Archives of Biochemistry and Biophysics 481 (2009) 177–182
0003-9861/$ - see front matter © 2008 Published by Elsevier Inc.
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Intro duc tion
The Ginkgo bi loba extract EGb761 is com monly used in the
pre ven tion and ther apy of vas cu lar and cere bral dis or ders includ-
ing Alz hei mer’s dis ease (AD) . AD, a pro gres sive neu ro de gen er-
a tive dis or der, is char ac ter ized by extra cel lu lar amy loid-b (Ab)1
plaques, intra cel lu lar neu ro fi bril lary tan gles and mas sive neu ro nal
loss in the cor tex and hip po cam pus [2–4]. EGb761 is a stan dard-
ized extract of the leaves of the Ginkgo bi loba tree and is char ac-
ter ized by its main frac tions, the flavo nols (mainly isorh am ne tin,
ka empf er ol and quer ce tin: 22–27%) and the ter pen e lac tone (5–7%).
These two frac tions are thought to be, at least partly, respon si ble
for potential neu ro pro tec tive prop er ties of EGb761 [5–7]. Sev eral
human inter ven tion tri als reported ben e fi cial effects of Ginkgo
bi loba in the pre ven tion and ther apy of neu ro de gen er a tive dis-
or ders [7–12]. These stud ies revealed a sig nifi cant enhance ment
in cog ni tive func tions [9–11], mem ory  and con cen tra tion in
response to EGb761 treat ment [7,8]. How ever, the pla cebo con-
trolled dou ble blind tri als by Dodge et al. (2008) and van Don-
gen et al. (2003) did not show ben e fi cial effects of EGb761 in
age asso ci ated mem ory impair ment in elderly people [13,14]. In
spite of these con trary out comes, meta-ana lyzes revealed mod-
est ben e fi cial effects of EGb761 on cog ni tive func tions in patients
with AD [5,15]. It has been shown in stud ies in cul tured cells as
well as in lab o ra tory ani mals that EGb761 exhib its anti ox i dant
[16–18] and anti-inflam ma tory activ ity , mod u lates neu ro-
trans mit ters activ ity, and increases cere bral blood flow [6,20,21].
Fur ther more, recent ani mal stud ies have shown that short-term
treat ment with EGb761 influ ences mRNA and pro tein expres sion
of genes encod ing for pro teins involved in the path o gen e sis of AD
[22,23]. Addi tion ally, in trans genic cell and ani mal mod els for AD
it has been shown that Ab aggre ga tion [24,25] and Ab induced
oxi da tive stress was atten u ated by EGb761 treat ment [17,26]. Ab
is a cleav age prod uct of the trans mem brane amy loid pre cur sor
pro tein (APP). Two dif fer ent pro te ases can cleave APP in dif fer ent
posi tions. While the cleav age by a-secre tase occurs within the Ab
sequence and releases aAPP and a C83 frag ment, the b-secre tase
gen er ates bAPP and a C99 frag ment con tain ing the N-ter mi nus of
Ab. Fur ther cleav age of C83 and C99 by a c-secre tase leads to sol-
u ble, non toxic P3 and P6 frag ments and Ab and the P6 frag ment,
Effect of a short- and long-term treatment with Ginkgo biloba extract
on Amyloid Precursor Protein Levels in a transgenic mouse model relevant
to Alzheimer’s disease
Sabine Augustin a, Gerald Rimbach b, Kay Augustin b, Reinhard Schliebs c, Siegfried Wolffram a,*,
Rainer Cermak d
a Insti tute of Ani mal Nutri tion and Phys i ol ogy, Chris tian-Al brechts-Uni ver sity of Kiel, Her mann-Rode wald-Strasse 9, 24098 Kiel, Ger many
b Insti tute of Human Nutri tion and Food Science, Chris tian-Al brechts-Uni ver sity of Kiel, Kiel, Ger many
c Depart ment of Neu ro chem is try, Uni ver sity of Leip zig, Insti tute for Brain Research, Leip zig, Ger many
d Uni ver sity of Leip zig, Insti tute for Vet er i nary Phys i ol ogy, Leip zig, Ger many
a r t i c l e i n f oa b s t r a c t
Received 9 September 2008
and in revised form 13 October 2008
Available online 30 October 2008
Sev eral clin i cal tri als have reported ben e fi cial effects of the Ginkgo bi loba extract EGb761 in the pre ven tion
and ther apy of cog ni tive dis or ders includ ing Alz hei mer’s dis ease (AD). The aim of the pres ent long-term
feed ing trial was to study the impact of die tary EGb761 on Amy loid pre cur sor pro tein (APP) metab o lism in
mice trans genic for human APP (Tg2576). Tg2576 mice were fed diets with and with out EGb761 (300 mg/kg
diet) for 1 and 16 months, respec tively. Long-term treat ment (16 months) with EGb761 sig nifi cantly low ered
human APP pro tein lev els by »50% as com pared to con trols in the cor tex but not in the hip po cam pus. How-
ever, APP lev els were not affected by EGb761 in young mice. Cur rent data indi cate that APP seems to be an
impor tant molec u lar tar get of EGb761 in rela tion to the dura tion of the Ginkgo bi loba treat ment and/or the
age of the ani mals. Potential neu ro pro tec tive prop er ties of EGb761 may be, at least partly, related to its APP
low er ing activ ity.
© 2008 Published by Elsevier Inc.
Ginkgo bi loba
Alz hei mer’s dis ease
Amy loid pre cur sor pro tein
* Cor re spond ing author. Fax: + 49 431 880 152.
E-mail address: wolf fram@a ninu t.u ni-kiel.de (S. Wolffram).
1 Abbre vi a tions used: Ab, amy loid beta; AD, Alz hei mer’s Dis ease; ADAM-10, a dis-
in te grin and me tal lo pro tease-10; APP, amy loid Pre cur sor pro tein; AP Pbp1, amy loid
pre cur sor pro tein bind ing pro tein 1; BACE-1, beta site amy loid pre cur sor pro tein
cleav ing enzyme-1; C, con trol diet; EGb761, stand ard ised Ginkgo bi loba extract, G-
EGb761 sup ple mented diet; hPrP, h amster prion pro tein; huA PP, human amy loid
pre cur sor pro tein; msAPP, mouse amy loid pre cur sor pro tein; NEP, nep ri ly sin; o,
mice fed for 16 months; y, mice fed for 1 month.
178 S. Au gu stin et al. / Archives of Biochemistry and Biophysics 481 (2009) 177–182
respec tively (Fig. 1). Released Ab can undergo pro te o lytic deg ra da-
tion by sev eral pro te ases includ ing nep riyl sin (NEP) [4,27]. How-
ever, little is known how EGb761 affects the pro cess ing of APP, a
cru cial step in the path o gen e sis of AD, gen er at ing neu ro toxic Ab.
Thus, the aim of the pres ent study was to inves ti gate the short (1
month) and long-term (16 months) effect of die tary EGb761 sup-
ple men ta tion on APP metab o lism in a trans genic mouse model rel-
e vant for Alz hei mer dis ease.
Mate ri als and meth ods
Tris (hydroxy methyl) ami no meth ane (TRIS), sodium chlo ride,
sodium dode cyl sul fate (SDS) and Tween 20 were obtained from
Carl Roth (Kar lsruhe, Ger many). Sodium deoxy cho late and no nyl-
phe nyl-poly eth yl ene gly col (NP-40) were obtained from Sigma
Aldrich (Stein heim, Ger many).
The stan dard ized Ginkgo bi loba extract (EGb761) was a kind gift
from Dr. Schw a be phar ma ceu ti cals (Kar lsruhe, Ger many). EGb761
was man u fac tured accord ing to the Ger man Fed eral Health Author-
ity (BGA/BfArM Kom mis sion E, 1994) using ace tone/water as extrac-
tion sol vent and subsequent puri fi ca tion steps. The com po si tion of
the used extract is described in Au gu stin et al. 2008 .
Trans genic Tg2576 mice express ing the Swed ish muta tion of
human APP695 (APPK670N/M671L) at high lev els under the con-
trol of the ham ster prion pro tein pro moter were used in this study.
Trans genic mice were bred by mat ing Tg2576 males with C57B6/SJL
F1 females. Trans ge ni ty of the ani mals was tested for at 3 weeks of
age by PCR using tail biopsy sam ples as described in Hsiao et al.
At the age of 4 months, female Tg2576 were ran domly divided
into four groups (n = 10 for the short-term treat ment of 4 weeks
and n = 6 for the long-term treat ment of 16 months; body weight
21.5 ± 1.2 g; mean ± SEM).
Prior to the inter ven tion period, mice were adapted to a
low-fla vo noid con trol diet (C1000, Al tro min; Lage, Ger many) for 1
week. Dur ing inter ven tion (4 weeks and 16 months, respec tively),
mice were fed either the con trol diet (C) or EGb761 sup ple mented
(300 mg/kg diet) diet (G). Mice were housed under stan dard
con di tions (21–23 °C, 50–60% humid ity, 14-h light/10-h dark
cycle) and body weights were recorded weekly. Ani mal care and
exper i men tal pro ce dures were con ducted accord ing to the Ger man
Guide lines and Reg u la tions on Ani mal Care (De uts ches Tiers-
chutzge setz, 2006) and were approved by the Uni ver sity of Kiel
Com mit tee on Ani mal Care. At the age of 5 and 20 months,
respec tively, mice were decap i tated and brain tis sues (cor tex and
hip po cam pus) rap idly dis sected. Blood was taken from all ani mals
and sam ples from each die tary group were pooled.
Deter mi na tion of fla vo noid con tent in plasma
Plasma was obtained after cen tri fu ga tion (2000g for 10 min,
4 °C) and stored at ¡80 °C until anal y sis. Flavo nols were ana lyzed
in plasma ali quots by HPLC with post col umn deriv a ti za tion as
described pre vi ously . All plasma sam ples were treated with
b-glu cu ron i dase/sul fa tase (Carl Roth, Kar lsruhe, Ger many) prior to
extrac tion of flavo nols.
Gene expres sion anal y sis
Half of each cor tex and hip po cam pus (short-term experiment:
n = 10 and long-term experiment: n = 6) were imme di ately sus-
pended in RNAlater™ RNA sta bi li sa tion reagent (Qiagen, Hil den,
Ger many) and incu bated over night. Total RNA was extracted
accord ing to the RNeasy™ Lipid Tis sue Pro to col (Qiagen). RNA
integ rity, con cen tra tion and purity were checked as pre vi ously
described in Au gu stin et al. (2008) . RNA ali quots were stored
at ¡80 °C until anal y sis.
Mouse APP (msAPP), ADAM10 a pro te ase with a-secre tase
activ ity, NEP, APP bind ing pro tein (AP Pbp1) and human APP
(huA PP) primer pairs were designed to the cor re spond ing mRNA as
shown in Table 1 using Primer3 soft ware (http://fok ker.wi.mit.edu/
primer3/input.htm). The Gen Bank Acces sion Nos. are: P05067
for the human APP, P12023 for mouse APP, O35598 for ADAM10,
Q8VBW6 for AP Pbp1 and Q61391 for NEP. The mRNA and primer
sequences are given in Table 1. Primer pairs were obtained from
MWG (Ebers berg, Ger many). Quan ti TectP™ Primer Assay (Qiagen)
was used for 18S rRNA ampli fi ca tion, with a prod uct of 149 bp. For
one-step quan ti ta tive reverse trans crip tase poly mer ase chain reac-
tion (one-step qRT-PCR) two ali quots of RNA were ampli fied using
Quan ti Tect™SYBR™Green RT-PCR Kit (Qiagen). Exter nal rel a tive
stan dard curves of total RNA were deter mined with each run. Data
was nor mal ized by divid ing the expres sion level of tar get genes
by the level of 18S rRNA. Each PCR reac tion (final vol ume 20.0 lL)
Fig. 1. Scheme of APP pro cess ing.
S. Au gu stin et al. / Archives of Biochemistry and Biophysics 481 (2009) 177–182 179
Nucle o tide sequences of PCR prim ers used in real-time qRT-PCR.
Gene RNA sequenceSenseAnti sense Prod uct
gctgccaggt attg gat cat
cat tttgaccagcctcg act
ac cgctgcttagttggt gag
ggtgtgccagtga a gat gag
was per formed as described in the sup pli ers pro to col. Real-time
cycler con di tions were set accord ing to the Qiagen pro to col for 40
cycles. The anneal ing tem per a ture was 55 °C for msAPP, huA PP,
NEP and AP Pbp1 and 58 °C for ADAM10. “No tem plate” and “no
reverse trans crip tase” reac tions were used as neg a tive con trols.
Quan ti fi ca tion and melt ing curves of the ampli fied prod ucts were
ana lyzed using the Ro tor Gene3000 soft ware ver sion 6.0 (Cor bett
Life science; Syd ney, Aus tra lia).
Immu no blot ting
Resid ual cor ti ces and hip po campi were fro zen in liquid nitro gen
and stored at ¡80 °C until Western blot anal y sis. For iso la tion of
mem brane-bound pro teins Radio Immuno Pre cip i ta tion Assay
(RIPA)-lysis-buffer (150 mM sodium chlo ride, 1.0% NP-40, 0.5%
sodium deoxy cho late, 0.1% SDS, 50 mM Tris, pH 8.0) con tain ing pro-
te ase-inhib i tors (Com plete Mini; Roche, Mann heim, Ger many) was
used. Tis sue sam ples (25 ± 5 mg) were homog e nized in RIPA-buffer
(200 ll per 5 mg) with a rotor-sta tor homog e nizer for 45 s, agi tated
for 2 h at 4 °C, and cen tri fuged (20 min, 15300g, 4 °C). The super na-
tant was removed and the pro tein amount was mea sured with the
BCA kit (Pierce Bio tech nol ogy, Rock ford, USA). Pro tein homog e nates
were diluted 1:1 with Lae mmli sam ple buffer (Bio Rad, Lab o ra to ries;
Munich, Ger many) con tain ing 5% b-mercap toethanol and dena tured
for 5 min at 95 °C. Elec tro pho re sis was per formed on 10% Tris–HCl gel
(15 lg pro tein per lane; Cri te rion Gel Sys tem, Bio Rad) and pro teins
were trans ferred onto Im mo bi lion P-PVDF mem branes (Mil li pore,
Bed ford, USA). Mem branes were blocked in 3% non-fat milk (Bio mol,
Ham burg, Ger many) in Tris-buf fered saline con tain ing 0.5% Tween
20 (TBST) at room tem per a ture for 6 h. Sub se quently, blots were
incu bated with the primary anti bod ies for 12 h at room tem per a ture
fol lowed by incu ba tion with horse rad ish per ox i dase-con ju gated sec-
ond ary anti bod ies (1:10,000; Ab cam, Cam bridge, UK) for 1 h at room
tem per a ture. Immu no de tec tion ana lyzes were per formed with the
Imm un Star™ We sternC Kit (Bio Rad). All quan ti ta tive ana lyzes were
per formed with the Chem i Doc XRS using Quan ti ty One™ soft ware
(ver sion 4.6.5; Bio Rad).
When nec es sary, blots were stripped (62.5 mM Tris–HCl, 2%
SDS, and 1% b-mercap toethanol, pH 7.6) for 1 h at 55 °C fol lowed by
wash ing in water and TBST.
Anti bod ies
The fol low ing primary anti bod ies were used in this study:
Mouse mono clo nal anti-huA PP (clone 6A6; 1:2000; Bio mol, Ham-
burg, Ger many), mouse mono clo nal anti-APP (clone 22C11; 1:2000;
Chem icon, Teme cu la, USA), rab bit poly clonal anti ADAM10 (1:1000;
Dia no va, Ham burg, Ger many) and rab bit poly clonal anti-actin as an
inter nal ref er ence con trol (1:2000; Ab cam). All anti bod ies were
diluted in TBST.
Sta tis ti cal anal y sis
Graph pad Prism 4 soft ware (Graph pad Soft ware Inc., San Diego,
CA, USA) was used for sta tis ti cal anal y sis in the pres ent study. Data
for rel a tive huA PP mRNA lev els were ana lyzed by one-way ANOVA
fol lowed by Tu key–Kramer test for pair wise com par i son of group
means. Data for msAPP, ADAM10, AP Pbp1 and NEP mRNA lev els
in long-term treated ani mals and for huA PP and ADAM10 pro tein
expres sion were ana lyzed by a t-test for inde pen dent sam ples. In
the absence of nor mally dis trib uted data the non para met ric Mann-
Whit ney-U test was con ducted. Data are pre sented as mean ± SEM,
and sig nifi cance was accepted at P < 0.05.
In order to deter mine whether a long-term treat ment (16
months) with EGb761 influ ences genes and cor re spond ing pro teins
lev els of molec u lar tar gets, which are involved in APP metab o lism
qRT-PCR and Western Blot anal y sis were per formed in the cor tex
and hip po cam pus from trans genic Tg2576 mice fed with and with-
out Ginkgo bi loba extract over 1 and 16 months, respec tively. Mouse
cor tex and hip po cam pus RNA qual ity was good as indi cated by the
ratio between A260 nm and A280 nm which was 71.8. The mRNA lev-
els of the house keep ing gene 18 s rRNA in cor tex and hip po cam pus
were at sim i lar lev els in all ani mals (data not shown).
Plasma con cen tra tion of flavo nols
Con cen tra tions of flavo nols in plasma of mice treated with
EGb761 were 0.32 lmol/L, 0.076 lmol/L and 0.048 lmol/L for isorh-
am ne tin, ka empf er ol and quer ce tin, respec tively. The cor re spond-
ing val ues in the con trol group were 0.019 lmol/L (isorh am ne tin),
0.028 lmol/L (ka empf er ol) and 0.040 lmol/L (quer ce tin). Thus, the
sum of all three flavo nols was more than five-fold higher in the
group treated with EGb761. Plasma con cen tra tions were not sta tis-
ti cally ana lyzed since pooled sam ples were used.
Cor tex data
The rel a tive mRNA and pro tein lev els of huA PP are shown in Fig.
2A and B. The rel a tive cor tex mRNA lev els of genes involved in APP
pro cess ing after long-term treat ment with and with out EGb761 are
sum ma rized in Fig. 3A–D. In the cor tex of mice fed EGb761 over 16
months huA PP mRNA lev els were sig nifi cantly decreased com pared
to the mice fed the con trol diet for 1 month (p < 0.01, Fig. 2A). Fur-
ther, in the cor tex of long-term treated mice (oG) huA PP mRNA lev els
were sig nifi cantly dimin ished com pared to the con trol mice (oC) at
the same age (p < 0.05, Fig. 2A). Thus, rel a tive mRNA lev els in cor tex
of the long-term treated EGb761 mice (oG) were at the same level
as in the young ani mals on the con trol diet (yC) (Fig. 2A). Fur ther-
more, EGb761 sig nifi cantly low ered (p < 0.01) huA PP pro tein lev els
by »50% as com pared to the con trols after 16 months of treat ment
(Fig. 2B). How ever, in accor dance to the mRNA data, one month of
treat ment with EGb761 treat ment had no effect on huA PP pro tein
lev els in cor tex as well (data not shown). Fur ther more, in young mice
treated only for 1 month with EGb761, no sig nifi cant effect on cor ti cal
mRNA lev els of genes involved in APP pro cess ing (msAPP, ADAM10,
AP Pbp1 and NEP) was observed (data not shown). Long-term treat-
ment with EGb761 affected nei ther mRNA lev els of msAPP (Fig. 3A),
180 S. Au gu stin et al. / Archives of Biochemistry and Biophysics 481 (2009) 177–182
ADAM10 (Fig. 3B), AP Pbp1 (Fig. 3C) not of NEP (Fig 3D), sig nifi cantly.
Addi tion ally, pro tein lev els of ADAM10 remained largely unchanged
in cor ti ces of Tg2576 mice, in response to long-term die tary EGb761
treat ment (data not shown).
Hip po cam pus data
Lev els of huA PP mRNA were increased in hip po campi of
20-month-old mice who had been on the con trol diet com pared
to young ani mal on the same diet (Fig. 4A). Fur ther, EGb761 treat-
ment (oG) showed a trend (p < 0.1) towards lower huA PP mRNA
lev els after long-term treat ment (16 months) com pared to mice
of the same age on the con trol diet (oC) (Fig. 4A). Long-term die-
tary treat ment with EGb761 also decreased huA PP pro tein lev els
by »20% in hip po cam pus. How ever, this was also not sig nif i-
cant (p < 0.1, Fig. 4B).
Sim i lar to cor tex, rel a tive mRNA lev els of msAPP nor ADAM10,
AP Pbp1 and NEP were not sig nifi cantly altered by long-term
die tary sup ple men ta tion with EGb761 in hip po campi (Fig. 5A–D).
Hip po campi of mice fed EGb761 over a period of 16 months also
did not exhibit any sig nifi cant dif fer ences in ADAM10 pro tein lev-
els com pared to con trols (data not shown).
Fur ther more, no sig nifi cant effects of EGb761 on rel a tive mRNA
lev els of msAPP, ADAM10, AP Pbp1 and NEP in hip po campi of young
mice were observed (data not shown).
Dis cus sion
The pres ent study aimed to inves ti gate the effect of chronic
EGb761 treat ment on APP gen er a tion and pro cess ing in a trans genic
mouse model of AD. The results indi cate that a long-term die tary
sup ple men ta tion with EGb761 dif fer en tially affected mRNA and
pro tein lev els in brain tis sue of trans genic Tg2576 mice. The Tg2576
mice, as used in the pres ent study, con tain the human APP with the
dou ble muta tion (K670N/M671L) inserted into a ham ster prion pro-
tein (hPrP) cos mid vec tor [29,30]. Tg2576 mice develop increased
lev els of Ab and hence, senile Ab plaques in cor tex and hip po cam-
pus [29–33]. The Ab accu mu la tion, as depos its and plaques in the
brain, is a trig ger ing event in AD [4,27]. Thus, APP, sec re ta ses and Ab
degrad ing enzymes are potential molec u lar tar gets in ther apy of AD
and are cur rently in focus of AD research [3,34,35]. Our results show
a tis sue depen dent effect of EGb761 on APP gen er a tion. In cor tex
EGb761 sig nifi cantly decreased huA PP mRNA and pro tein lev els after
16 months of die tary inter ven tion. In hip po cam pus huA PP mRNA
and pro tein lev els were only mod er ately decreased by the EGb761
treat ment. In this con text it has been shown by Lah ir i and Ge (2004)
that gene expres sion of APP is cell and tis sue spe cific . In fact APP
expres sion in rat glia cells was lower as com pared to neu rons .
More over, Wa tan a be et al. have shown in mice that die tary treat-
ment with EGb761 dif fer ently affected gene expres sion in cor tex
and hip po cam pus . In their study, nine genes were up reg u lated
due to EGb761 in cor tex whereas in hip po cam pus only one gene was
Fig. 2. (A) Effect of EGb761 on rel a tive huA PP mRNA lev els in Tg2576 mouse cor tex after short (1 month) and long-term (16 months) of treat ment with EGb761. Results are
expressed as mean + SEM of six cor ti ces. (B) Rel a tive huA PP pro tein lev els in Tg2576 mouse cor tex after long-term (16 month) treat ment (n = 6). Actin served as load ing con-
trol. Western blots of indi vid ual ani mals are pre sented. *p < 0.05, **p < 0.01; yC-young trans genic ani mals treated for 1 month with the con trol diet, yG-trans genic ani mals
treated for 1 month with the EGb761 sup ple mented diet, oC-trans genic ani mals treated for 16months with the con trol diet, oG-trans genic ani mals treated for 16 months
with the EGb761 sup ple mented diet.
Fig. 3. Effect of EGb761 on rel a tive mRNA lev els of msAPP (A), ADAM10 (B), AP Pbp1
(C), and NEP (D) in cor ti ces of Tg2576 mice treated over 16 months with EGb761 (G)
or con trol diet (C). Results are expressed as mean + SEM of six cor ti ces.
S. Au gu stin et al. / Archives of Biochemistry and Biophysics 481 (2009) 177–182 181
Fig. 5. Effect of long-term EGb761 treat ment (16 months) on rel a tive mRNA lev els
of msAPP (A), ADAM10 (B), AP Pbp1 (C), and NEP (D) in hip po campi of tg2576 mice.
Results are expressed as mean + SEM of six cor ti ces.
Fig. 4. (A) Effect of EGb761 on rel a tive huA PP mRNA lev els in hip po campi of Tg2576 mice after short (1 month) and long-term (16 months) of treat ment with EGb761. Results
are expressed as mean + SEM of six hip po campi. (*)p < 0.1. (B) Rel a tive huA PP pro tein lev els in hip po campi of Tg2576 mice after long-term (16 month) treat ment. Actin served
as load ing con trol. Western blot of indi vid ual ani mal are pre sented. (*)p < 0.1, *p < 0.05, **p < 0.01; yC-young trans genic ani mals treated for 1 month with the con trol diet,
yG-trans genic ani mals treated for 1 month with the EGb761 sup ple mented diet, oC-trans genic ani mals treated for 16 months with the con trol diet, oG-trans genic ani mals
treated for 16 months with the EGb761 sup ple mented diet.
sig nifi cantly affected by EGb761 treat ment . Thus, our data and
pre vi ously pub lished stud ies indi cate that cor tex and hip po cam pus
respond dif fer ently to EGb761 as far as APP gen er a tion and its metab-
o lism is con cerned [36–38].
The trans genic huA PP in Tg2576 is under the con trol of the hPrP
pro moter [29,30]. The hPrP pro moter con tains var i ous reg u la tory
regions for tran scrip tion fac tors e.g. SP1 and AP1 bind ing sites as
well as a CCAAT-box . In vitro and ani mal stud ies have shown
that EGb761 low ers mRNA and pro tein lev els of pro in flam ma-
tory cyto kines such as inter leu kin-1b (IL-1b) and tumor necro sis
fac tor-a known to acti vate AP1 and SP1 gene expres sion [20,40,41].
Inter est ingly, Apelt et al. (2004) have shown that Tg2576 mice, as
used in the pres ent study, exhibit sig nifi cantly increased lev els
of IL-1b between the age of 12 and 21 month com pared to young
mice (3 month) . Hence, in our study EGb761 or one of its frac-
tions may have influ enced huA PP expres sion via down reg u la tion
of pro in flam ma tory cyto kine expres sion. There fore, stud ies in
trans genic ani mals con tain ing both the huA PP pro moter and the
huA PP open read ing frame were use ful to inves ti gate a pos si ble
influ ence of EGb761 on APP pro moter activ ity Another find ing of
the pres ent study is that EGb761 affected APP pro tein lev els in old
but not in young mice indi cat ing that either the age and/or the
dura tion of the EGb761 treat ment are impor tant deter mi nants of
Ginkgo bi loba effi cacy.
Fur ther more, we inves ti gated the impact of EGb761 on
ADAM10 mRNA and pro tein lev els. ADAM10, a pro te ase with
a-secre tase activ ity, cleaves APP within the Ab sequence gen er at-
ing non path o genic prod ucts (Fig. 1) [43,44]. Our results sup port
the data by Col ciaghi et al. (2004) indi cat ing that nei ther ADAM10
gene nor pro tein lev els are influ enced by EGb761 treat ment .
These authors reported that Ginkgo bi loba reg u lates APP pro cess-
ing towards the a-secre tase path way with out alter ing ADAM10
mRNA lev els after 5 days of treat ment . Thus, nei ther short nor
long-term treat ment with EGb761 might affect mRNA and pro tein
lev els of ADAM10 in rats and Tg2576 mice, respec tively. Col ciaghi
et al. have fur ther shown that APP cleav age prod ucts gen er ated
by the a-secre tase were increased in rat hip po cam pus. Thus it is
pos si ble that ADAM10 enzyme activ ity lev els are increased due
to EGb761 treat ment . As far as Ab for ma tion due to b-secre-
tase is con cerned, we have recently shown that nei ther mRNA nor
enzyme activ ity lev els of b site amy loid pre cur sor pro tein cleav ing
enzyme-1 (BACE-1) in Tg2576 mice cor tex was influ enced by long-
term treat ment (16 months) with EGb761 .
Over all, cur rent data indi cate that long-term die tary treat ment
with EGb761 over 16 months sig nifi cantly decreased huA PP pro-
tein lev els in the cor tex of Tg2576 mice. Thus, APP seems to be
an impor tant molec u lar tar get of EGb761. More over, the effects of
EGb761 might be rather tis sue spe cific since the cor tex seems to be
182 S. Au gu stin et al. / Archives of Biochemistry and Biophysics 481 (2009) 177–182 Download full-text
more respon sive towards EGb761 than the hip po cam pus. Fur ther
stud ies are war ranted to prove whether the changes in APP lev els
in response to EGb761 are also asso ci ated with changes in neu ro-
toxic Ab lev els and Ab induced senile plaques.
Acknowl edg ments
We are grate ful to the Ger man Research Foun da tion (DFG) for
fund ing this study by grant no. CE 59/2-1. The authors would like
to express their grat i tude to Dr. Karen Hsiao for kindly pro vid ing
Tg2576 F1 mice which were back crossed to breed N2 gen er a tion
mice used in this study. Data of this arti cle were awarded the Les-
ter Packer Young Inves ti ga tor award at the Oxy gen Club of Cal i for-
nia World Con gress 2008 (St. Bar bara, USA).
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