitative analysis of A25-35-, TM- and A+TM-induced neuronal death in areas CA1, CA3 and the DG. Fluorescence intensity (data shown in Fig. 2A) was analyzed quantitatively using the Scion image software package. Each value represents the mean ± S.E.M. for four (B and D) and five (C) different experiments. ^#P < 0.001, ^$P < 0.001, ⁺P < 0.001 when compared with each control. *P <

0.05, **P < 0.01.㻌

17

2. A25-35

ࠊ

࠾ࡼࡧࡑࡢే⏝ࡀᇵ㣴ᾏ㤿ษ∦ࡢᑠ⬊యࢫࢺࣞࢫ㛵㐃ࢱࣥࣃࢡ

㉁

࠾ࡼࡧ

ࡢⓎ⌧࡟ཬࡰࡍᙳ㡪

A

ࡼࡿ TM ㄏⓎ⣽⬊Ṛࡢቑᙉ࣓࢝ࢽࢬ࣒ࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵ࡟ࠊ A

ࠊ

TM ࠾ࡼࡧ㢧ⴭ࡞⣽⬊Ṛቑᙉࡀㄆࡵࡽࢀࡓ A

25 M ࡜ TM 40 g/mL ࡢే⏝

ᭀ㟢ࡀࠊᑠ⬊యࢫࢺࣞࢫ㛵㐃ࢱࣥࣃࢡ㉁ GRP94 (94 kDa)ࠊGRP78 (78 kDa)࠾ࡼ

ࡧ CHOP (26 kDa)ࡢⓎ⌧࡟ཬࡰࡍᙳ㡪࡟ࡘ࠸᳨࡚ウࡋࡓࠋA

25 M ༢⊂ᭀ

㟢࡛ࡣࠊ GRP94ࠊ GRP78 ࠾ࡼࡧ CHOP ࡢⓎ⌧ࣞ࣋ࣝ࡟ኚ໬ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ

ࡀࠊ TM ༢⊂ᭀ㟢࡟ࡼࡾࡇࢀࡽࡢⓎ⌧ࣞ࣋ࣝࡢ᭷ព࡞ୖ᪼ࡀㄆࡵࡽࢀࡓ (Fig. 3)ࠋ ࡋ࠿ࡋࠊA

ࡣ TM ࡟ࡼࡿࡇࢀࡽࡢࢱࣥࣃࢡ㉁Ⓨ⌧ୖ᪼࡟ᑐࡋ࡚ᙳ㡪ࢆཬࡰ

ࡉ࡞࠿ࡗࡓ (Fig. 3)ࠋ

(A) GRP94 (B) GRP78 (C) CHOP

TM 40 g/mL

A25 M - + - +

TM 40 g/mL

A25 M - + - +

TM 40 g/mL

A25 M - + - +

94 kDa

# -actin

Relative level ^#

78 kDa -actin

# #

Relative level

26 kDa -actin

#

Relative level

#

3. A25-35

ࠊ

࠾ࡼࡧࡑࡢే⏝ࡀᇵ㣴ᾏ㤿ษ∦ࡢ

άᛶ࠾ࡼࡧ

࡜

ࡢάᛶ໬యࡢⓎ⌧࡟ཬࡰࡍᙳ㡪

A

ࠊTM ࠾ࡼࡧࡑࡢే⏝ࡀ calpain άᛶ࠾ࡼࡧ caspase-12ࠊcaspase-3 ࡢά

ᛶ໬యࡢⓎ⌧࡟ཬࡰࡍᙳ㡪࡟ࡘ࠸᳨࡚ウࡋࡓࠋ Calpain άᛶࡣࠊ calpain ࡢάᛶ໬

Fig. 3. Effects of A25-35, TM and A+TM on ER chaperone induction and CHOP expression in OHCs.

(A) The expression of GRP94 (A), GRP78 (B) and CHOP (C) was investigated by Western blotting after exposure to A25-35, TM or A+TM for 48 h. Amounts of GRP94 (94 kDa), GRP78 (78 kDa) and CHOP (26 kDa) were assessed by densitometric analysis, and the relative level of each band was calculated as described in Materials and Methods. Each value represents the mean ± S.E.M. for four different experiments. ^#P < 0.05 as compared with the control.㻌

18

࡟ࡼࡾศゎࢆཷࡅࡿ⚄⤒≉␗ⓗ⣽⬊㦵᱁ࢱࣥࣃࢡ㉁ࡢ full-length -spectrin (240 kDa)ࡢศゎ⏘≀ࡢⓎ⌧ࣞ࣋ࣝࢆᣦᶆ࡜ࡋࡓࠋ A

25 M ༢⊂ᭀ㟢ࡣࠊ -spectrin ศゎ⏘≀ (145 kDa)࠾ࡼࡧ caspase-12 ࡢάᛶ໬య (42 kDa)ࠊcaspase-3 ࡢάᛶ໬

య (29 kDa)ࡢⓎ⌧ࣞ࣋ࣝ࡟ᙳ㡪ࢆཬࡰࡉ࡞࠿ࡗࡓࡀࠊTM ༢⊂ᭀ㟢ࡣࡑࢀࡽࡍ

࡭࡚ࡢⓎ⌧ࣞ࣋ࣝࢆ᭷ព࡟ୖ᪼ࡉࡏࡓ (Fig. 4)ࠋࡲࡓࠊ A

ࢆ TM ࡜ే⏝ᭀ㟢

ࡍࡿ࡜ࠊ GRP94ࠊ GRP78 ࠾ࡼࡧ CHOP ࡢሙྜ࡜ࡣ␗࡞ࡾࠊ TM ༢⊂࡜ẚ㍑ࡋ࡚ࠊ

᭷ព࡞ -spectrin ศゎ⏘≀࠾ࡼࡧ caspase-12ࠊ caspase-3 ࡢάᛶ໬యࡢⓎ⌧ୖ᪼ࡀ ㄆࡵࡽࢀࡓ (Fig. 4)ࠋTM ࠾ࡼࡧ A

࡜ TM ే⏝ (A +TM)ࡀㄏⓎࡍࡿ⣽⬊Ṛ

࡟࠾ࡅࡿ caspase-12 ࡢάᛶ໬ࡢ㛵୚ࢆࡉࡽ࡟᫂ࡽ࠿࡟ࡍࡿࡓࡵࠊ caspase-12 ࡢ≉

␗ⓗ㜼ᐖ⸆࡛࠶ࡿ z-ATAN-fmk (ATAN)๓ฎ⨨ࡀ TM ࠾ࡼࡧ A +TM ㄏⓎ⣽⬊Ṛ

࡟ཬࡰࡍᙳ㡪ࢆ᳨ウࡋࡓࠋATAN 50 M ࡣ TM ࠾ࡼࡧ A +TM ᭀ㟢 1 ᫬㛫๓࡟

ฎ⨨ࡋࡓ࡜ࡇࢁࠊTM ࠾ࡼࡧ A +TM ㄏⓎ⣽⬊Ṛࢆᾏ㤿ࡢ඲㡿ᇦ࡟࠾࠸࡚㢧ⴭ

࡟ᢚไࡋࡓ (Fig. 5)ࠋ

TM 40 g/mL

A25 M - + - +

145 kDa -actin

*

#

Relative level

-Spectrin breakdown products

42 kDa -actin

(B) Caspase-12

#

*

Relative level

TM 40 g/mL

A25 M - + - +

(C) Caspase-3

TM 40 g/mL

A25 M - + - +

29 kDa -actin

Relative level

#

*

Fig. 4. Effects of A25-35, TM and A+TM on calpain activity and levels of the cleaved forms of caspase-12 and caspase-3 in OHCs. Calpain activity was assessed by the amount of -spectrin breakdown products. -Spectrin breakdown products (A), and levels of the cleaved forms of caspase-12 (B) and caspase-3 (C), were investigated by Western blotting after exposure to A25-35, TM or A+TM for 48 h. Amounts of -spectrin breakdown products (145 kDa), caspase-12 (42 kDa) and caspase-3 (29 kDa) were assessed by densitometric analysis. Each value represents the mean ± S.E.M. for three or four different experiments. ^#P<0.05 as compared with the control. *P < 0.05 as compared with TM alone.㻌

19

TM 40 g/mL TM + A25M

(A)

None

+ ATAN 50 M

Fluorescence (% of maximum cell death)

(B)

CA1 CA3 DG

*** ***

** **

**

*

4. A25-35

ࡢ ㄏⓎ⣽⬊Ṛቑᙉ࡟ཬࡰࡍ

࠾ࡼࡧ

ࡢᙳ㡪

Fig. 4 ࡛♧ࡋࡓࡼ࠺࡟ࠊA

࡜ TM ࡢే⏝ࡣ TM ༢⊂ࡼࡾࡶ calpain άᛶࡢ

ࣞ࣋ࣝࢆୖ᪼ࡉࡏࡓࡇ࡜࠿ࡽࠊࡇࡢ calpain ࡢάᛶ໬ࡢஹ㐍࡟⣽⬊ෆ Ca

⃰ᗘࡢ

ୖ᪼ࡀ㛵୚ࡍࡿ࠿ྰ࠿ࢆࠊ⣽⬊ෆ Ca

࣮࢟ࣞࢺ⸆ࡢ BAPTA-AM ࠾ࡼࡧ L ᆺ Ca

ࢳࣕࢿࣝ㐽᩿⸆ࡢ nifedipine ࡀ TM ࠾ࡼࡧ A

࡜ TM ే⏝ (A +TM)ㄏⓎ⣽⬊

Ṛ࡟ཬࡰࡍᙳ㡪࡟ࡼࡾ᳨ウࡋࡓࠋ BAPTA-AM 10 M ࢆ TM ࠾ࡼࡧ A +TM ᭀ㟢 1 ᫬㛫๓࡟ฎ⨨ࡋࡓ࡜ࡇࢁࠊ TM ㄏⓎ⣽⬊Ṛ࡟ࡣᙳ㡪ࢆཬࡰࡉ࡞࠿ࡗࡓࡀࠊ CA3 㡿ᇦ࡟࠾࠸࡚ㄆࡵࡽࢀࡿ A

ࡢ TM ㄏⓎ⣽⬊Ṛቑᙉࢆ㢧ⴭ࡟ᢚไࡋࡓ (Fig.

Fig. 5. Effects of the caspase-12-specific inhibitor, z-ATAN-fmk (ATAN), on TM- and A+TM-induced neuronal death in OHCs. Cells were exposed to TM (40 g/mL) in the absence or presence of ATAN (50 M). (A) Representative photomicrographs of neuronal death assessed by PI fluorescence in OHCs. PI fluorescence images of OHCs were examined 48 h after the treatment.

(B) Quantitative analysis of neuronal death in areas CA1, CA3 and the DG. Fluorescence intensity (data shown in Fig. 5A) was analyzed quantitatively using the Scion image software package. Each value represents the mean ± S.E.M. for four different experiments. *P < 0.05, **P < 0.01, ***P <

0.001.㻌

20

6A, B)ࠋBAPTA-AM ฎ⨨ࡢᙳ㡪࡟ࡘ࠸࡚ LDH release ἲࢆ⏝࠸᳨࡚ウࡋࡓ࡜ࡇ

ࢁࠊBAPTA-AM 10 M ࡣࠊA +TM ࡟ࡼࡿ LDH 㐟㞳㔞ࡢቑຍࢆ᭷ព࡟ᢚไࡋ ࡓ (Fig. 6C)ࠋNifedipine ๓ฎ⨨ࡢᙳ㡪࡟ࡘ࠸࡚ࡶ᳨ウࡋࡓ࡜ࡇࢁࠊnifedipine 10 M ࡣࠊCA3 㡿ᇦ࡟࠾࠸࡚ㄆࡵࡽࢀࡿ A +TM ࡟ࡼࡿ⣽⬊Ṛࡢቑᙉࢆ㢧ⴭ࡟ᢚ ไࡋ (Fig. 6A, B)ࠊ A +TM ࡟ࡼࡿ LDH 㐟㞳㔞ࡢቑຍࢆ᭷ព࡟ᢚไࡋࡓ (Fig. 6C)ࠋ

TM 40 g/mL TM + A25M

+ Nifedipine 10 M None

(A)

+ BAPTA-AM 10 M

(B)

Fluorescence (% of maximum cell death)

CA1 CA3 DG

(C)

****

**

LDH release (% of control)

BAPTA-AM Nifedipine

TM TM + A

- + + +

+ + +

- - -

- - - - -

- - -

* *

*

Fig. 6. Effects of BAPTA-AM and nifedipine on TM- and A+TM-induced neuronal death in OHCs.

(A) Aliquots (100 L) containing TM or A+TM with or without BAPTA-AM and nifedipine were applied directly to the top of each slice. PI fluorescence was measured 48 h after addition of each drug solution. (B) Quantitative analysis of the effects of BAPTA-AM and nifedipine on TM- and A+TM-induced neuronal death in areas CA1, CA3 and the DG. Fluorescence intensity (data shown in Fig. 6A) was analyzed quantitatively using the Scion image software package. (C) After exposure, the amount of LDH released into the medium was assayed as described in Materials and Methods. Each value represents the mean ± S.E.M. for four different experiments. *P < 0.05, **P < 0.01.

21

5.

ᇵ ᇵ㣴ᾏ㤿ษ∦࡟࠾ࡅࡿ

ࡢ

ㄏⓎ⣽⬊Ṛቑᙉ࡟ཬࡰࡍ

ࡢᙳ㡪 ᇵ㣴 3 㐌㛫ᚋࡢᾏ㤿ษ∦࡟࠾ࡅࡿ TM ࠾ࡼࡧ A

࡜ TM ే⏝ (A +TM)ㄏ

Ⓨ⣽⬊Ṛ࡟ཬࡰࡍ SAC ࡢᙳ㡪࡟ࡘ࠸᳨࡚ウࡋࡓࠋᇵ㣴ᾏ㤿ษ∦ࡢ DG 㡿ᇦ࡟࠾

࠸࡚ࠊSAC ࡣ TM ㄏⓎ PI 㝧ᛶ⣽⬊ᩘࢆ᭷ព࡟ῶᑡࡉࡏࡓ (Fig. 7A, B)ࠋࡲࡓࠊ A +TM ㄏⓎ⣽⬊Ṛ࡟ᑐࡋ࡚ CA3ࠊDG 㡿ᇦ࡟࠾࠸࡚ࠊ᭷ព࡟ PI 㝧ᛶ⣽⬊ࢆῶ ᑡࡉࡏࡓࠋTM ࠾ࡼࡧ A +TM ㄏⓎ⣽⬊Ṛ࡟ᑐࡍࡿ SAC ࡢ⣽⬊ಖㆤస⏝ࢆホ౯ ࡍࡿࡓࡵࠊLDH 㐟㞳㔞ࡢኚ໬ࢆㄪ࡭ࡓ࡜ࡇࢁࠊSAC ࡣ TM ࡟ࡼࡿ LDH 㐟㞳㔞 ࡢቑຍ࡟ᑐࡋ࡚ᢚไഴྥࢆ♧ࡋࠊA +TM ࡟ࡼࡿ LDH 㐟㞳㔞ࡢቑຍࢆ᭷ព࡟ᢚ ไࡋࡓ (Fig. 7C)ࠋ

TM 40 g/mL TM + A25M

+ SAC 100 M

None

(A)

(B)

Fluorescence (% of maximum cell death)

CA1 CA3 DG

(C)

LDH release (% of control)

SAC - + - + - +

TM TM + A

**

** ***

*** ***

*

Fig. 7. Effects of SAC on TM- and A+TM-induced neuronal death in OHCs. (A) Aliquots (100 L) containing TM or A+TM with or without SAC were applied directly to the top of each slice. PI fluorescence was measured 48 h after addition of each drug solution. (B) Quantitative analysis of the effects of SAC on TM- and A+TM-induced neuronal death in areas CA1, CA3 and the DG.

Fluorescence intensity (data shown in Fig. 7A) was analyzed quantitatively using the Scion image software package. (C) After exposure, the amount of LDH released into the medium was assayed as described in Materials and Methods. Each value represents the mean ± S.E.M. for four different experiments. *P < 0.05, **P < 0.01, ***P < 0.001.㻌

22

6.

ᇵ ᇵ㣴ᾏ㤿ษ∦࡟࠾ࡅࡿᑠ⬊యࢫࢺࣞࢫ㛵㐃ࢱࣥࣃࢡ㉁ࡢⓎ⌧࡟ཬࡰࡍ

ࡢᙳ㡪

A

࡟ࡼࡿ TM ㄏⓎ⣽⬊Ṛቑᙉ࡟ᑐࡍࡿ SAC ࡢᢚไస⏝ࡢ࣓࢝ࢽࢬ࣒ࢆ᫂

ࡽ࠿࡟ࡍࡿ┠ⓗ࡛ࠊᑠ⬊యࢫࢺࣞࢫ㛵㐃ࢱࣥࣃࢡ㉁ࡢⓎ⌧ࣞ࣋ࣝࡢୖ᪼࡟ཬࡰ

ࡍ SAC ࡢᙳ㡪࡟ࡘ࠸᳨࡚ウࡋࡓࠋFig. 8 ࡟♧ࡋࡓࡼ࠺࡟ࠊSAC ࡣ TM ࠾ࡼࡧ

A

࡜ TM ే⏝ (A +TM)ᭀ㟢࡟ࡼࡾୖ᪼ࡋࡓ GRP94ࠊ GRP78ࠊ CHOP ࡢⓎ⌧

ࣞ࣋ࣝ࡟ࡣᙳ㡪ࢆཬࡰࡉ࡞࠿ࡗࡓࠋ୍᪉ࠊSAC ࡣ TM ᭀ㟢࡟ࡼࡾୖ᪼ࡋࡓ -spectrin ศゎ⏘≀࠾ࡼࡧ caspase-12 ࡢάᛶ໬యࡢⓎ⌧ࢆ᭷ព࡟ᢚไࡋࠊ A +TM

ᭀ㟢࡟ࡼࡾୖ᪼ࡋࡓ -spectrin ศゎ⏘≀࠾ࡼࡧ caspase-12ࠊ caspase-3 ࡢάᛶ໬య ࡢⓎ⌧ࡶ᭷ព࡟ᢚไࡋࡓ (Fig. 9)ࠋ

(A) GRP94 (B) GRP78 (C) CHOP

TM 40 g/mL SAC 100 M

TM 40 g/mL TM 40 g/mL

Relative level Relative level Relative level

TM + A

94 kDa 78 kDa

- + + ^{SAC 100 M} + + ^{SAC 100} M + +

TM + A TM + A

- - - - -

29 kDa

-actin -actin -actin

Fig. 8. Effects of SAC on ER chaperone induction and CHOP expression in OHCs. The expression of GRP94 (A), GRP78 (B) and CHOP (C) was measured after exposure to TM, A+TM or SAC for 48 h. Relative amounts of GRP94 (94 kDa), GRP78 (78 kDa) and CHOP (26 kDa) were assessed by densitometric analysis. Each value represents the mean ± S.E.M. for four different experiments.㻌

23

*

*

**

*

**

Relative level Relative level

-actin

Relative level

TM 40 g/mL SAC 100 M

TM 40 g/mL TM 40 g/mL

TM + A

- + + ^{SAC 100 M} + + ^{SAC 100} M + +

TM + A TM + A

- - - - -

-actin -actin

-Spectrin

breakdown products (B) Caspase-12 (C) Caspase-3

145 kDa 42 kDa 29 kDa

Fig. 9. Effects of SAC on ER stress-induced calpain activity and levels of the cleaved forms of caspase-12 and caspase-3 in OHCs. Calpain activity was assessed by the amount of -spectrin breakdown products. -Spectrin breakdown products (A) and levels of the cleaved forms caspase-12 (B) and caspase-3 (C) were measured after exposure to TM or A+TM with or without SAC for 48 h.

Relative amounts of -spectrin breakdown products (145 kDa), caspase-12 (42 kDa) and caspase-3 (29 kDa) were assessed by densitometric analysis, and the relative levels were calculated as described in Materials and Methods. Each value represents the mean s S.E.M. for three or four different experiments. *P < 0.05, **P < 0.01.㻌

24

2-4 ⪃ ⪃ᐹ

⪁ேᩬࡢ୺せᡂศ࡛࠶ࡿ A

ࡿ (18)ࠋ୍᪉ࠊᑠ⬊యࢫࢺࣞࢫࡀ AD Ⓨ⑕࡟࠾࠸࡚㔜せ࡞ᙺ๭ࢆᢸࡗ࡚࠸ࡿࡇ

࡜ࡀࠊᙜ◊✲ᐊࢆྵࡵࡓᩘከࡃࡢ◊✲⪅࡟ࡼࡗ࡚ሗ࿌ࡉࢀ࡚࠸ࡿ (11, 15, 16, 35,

51)ࠋࡲࡓࠊᑠ⬊యࢫࢺࣞࢫࡣࠊAD Ⓨ⑕ࡢ༴㝤ᅉᏊ࡜࡞ࡿ⚄⤒⅖⑕ࡸ⪁໬࡞࡝

࡟ࡶᐦ᥋࡟㛵ಀࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (13, 14)ࠋࡋ࠿ࡋࠊA

ࣞࢫㄏⓎ⣽⬊Ṛ࡟࡝ࡢࡼ࠺࡞ᙳ㡪ࢆཬࡰࡍࡢ࠿࡟ࡘ࠸࡚ࡣ୙᫂࡞Ⅼࡀከ࠸ࠋࡑ ࡇ࡛ᮏ❶࡛ࡣࠊA ࡢࢥ࢔㓄ิ࡛࠶ࡿ A

ࡀᑠ⬊యࢫࢺࣞࢫㄏⓎᾏ㤿⚄⤒⣽

⬊Ṛ࡟ᑐࡋ࡚࡝ࡢࡼ࠺࡞ᙳ㡪ࢆཬࡰࡍࡢ࠿ࠊᾏ㤿ࡢ⚄⤒ᵓ㐀ࢆಖᣢࡋࡓᾏ㤿ษ

∦ᇵ㣴⣔ࢆ⏝࠸᳨࡚ウࢆ⾜ࡗࡓࠋA

ࡣࠊึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊ࡸ 7 㐌㛫ᇵ 㣴ࡋࡓᾏ㤿ษ∦࡟࠾࠸࡚⚄⤒ẘᛶࢆ♧ࡍࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀ (16, 17)ࠊ3 㐌㛫ᇵ㣴ࡋࡓᾏ㤿ษ∦࡛ࡣࠊ A

25 Mࠊ 50 M ࡢ 48 ᫬㛫ᭀ㟢࡟ࡼࡿ⣽⬊Ṛ ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓ (Fig. 2)ࠋ୍᪉ࠊᑠ⬊యࡢ N-glycosylation 㜼ᐖ࡟ࡼࡾᑠ⬊య ࢫࢺࣞࢫࢆច㉳ࡍࡿ TM (52)༢⊂ᭀ㟢࡛ࡣࠊ⃰ᗘ౫Ꮡⓗ࡞⣽⬊ṚࡀㄏⓎࡉࢀࡓ

(Fig. 2)ࠋᙜ◊✲ᐊ࡟࠾࠸࡚ࡣ௨๓࡟ࠊึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊࡟࠾࠸࡚ TM ᭀ㟢

࡟ࡼࡾ⃰ᗘ౫Ꮡⓗ࡞⚄⤒⣽⬊Ṛࡀ㉳ࡇࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿࡀ (16)ࠊᮏ◊✲࡟

࠾ࡅࡿᾏ㤿ษ∦ᇵ㣴⣔࡟࠾࠸࡚ࡶ TM ࡣ⣽⬊ṚࢆㄏⓎࡍࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋ

ࡲࡓࠊFig. 2 ࡟♧ࡋࡓࡼ࠺࡟⣽⬊ṚࢆㄏⓎࡋ࡞࠸⃰ᗘࡢ A

25 M ࡜ TM ࡢ

ే⏝ᭀ㟢࡟ࡼࡾࠊTM ㄏⓎ⣽⬊ṚࡢቑᙉࡀㄆࡵࡽࢀࡓࠋA

࡜ TM ే⏝࡟ࡼ

ࡿ⣽⬊Ṛቑᙉస⏝ࢆᾏ㤿ࡢ㡿ᇦ (CA1ࠊCA3ࠊDG)ู࡟ゎᯒࡋࡓ࡜ࡇࢁࠊA

࡜ TM 20 g/mL ࡢే⏝ࡣ DG ࠾ࡼࡧ CA1 㡿ᇦࡢࠊ TM 40 g/mL ࡜ࡢే⏝ࡣ CA3 㡿ᇦࡢ⣽⬊Ṛࢆቑᙉࡉࡏࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓ (Fig. 2D)ࠋࡇࢀࡽࡢ⤖ᯝࡣࠊ

A

ࡢ TM ㄏⓎ⣽⬊Ṛቑᙉస⏝࡟ࡣᾏ㤿ࡢ㡿ᇦ≉␗ᛶࡀㄆࡵࡽࢀࡿࡇ࡜ࢆ♧

၀ࡋ࡚࠸ࡿࠋᙜ◊✲ᐊ࡛ࡣࠊෆᅾᛶࡢ pro-caspase-12 ࡢⓎ⌧㔞ࡀࠊᾏ㤿ࡢྛ㡿 ᇦ࡛␗࡞ࡾࠊ CA3 ࡟࠾ࡅࡿⓎ⌧㔞ࡣ CA1 ࡸ DG ࡜ẚ㍑ࡍࡿ࡜㢧ⴭ࡟ప࠸ࡇ࡜ࢆ

ሗ࿌ࡋ࡚࠸ࡿ (53)ࠋࡇࡢࡇ࡜ࡀ TM ㄏⓎ⣽⬊Ṛ࡟ᑐࡍࡿឤཷᛶࡢ㡿ᇦ≉␗ᛶ࡜

25

ᑡ࡞ࡃ࡜ࡶ୍㒊㛵㐃ࡍࡿࡶࡢ࡜⪃࠼ࡽࢀࡿࠋࡲࡓࠊᮏ◊✲࡛ㄆࡵࡽࢀࡓ A

࡟ࡼࡿ TM ㄏⓎ⣽⬊Ṛࡢቑᙉࡣࠊ pro-caspase-12 ࡢⓎ⌧ࣞ࣋ࣝࡢ㧗࠸ DG ࡲࡓࡣ CA1 㡿ᇦ࡛᪩ᮇ࡟ㄆࡵࡽࢀ࡚࠸ࡿࡇ࡜࠿ࡽࠊ A

࡟ࡼࡿ⣽⬊Ṛቑᙉࡣࠊ

pro-caspase-12 ࡢάᛶ໬ࡀ㛵୚ࡍࡿ࢔࣏ࢺ࣮ࢩࢫ⤒㊰ࢆ௓ࡋ࡚࠸ࡿࡇ࡜ࡀ♧၀

ࡉࢀࡓࠋ

A

࡟ࡼࡿ TM ㄏⓎ⣽⬊Ṛቑᙉᶵᵓࢆࡉࡽ࡟᫂ࡽ࠿࡟ࡍࡿࡓࡵࠊᑠ⬊యࢫ

ࢺࣞࢫ㛵㐃ࢱࣥࣃࢡ㉁ࡢᣲື࡟ࡘ࠸࡚ Western blot ἲࢆ⏝࠸᳨࡚ウࡋࡓࠋศᏊ ࢩ࡛ࣕ࣌ࣟࣥ࠶ࡿ GRP94ࠊGRP78 ࠾ࡼࡧ㌿෗ᅉᏊ࡛࠶ࡿ CHOP ࡢⓎ⌧ࡣࠊTM ༢⊂ᭀ㟢࡟ࡼࡾⓎ⌧ࣞ࣋ࣝࡀⴭࡋࡃୖ᪼ࡋࡓࡀࠊA

ࢆే⏝ࡋ࡚ࡶ TM ༢⊂

࡜ࡢ㛫࡟᭷ព࡞ᕪࡣㄆࡵࡽࢀࡎࠊቑᙉຠᯝࢆ♧ࡉ࡞࠿ࡗࡓ (Fig. 3)ࠋ CHOP ࣀࢵ

ࢡ࢔࢘ࢺ⣽⬊࡛ࡣࠊTM ࡟ࡼࡾㄏᑟࡉࢀࡿ࢔࣏ࢺ࣮ࢩࢫࡀᢚไࡉࢀࡿࡇ࡜࠿ࡽ

(54)ࠊCHOP ࡣ TM ࡟ࡼࡿᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛ࡟῝ࡃ㛵୚ࡍࡿࡇ࡜ࡀ♧

၀ࡉࢀ࡚࠸ࡿࠋᮏ◊✲࡟࠾࠸࡚ࡶࠊTM ᭀ㟢࡟ࡼࡾ CHOP ࡀㄏᑟࡉࢀࡓࡇ࡜࠿

ࡽࠊ⣽⬊Ṛ࡟῝ࡃ㛵୚ࡍࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡋ࠿ࡋࠊ A

ࡢే⏝ࡀࠊ TM ༢⊂ᭀ㟢࡟ࡼࡿ CHOP Ⓨ⌧ୖ᪼࡟ᙳ㡪ࢆཬࡰࡉ࡞࠿ࡗࡓࡇ࡜࠿ࡽࠊA

ࡢ TM ㄏⓎ⣽⬊Ṛቑᙉᶵᵓ࡟ࡣࠊศᏊࢩࣕ࣌ࣟࣥ࠾ࡼࡧ CHOP ࡣ㛵୚ࡋ࡞࠸ࡇ࡜

ࡀ♧၀ࡉࢀࡓࠋ Caspase-12 Ḟᦆ⬇ᛶ⥺⥔ⱆ⣽⬊ࡣࠊTM ㄏⓎ⣽⬊Ṛࡸ A ㄏⓎ⣽

⬊Ṛ࡟᢬ᢠᛶࢆ♧ࡍࡇ࡜(15)ࠊᇵ㣴ᾏ㤿⚄⤒⣽⬊࡟࠾࠸࡚ࠊA

caspase-12 ࡀ㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (16)ࠋࡑࡇ࡛ࠊA

࡟ࡼࡿ

TM ㄏⓎ⣽⬊Ṛቑᙉᶵᵓ࡬ࡢ calpainࠊcaspase-12 ࠾ࡼࡧᐇ⾜ caspase ࡛࠶ࡿ

caspase-3 ࡢ㛵୚࡟ࡘ࠸᳨࡚ウࢆ⾜ࡗࡓࠋ TM ᭀ㟢࡟ࡼࡾㄏⓎࡉࢀࡓ -spectrin ศ ゎ⏘≀ࡢ⏘⏕ (calpain άᛶ)࠾ࡼࡧ caspase-12ࠊcaspase-3 ࡢάᛶ໬యࡢⓎ⌧ࡣࠊ

A

࡜ࡢే⏝࡟ࡼࡾ᭷ព࡟ቑຍࡋࡓ (Fig. 4)ࠋࡲࡓࠊcaspase-12 ≉␗ⓗ㜼ᐖ๣

࡛࠶ࡿ ATAN ࡣࠊᾏ㤿඲㡿ᇦ࡟࠾࠸࡚ TM ࠾ࡼࡧ A +TM ࡟ࡼࡾᘬࡁ㉳ࡇࡉࢀ

ࡿ⣽⬊Ṛ࡟ᑐࡋ࡚ࠊ㢧ⴭ࡞ᢚไస⏝ࢆ♧ࡋࡓ (Fig. 5)ࠋࡇࢀࡲ࡛࡟ࠊኚ␗ PS1

ࢆ㐣๫Ⓨ⌧ࡉࡏࡓ PC12 ⣽⬊ࡣࠊcalpain ࡜ caspase-12 ࡢάᛶቑ኱࡟ࡼࡾ TM ㄏ

26

Ⓨ⣽⬊Ṛࡀቑᙉࡍࡿࡇ࡜ (55)ࡸࠊኚ␗ PS1 ࢆࣀࢵࢡ࢖ࣥࡋࡓึ௦ᇵ㣴኱⬻⓶㉁

⚄⤒⣽⬊࡛ࡣࠊcaspase-12 ࡢάᛶ໬ࡢቑ኱ࢆ௓ࡋ࡚ TM ㄏⓎ⣽⬊Ṛࡀቑᙉࡍࡿ

ࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (56)ࠋࡇࢀࡽࡢሗ࿌࡜ᮏ◊✲ࡢ⤖ᯝ࠿ࡽࠊA

ࡣ TM ㄏⓎ⣽⬊Ṛ࡟ᑐࡋ࡚ࠊcalpain ࡢάᛶࡢஹ㐍ࢆ௓ࡋ࡚ୗὶࡢ caspase-12 ࡢάᛶ໬

ࢆቑᙉࡋࠊࡑࡢ⤖ᯝࠊ⣽⬊Ṛᐇ⾜ᅉᏊ࡛࠶ࡿ caspase-3 ࡢάᛶ໬ࡢቑ኱ࢆᘬࡁ㉳

ࡇࡍࡇ࡜࡟ࡼࡾ⣽⬊Ṛࢆቑᙉࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋA

࡟ࡼࡿ⣽⬊Ṛቑᙉ ᶵᵓ࡟ calpain ࡢ㛵୚ࡀ♧၀ࡉࢀࡓࡇ࡜࠿ࡽࠊ calpain ࡢάᛶࡢஹ㐍࡟⣽⬊ෆ Ca

⃰ᗘࡢୖ᪼ࡀ㛵୚ࡍࡿ࠿ྰ࠿ࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵࠊBAPTA-AM ࡜ nifedipine ๓ ฎ⨨ࡢຠᯝࢆ᳨ウࡋࡓࠋ BAPTA-AM ࡣࠊTM ㄏⓎ⣽⬊Ṛ࡟ᑐࡋ࡚ᙳ㡪ࢆཬࡰࡉ

࡞࠿ࡗࡓࡀࠊA

࡟ࡼࡿ CA3 㡿ᇦࡢ⣽⬊Ṛቑᙉࢆ㢧ⴭ࡟ᢚไࡋࡓ (Fig. 6)ࠋ

ࡲࡓࠊnifedipine ࡣࠊBAPTA-AM ࡜ྠᵝ࡟ A

࡟ࡼࡿ CA3 㡿ᇦࡢ⣽⬊Ṛቑ ᙉࢆ㢧ⴭ࡟ᢚไࡋࡓ (Fig. 6)ࠋAD ᝈ⪅⬻(57)ࡸ APP ࢺࣛࣥࢫࢪ࢙ࢽࢵࢡ࣐࢘

ࢫ⬻ (58)࡟࠾࠸࡚ࠊ calpain άᛶࡢஹ㐍ࡀㄆࡵࡽࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡲ

ࡓࠊA

Ca

ࢳࣕࢿࣝ (60)࠾ࡼࡧᑠ

⬊యࡢࣜ࢔ࣀࢪࣥཷᐜయࡸ࢖ࣀࢩࢺ࣮ࣝ୕ࣜࣥ㓟ཷᐜయ (61)࡟స⏝ࡋ࡚⣽⬊

ෆ Ca

⃰ᗘࢆୖ᪼ࡉࡏ⣽⬊Ṛࢆᘬࡁ㉳ࡇࡍࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡇࢀࡽࡢ▱

ぢ࡜ᮏ◊✲ࡢ⤖ᯝࡼࡾࠊ A

ࡣ L ᆺ Ca

ࢳࣕࢿࣝ࠿ࡽ Ca

ࡢὶධࢆಁ㐍ࡉࡏ

࡚⣽⬊ෆ Ca

⃰ᗘࡢୖ᪼ࢆᘬࡁ㉳ࡇࡋࠊᑠ⬊యࢫࢺࣞࢫ࡛άᛶ໬ࡉࢀࡿ calpain άᛶࢆቑᙉࡍࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋᮏᐇ㦂⣔࡟࠾࠸࡚ࠊBAPTA-AM ࡀ A

࡟

ࡼࡿ TM ㄏⓎ⣽⬊Ṛቑᙉࢆ CA3 㡿ᇦ࡛ࡢࡳᢚไࡋࠊCA1 ࠾ࡼࡧ DG 㡿ᇦ࡛ࡣ ᢚไࡋ࡞࠿ࡗࡓࠋࡇࡢࡇ࡜࠿ࡽࠊCA1 ࠾ࡼࡧ DG 㡿ᇦࡣ BAPTA-AM ࡛ࡣ࢟ࣞ

࣮ࢺ࡛ࡁ࡞࠸㔞ࡢ⣽⬊ෆ Ca

ࡀ㛵୚ࡋ࡚⣽⬊Ṛࢆᘬࡁ㉳ࡇࡋ࡚࠸ࡿࡇ࡜ࡀ♧၀ ࡉࢀࡿࡀࠊᾏ㤿ࡢ㡿ᇦ≉␗ᛶ࡟ࡘ࠸࡚ࡣ௒ᚋࡉࡽ࡞ࡿ᳨ウࡀᚲせ࡜⪃࠼ࡿࠋ

ḟ࡟ࠊ TM ࠾ࡼࡧ A

ࡢ TM ㄏⓎ⣽⬊Ṛቑᙉ࡟ᑐࡍࡿ SAC ࡢಖㆤస⏝࡟ࡘ

࠸᳨࡚ウࡋࡓࠋࡑࡢ⤖ᯝࠊSAC ࡣ TM ༢⊂࡛ࡣ DG 㡿ᇦࠊA +TM ࡛ࡣ CA3 㡿

ᇦ࠾ࡼࡧ DG 㡿ᇦ࡟࠾࠸࡚ࠊ㢧ⴭ࡞⣽⬊Ṛᢚไస⏝ࢆ♧ࡋࡓ (Fig. 7A, B)ࠋᙜ◊

27

✲ᐊ࡛ࡣࠊࡇࢀࡲ࡛࡟ึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊࡟࠾࠸࡚ࠊ SAC ࡣ TM ㄏⓎ⣽⬊Ṛ

ࢆᢚไࡍࡿࡇ࡜ࢆሗ࿌ࡋ࡚࠸ࡿ (16)ࡀࠊᮏ◊✲࡟ࡼࡾࠊ SAC ࡣᾏ㤿ษ∦ᇵ㣴⣔

࡟࠾࠸࡚ࡶ TM ㄏⓎ⣽⬊Ṛ࠾ࡼࡧ A

ࡼࡿ⣽⬊Ṛቑᙉࢆ㡿ᇦ≉␗ⓗ࡟ᢚไࡍ

ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋḟ࡟ࠊSAC ࡀ♧ࡍ TM ࠾ࡼࡧ A +TM ㄏⓎ⣽⬊Ṛᢚ ไస⏝ࡢ࣓࢝ࢽࢬ࣒ࢆ᫂ࡽ࠿࡟ࡍࡿࡓࡵ࡟ࠊᑠ⬊యࢫࢺࣞࢫ㛵㐃ࢱࣥࣃࢡ㉁ࡢ

Ⓨ⌧ኚ໬࡟ࡘ࠸᳨࡚ウࢆ⾜ࡗࡓࠋSAC ࡣ TM ࠾ࡼࡧ A +TM ࡢᭀ㟢࡟ࡼࡾୖ᪼

ࡋࡓ GRP94ࠊ GRP78ࠊ CHOP ࡢⓎ⌧ୖ᪼࡟ࡣᙳ㡪ࢆཬࡰࡉ࡞࠿ࡗࡓࡀ (Fig. 8)ࠊ

-spectrin ศゎ⏘≀࠾ࡼࡧ caspase-12 ࡢάᛶ໬యࡢⓎ⌧ࢆ᭷ព࡟ᢚไࡋࡓ (Fig.

9)ࠋ௨ୖࡢ⤖ᯝࡼࡾࠊSAC ࡢᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛ࡟ᑐࡍࡿ⚄⤒⣽⬊ಖㆤ

స⏝࡟ࡣࠊcalpain άᛶࡢ㜼ᐖࢆ௓ࡋࡓ caspase-12 ࡢᢚไࡀ㛵୚ࡋ࡚࠸ࡿࡇ࡜ࡀ

♧၀ࡉࢀࡓࠋ

28

2-5 ᑠ ᑠᣓ

3 㐌㛫ᇵ㣴ࡋࡓᾏ㤿ษ∦࡟࠾࠸࡚ࠊ

1. A

25 Mࠊ 50 M ࡣࠊ⣽⬊ṚࢆㄏⓎࡋ࡞࠿ࡗࡓࡀࠊ TM 20ࠊ 40ࠊ 80 g/mL

ࡣ⃰ᗘ౫Ꮡⓗ࡟⣽⬊ṚࢆㄏⓎࡋࡓࠋࡲࡓࠊA

25 M ࡣ TM ㄏⓎ⣽⬊Ṛ

ࢆ㢧ⴭ࡟ቑᙉࡋࡓࠋ

2. A

ࡣࠊTM ࡟ࡼࡿ GRP94ࠊGRP78ࠊCHOP ࡢⓎ⌧ୖ᪼࡟ᙳ㡪ࢆཬࡰࡉ

࡞࠿ࡗࡓࡀࠊ -spectrin ศゎ⏘≀࠾ࡼࡧ caspase-12ࠊcaspase-3 ࡢάᛶ໬యࡢ

Ⓨ⌧ࢆⴭࡋࡃୖ᪼ࡉࡏࡓࠋ

3. Caspase-12 ≉␗ⓗ㜼ᐖ⸆ ATAN ࡣࠊTM ࠾ࡼࡧ A

࡜ TM ే⏝࡟ࡼࡾㄏ

Ⓨࡉࢀࡿ⣽⬊Ṛࢆᾏ㤿ࡢ඲㡿ᇦ࡟࠾࠸࡚㢧ⴭ࡟ᢚไࡋࡓࠋ

4. BAPTA-AM ࡜ nifedipine ࡣࠊA

࡟ࡼࡿ TM ㄏⓎ⣽⬊Ṛቑᙉࢆ CA3 㡿ᇦ

࡛㢧ⴭ࡟ᢚไࡋࡓࠋ

5. SAC ࡣࠊA

ࡢ TM ㄏⓎ⣽⬊Ṛቑᙉࢆ㢧ⴭ࡟ᢚไࡋࡓࠋࡲࡓࠊSAC ࡣࠊ

TM ࠾ࡼࡧ A

࡜ TM ే⏝࡟ࡼࡿ GRP94ࠊGRP78ࠊCHOP ࡢⓎ⌧ୖ᪼࡟

ࡣᙳ㡪ࢆཬࡰࡉ࡞࠿ࡗࡓࡀࠊ -spectrin ศゎ⏘≀ࠊ caspase-12 ࠾ࡼࡧ caspase-3 ࡢάᛶ໬యࡢⓎ⌧ࢆᢚไࡋࡓࠋ

௨ୖࡢ⤖ᯝࡼࡾࠊA

༢⊂࡛ࡣ⣽⬊ṚࢆㄏⓎࡋ࡞࠸ᇵ㣴 3 㐌㛫ᚋࡢᡂ⇍ᾏ 㤿ษ∦࡟࠾࠸࡚ࠊA

ࡀ TM ࡟ࡼࡾㄏⓎࡉࢀࡿᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛࢆ

ቑᙉࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡑࡢ⣽⬊Ṛቑᙉᶵᵓ࡟ࡣࠊL ᆺ Ca

ࢳࣕࢿࣝ

ࢆ௓ࡋࡓ ⣽⬊ ෆ Ca

⃰ᗘࡢୖ ᪼࡟ ࡼࡿ calpain ࡢάᛶ ໬࡟ ࡼ ࡾ ࠊୗὶࡢ

caspase-12 ࠾ࡼࡧ caspase-3 ࡢάᛶࢆࡉࡽ࡟ஹ㐍ࡍࡿᶵᵓࡀ㛵୚ࡍࡿࡇ࡜ࡀ♧၀

29

ࡉࢀࡓࠋࡲࡓࠊSAC ࡢᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛ࡟ᑐࡍࡿಖㆤస⏝ࡣ calpain

άᛶࡢᢚไࡀ㛵୚ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ

30

➨ 3 ❶

ᑠ⬊యࢫࢺࣞࢫㄏⓎᾏ㤿⚄⤒⣽⬊Ṛ࡟࠾ࡅࡿ

calpain ࡟ᑐࡍࡿ SAC ࡢస⏝

31

3-1 ᗎ ᗎㄽ

๓❶ࡢ⤖ᯝࡼࡾࠊᾏ㤿ษ∦ᇵ㣴⣔࡟࠾࠸࡚ࠊ SAC ࡣ A

࡟ࡼࡿ TM ㄏⓎ⣽

⬊Ṛቑᙉࢆ㢧ⴭ࡟ᢚไࡋࠊࡑࡢಖㆤస⏝࡟ࡣ calpain άᛶࡢᢚไࡀ㛵୚ࡍࡿࡇ࡜

ࡀ♧၀ࡉࢀࡓࠋ Calpain ࡣ⣽⬊ෆ Ca

⃰ᗘࡢୖ᪼࡟ࡼࡾάᛶ໬ࡉࢀࠊᇶ㉁ศᏊࡢ ᵓ㐀ࡸᶵ⬟ࢆኚ໬ࡉࡏࠊ⣽⬊㦵᱁ࡢ⥔ᣢࠊ⣽⬊ቑṪࡸ⣽⬊࿘ᮇࡢไᚚ➼ࡢᵝࠎ

࡞⏕⌮⌧㇟࡟㛵୚ࡋ࡚࠸ࡿ(62)ࠋ⤌⧊ᬑ㐢ⓗ࡟Ⓨ⌧ࡋ࡚࠸ࡿ calpain ࡟ࡣ 2 ✀ ࡢ࢔࢖ࢯࢨ࢖࣒ࡀᏑᅾࡋࠊin vitro ࡛ࡢάᛶ໬࡟ᚲせ࡞ Ca

⃰ᗘࡀ M ࣞ࣋ࣝࡢ -calpain ࡜ mM ࣞ࣋ࣝࡢ m-calpain ࡟༊ูࡉࢀࡿ(62)ࠋ㏻ᖖࠊ⏕యෆ࡛ࡣࠊෆ

ᅾᛶ calpain 㜼ᐖࢱࣥࣃࢡ㉁࡛࠶ࡿ calpastatin ࡀᏑᅾࡋ࡚࠾ࡾࠊ⏕⌮ⓗ᮲௳ୗࡢ

⣽⬊ෆ࡛ࡣࠊ -࠾ࡼࡧ m-calpain ࡣ calpastatin ࡟ࡼࡾάᛶࡀㄪ⠇ࡉࢀࠊ㐣๫࡞ά ᛶ໬ࡀ㉳ࡁ࡞࠸ࡼ࠺࡟ไᚚࡉࢀ࡚࠸ࡿ (62)ࠋࡋ࠿ࡋࠊAD ࡢᝈ⪅࡟࠾࠸࡚ࡣ

calpastatin ࡢⓎ⌧ࡀపୗࡋ࡚࠸ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠾ࡾ (63)ࠊAD ᝈ⪅⬻ (57)

ࡸ APP ࢺࣛࣥࢫࢪ࢙ࢽࢵࢡ࣐࢘ࢫ (58)࡟࠾࠸࡚ࠊcalpain άᛶࡀஹ㐍ࡋ࡚࠸ࡿ

ࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡲࡓࠊcalpain ࡣ tau ࡢࣜࣥ㓟໬ࢆஹ㐍ࡋ࡚⚄⤒⣽⬊ࡢ ኚᛶ࣭⬺ⴠࢆᘬࡁ㉳ࡇࡍࡇ࡜ࡶሗ࿌ࡉࢀ࡚࠾ࡾ (64)ࠊAD ࡢ⑓ᅉ࡟࠾࠸࡚㔜せ

࡞ᙺ๭ࢆ₇ࡌࡿࢱࣥࣃࢡ㉁࡛࠶ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋࡲࡓࠊᑠ⬊యࢫࢺࣞࢫ㈇

Ⲵ᫬࡟࠾࠸࡚ࡣࠊ⣽⬊ෆ Ca

⃰ᗘࡢୖ᪼ࡀ㉳ࡇࡾࠊ㐣๫࡞ -࠾ࡼࡧ m-calpain ࡢάᛶ໬ࡀᘬࡁ㉳ࡇࡉࢀࠊcaspase-12ࠊcaspase-3 ࢆ௓ࡋࡓ⚄⤒⣽⬊ṚࢆㄏⓎࡍ

ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (43)ࠋࡉࡽ࡟ࠊ calpain Ḟᦆ࣐࢘ࢫ࠿ࡽసᡂࡋࡓ⬇ᛶ⥺

⥔ⱆ⣽⬊ࡣࠊcaspase-12 ࡢάᛶ໬ࡢᢚไࡸᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛ࡟᢬ᢠᛶ

ࢆ♧ࡍࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠾ࡾ (65)ࠊ calpain ࡣᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛ࡟࠾ࡅ

ࡿ caspase-12 ࡢάᛶ໬࡟㔜せ࡞ᙺ๭ࢆᯝࡓࡍ࡜⪃࠼ࡽࢀࡿࠋࡇࡢࡼ࠺࡟ࠊᑠ⬊

యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛ࡟࠾࠸࡚ calpain ࡢ㛵୚ࡀ♧၀ࡉࢀ࡚࠸ࡿࡶࡢࡢࠊSAC

ࡀ calpain ࡢάᛶ໬࡟ཬࡰࡍᙳ㡪ࡸ⣽⬊ಖㆤస⏝ࡢヲ⣽࡟ࡘ࠸࡚ࡣ୙᫂࡞Ⅼࡀ

ከ࠸ࠋ

ࡑࡇ࡛ᮏ❶࡛ࡣࠊ SAC ࡢᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛ࡟ᑐࡍࡿಖㆤస⏝ࡢᶵᗎ

32

ࢆ᫂ࡽ࠿࡟ࡍࡿ┠ⓗ࡛ࠊᾏ㤿ึ௦ᇵ㣴⣔࠾ࡼࡧ

ࡢ calpain άᛶ ᐃ⣔ࢆ

⏝࠸࡚ࠊcalpain ࡟ᑐࡍࡿ SAC ࡢస⏝࡟ࡘ࠸᳨࡚ウࡋࡓࠋ

33

3-2 ᐇ ᐇ㦂᪉ἲ

1.

౑⏝⸆≀

ᮏ ◊ ✲ ࡛ ౑ ⏝ ࡋ ࡓ ⸆ ရ ࡣ ௨ ୗ ࡢ ఍ ♫ ࠿ ࡽ ㉎ ධ ࡋ ࡓ ࡶ ࡢ ࢆ ⏝ ࠸ ࡓ ࠋ N-Acetyl-Leu-Leu-Nle-CHO (ALLN)ࠊprotease inhibitor cocktail ࠊtunicamycinࠊ

-cysteineࠊN -acetylcysteineࠊ[3-(4,5)-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT)ࠊ

Sigma-Aldrich ࡼࡾࠊ bovine serum albumin (BSA)ࡣ࿴ග⣧⸆ᕤᴗᰴᘧ఍♫ࡼࡾࠊ TrypLE Express

ࠊ Neurobasal mediumࠊ FBSࠊ B27 supplement ࡣ Invitrogen ࡼࡾࠊHEPES ࡣྠோ໬Ꮫ◊✲ᡤࡼࡾࠊTween 20 ࡣ National Diagnostics ࡼࡾࠊ calpeptinࠊ anti- -spectrin monoclonal antibodyࠊ calpastatinࠊ purified -calpainࠊ purified m-calpainࠊ 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606)ࡣ CHEMICOM ࡼࡾࠊ anti-mouse-IgG-horseradish peroxidas ࡣ Santa Cruz Biotechnology, Inc ࡼࡾࠊ ECL ࡣ Amersham Biosciences ࡼࡾ㉎ධࡋࡓࠋࡲࡓࠊ ࡑࡢ௚ࡢ⸆ရࡣヨ⸆≉⣭ࢆ㉎ධࡋ࡚౑⏝ࡋࡓࠋ

2.

ึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊ࡢㄪ〇

ᾏ㤿⚄⤒⣽⬊ࡢึ௦ᇵ㣴ἲࡣࠊ௨๓ࡢሗ࿌ (16)࡟ᚑࡗࡓࠋື≀ᐇ㦂ࡣࠊ᪥ᮏ

኱Ꮫື≀ᐇ㦂㐠Ⴀෆつ࡟ᇶ࡙࠸࡚⾜ࡗࡓ(ᢎㄆ␒ྕ AP13P002)ࠋWistar ⣔㞤㞝

ࣛࢵࢺࡣࠊ୕༠ࣛ࣎ࢧ࣮ࣅࢫᰴᘧ఍♫ࡼࡾ㉎ධࡋࠊ᪥ᮏ኱Ꮫ⸆Ꮫ㒊ᐇ㦂ື≀ࢭ

ࣥࢱ࣮࡛㣫⫱࣭஺㓄ࡋࠊዷፎࣛࢵࢺࢆᚓࡓࠋᐇ㦂࡟ࡣዷፎ 18 ᪥┠ࡢ⫾ඣ (⫾⏕

18 ᪥㱋)ࢆ⏝࠸ࡓࠋ⫾⏕ 18 ᪥㱋ࡢ Wistar ⣔ࣛࢵࢺࡼࡾᾏ㤿ࢆ᦬ฟࡋࠊTrypLE Express

࠾ࡼࡧ 0.01% deoxyribonuclease ࡟ࡼࡾ㓝⣲ฎ⌮ࡋࡓᚋࠊ➼㔞ࡢ FBS

ࢆῧຍࡋ཯ᛂࢆ೵Ṇࡋࠊ1000 rpmࠊ10 ศ㛫㐲ᚰศ㞳ࡋࡓࠋୖΎࢆ㝖ཤࡋࡓᚋࠊ ணࡵ poly-

-lysine ฎ⌮῭ࡳࡢ culture plate (ASAHI GLASS)࡟ 5.0×10

ಶ / cm

ࡢ ᐦᗘ࡛⣽⬊ࢆ᧛✀ࡋࡓࠋ᧛✀ᚋ 48 ᫬㛫ࡲ࡛ࡣ B27 supplement ࢆྵࡴ DMEM/F12

࡛ࠊࡑࡢᚋࡣ B27 supplement ࢆྵࡴ Neurobasal medium ࡛ࠊ5% CO

/ 95% airࠊ

37

Cࠊຍ‵᮲௳ୗࡢ࢖࣮ࣥ࢟ࣗ࣋ࢱ࣮ෆ࡛ 5~6 ᪥(ྜィ 7~8 ᪥)㛫ᇵ㣴ࡋࡓࠋᇵ

34

㣴ᮇ㛫୰ࡣ 2~3 ᪥ẖ࡟ medium ࡢ஺᥮ࢆ⾜ࡗࡓࠋ

3.

⣽ ⣽⬊Ṛࡢホ౯ἲ

⚄⤒⣽⬊ࡢ⣽⬊Ṛࡢホ౯࡟ࡣࠊ MTT ἲࢆ⏝࠸ࡓࠋ MTT ἲࡣࠊ௨๓ࡢሗ࿌(16)

࡟ᚑࡗࡓࠋMTT ⁐ᾮ (60 g/mL)࡛ 2 ᫬㛫ฎ⨨ࡋࡓᚋࠊ࣐࢖ࢡࣟࣉ࣮ࣞࢺ࣮ࣜ

ࢲ࣮ (BIO-RAD)࡟ࡼࡾ྾ගᗘ (Test 570 nmࠊReference 640 nm)ࢆ ᐃࡋࡓࠋ

4.

ࢱࣥࣃࢡ㉁Ⓨ⌧ࡢホ౯

ࢱࣥࣃࢡ㉁Ⓨ⌧ࡢホ౯࡟ࡣࠊ Western blot ἲࢆ⏝࠸ࡓࠋྛ⸆≀ฎ⨨ᚋࡢึ௦ᇵ 㣴ᾏ㤿⚄⤒⣽⬊࠿ࡽ sample ࢆㄪ〇ࡋࠊ Western blot ࢆ⾜ࡗࡓࠋ᪉ἲ࡟ࡘ࠸࡚ࡣࠊ

➨ 2 ❶࡟‽ࡌ࡚⾜ࡗࡓࠋ

5. Calpain

άᛶࡢ ᐃ

Calpain άᛶࡢ ᐃࡣࠊࢫࢡࢩࢽࣝ໬ calpain ᇶ㉁ࢆ౑⏝ࡋࡓ Calpain-Glo™

Protease Assay kit (Promega)ࢆ⏝࠸࡚ ᐃࡋࡓࠋ Calpain-Glo™ Reagent 50 µL ࡜ࢩ ࢫࢸ࢖ࣥ㛵㐃໬ྜ≀ࠊcalpain 㜼ᐖ⸆࠾ࡼࡧ Ca

ࡢ⤊⃰ᗘࡀ -calpain ࡣ 1 Mࠊ m-calpain ࡣ 1mM ࡟࡞ࡿࡼ࠺࡟ CaCl

ࢆຍ࠼ࡓࢧࣥࣉࣝ 50 µL ࢆΰྜࡋࡓࠋ10 ศᚋ࡟ࠊ࣑ࣝࣀ࣓࣮ࢱ࣮(FlexStation3, Molecular Devices)࡟ࡼࡾⓎගᙉᗘࢆ ᐃ ࡋࡓࠋྛ⸆≀ฎ⨨࡟ࡼࡿ ᐃ್࠿ࡽ⸆≀↓ฎ⨨ࡢ ᐃ್ࢆᘬ࠸ࡓ್ࢆ⏝࠸ࠊ⁐

፹ࢆ control ࡜ࡋ࡚ࠊcontrol ࡟ᑐࡍࡿ๭ྜ࡛⾲ࡋࡓࠋCurve fitting ࡣࠊGraphPad Prism ࡢ sigmoidal dose-response (variable slope)ࢆ౑⏝ࡋࠊ㓝⣲άᛶࢆ 50%㜼ᐖࡍ

ࡿࡢ࡟ᚲせ࡞⃰ᗘ (IC

)ࢆィ⟬ࡋࡓࠋ

6.⤫ィᏛⓗฎ⌮

ࡍ࡭࡚ࡢ್ࡣᖹᆒ್ ± ᶆ‽ㄗᕪ࡛♧ࡋࡓࠋ᭷ពᕪ᳨ᐃࡣ୍ඖ㓄⨨ศᩓศᯒ

ἲ(one-way ANOVA)࡛ゎᯒᚋࠊTukey

s multiple test ࢆ⏝࠸ࡓࠋ