HOI NGH! KHOA HOC C O N G NGHf SINH HOC TC, :: ~ ~ •""
Bieu HlEN DINH HLfdNG ha-SYNUCLElN (HUMAN a-SYNUCLElN) TAI CAC Tg BAO THAN KINH S A N SINH DOPAMINE C O A RUdl G I A M CHUVeN GEN VA NGHlfiN CCrU T A C O Q N G C O A a-SYNUCLEIN TRONG B^NH PARKINSON
Nguyen Thj Tudng Vy, Dang Thj Phuvng Thdo, Trin Linh T h u ^ Tnfdng D^i hgc Ktioa hgc Tg nhien, D^i hgc Quoc gia Tp. Hd Chl Minh
T6M TAT
Sir tich tu a-synuclein, do gen SNIOA ma ho^ vd c^c dfmg d6t bien a-synuclein nhu A30P, AS3T, E46K co lien quan m|t thiet dSn svt phit sinh benh Parkinson. Trong nhieu mo hinh nghien cihi bfnh thoSi hoS than kinh, ruoi giam Drosopkila melanogaster la mo hinh CO nhi^u uu di£m vtrgl troi voi khoang 70% gen dugc tim thiy co liec quan den cac benh thoai h6a thin kinh d ngirdi. Trong nghien ci^ nAy, chiing toi tao md hinh moi giam chuyen gen va bt6u hifn ha-synuclein dinh huong tr\rc tiq) tai cac tl bio thin kinh sia sinh dopamine nhSm budc dAu khao sat su Anh huong cua a-s^uclein den qud trinh sdn sinh dopamine. Cac ket qui khio sdt cho thiy sir tang bieu hi?n vugt miic a-synuclein va hai d^ing dgt bien A30P, A53T trong tc bao thin kinh san sinh dopamme da lam giam kba nang leo tree ciia nidi giam. Chung toi cung da kiem tra miic dg bieu hifn cua tyrosine hydroxylase (TH), m$t enzyme co vai IT6 quan trgng doi voi sir sinh tong hgp dopamine msng nao ruoi va nhSn llily hiSn tugng giam cujmg dg bieu hifn TH trong mdi giim chuy^ gen bi^u hifn a-synuclein t^i cdc te bilo thin kinh san sinh dopamine. Cdc k^ qua thvrc n g h i ^ cho thiy chung toi dA tfadnh cong trong vifc sur dung md hinh rudi giam chuyen gen trong nghidn cihi co che phan tii bdnh Padcmson.
Til khoa Alpha-synuclein, braih Paridnson, dopamine, Drosophila melanogaster, kha ndng v^n dgng, UAS-GAL4.
MdeAu
Bdnh Pahdnson Id b^nh thodi h6a hd th6ng thdn kinh trung \xang phd bi4n di>ng thii- hai sau b^nh Alzheimer du'VC bdc sy James Parkinson md td l l n d4u tidn ndm 1817. Bdnh gdy ra cdc r6i logn chijc nang vdn dOng do si/ ^odi hda din cdc t^ bdo thin kinh san sinh dopamine tgi vCing chit den thu$c ndo giOa cung viti sg xuit hl$n cua cau tnic bit thuang thi vLii Lewy (Gandhi et al., 2005). Dopamine Id mgt chit din truyin than kinh quan hQng co vai tr6 trong truyin tin hidu giu-a cdc t i bdo thin kinh tiii cdc ca quan d i kfch thich v$n d$ng. B$nh thu'ong xuit hi#n it nhiJng ngu'di b^n 60 tu6i.
Nhiiu nghidn ciiu cho ring nguydn nhan chinh gay ra b?nh Id do sy anh hud'ng cua viec tiep xilc v&\ cac dOc t6 trong m6i tnfdng sing theo thCri gian da gdy cdc biin d i i v i gen di truyin ir ngu'di b?nh, Hidn nay cac nghidn ciiu dd tim thiy nhO-ng gen c6 lien quan mdt thiit vdi ca chi phdn ti> gdy ra b#nh Paridnson nhu': alpha-synuclein, parkin, LRRK2, DJ-T. . UCHL1, PINK1 (Whihworth, 2011). Alpha-synuclein la protein dau tidn du-^c tim thiy c6 lidn quan mdt thiet vm b^nh Parkinson vd mOt s i bdnh thodi hda thin kinh khac i> ngu'di. & hiu het cdc bdnh nhdn Parkinson deu thiy c6 s^f bieu hi§n cua cdc thi vui Lewy tgi cdc vung t i bdo thin kinh sdn sinh dopamine. Cdc dgng t h i vili ndy hlnh thdnh do sg tich ty biiu hi^n vvgt mire protein o-synudein va cac dgng d6t biin A30P, A53T, E46K (Polymeropoulos et al., 1997;
Lucking et al., 2000). Vai frd ciia a-synuclein van chya dup'c biet ro, mdt vdi nghien ciiu cho rang a-synuclein cung dong vai trd quan frgng trong vi#c hlnh thdnh cdc tiii synap vd tham gia vdo qua trinh diiu hoa tiet dopamine du'di sg xiic tdc cOa enzyme tyrosine hydroxylase vd DOPA decarboxylase (Yu et al., 2005).
Nghidn ciiu cdc b?nh di fruyin cOng nh\r cdc bdnh frioai hoa thin kinh cua ngudi gdp nhiiu khd khdn vd hgn chi ve phdn tich tdc dgng cila cdc gen di fruyin len cdc ca chi gdy b?nh; vi vgy vide tim mo hinh bdnh d i nghidn ciiu cdc khia cgnh chi>c ndng tdc d^ng cua gen hogc protein Idn ca chi phdn tir cua b§nh ngdy cdng frd ndn quan tnpng. Tii' ndm 1910, Morgan vd cdc cOng sy dd xay dyng thdnh cong cdc thuyet di tnjyin nhiim sdc t h i trdn doi tuvng m i l giim DrosoDhiia melanogaster dd md ra nhiiu hudng nghien cdu cdc b?nh thoai hoa thin kinh trdn m6 hinh ndy. Cdc nghi&i cuu vd cdc bdnh thodi h6a than kinh frdn md hinh Dnssophila melanogater Oirgc su dyng ngdy cdng pho biin vdi nhiiu uu diim vugl trfy nhu chu ky song ngdn, dd nuoi, bO gen don gidn vd co khodng 70% gen gdy b#nh d ngudi frjong dflng vd dup'c tim thiy d njoi giam (Whitworth, 2011). De nghien ciiu chiic nang cCia mpt gen trong cdc b^nh di tmyin cung nhu bgnh Parkinson, cac nhd khoa hgc thudng ngiiien ciiu dnh hudng cua gen d ruii giim bing phuang phdp bidu hign vugt miic gen 06 su dyng hg friing UAS-GAL4 va gay mit chdc nang cua gen ttiing qua ky thudt knockdown mpt gen bang RNAi (HirUi. 2010). Trong mOt s6 nghidn cuu trudc ddy. cdc nhd khoa hpc da Uidnh cdng frong vide tgo m6 hlnh ruoi chuyen gen mang kiiu hinh bgnh Parkinson thong qua biiu hidn protein ha-synuclein trong ruoi giim, cdc kdt qud nghidn cuu nay cho thiy vigc biiu hidn ho-synuclein gdy tich ty t h i Lewry trong ndo mii vd Idm gidm khd ndng vdn dflng cua mOi chuyin gen (Feany & Bender, 2000; Park., 2006).
Vdi cdc kit qua nghien cuu dd cflng b i . ha-synuclein dupe biit din nhu Id mfll trang cac protein lien quan din bdnh Parttinson. Sy tich ty pratein nay trong ndo dupe hiiu nhu mflt bilu hiin cua frldu chiing bgnh. Tuy nhidn, vai fr6 diiu hod cy ttie cua ha-synuclein frong co chi bflnh Parkinson vin c6n dang tiip tyc dupe ldm r6. Trong nghien cdu nSy, Chung tcH su dyng hg thing UAS-GAL4 nham bilu hien dinh hudng ho-synuclein tgi t i bdo thin kmh sdn sinh dopamine S t h^n A?np 1^?T -^^" "t ^ ^ " ^ ^ ^ ^ ^^"^ 9ia sy bilu hign vupl mdc protein ha-synuclein dgng hoang dgi vd d^ng dflt biin A30P, A53T cung nhu tdc dflng cCia sy bilu hifln ndy tren khd ndng vgn dflng ciia ruii giim vd sy sdn sinh P S ! ^ T ! K ^ ^^^1°"^^^^ ^^^- ""^^ ^"^y""® ^ " ^ " ^^^"9 ^ ° " 9 s^ ^'"^^ '^"9 fi^P cfi^t <^^" truyin thin kinh dopamine.
K . f « • "^P '^" " ' ' ^ ''^^"9 ^^^ ^3' 'r^ '^'Ja ha-synuclein trong sy phdt sinh benh Parkinson vd Idm sdng 16 hon tdc dflng cua sy bieu hign protein ndy trong su tich ty TH vd thflng qua do diiu hod sy smh ting hpp dopamine.
NGUYEN LffU VA PHlfCNG P H A P Cdc ddng ruoi
Cdc ddng roii mang GAL4 dupe si> dyng cd nguin goc tu ngdn hang njoi Bloomington: P(+;Elav GAL4/Gvo-+} aiOp bidu hifln tai mo nao. dflng P{+:+,Ddc-GAL4} chda promoter ciia gen DOPA decarboxylase giup bieu hidn gen tgi cdc t*
,;:,,, 3C CONG NGHE SINH HQC T O A N Q U 6 C 2013
bdo sinh dopamine.
Cdc ddng ruii giim ehuyin gen mang gen UAS-SWCA ma hda cho ha-synuclein dgng hoang dgi (WT) va 2 dgng dflt biin A30P, A53T Ovgc thilt Igp tgi Phdng thi nghidm Cong nghd Sinh hpc Phdn ti>, Trudng Dgi hpe Khoa hpc Ty nhidn Tp. H i Chi Minh ed kiiu gen tuong ung nfiu sau: P(+;UAS-WT SNCA;+}, P{+;UAS-A30P SNCA+) PfUAS- A53T_SNCA;+;+}.
Cdc ddng ruoi doi ehung: dflng ruii hoang dai CantonS P {+;+;+}, ddng bieu hiin P-galactosidase P{+;+;UAS-LacZ}.
Tit cd cdc dflng ruii duoc nuoi trdn mdi frudng gidu ndm men, dudng vd sua d nhiit dp 25°C.Cdc phdp lar giiia cac ding roii duvc khao sdt d diiu kidn nhiflt dfl 28°C.
Kiem tra SLP bleu hi$n protein ha-synuclein ciia c:ac dong ruoi chuyen gen (gi mo nao
Cdc ddng ruii giam chuyin gen ha-synuclein Ok duyc tgo vd kilm bB se duac tiin hanh chgn lgc vd thu cdc njii cai c6n trinh, sau dd lai vdi ddng bilu hidn protein GAL4 nhdm kiim tra sy bieu hi^n ciJa protein ha-synuclein tai md nao.
Si> dyng ddng moi Elav-GAL4/Cyo Id dflng ruii mang promoter chuyen blflt cho mfl nao cd kiiu hinii cdnh cong do gen cyo quy djnh, do dd khi lai dong ruii ndy vdi cdc ddng UAS-SNCA ttil chi nhiing te bdo Hian kinh mdi biiu hign protein ha-synuclein. Thi hd con lai duac chpn lpc dya vdo kilu hinh do cdc nhiim sac t h i cdn bdng (balancer) quy ^nh, vi vdy edc cd t h i m i i lai FI mang vd bilu hign dflng thdi UAS-SNCA va Elav-GAL4 dupe lya chpn se cd kiiu hlnh cdnh thing.
Sau dd ehung tdi ttiu nhgn protein tong tir edc mdu diu m i i vd tiln hdnh didn di plian tfch protein bang SDS-PAGE vd kiim chdng bdng Westem blot vdi khang t h i ddc hidu khdng a-synuclein.
Ddng ruii Dde-GAL4 khdng mang balancer vd Id ddng mang promoter chuydn biet giijp biiu hign tai edc t i bdo ttiin kinh sdn sinh dopamine. Cdc t h i hfl m i i lai FI duvc sd dyng khdo sat sy bilu hign protein a-synuclein tgi cdc mfl than kinh diuyfln bi$t san sinh dopamine bdng phuang phdp hda md miln djeh huynh quang va khao sdt sy biiu hifln ve mgt kiiu hlnh
Nhupm hoa md miSn djch hujmh quang (Immunohistochemistry)
Ruii lai FI dug^ 25 ngdy tuii d\rgc chpn dCing d i tdch nao va nhuflm hda mfl miln djch huSmh quang. Ndo m i l duyc tdch trong dung djch PBS vd c i d[nh md bang dung djeh parafdmiald^yde 4% pha trang PBS d nhidt dfl phdng 30 phiiL Sau dfl rda bing dung djch PBS-0.3% TritonXlOO (v/v) vd khda m i u md bdng dung djch huyit thanh dd (Nonnal goat semm) 10% trong PBS-0.15% TritonXlOO (v/v) d nhidt dp phwig 30 phiit Miu md ndo duyc u vdi khdng t h i sa cip khdng a-synuclein ho§c khdng t h i khdng tyrosine hydroxylase d 4''C qua ddm Sau dd mau mfl dupe rda 4-5 lln bang dung djeh PBS-0.3% TritonXlOO vd^duyc ii vdl khdng ttie thu d p phdt huynh quang FITC 484nm hodc Rhodamine 580nm d nhiflt dfl phdng 2-3 gid. Mau nao Oiigc fvra sgeh vd tdch nao d djnh len lame vd quan sdt dudi kinh hiin vi huynh quang. Htnh dnh dupe phdn tich bing chuang trinh ImageJ, thflng kd bing kilm djnh ANOVA-test vd t-test eOa Microsoft Excel.
Khdo sdt khd ndng leo trdo ciia r u i i giim
Nhim ddnh gid sy biiu hidn ciia ha-synuelein Idn cdc t i bdo thin kinh sdn sinh dopamine ed anh hudng nhu thi ndo din mil giam ehuyin gen, chiing tfli tiin hdnh cdc thd nghigm khdo sat v l khd nang vgn dflng leo treo cua mil giim ttieo ngdy tuii (Minois et al., 2001; Pendleton et al., 2011). Bdt ngiu nhidn 30 m i i dye vd 30 aioi cdi ciJa cd ttii m i i FI d mil dflnp thi nghiflm va ddng d i i chung, cho vdo mii flng 10 cd t h i . Cdc ddng mot duyc khao sdt khd ndng leo frdo trong cdc flng trfln dudng kfnh Scm vd ddnh diu chiiu ddi din lOem. Go nhg cde ong khdo sat d i cdc cd ttil m i l v l vj tri Ocm, vd trong 10 gidy ghi nhdn s i ca t h i mil vugl qua mdc 10cm. Ldp Igi 3 lin, vd cdc thi nghiflm khdo sdt lien tyc cdch 5 ngdy/lin trong cClng diiu ki$n. Cdc sfl ligu dupe xu ly thong ke.
K£T QUA VA THAO LUAN
Biiu hign djnh hudng ha-synuclein trong md ndo r u i i g i i m chuyen gen
D i nghidn cuu chue ndng eua ha-synuclein trdn md hlnh m i i giim chuyin gen, chiing toi sd dyng hd thing biiu hien UAS-GAL4 (Hlnh 1. A) nhim biiu hien vuyt mdc protein ha-synuclein tgi vj tri cac md cin khdo sdt. Bflnh Paddnson nhy da gidi thi§u Id binh thodi hfla than kinh, do do cdc thi nghiem khdo sdt trong nghidn cdu ndy chiing tfli biiu hien djnh hudng protein ha-synuclein (SNCA) tgi md nao cCia mil giam chuyen gen bing hfl thong UAS-GAL4 vdi promoter chuydn bidt mfl ndo Elav vd kilm tra sy biiu hifln tren dflng mdi mang Elav-GAL4>UAS-5NCA. H$ thing ndy g i m 2"
phin, UAS (upstream activation sequence) Id dogn frinh ty duac thilt k l trudc gen mpc tiflu, vd GAL4 dupe thiet ke nim sau promoter bilu hifln djnh hudng md. Trong ddng moi lai FI mang GAL4 vd UAS, GAL4 dupe ting hap se gan Idn frinh ty UAS, va gen myc tieu dudi sy kiim sodt cOa UAS se dupe phien m§ giiJp bieu hign dinh hudng protein t^i cdc mfl khao sdt.
Kit qua khdo sdt protein tong s i td dau ruoi lai FI mang Elav-GAL4>UAS-SNCA d cae dong thi nghigm bang SDS- PAGE vd Westem blot vdi khang thi khdng ha-synuclein cho friiy cd sy biiu hign protein ha-synuclein (Hinh 1. B, giing 3, 4, 5) khi so sanh vdi mdu dil chdng ttiyc hign tren ddng Elav-GAL4 (Hinh 1. B, gieng 2) Nhu vdy, protein ho- synucleln d dgng hoang dai (WT) vd 2 dgng dflt biln A30P, A53T da duyc bilu hidn djnh hudng tai md nao cua ca ttil mil lai F1.
HOI NGHI KHOA HOC CONG NGHE SINH Hpc ToS
lUaUhfaiai OAU
Hlnh 1 Brtu xm diim hirtrg protein hoHiynucleln M n ding nioi giim Eiav-GAL4/Cyot;l mS nSo ft MOhirti bl4u W«ndl*
tbS 1 * ^ n-synudtfn; 1; Tiiang proldn; 2: D6| ctiimg FH*. Elav-GAL4;.,*); 3: F1<*;UAS-Wr_SNCA/Elav-GA1.4,t). 4. F1(*.UAS- A30P_SNCA/Elav-GAL4:+}; 5: F1{UAS-A53T_SNCA/+;Eiav-GAL4/+;+}.
Bilu hi$n dinh h i r i n g ha.synuciein trong t& bao t h i n kinh san sinh dopamine
BS danh ga inh hirftng ctia sif Wiu hi#n ha-synudein <A (dc dsng d^t biSn tr6n cSc fi bao thin kmh s i n sinh dopamine dira tr6n co chl ting h w dopamine (Yu et al., 2005), chung 161 bilu hi«n dinh hu6ng ho-synuclein Irong t t bSo thin kinh a n sinh dopamine du*i su diiu hoS cGa promoter Ddc (DOPA decarboxylase), m«t promoter diSu hoi t6nB hop DOPA decarboxylase - enzyme cd vai tro trong con dudng sinh t6ng hop dopamine. Chung t6i thl6t l$p cic ddng niS chuyin gen mang phirc hop gen Ddc-GAL4>UAS.SNCA v i thu nh^n m6 nao ni6i chuyen gen, nhugm hite m6 miin dich hu>nh quang vfti khing t h i khing o-synudein. Kit qui kiim tia biiu hi$n protein ho-simudem trong t« bio thin kinh sin sinh dopamine (Hlnh 2. A) cho thiy mOt so viing tren m i nao 6 ni6i mang gen bi6u hiJn ho-synudein dgng WT v i d?ng ilOt biin A30P, A53T cho tin hi#u huirnh quang ir cic ciim t i bio sinh dopamine (vung du'oc khoanh trtn) khi dii chiiu ti^n m i hinh cic vilng sin sinh dopamine duoc cfing b i b&i White v i cing sv nam 2010 (Hinh 2, A3). Cic ding m i l dii chimg Ddc-GAL4 v i Ddc-GAL4>UAS-L.acZ khing cho tin hiiu lai vdri khing t h i khing ho- synudeln(Hlnh2.A1,2.A2).
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nefiS-VTjuctifflSfGAUif ilU*SA3W.SKCA^JMMUL4H- UASASJT_SNCA/+;i'J»*«"*>
y
hfinh 2. Biiu hi^n djnh hudng ha-$ynuclein trdn mo nao ruoi t^i cdc vdng t i bao thin kinh dopamine. A: Nhuflm Ii6a mfl miln djch huynh quang md nio vdi khdng thS khdng a-synucleln; B: Bleu d i cu^g dfl tin hi^u a-synuclein/pixel.
Kit qud nhuflm md duyc xdc ^nh cudng dfl tin hiflu bang chuong ttlnh ImageJ vd thong ke ttil hifln qua bilu d i Hlnh 2. B cho thiy cudng dfl tin hiflu hujnh quang thu duyc td cac ddng ruii cd biiu hien protein a-synuclein eao hon nhiiu SO vdi cdc dflng dii chdng vd cd !^ nghTa ttiing kS khi phdn tieh bing kilm dinh t-test vdi cdc gia trj p<0.05, n=3.
Anh hudng cua bieu hien wgt mdc hO'Synuclein trong t i bdo than kinh sdn sinh dopamine ^ Bflnh Parkinson d ngudi cd cdc triflu chdng chinh ve r i i logn chdc ndng vdn dflng. gii> thang bdng ecr t h i vd thums
xay ra nhat d ngudi td 60 tuii trd len. Do do, trdn md hlnh nghien cdu nay, chdng tdi cQng thyc hl^n cdc th( n ^ khdo sat ddnh gid sy biiu hiin cOa ha-synuclein dnh hudng din hfl vfln ddng vd sdc sing theo ngdy tuii eOa cdc (^
ruii da trudng thdnh tinh td ngdy nd diu ttdn. Khd ndng vdn dflng cda mii duyc ddnh gid thflng qua tic dg leo trdd"
ruii giam (climbing assay-Minois eta!., 2001; Pendleton etal., 2011). Chung tfli nhdn Uiiy cdc dflng ruii c d s y f i hifln a-synuclein tgi t i tiao ttiin kinh san sinh dopamine di chuyin ehflm hon so vdi cdc dflng^doi chdng d ed ruoi ij va mii cdi (Hlnh 3). Sy bilu hign a-synuclein Idm gidm mgnh khd nang leo tteo d cde ddng ruoi Ddc-GAL4>UAS-Sf!
sau 20 ngdy tuii so vdi cdc dflng doi ehdng Ddc-GAL4 vd Ddc-GAL4>UAS-LacZ. Su khdc bigt v i khd ndng vdn 6
PC CONG NGHE SINH HQC TOAN QUOC 2013
ndy c6 y nghTa thing kd khi phan tfch bing ANOVA-test vdi edc gid trj p<0.05 d ca hai quan ttii njoi dye vd m i i cdi.
-^+T<^Ddc-GAI.4/••
-•-+•, I^UAS-LacZ/Ddc^ AL4 - * - UAS-A53T_SNCA/*;-l%I>dc-<lAI./*/+
+-,UAS-Wr_SNCAy+J>do-GAI>l/+
Hinh 3. Do ttij khdo sdt svr dnh hudng cda ha-synuclein Ifin khd ndng v,
synuclein tgi t i bdo thin kinh sdn sinh dopamine. 1 d$ng leo trio cua ruoi glim chuydn gen biiu hi$n a- Biiu hi$n wgt mdc ha-synuclein Idm gidm b i l u hifn enzyme TH trong cdc t i bdo than kinh sdn sinh dopamine Td cdc kit qud khdo sdt khd nang vfln dflng cda aiii giim ehuyin gen, d i tim hiiu sdu hon mii lidn hfl chue nang gii>a ha-synudein vd bflnh Parkinson ttdn mfl hlnh mfli, chOng tdi ttin hdnh khdo sdt sy bilu hidn cda cde tin hiflu thin kinh dopamine ttiing qua marker tyrosine hydroxylase (TH) d moi lai FI Ddc-GAL4>UAS-SNCA. Tynisine hydnaxylase Id mflt enzyme cd vai ttfl quan trgng ttong co c h i sinh ting hyp dopamine, theo cdc nghidn cuu da edng b i thi ddy Id mflt maricer htKi hiflu dCing tanng khdo sdt trgng thdi sinh ti cua te bdo ttiin kinh sdn sinh dopamine d cdc md hinh lien quan din bflnh thin kinh (Nassel., 1992; Friggi-Grelin et al., 2003). Do dd ehung tdi tiin hdnh thu md ndo ruii giim trudng thdnh vd nhuflm hda mfl miln djch hujfnh quang vdi khdng the khdng tyrosine hydroxylase.
/ / / / /
• / / • / /
^ • * ' « * /
*% UAS-Wr.SnCA/f. tUt^iAhU* *•, UASvUtr.SNCVf, Mt^SALUt VAS^STJMIM*-, *i I M t G A L V t
Hlnh 4. Sy bidu hi$n tin hif u tyrosine hydroxylase tgi cac t i bao thin kinh sdn sinh dopamine cda cdc dong ruii biiu hiin ha- synuclein trSn d6ng Ddc-GAL4. A. Nhuflm hoa mo miin d|ch hu^nh quang mo ndo vdi khdng thi khang tyrosine hydroxylase; B: Bilu do cudng dp tin higu TH/pixel.
Cde miu md nao eOa cdc ddng m i l lai Dde-GAL4>UAS-SNCA cho thiy cd tin higu TH tgi cdc vung te bdo san sinh dopamine (Hlnh 4. A4, A5, A6) thip han so vdi cdc dong doi ehdng Ddc-GAL4 vd Ddc-GAL4>UAS-LaeZ (Hlnh 4. A l , A2). Cdc vung bilu hign cung tuong dng vdi cdc vung thin kinh san sinh dopamine duyc scr d i hda trdn hllnh 4. A3.
(White et al., 2010). Dong thdi khi phdn tich cudng dg tin hidu huynh quang cOa TH bing chuong trinh ImageJ d biiu d i Hlnh 4. B cung cho thay lugng TH trong mfl ndo cua cdc dong mdi ha-synuclein tai md nao cung thip hon so vdl edc ddng d6i ehung. Cdc kit qud duyc xd ly thflng ke bdng hdm t-test vdi cdc gia tri p<0.65, n=3.
K E T L U A N
Chung tfli da thdnh cflng ttong vide su dgng h§ thing GAL4-UAS d i biiu hien a-synuelein dinh hudng trong md nao vd t i bdo thin kinh sdn sinh dopamine trong ruoi giam diuyin gen. Sy bilu hien vuyt mdc ciia ho-synueleln vd hai dgng a- synuclein dflt biin A30P, A53T Idm gidm khd ndng van dflng cua m i i glim chuyin gen va lam giam biiu hifln enzyme tyrosine hydroxylase trong t i bdo thin kinh sdn sinh dopamine. Day cd t h i Id nguyen nhdn chinh gdy giam tong hyp
H O I N G H ! K H O A HQC C O N G N G H t S t N h l H g C T _ T '^
dopamine b ^ n g n d o v d gdy b g n h P a r k i n s o n . T A I L i e u T H A M K H A O
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EXPRESSION OF ha-SYNUCLEIN (HUMAN a-SYNUCLEIN) IN TRANSGENIC DROSOPHILA MELANOGASTER DOPAMINERGIC CELLS AND ANALYSIS ROLES OF a-SYNUCLEIN IN PARKINSON DISEASE
N g u y e n T h i T u o n g V y , D a n g T h i P h u o n g T h a o , T r a n L i n h T h u o c University of Science, VNU-HCM
SUMMARY
The human gene encoding a-synuclein protein is SNCA, a 140 amino acid protein richly expressed in presynaptic terminals in Ox central nervous system. Over-expression and accumulation of normal a-synuclein protem as well as mutant forms of a-synucleii such as A30P, A53T, E46K cause to form Lewy bodies and are major reason linked to Parkinson's disease (PD). The major cause aF PD IS the degeneration of dopanunergic neurons that the neural transmitter dopamine has a role of locomotor control. Out of sevenl neurodegencration research models, Drosophila melanogaster is a powerliil organism model in which about 70% of genes is homologous to human neurodegenerative genes; thus it is widely used for studying molecular pathomechanism and therapies of neurodegenerative diseases. Previous reports demonstrated that over-expression of a-synuclein in Drosophila brain reduced fly locomotor ability and form of Lewy bodies as siiown in PD patients. In this study, we established transgenic fly model K | orientatively eicpressed ha-synucleiu in dopaminergic cells of those transgenic flies in order to investigate the eflecls of a-synoSp protein in dopamine synthesis. Our data revealed that over-expression of a-synuclein and its delivered mutant type A30P, A51l weakened fly locomotor ability. Furthemiore, our immunostain with anti tyrosine hydroxylase also showed diat the a-synudcB expression gave effect as reduction of tyrosine hydroxylase (TH) in Drosophila brain. Taken together, the data strongly demoostntcd I about the role of a-synuclein in reduction of dopamine synthesis and causing Parkinson's disease.
Keywords: Alpha-synuclein, climbing ability, dopamine, Drosophila melanogaster, Parkinson's disease, UAS-GAL4.
Author for corresspondence- Tel: +84 -8 38307079; Email: lhaodD<5?hcmus-edu vn
