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a. ãã«ãã³ãšçŽ°èã«å¯Ÿãããããã¯ã©ã³ãã«ããå¹³è¡ç·ç¶ã·ããã¹åŸé»æµÂ (PF-EPSC)ãèšæž¬ããéã®æ³¢åœ¢ãã·ã¯ããã¢ãžã (CTZ, éç·)ãçšããããšã§ãã·ããã¹ééã®éé¢ã°ã«ã¿ãã³é žã®éãçŽæ¥çã«æ³¢åœ¢ã«åæ ãããã
b. CTZæ·»å æã®EPSCã®å¢å å²åã瀺ããã°ã©ããDscamdel17del17ããŠã¹ã§ã¯éçå (WT)ã«æ¯ã¹ãŠå¢å å²åã倧ããããšãããã·ããã¹éã®éé¢ã°ã«ã¿ãã³é žéãå€ããªã£ãŠãããšèããããã
c. é»åé¡åŸ®é¡ã«ããå¹³è¡ç·ç¶ã·ããã¹ã芳å¯ããç»åãæäœã«ãã£ãŠGLASTã®ååžãå¯èŠåããŠãã(é»ç¹, 玫ç¢é )ãæ£åžžããŠã¹(WT)ã§ã¯ã·ããã¹è¿å(é»è²ç¢é )ã«éç©ããGLASTããDscamæ©èœåªå€±ããŠã¹ã§ã¯ããé¢ããäœçœ®ã«ååšããã
d. GLASTååãšã·ããã¹(ã¢ã¯ãã£ãŽãŸãŒã³ãšåŒã°ããéšäœã®æ«ç«¯)ã®è·é¢ãèšæž¬ããçµæã环ç©åºŠæ°ååžã§è¡šããå³ã暪軞ãè·é¢ã瀺ããŠãããã·ããã¹ããé¢ããGLASTãå€ãã»ã©ã°ã©ãã¯å³å¯ãã«ãªãã
3. DSCAMã¿ã³ãã¯è³ªã¯GLASTãšçµåããå°è³ã·ããã¹ã«å ±å±åšãã
ã次ã«ãå°è³ã®æœåºæ¶²ããDSCAMæäœãçšããŠDSCAMã¿ã³ãã¯è³ªãšããã«çµåããåå矀ããŸãšããŠåé¢ããŸããããã®åé¢ç£ç©ãããŠãšã¹ã¿ã³ããããæ³ã§èª¿ã¹ããšããããã®äžã«GLASTååãå«ãŸããŠããããšã芳å¯ãããŸãã(å³4a)ãã€ãŸããå°è³ã«ãããŠDSCAMãšGLASTãçµåããŠããããšã瀺åãããŸããã次ã«ãå¹é€çŽ°èãçšããŠçµåéšäœã調ã¹ããšãããDSCAMã¿ã³ãã¯è³ªã®çŽ°èå€é åãšGLASTãçµåããããšãããããŸãã(ããŒã¿çç¥)ãããã«ãå è¿°ã®DSCAMã¿ã³ãã¯è³ªã«ALFAã¿ã°ãã€ããéºäŒåæ¹å€ããŠã¹(DscamALFA/ALFA)ã®å°è³ã芳å¯ãããšãããDSCAMãšGLASTãåãå Žæã«å ±å±åšããŠããããšã芳å¯ãããŸãã(å³4b)ãGLASTã¯è奮æ§ã·ããã¹ã«åŒ·ãå±åšããããšãç¥ãããŠããŸããããŸãå³2ã§DSCAMãã·ããã¹ã«æ¿çž®ããããšãããã£ãã®ã§ããã®äºã€ã®ã¿ã³ãã¯è³ªãå°è³ã®è奮æ§ã·ããã¹ã«ãããŠçµåããŠããã®ã§ã¯ãªãããšããããšããããã«ç€ºåãããŸããã
å³4: DSCAM-GLASTçµåãšå°è³çµç¹å ã§ã®å ±å±åš
a. æäœå°è³ã®æœåºæ¶²ã«å¯ŸããŠDSCAMæäœãçšããå ç«æ²éæ³Â (IP: Immunoprecipitation)ãè¡ããDSCAMããã³ãã®çµåã¿ã³ãã¯è³ªãéãããã®ç²Ÿè£œç©ã«å¯ŸããŠãŠãšã¹ã¿ã³ããããæ³ãè¡ã£ããGLASTæäœã§åŒ·ãã·ã°ãã«ãèŠãããã®ã§(äžã®ããã«)ãDSCAMãšGLASTãå°è³çµç¹å ã§ãäºãã«çµåããŠããããšã瀺åãããã
b. DSCAM-ALFAã¿ã³ãã¯è³ªãçºçŸããã²ãã ç·šéããŠã¹(DscamALFA/ALFA)ã®çåŸ14æ¥ç®å°è³ã«å¯ŸããŠè¡ã£ãå ç«çµç¹ååŠæè²ãALFAã¿ã°ãšGLASTã®ã·ã°ãã«ãé£æ¥ããŠããããšãããDSCAM-ALFAãšGLASTãå°è³çµç¹ã§å ±å±åšããããšãããã(é»è²ç¢é )ãGLASTã¯ã·ããã¹ã«åŒ·ãå±åšããããšãç¥ããããããŸãå³2ã§DSCAMãã·ããã¹ã«æ¿çž®ããããšãããã£ãã®ã§ããã®äºã€ã®ã¿ã³ãã¯è³ªã¯å°è³ã·ããã¹ã«ãããŠçµåããŠããã®ã§ã¯ãªãããšèããããã
ãDSCAMã¯ç¥çµçŽ°èåŽã§çºçŸã(å³1)ãGLASTãããŒã°ãã³ã°ãªã¢åŽã§çºçŸããããšãèãããšãDSCAMãšGLASTãå°è³ã®è奮æ§ã·ããã¹ã«ãããŠçŽ°èãè¶ ããŠ(ç¥çµçŽ°èãšããŒã°ãã³ã°ãªã¢)ããäºãã«çµåããŠããããšã瀺åãããŸãã以äžãããã·ããã¹ã«ãããŠãç¥çµçŽ°èèäžã®DSCAMããã®çŽ°èå€ãã¡ã€ã³ãä»ããŠããŒã°ãã³ã°ãªã¢çŽ°èèäžã®GLASTãšçµåããããšã«ãã£ãŠãGLASTãã·ããã¹åŽãžéç©ãããŠããããšã瀺åãããŸããããã®ã¡ã«ããºã ã«ãã£ãŠãGLASTãã·ããã¹è¿åãžãšéç©ããããšã«ãªããçµæçã«ã·ããã¹å ã®éé¢ã°ã«ã¿ãã³é žã®å¹ççãªé€å»ãå¯èœãšãªã£ãŠãããšèããããŸãã(å³5)ãéã«ãDSCAMã®æ©èœã倱ããããšGLASTãã·ããã¹ã«éç©ãã«ãããªããå¹ççãªã°ã«ã¿ãã³é žã®é€å»ãé»å®³ãããããã«ãé©åãªã·ããã¹æ©èœãé害ãããããšã«ãªããŸã
å³5: DSCAMã«ããGLASTã®ã·ããã¹ãžã®éç©ã¡ã«ããºã ãšç æ
(å·Šå³)æ£åžžããŠã¹(éçå)ã§ã¯ãã·ããã¹ã®ãã«ãã³ãšçŽ°èèäžã«é 眮ãããDSCAMãããã®çŽ°èå€é åã䜿ã£ãŠããŒã°ãã³ã°ãªã¢çŽ°èèäžã®GLASTãšçµåããGLASTãã·ããã¹ãžãšéç©ããããããã«ãã£ãŠãã·ããã¹ééã®äœå°ã°ã«ã¿ãã³é žãå¹çããé€å»ãããããã«ãã³ãšçŽ°èäžã®DSCAMãåããšç¹å®ãããçç±ã«ã€ããŠã¯ããç 究ã®æ矩ã»ä»åŸã®å±æãã®é ãåç §ã
(å³å³)DSCAMã®æ©èœã倱ããããšGLASTã®ã·ããã¹ãžã®éç©ãæãªããããããã·ããã¹ããã³ãã®åšèŸºã«éå°ã°ã«ã¿ãã³é žãæŒåºããé©åãªã·ããã¹æ©èœãé害ãããã
4.DSCAMã«ããäœå°ã°ã«ã¿ãã³é žå¶åŸ¡ã¯ãå°è³ã®è奮æ§ã·ããã¹ã®çºéã«å€§ããé¢äžãã
ãäžè¬çã«ãã·ããã¹ã«ãããäœå°ãªéé¢ã°ã«ã¿ãã³é žã®ã³ã³ãããŒã«ã¯ãã·ããã¹ã®é©åãªæ©èœã«å¿ èŠãšãããã ãã§ãªããç¥çµç³»ã®çºçã»çºéã«ã倧ããé¢ãã£ãŠãããšèããããŠããŸããããã§æã ã¯ããã«ãã³ãšçŽ°èãžãšæå°ãã2çš®é¡ã®è奮æ§ã·ããã¹(å¹³è¡ç·ç¶ã·ããã¹ãšç»äžç·ç¶ã·ããã¹)ã®çºéã«ã€ããŠèª¿ã¹ãŸããããã®2çš®é¡ã®ã·ããã¹ã¯ããäºãã«ç«¶åããŠããªããªãŒã奪ãåããªããçºéããã®ã§ãããç»äžç·ç¶ã·ããã¹ãçºéã«äŒŽã£ãŠå°ããã€ãã«ãã³ãšçŽ°èã®åºéšããæš¹ç¶çªèµ·ã®æ«ç«¯æ¹å(äžæ¹)ãžãšæ°ãå¢ãããŠããããšãç¥ãããŠããŸããæ£åžžãªããŠã¹ãšæ¯ã¹ãŠãDscaméºäŒåã®æ©èœåªå€±ããŠã¹(Dscamdel17/del17)ã§ã¯ãç»äžç·ç¶ã·ããã¹ã®äžæ¹æ¡å€§ã極端ã«æãªãããããšã芳å¯ãããŸãã(å³6a, b)ããã®çŸè±¡ã¯ãDscaméºäŒåããã«ãã³ãšçŽ°èã ãã§é»å®³ããããã¯ã¢ãŠãããŠã¹(Dscamflox/flox; Pfcp2Cre)ã§ãèªãããããã(å³6cãå³äžvehicleåç §)ããã«ãã³ãšçŽ°èã§çºçŸããDSCAMã¿ã³ãã¯è³ªãããããã®ã·ããã¹çºéã«éèŠãªåœ¹å²ãæãããŠããããšãæããã«ãªããŸããã
äžè¿°ã®ããã«ãDSCAMã倱ããããšå¹³è¡ç·ç¶ã·ããã¹ã§ã®ã°ã«ã¿ãã³é žååãé害ãããŸãã®ã§(å³2)ãããããæŒãåºãéé¢ã°ã«ã¿ãã³é žã競åããç»äžç·ç¶ã·ããã¹ã®çºéãé»å®³ãããšããå¯èœæ§ãæµ®äžããŠããŸãããããã§ãäžèšã®DSCAMéºäŒåããã«ãã³ãšçŽ°èã ãã§ããã¯ã¢ãŠãããããŠã¹(Dscamflox/flox; Pfcp2Cre)ã«å¯ŸããŠãGLASTã®ã°ã«ã¿ãã³é žåã蟌ã¿ä¿é²å€(ãªã«ãŸãŒã«)ãæäžãããšãããç»äžç·ç¶ã·ããã¹ã®çºéç°åžžãããªã軜æžãããŸãã(å³6c-e)ã以äžãããDSCAMæ¬ æã«ããç»äžç·ç¶ã·ããã¹ã®çºéç°åžžããGLASTã«ããéé¢ã°ã«ã¿ãã³é žã®åã蟌ã¿é害ã®ããã§ããããšã瀺åãããŸããããŸããDSCAMã®æ©èœç°åžžã«ããç»äžç·ç¶ã·ããã¹ã®é害ã«ã€ããŠãæš¡åŒå³ã«ãŸãšããŸãã(å³6f)ã
å³6: DSCAMæ¬ æåã³ã°ã«ã¿ãã³é žååäžå šã«ããç»äžç·ç¶ã·ããã¹ã®çºéé害
a. Dscamæ©èœæ¬ 倱ããŠã¹(Dscamdel17del17)ã®çåŸ30æ¥ç®ã®å°è³çµç¹ããã«ãã³ãšçŽ°èããŒã«ãŒ(Calbindin)ãšç»äžç·ç¶ã·ããã¹ããŒã«ãŒ(vGluT2)ã§å ç«æè²ããç»åã
b. ããŠã¹ã®çåŸ15æ¥(P15), 30æ¥(P30), 60æ¥(P60)ã«ãããç»äžç·ç¶ã·ããã¹å¯åºŠã®èšæž¬çµæã瀺ããã°ã©ãã
c. ãªã«ãŸãŒã«æäžå®éšã®ãã¶ã€ã³ã
d. å¯Ÿç §çŸ€(Control)ããŠã¹ãšããã«ãã³ãšçŽ°èã ãã§DscaméºäŒåãããã¯ã¢ãŠãããããŠã¹(Pcp2cre-cKOãšè¡šèšãæ£ç¢ºã«ã¯Dscamflox/flox; Pcp2Cre)ã®çåŸ30æ¥ã«ãããvGluT2å ç«æè²ã®ç»å(Vehicle: ççé£å¡©æ°Žæäž, Riluzole: ãªã«ãŸãŒã«æäž)ã
e. ãªã«ãŸãŒã«æäžåŸã®ç»äžç·ç¶ã·ããã¹å¯åºŠã®èšæž¬çµæã瀺ããã°ã©ã(æç·å ¥ã: ãªã«ãŸãŒã«æäžçŸ€)ãã°ã©ãå·ŠåŽã¯ãã«ãã³ãšçŽ°èäžæ¹(proximal), å³åŽã¯ãã«ãã³ãšçŽ°èäžæ¹(distal)ã
f. DSCAMæ¬ æã«ããå°è³ã®ã·ããã¹åœ¢æé害ã®æš¡åŒå³ã
5.DSCAMã¯ããŠã¹ã®éååŠç¿ã«å¿ èŠãšããã
ããã§ãã¯æš¡æ§ã®å£ã®å åŽã«ããŠã¹ãåºå®ãããã®å£ã15床ãã€å·Šå³ã«åšæçã«åãããšãããŠã¹ã¯ç®ã§ãã®åããè¿œããŸãããèšç·ŽãéããŠåŠç¿ãããšãã ãã ããã®åããäžæã«ãªã£ãŠããŸããããããDscaméºäŒåããã«ãã³ãšçŽ°èã ãã§ããã¯ã¢ãŠãããããŠã¹(Dscamflox/flox; Pfcp2Cre)ã§ã¯ããã®åŠç¿èœåã極端ã«äœäžããããšãããããŸãããç»äžç·ç¶ã·ããã¹ã®çºéãããã®éååŠç¿(OKRãšåŒã°ããŸã)ã«é¢ããããšãç¥ãããŠããŸãã®ã§ãDSCAMãç»äžç·ç¶ã·ããã¹ã®çºéå¶åŸ¡ãä»ããŠãéååŠç¿ã«é¢äžããããšã瀺åããããšèšããŸãã
å³6: DSCAMæ©èœæ¬ æããŠã¹ã§ã¯å°è³ãé¢äžããéååŠç¿ãé害ããã
a.æ°Žå¹³æ§èŠæ©æ§çŒçåå¿(hOKR)ã®æš¡åŒå³ã
b.60åéã®hOKRèšç·Žäžã«å¢å ããçŒçåå¿ã®å²å(10åéæ¯ã®å¹³åå€)ã瞊軞(Gain)ã®å€ãäœãã»ã©ãæš¡æ§ãè¿œãç®ã®åããå°ãªãããšã瀺ã
ç 究ã®æ矩ã»ä»åŸã®å±æ
ãã·ããã¹ã«ãããéé¢ã°ã«ã¿ãã³é žã®ã³ã³ãããŒã«ããã·ããã¹æ©èœã»è³ç¥çµåè·¯æ©èœãè³ã®çºéã«æ¥µããŠéèŠãªåœ¹å²ãæããããšãç¥ãããŠããŸããããã®é€å»ã«é¢ããã°ã«ã¿ãã³é žãã©ã³ã¹ããŒã¿ãŒããã©ã®ããã«ã·ããã¹ãèŠã€ãåºããããã«å¹ççã«éç©ããã®ãããã®ååæ©æ§ã«ã€ããŠã¯ããã£ãŠããŸããã§ããã
æ¬ç 究ã¯ãããŠã³çé¢é£ååã§ããDSCAMãã·ããã¹ã®ç¥çµçŽ°èåŽã®çŽ°èèã«å±åšãã现èå€ãã¡ã€ã³ãä»ããŠããŒã°ãã³ã°ãªã¢çŽ°èèäžã®ã°ã«ã¿ãã³é žãã©ã³ã¹ããŒã¿ãŒ(GLAST)ãã·ããã¹ãžãšåŒãã€ããããšãæããã«ãããã®ã§ããããã°ã«ã¿ãã³é žãã©ã³ã¹ããŒã¿ãŒã®ã·ããã¹ãžã®å±åšã¡ã«ããºã ããšããç¥çµç§åŠäžã®å€§ããªèª²é¡ã解æãããã®ã§ãããŸãã
ãŸãããã«ãã³ãšçŽ°èã ãã§Dscamãããã¯ã¢ãŠãããããŠã¹ã§ãã·ããã¹ã®çºéç°åžžã芳å¯ãããŸããã®ã§ãå°ãªããšãDSCAMã¿ã³ãã¯è³ªã¯åŸã·ããã¹åŽ(ãã«ãã³ãšçŽ°èåŽ)ã®çŽ°èèã«å±åšããããã§ããŒã°ãã³ã°ãªã¢çŽ°èèäžã®GLASTãšçµåããŠãããšèããããŸãã
ããã«ãDSCAMãGLASTã®ã·ããã¹ãžã®éç©æ©æ§ãä»ããŠãã·ããã¹ã®æ£åžžãªæ©èœãçºéãå¶åŸ¡ããããã«éååŠç¿ã«ãé¢ããããšã瀺ãããŸãããä»åã®çºèŠã¯ãããŠã³çã®ç²Ÿç¥ã»ç¥çµçç¶ããDSCAMéºäŒåã®å€ç°ã«ãã粟ç¥çŸæ£ã®ç æ ã®ç解ã«ã€ãªãããšæåŸ ãããŸãã
çšèªè§£èª¬
1) DSCAM/DscaméºäŒå
Down syndrome cell adhesion molecule (DSCAM) ã¯1998幎ã«ãã21çªæè²äœã®ããŠã³ç責任é åã§èŠã€ãããããŠã³çã®é¢é£éºäŒåãšããŠæ³šç®ããããããã«ãããŸããŸãªç²Ÿç¥çŸæ£ã«é¢é£ããããšã瀺åãããŠããããã®èåŸã®ç æ æ©åºã¯äžæã§ããããã®éºäŒåã¯ãããã»ããŠã¹ãªã©ã®èæ€åç©ã ãã§ãªããã·ã§ãŠãžã§ãŠããšçã®ç¡èæ€åç©ã«ãããŠããç¥çµç³»ã®çºçã«éèŠãªåœ¹å²ãæããããšãå ±åãããŠããŠããã
2) ãã«ãã³ãšçŽ°è
å°è³ç®è³ªãæ§æããæå¶æ§ç¥çµçŽ°èã®äžã€ã§ã倧ããªçŽ°èäœãšè€éã«æåããããæš¹ç¶çªèµ·ãæã€ç¹åŸŽçãªåœ¢æ ãåãããé¡ç²çŽ°èããå¹³è¡ç·ç¶ãäžãªãªãŒãæ žç¥çµçŽ°èããç»äžç·ç¶ãšãã2çš®é¡ã®è奮æ§ç¥çµç·ç¶ã®æå°ãåããŠãè奮æ§ã·ããã¹(å¹³è¡ç·ç¶ã·ããã¹ãç»äžç·ç¶ã·ããã¹)ã圢æããã
3) ããŒã°ãã³ã°ãªã¢
å°è³ã ãã«ååšãããã¢ã¹ãããµã€ãã®äžçš®ããã«ãã³ãšçŽ°èã«æå°ããã·ããã¹ãå ã¿èŸŒã¿ãã°ã«ã¿ãã³é žãã©ã³ã¹ããŒã¿ãŒãä»ããŠæŸåºãããç¥çµäŒéç©è³ª(ã°ã«ã¿ãã³é ž)ãåã蟌ããªã©ãç¥çµçŽ°èã®æŽ»åããµããŒããã圹å²ãæã€ã
4) ã°ã«ã¿ãã³é žãã©ã³ã¹ããŒã¿ãŒãšGLAST
è奮æ§ã·ããã¹ã«ãããŠæ©èœããç¥çµäŒéç©è³ªã§ããã°ã«ã¿ãã³é žã¯ãç¥çµæŽ»åäŸåçã«æŸåºãããåŸãã°ã«ã¿ãã³é žãã©ã³ã¹ããŒã¿ãŒã«ãã£ãŠã·ããã¹ééããååãããããšã«ãã£ãŠéå°ãªå ¥åãèµ·ãããªãããå¶åŸ¡ãããŠãããGLASTã¯ããŒã°ãã³ã°ãªã¢ãå«ãã¢ã¹ãããµã€ãã«çºçŸããã°ã«ã¿ãã³é žãã©ã³ã¹ããŒã¿ãŒã§ãããã·ããã¹ééã«ãããã°ã«ã¿ãã³é žæ¿åºŠãé«ãå Žåã«ã®ã¿å¿çããã°ã«ã¿ãã³é žã现èå ãžãšåã蟌ãã