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Journal of Alzheimer’s Disease 21 (2010) 263–276 263
DOI 10.3233/JAD-2010-091528
IOS Press
Severe Motor Neuron Degeneration in the
Spinal Cord of the Tg2576 Mouse Model of
Alzheimer Disease
Ji-Seon Seoa, Yea-Hyun Leema, Kang-Woo Leea, Seung-Woo Kimb, Ja-Kyeong Leeband
Pyung-Lim Hana,∗
aDepartments of Brain & Cognitive Sciences and Chemistry and Nano Science, Ewha Womans University, Seoul,
Republic of Korea
bDepartment of Anatomy, Inha University School of Medicine, Inchon, Republic of Korea
Accepted 4 March 2010
Abstract. The transgenic mouse Tg2576 is widely used as a murine model of Alzheimer’s disease (AD) and exhibits plaque
pathogenesis in the brain and progressive memory impairments. Here we report that Tg2576 mice also have severe spinal cord
deficits. At 10 months of age, Tg2576 mice showed a severe defect in the hindlimb extension reflex test and abnormal body
trembling and hindlimb tremors when suspended by the tail. The frequency and severity of these abnormalities were overt at
10 months of age and became gradually worsened. On the foot-printing analysis, Tg2576 mice had shorter and narrower strides
than the non-transgenic control. Histological analyses showed that neuronal cells including cholinergic neurons in the lumbar
cord of Tg2576 mice were severely reduced in number. At 16 months of age, Tg2576 mice showed high levels of amyloid-β
accumulation in the spinal cord. Consistent with this, Tg2576 mice showed that lipid peroxidation levels were increased and
mitochondrial metabolic activity were significantly reduced in the spinal cord. Administration of curcumin, a natural compound
that has antioxidant properties, notably reversed motor function deficits of Tg2576 mice. The enhanced lipid peroxidation and
neuronal loss in the lumbar cord was also partially suppressed by curcumin. Electron microscopic analysis revealed that the
sciatic nerve fibers were severely reduced in number and were demyelinated in Tg2576 mice, which were partially rescued by
curcumin. These resultsshowed that Tg2576 mice display severe degeneration of motor neurons in the spinal cord and associated
motor function deficits.
Keywords: Motor neurons, reactive oxygen species, spinal cord, Tg2576
Supplemental data available online: available online: http://www.j-alz.com/issues/21/vol21-1.html#supplementarydata
INTRODUCTION
The transgenic mouse Tg2576 is widely used as a
murine model of Alzheimer’s disease (AD). Tg2576
mice were generated to express human AβPP695 with
the Swedish familial AD double mutation K670N-
∗Correspondence to: Pyung-Lim Han, Ph.D., Department of Brain
& Cognitive Sciences, Ewha Womans University, 11-1 Daehyun-
Dong, Seodaemoon-Gu, Seoul, 120-750, Republic of Korea. Tel.:
+82 2 3277 4130; Fax: +82 2 3277 3419; E-mail: plhan@ewha.ac.kr.
M671L (hAβPPswe) in an attempt to mimic familial
cases of AD [1]. Tg2576 mice show AD-like patho-
physiological changes, such as amyloid plaque de-
posits and gliosis in the brain starting at 9–12 months
of age [1–3]. Moreover, Tg2576 mice display mem-
ory impairments in several behavioral tests, including
Morris water maze, Y-maze, passive avoidance, active
avoidance, and circular platform [4]. Thus, it has been
thought that Tg2576 mice display various neurologi-
cal and behavioral deficits that resemble those of hu-
man AD patients in terms of age-dependent cognitive
ISSN 1387-2877/10/$27.50 2010 – IOS Press and the authors. All rights reserved
264 J.-S. Seo et al. / Spinal Cord Phenotypes of Tg2576 Mice
deficits and plaque pathogenesis. More importantly,
Tg2576 mice are readily available from a commercial
source. Therefore, this model has been used extensive-
ly worldwide in the past decade and has contributed to
our understanding of AD-like pathophysiology in the
brain.
The AD-like pathology displayed by Tg2576 mice
is produced by the transgenic expression of a mutant
form of human AβPP (hAβPPswe) in the brain under
the control of the 6.9-kb hamster prion protein (PrP)
promoter. The PrP promoter is able todirect expression
of a target gene at high levels in neuronal cells in the
central nervous system(CNS). While PrP is pathogenic
and causes scrapie in sheep and cow [5], the endoge-
nous cellular prion protein (PrPc) is present in various
regions of the body, including the spinal cord, brain
stem, and even epithelial cells of the coroid plexusand
intestine [6]. Although the ability of the 6.9-Kb PrP
promoter has not been characterized in detail, it likely
drives expression of the target gene in a distribution
pattern similar to that of the endogenous PrPc, i.e., not
only in the brain, but also in other regions of the body,
including the spinal cord. Recently, it was reported that
Tg-AβPP/PS1 AD model mice showed motor function
deficitsand spinalcord defects[7–9]. It istherefore im-
portant to understand whether Tg2576 mice have any
spinal cord problems. However, the transgenic effect
of the hAβPPswe on the spinal cord of Tg2576 mice
has not been studied, and AD research has continued
to work with this model without knowingthe potential
pathophysiology in the spinal cord.
In the present study, we demonstrate for the first
time that Tg2576 mice exhibit severe motor neuron
degeneration in the spinal cord and associated motor
function deficits.
MATERIALS AND METHODS
Animals and curcumin administration
The transgenic Tg2576 mouse is a murine model of
AD generated by overexpressing a mutant form of hu-
man AβPP (K670M/N671L) [1]. Tg2576 mice used in
this study were purchased mostly from Taconic Farms
Inc. (Germantown, NY, USA) or obtained from the
breeding with maintaining the genetic background in
C57BL6 x SJL F1 hybrid as described previously [1].
After weaning, 2–3 animals were housed per cage in a
temperature- and humidity-controlled environment un-
der a 12 h light/dark cycle (lights on at 7 a.m.), and
were maintained on a diet of lab chow and water ad
libitum. Curcumin was purchased from Sigma-Aldrich
Inc. (St. Louis, MO), and administered in a lab chow
form(500 ppm) for6 monthsbeginningat 10months of
age. All animals were handled in accordance with the
animalcare guidelinesof theEwha WomansUniversity
School of Medicine.
Assessment of Amyloid-βlevels
Aβ1−40 and Aβ1−42 ELISA assays were performed
as described previously [10,11]. The L3-5 spinal cord
was individually homogenized in Tris-buffered saline
(20 mM Tris and 137 mM NaCl, [pH 7.6]) supple-
mentedwith proteaseinhibitormixtures (CompleteMi-
ni; Roche, IN, USA). Homogenates were centrifuged
at 100,000 g for 1 h at 4◦C, and the supernatant was
used to measure soluble forms of Aβ1−40 and Aβ1−42.
The pellet was sonicated in 70% formic acid and cen-
trifuged as above, and the supernatant was collected.
The formic acid extract was thenneutralized with 1 M
Tris buffer (pH 11) by 1:20 dilution, and the diluted
formic acid extract were used for each assay. Human
Amyloid Aβ1−40 and Aβ1−42 colorimetric sandwich
ELISA kits (BioSource, Camarillo, CA, USA) were
used according to the manufacturer’s instructions.
Measurement of lipid peroxidation by
malondialdehyde (MDA)assay
Lipid peroxidation was assessed bymeasuring MDA
levels using the Bioxytech MDA-586 kit (Oxis Re-
search, Portland, OR, USA) as described previous-
ly [10,12]. Briefly, spinal cords (L3-5) were homog-
enized in 4 vol. of ice-cold 20 mM PBS (phosphate
buffered saline; pH 7.4) containing 5 mM butylat-
ed hydroxytoluene. Homogenates were centrifuged at
3,000 g for 10 min at 4◦C, and the supernatant was
used for each assay. The protein concentration of the
supernatant was determined by the Bradford method
(Bio-RadLaboratories,USA). For eachreaction,200µl
of the supernatant was mixed with 10 µl of probu-
col, 640 µl of diluted R1 reagent (1:3 of methanol:N-
methyl-2-phenylindole),and150 µl of 12 N HCl. Each
reaction was incubated at 45◦C for 60 min and cen-
trifuged at 10,000 g for 10 min. The absorbance of
the supernatant was then measured at 586 nm. MDA
data were normalized to the protein concentration and
expressed as a percentage of the sham control value.
Protein-bound MDA content was obtained by subtrac-
tion.
J.-S. Seo et al. / Spinal Cord Phenotypes of Tg2576 Mice 265
Mitochondria preparation
Mitochondria in the lumbar cord were isolated using
a QProteome Mitochondria Isolation Kit (QIAGEN,
Hilden, Germany) by following the manufacturer’s in-
structions. Briefly, the lumbar cord was homogenized
in 500 µl ice – cold lysis buffer containingprotease in-
hibitors. Homogenates were centrifugedat 1,000 g for
10 min at 4◦C, and the pellets were collected and resus-
pended in 1.5 ml lysis buffer. After passing through a
syringe 10 times, they were then centrifuged at 1,000 g
for 10 min at 4◦C, and the supernatants containing the
mitochondriawere collectedand recentrifugedat 6,000
g for 10 min at 4◦C. The pellets were resuspended in
1 ml of 10 mM Tris buffer(pH 7.6). After assay of pro-
teinconcentration, they weresubjected to mitochondria
activity assay.
Assessments of mitochondria metabolic function
Metabolic activities of mitochondrial complex I, II,
or IV was measured by following the procedures de-
scribed previously [13,14], with some modifications.
For complex I assay, 1 µg of the mitochondria prepara-
tion was preincubated at 37◦C in 240 µL of the incuba-
tion mixture (5 mM potassium phosphate, 70 g/L BSA,
60 mM DCIP, 17.5 mM decylubiquinone,and 1.0 mM
antimycine-A, pH 7.8). After 3 min, 5 µL of 160 mM
NADH was added andthe absorbance was measured at
600nm usingSpectra 190max UV detector(Molecular
devices, Sunnydale, CA, USA) at every 10-s intervals
for 5 min at 37◦C. After 5 min, 2.5 µLof100µM
rotenone in DMSO was added and the absorbancewas
measured again at 10-s intervals for 5 min.
For Complex II assay, the incubation mixture con-
tained 5 mM potassium phosphate, 70 g/L BSA,
0.25 mM EDTA, 0.1 mM ATP, 1 M succinate, 0.1 M
potassium cyanide (KCN), 30 mM DCIP, 17.5 mM
decylubiquinone, 1 mM antimycine-A, and 3 mM
rotenone, pH 7.8. One µg of the mitochondria fraction
was preincubated at 37◦C in 240 µl of the incubation
mixture without KCN and succinate. After 10 min,
KCNand succinatewere addedand theabsorbance was
measured at 600 nm at 10-sec intervals for 5 min at
37◦C. Blank absorbance was measured in the presence
of 500 mM malonate.
For Complex IV assay, the incubation mixture con-
tained cytochorome c solution (17 µM, cytochrome c,
0.03 M potassium phosphate (pH 7.4), and 1.2 M sodi-
um hydrosulfite. One µg of the mitochondria fraction
was preincubated at 30◦C in 300 µl of the incubation
mixture. After 3 min, KCN was added to the final con-
centration of 0.1 M and the absorbance was measured
at 550 nm at 10-sec intervals for 5 min at 30◦C.
Citrate synthase (CS) activity was measured by
following the procedure described previously [15].
Briefly, the incubation mixture contained50 mM Tris-
HCl (pH 7.5), 10 mM Acetyl CoA, and 10 mM DNTB.
One µg of the mitochondria fraction was preincubat-
ed at 30◦C in 240 µl of the incubation mixture with-
out KCN. After 10 min, KCN was added and the ab-
sorbance was measured at 410 nm at 10-sec intervals
for 5 min at 30◦C.
Hindlimb extension reflexes
Hindlimb extension reflexes were evaluated accord-
ing to the procedure and scoring system described
by [16,17]. Briefly, mice were suspended by the tail,
and the degree of motor deficit was scored on a 0 to
2 scale: a normal extension reflex in both hindlimbs
was scored as 2; imbalanced extension in thehindlimbs
as 1.5; extension reflex in only one hindlimb as 1.0;
the absence of any hindlimb extension as 0.5; and total
paralysis as 0.
Body trembling and hindlimb tremor assessments
Body trembling and hindlimb tremors were evalu-
ated according to the following procedure and scor-
ing system. Briefly, mice were suspended by the tail,
and the degree of body trembling and hindlimbtremors
was scored using a 0–4 rating scale: 0, normal in both
hindlimbs and body; 1, weak tremors (1–5 frequen-
cy/10 sec) of the hindlimbs and body; 2, intermediate
tremors (6–10 frequency/10 sec) of the hindlimbs and
body; 3, severe tremors (11–15 frequency/10 sec) of
the hindlimbs and body; very severe (16 and higher
frequency/10 sec) of the hindlimbs and body.
Footprint analysis
Footprint analysis was performed as described pre-
viously [18] with a minor modification. Briefly, mice
were trained to cross an illuminated alley (10 cm wide
and 33 cm long) and to go straight to the opposite side
located at the end of the alley. Their hindlimbs were
then coated with Chinese ink, and the mice were al-
lowedto walk alongthe corridor,the floor of which was
covered with a sheet of an oriental white paper. This
task was recorded in 1–2 trials until 10 clearly visible
footprints per animal were obtained. The footprints
were then scanned and the stride intervals were evalu-
ated using TOMORO ScopeEye 3.6 program (Techsan
Community, Seoul, Korea).
266 J.-S. Seo et al. / Spinal Cord Phenotypes of Tg2576 Mice
Immunohistochemistry and cresyl violet staining
Cresyl violet staining was performed as previously
described [19]. Mice were sacrificed and perfused with
4% paraformaldehyde in 0.1 M phosphate buffer (PB,
pH 7.4). The spinal cord was removed and post-fixed
in the same fixative at 4◦C overnight. Lumbar cord
(L3-L5) was coronally cut into 40-µm-thick sections
with a vibratome (Leica VT 1000S; Leica Instruments,
Nussloch,Germany). Spinal cordsections werestained
with 0.5% cresyl violet.
For immunohistochemistry, free-floating sections
were incubated with 4% bovine serum albuminin PB-
ST (PBS containing 0.1% Triton x-100; pH 7.4) for
1 h, and then with anti-ChAT (Chemicon, Temecula,
CA, USA), monoclonal anti-COX (cytochrome c oxi-
dase) complex IV subunit I (Mitosciences Inc, Eugene,
OR), or polyclonal anti-HNE (Alpha Diagnostic Intl.
Inc, Texas, USA) at 4◦C overnight. The sections were
washed with PBST and were reacted with biotinylat-
ed secondary antibodies diluted at 1:200 in PBST and
visualized by using the ABC Elite kit (Vector Labo-
ratories; Burligame, CA, USA). Stained images were
analyzed using Olympus BX 51 microscope equipped
with a DP71 camera and DP-B software (Olympus Co.,
Tokyo, Japan). Cell counts were performed in a blind
manner without knowledgeof genotypes. Cell density
of cresyl violet-stained cells or ChAT-positive cells in
the lumbar cord was counted using TOMORO Scope-
Eye 3.6 program (Techsan Community, Seoul, Korea)
after converting captured images of stained areas into
digital images.
Resin histology and electron microscopy
Mice were transcardially perfused with PBS, pH 7.4,
containing 4% paraformaldehyde and 2.5% glutaralde-
hyde. After washing in PBS three times, the sciatic
nerve and the ventral root at L5 were carefully dis-
sected and post-fixed at 4◦C for 1 h with 1% osmium
tetroxide (0.1 M PBS, PH 7.4). After washing with
fresh 1% osmium tetroxide in PBS, the tissue samples
were stored at 4◦C until they were embedded in Epon
812 (Polysciences, USA). Semi-thin (1-µm thick) sec-
tions were cut from each block using an ultramicro-
tome(Reichert-Jung, USA),then stained with1% alka-
line toluidine blue, and examined by light microscopy
(Olympus BX51; Tokyo, Japan). Ultra-thin (70-nm
thick) sections were prepared, treated with lead citrate
and uranyl acetate to enhance contrast, and examined
using a Hitachi H-7650 (Hitachi Co., Tokyo, Japan).
Statistical analysis
Datawere analyzedusing GraphPad Prism4 (Graph-
Pad Software, San Diego, CA, USA). Two-sample
comparisons were carried out using the Student’s t-
test, while multiple comparisons were made using one-
way ANOVA followed by the Newman-Keulsmultiple
comparison test. All data were presented as means ±
S.E.M. and statistical difference was accepted at the
5% level unless otherwise indicated.
RESULTS
Transgenic Tg2576 mice show visible plaque de-
posits in the brain, startingat 9 months of age: after this
ageplaque deposition andAβlevelsare enhancedslow-
ly and exponentially withage [1,10,20]. While handing
Tg2576 mice in their home cages, we observed that
these mice displayed abnormal motor function pheno-
types at 10 months of age. It is somewhat surprising
that motor function phenotypes in this model have not
beendescribed thus far,giventhe factthat Tg2576mice
are so popular and have been widely used in various
behavioral studies. We observed that, while being sus-
pended by the tail, Tg2576 mice held and retracted the
hindlimbs in the inward direction; in starkly contrast to
the stretching of the hindlimbs in the outward direction
incontrol mice (Fig.1A; SupplementalFig. 1,available
online: http://www.j-alz.com/issues/21/vol21-1.html#
supplementarydata). Thus, the hindlimb extension re-
flex response in Tg2576 mice was severely defec-
tive, and these neurologic deficits were more severe at
16months of agethan that at10months of age(Fig.1C).
Tg2576 mice at 10–16 months of age also displayed
severe body trembling and hindlimb tremors when sus-
pended by the tail (Fig. 1B). The frequency and sever-
ity of the motor function abnormalities progressively
worsened after 10 months of age in both female and
male Tg2576 mice (Fig. 1D).
The distinct impaired motor function phenotypes of
Tg2576mice promptedus to investigatepotentialhisto-
logical defects in the spinal cord. Cresyl violet staining
showed that the numbers of cresyl violet-stained cells
in the ventral horn of the Tg2576 mice at 10 months
of age were reduced compared with that in the non-
transgenic control mice and further reduced to approx-
imately 52.5% of the non-transgenic control mice at
16 months of age (Figs 2A–G). Anti-ChAT-positive
neurons in the lumbar cord of Tg2576 mice were also
gradually reduced in number with aging, and remained
J.-S. Seo et al. / Spinal Cord Phenotypes of Tg2576 Mice 267
Fig. 1. Abnormal hindlimb extension reflex and body trembling phenotypes of Tg2576 mice. Photographs of the hindlimb extension reflex
induced by tail suspension of non-transgenic control (A) and Tg2576 mice (B). C) Quantification of the hindlimb extension reflex at 6, 11, or
16 months of age using a 0-3 numerical scale as described in Materials and Methods; 0, normal; 1, mild defect in hindlimb extension reflex; 2,
severe defect in hindlimb extension reflex. D) The body trembling and hindlimb tremor scoring system consisted of a 0–4 numerical scale: 0,
normal in both hindlimbs and body; 1, weak tremors of the hindlimbs and body; 2, intermediate tremors of the hindlimbs and body; 3, severe
tremors of the hindlimbs and body; 4, very severe tremors of the hindlimbs and body. The frequency of hindlimb tremor per 10 sec is indicated in
the table (top panel). Quantification of scored body trembling and hindlimb tremor of Tg2576 mice at 6, 11, or 16 months of age (bottom panel).
Data are presented as means ±SEM (n=5–8). ∗and ∗∗ denote a significant difference between groups at p<0.05 and p<0.01, respectively.
One-way ANOVA with Newman-Keuls multiple range test was applied.
neurons at 16 months of age expressed ChAT at much
lower levels (Figs 2H–N), indicating that cholinergic
neurons in the spinal cord of Tg2576 mice were affect-
ed. Consistent with these findings, histological analy-
sis of toluidine blue stained ventral root at the level of
L5 showed that the nerve fibers in Tg2576 mice had a
defect in myelination and were reduced in number at
10 months of age (Supplemental Fig. 2).
Immunohistological analysis with anti-Aβ1−42 anti-
body (Bam10) showed no evidence of plaque deposi-
tion in the lumbar cord of Tg2576 mice at 16 months of
age. However,ELISA assay revealed that the levels of
Tris-buffersoluble Aβ1−42 andAβ1−40 were markedly
enhanced in the lumbar cord (L3-L5) in an age depen-
dent manner (Figs 3A and B). Similarly, formic acid-
extractedinsolubleforms ofAβ1−42 and Aβ1−40 levels
were increased (Figs 3C and D). Because highlevels of
Aβ1−42 result in strong oxidative stress in the brains of
Tg2576 mice [10,11], we investigated whether Tg2576
mice have enhanced oxidative stress in the spinal cord.
Analysis of malondialdehyde (MDA) levels revealed
thatlipid peroxidation levelsinthe lumbar cord(L3-L5)
of Tg2576 mice at 6–14.5 monthsof age were marked-
ly enhanced compared to that in the non-transgenic
control, and the increase was age-dependent(Fig. 4A).
Consistent with this data, immunohistochemistry per-
formed with anti-HNE (4-hydroxy-2-nonenal) stain-
ing revealed that the levels of HNE, which is an ox-
idized by-product of lipid peroxidation, were notably
enhanced in the spinal cord of Tg2576 mice (Fig. 4B).
268 J.-S. Seo et al. / Spinal Cord Phenotypes of Tg2576 Mice
Fig. 2. Loss of cell density in the lumbar cord of Tg2576. A-F) Representative photomicrographs showing cresyl violet-stained lumbar L3-5
spinal cord of non-transgenic control (A-C) and Tg2576 mice (D-F) at 6 (A, D), 10 (B, E), and 16 (C, F) months of age. G) Quantification
of the numbers of cresyl-violet stained cells in the ventral horn of the lumbar L3-5 of non-transgenic control and Tg2576 mice at 6, 10, and
16 months of age. Cresyl violet-stained cells larger than 5 µm in diameter within the circled area (600 µm in diameter) were counted in both
ventral horns using TOMORO ScopeEye 3.6 program, as described in Materials and Methods. Counting of cresyl violet-positive cells with
larger than 15 µm within in the circled area in Fig. 2A showed similar results (data not shown). H-M) Representative photomicrographs showing
anti-ChAT-stained lumbar L3-5 spinal cord of non-transgenic control (H-J) and Tg2576 mice (K-M) at 6 (H, K), 10 (I, L) and 16 (J, M) months
of age. N) Quantification of the numbers of anti-ChAT-stained cells in the ventral horn of the lumbar L3-5 of non-transgenic control and Tg2576
mice at 6, 10, and 16 months of age. Cells within the circled area in Fig. 2A were counted as above using TOMORO ScopeEye 3.6 program.
Data represent means ±SEM (the numbers of animals included in each group were 4–8, and 2-3 sections for each animal were examined).∗
and ∗∗ indicate a difference between groupsat p<0.05 and p<0.01, respectively (one-way ANOVA with Newman-Keuls multiple range test).
The high oxidative stress levels in the spinal cord of
Tg2576 mice led us to test whether antioxidants can af-
ford a therapeutic effect against motor function deficits
of Tg2576 mice. To address this, we chose curcumin,
because it has a strong antioxidant property [21] and
confers anti-AD-like effects on the brains of Tg2576
mice [22]. Administration of curcumin (500 ppm) to
Tg2576 mice starting at 10 months of age partiallysup-
pressed the enhanced HNE levels inthe spinal cord of
Tg2576 mice at 18 months of age (Figs 4B–E). These
results suggest that the high oxidative stress pressure
correlated with the severity of motor function defects
and neuronal loss in the spinal cord of Tg2576 mice.
Next, we examined whether the mitochondriarespi-
ratory systems are affected in Tg2576 mice. The mi-
tochondria respiratory complex I activity in the lum-
bar cord of Tg2576 mice was comparable to that in
non-transgenic control. In contrast, the activities of
the complex II and complex IV were both reduced to
62.2% and 52.7%, respectively, of the non-transgenic
control (Figs 5A–C). The activity of citrate synthase
was reduced to 68.8% of the non-transgenic control,
implicating that mitochondrial metabolic activity in the
glucose metabolism is also impaired (Fig. 5D). Con-
sistent with this, an immunohistochemical study using
anti-COX (complex IV) subunit 1 against histological
sections of the spinal cord revealed that COX subunit 1
level was enhancedin Tg2576 mice, while it was par-
tially reduced in curcumin-treated mice. These results
support that Tg2576 mice showed impaired mitochon-
dria metabolic activity in the spinal cord and curcumin
partially suppressed the mitochondrial impairment.
Concerning behavioral phenotypes, administration
of curcumin starting at 10 months of age markedly
J.-S. Seo et al. / Spinal Cord Phenotypes of Tg2576 Mice 269
Fig. 3. Accumulation of Aβ1−42 and Aβ1−40 levels in the lumbar cord of Tg2576 mice. A-D) Amounts of Tris-buffer soluble (A, B) or formic
acid-extracted (C, D) Aβ1−40 (A, C) and Aβ1−42 (B, D) in the lumbar cord of non-transgenic control mice (Non-Tg, 16 months of age) and
Tg2576 mice (Tg) at 6, 10, or 16 months of age were measured by ELISA. Each group contained 4–6 animals and the ELISA was performed in
duplicate. ∗and ∗∗ indicate a difference between groups at p<0.05 and p<0.01, respectively (one-way ANOVA with Newman-Keuls multiple
range test).
improved hindlimb extension reflex defects (Fig. 6A)
and hindlimb tremors (Fig. 6B) as examined at 10–
14.5 months of age. Tg2576 mice at 16 months of age
were not able to walk normally on the floor. To quanti-
tatively assess their neurological deficits, we analyzed
thefootprintpatterns ofthe mice whenthey were forced
to walk along a narrow corridor (Figs 7A andB). In the
foot-printing test, the stride length of Tg2576 mice was
shorter than that of the non-transgenic control, while
the curcumin-fed group displayed no such phenotype
(Fig. 7C). Similarly, Tg2576 mice tended to show a
slightly narrower stride width than the non-transgenic
control, while curcumin administration prevented this
phenotype(Fig. 7D). These results suggest thatTg2576
mice have abnormalgaits, which were significantly im-
proved by curcumin.
Next,we examinedwhetherthetherapeutic effectsof
curcumin on the neurological deficits of Tg2576 mice
were produced via a neuroprotective mechanism. Cre-
syl violet staining of the lumbar cord L3-L5 showed
that cresyl violet-positive cells in the ventral horn of
the Tg2576 mice at 16 months of age were reduced in
numbercompared withthose in thenon-transgeniccon-
trol mice, and this reduction was inhibited by curcum-
in (Figs 8A–C,G). Anti-ChAT staining revealed that
cholinergic neurons in the lumbar cord of Tg2576mice
at 16 months of age were enhanced in number after
curcumin treatment (Figs 8D–F,H).
We continued to explore the nature of the neu-
ropathologic defects in the sciatic nerves of Tg2576
miceand theirpathologychange with curcumin. Tolui-
dine blue staining of semi-thin-sectioned sciatic nerves
showed that the number of nerve fibers in the sciatic
nervesofTg2576mice at16 monthsofage wasreduced
to 61.3% of that in the non-transgenic control, while
270 J.-S. Seo et al. / Spinal Cord Phenotypes of Tg2576 Mice
Fig. 4. Enhanced lipid peroxidation in the spinal cord of Tg2576 mice. A) MDA (malondialdehyde) levels in the lumbar cord of non-transgenic
control mice (Non-Tg) at 14.5 months of age and Tg2576 mice at 6, 10 and 14.5 months of age are presented. B-E) Quantification of (B), and
representative photomicrographs (C-E) showing, anti-HNE immunoreactivity in the ventral horn of the lumbar L3-5 of non-transgenic control
(nonTg; C), Tg2576 mice (Tg; D) and Tg2576 mice treated with curcumin (Tg+Cur; E), all at 16 months of age. The levels of anti-HNE
immunoreactivity within the circled area (600 µm in diameter) in Fig. 4C were quantified. Curcumin (500 ppm) was administered to Tg2576
mice starting at 10 months of age to 16 months of age. Both MDA and HNE are oxidized by-products of lipid peroxidation. Each group
contained 4–6 animals. ∗and ∗∗ denote differences between the indicated groups at p<0.05 and p<0.01, respectively (one-way ANOVA with
Newman-Keuls multiple range test).
curcumin treatment reversed the reduction to 90% of
the non-transgenic control. Moreover,severe demyeli-
nation phenotypes were evident in the nerve fibers of
Tg2576 mice and their deficits were partially reversed
by curcumin (Figs 9A–C; Supplemental Fig. 3). Con-
sistent with this, an EM study revealed that the ex-
tent of demyelination was severe in Tg2576 mice at
16 months of age, and these defects were partially sup-
pressed in Tg2576 mice fed curcumin(Figs 9D–I). Se-
vere demyelination in Tg2576 mice is consistent with
the neurological phenotypes of hindlimb tremors and
body trembling.
DISCUSSION
The present study demonstrates that Tg2576 mice
havevariousmotorfunctiondefects,includinghindlimb
extension reflex impairment, body trembling, hindlimb
tremors, and abnormal gaits. Moreover, Tg2576 mice
show severe neuronalloss in the spinal cord and axonal
degeneration of the sciatic nerves. Such spinal cord
phenotypes in Tg2576 mice raise some important is-
sues for AD-related behavioral studies and AD-related
pathology.
Our results suggest that behavioral analyses of
Tg2576 mice should be evaluated considering that this
model has spinal cord deficits. AD models have been
used to search for the mechanisms underlying patho-
logical changes, such as plaque pathology and behav-
ioral changes, such as memory impairment. Many be-
havioral studies of cognition are carried out under the
assumption that subject mice show normal movement
behaviors because cognitive functions of animals are
deduced from behavioral performance in behavioral
tests. For example, learning and memory measured by
J.-S. Seo et al. / Spinal Cord Phenotypes of Tg2576 Mice 271
Fig. 5. Mitochondria function was defective in the spinal cord of Tg2576 mice. A-D) Mitochondrial metabolic activities of the complex I
(A), complex II (B), complex IV (C), and citrate synthase (D) in the lumbar cord of Tg2576 mice at 16 months of age. ∗denotes differences
between the indicated groups at p<0.05 (Student’s t-test). E-J) Representative photomicrographs showing anti-COX (complex IV) subunit 1
immunoreactivity in the ventral horn of the lumbar L3-5 of non-transgenic control (non-Tg; E and H), Tg2576 mice (Tg; F and I) and Tg2576
mice treated with curcumin (Tg+Cur; G and J), all at 16 months of age. Photomicrographs of H-J represent a high magnification of the ventral
horn. K) Quantification of anti-COX (complex IV) subunit 1 immunoreactivity within the circled area (600 µm in diameter) in Fig. 4E in the
ventralhornofnon-transgeniccontrol(E),Tg2576mice(F)andTg2576micetreatedwithcurcumin(G).∗denotesdifferencesbetweenthe
indicated groups at p<0.05 (one-way ANOVA with Newman-Keuls multiple range test).
Fig. 6. Curcumin notably rescued motor extension reflex and body trembling phenotypes of Tg2576 mice. A, B) Abnormalities of tail
suspension-induced hindlimb extension reflex (A) and body trembling and hindlimb tremor phenotypes (B) of Tg2576 mice were rescued by
curcumin. Curcumin (500 ppm) was administered starting at 10 months of age and behavioral performance was assessed at 16 months of age.
Hindlimb extension reflex and body trembling and hindlimb tremor phenotypes were scored as described in Fig. 1 legend and Materials and
Methods. Data are presented as means ±SEM (n=5–8). ∗and ∗∗ denote a significant difference between groups at p<0.05 and p<0.01,
respectively (One-way ANOVA with Newman-Keuls multiple range test).
272 J.-S. Seo et al. / Spinal Cord Phenotypes of Tg2576 Mice
Fig. 7. Footprint analysis of Tg2576 mice and improvement of motor function deficits by curcumin. A) Experimental set-up of footprint analysis
(left panel). Stride length and stride width were defined as indicated (right panel). B) Representative footprints of each group. Individual mice
with hindlimbs coated with Chinese ink were allowed to walk on a sheet of oriental white paper. Non-Tg, non-transgenic control; Tg, Tg2576;
Tg-Cur, Tg2576 mice fed curcumin. C, D) Tg2576 mice had shorter (C) and wider (D) strides than non-transgenic control. Data are presented
as means ±SEM (n=5–8). ∗∗ denotes a significant difference between groups at p<0.01. One-way ANOVA with Newman-Keuls multiple
range test was applied.
the Morris water maze test are expressed as a function
of a distance swum in searchof a hidden platformin the
water maze. Poor swimming performance in the test is
regarded as an indication of cognitive defects. Howev-
er, any defects or potential weakness in the spinal cord
might influence the performance of the task in such
tests that require normal motor function. The Morris
water maze test is based on extensive repeated swim-
ming tests. Previous studies have intended to provide
evidence of normal motor function in that Tg2576 and
control non-transgenic mice show comparable swim-
ming velocities. However, knowing that Tg2576 mice
have spinal cord defects, it is now obvious that more
rigorous investigations of Tg2576 mice are needed.
Tg2576 mice show abnormal locomotoractivity in the
open field test. Because that reducedlocomotor activity
can be caused by problems in emotion [4,23], motor
function-drivenbehavioral tests need to be interpreted
with additional care.
Tg2576 mice are an excellent model of plaque
pathology in the AD brain, but do not clearly show
neurodegeneration in the brain, a true signature in the
brain with human AD pathology. Meanwhile, cogni-
tive impairments and psychiatric deficits are important
symptoms of AD, while motor function deficits are al-
so frequently observed in patients with AD [24]. In
this sense, Tg2576 mice may serve as an AD model
to explore Aβ-induced neurodegeneration in the CNS,
albeit in the spinal cord. In the past years, most studies
with murine models of AD have focused on functions
and dysfunctions of the AD brain, and consequently
less attention was paid to the spinal cord. The Tg2576
mice are very popular in AD research, and a phenotype
of Aβ-induced neurodegeneration in the spinal cord
might extend the usefulness of this model. Moreover,
J.-S. Seo et al. / Spinal Cord Phenotypes of Tg2576 Mice 273
Fig. 8. Curcumin suppressed neuronal loss in the spinal cord of Tg2576 mice. A-F) Representative photomicrographs showing cresyl violet
(A-C) or anti-ChAT (D-F) stained lumbar L3-5 spinal cord of non-transgenic control (non-Tg; A and D), Tg2576 mice (Tg; B and E), and Tg2576
mice fed curcumin (Tg-Cur; C and F) at 16 months of age. G, H) Quantification of the numbers of cresyl violet (G) or anti-ChAT (H) stained
cells in the ventral horn of the three mouse groups described above. Cells larger than 5 µm in diameter found within the circled area (600 µmin
diameter) in Fig. 8A in both ventral horns for each animal were counted using TOMOROScopeEye 3.6 program. Counting of cells with larger
than15µmwithininthecircledareainFig.8Ashowedsimilarresults(datanotshown).Datarepresentmeans±SEM(thenumbersofanimals
includedineachgroupwere4–8,and2–3sectionsforeachanimalwereexamined).∗∗indicatesadifferenceatthep<0.01levelineachgroup
(One-way ANOVA with Newman-Keuls multiple range test).
since human AD patients often show motor function
deficits [25–27], Tg2576 mice will bea useful model to
study how Aβ-induced neurodegeneration causes the
spinal cord pathology. Recently, it was reported that
transgenic Tg-AβPP/PS1 AD model mice showed mo-
tor function defects and/or spinal cord deficits [7–9].
These lines showed also axonal pathology in the spinal
cord, as Tg2576 mice displayed. Tg-AβPP/PS1 mice
also showed hyperphosphorylation of tau proteins, en-
hanced activity of GSK-3 in the spinal cord, and im-
pairments in axonal transport in sciatic nerves [7]. Al-
though the mechanisms for the spinal cord defects in
Tg-AβPP/PS1 and Tg2576 mice may not be identical,
the results of these studies, including ours, suggest that
a common mechanism, such as a mechanism requiring
Aβ-induced reactive oxygen species (ROS) accumu-
lation, is likely involved in the pathogenesis of spinal
cord deficits of Tg-AβPP/PS1 and Tg2576 mice.
We provide evidencethat high ROSlevels are related
to neuronal injury in the spinal cord of Tg2576 mice.
Because Tg2576 mice are transgenic animals express-
ing a mutant form of human AβPP under the control
of the PrP promoter [1] and Aβlevels are enhanced in
the spinal cord of Tg2576 mice (Fig.4), age-dependent
ROS accumulation in Tg2576mice is likely caused by
Aβ(Fig. 3). In fact, several lines of evidence support
thatAβ1−42 is neurotoxic, because it can induce oxida-
tivestress and increase lipid peroxidationin vitroand in
vivo[28–30]. Tg2576 mice at 14.5 months of age show
a significant level of visible plaques and at 16 months
of age they show more plaques, but no evidence for
neuronal loss, in the brain ([10] unpublished observa-
274 J.-S. Seo et al. / Spinal Cord Phenotypes of Tg2576 Mice
Fig. 9. Sciatic nerve degeneration of Tg2576 mice and partial suppression by curcumin. A-C) Transverse sections of toluidine blue-stained
sciatic nerves of non-transgenic control mice (non-Tg; A), Tg2576 mice (Tg; B), and Tg2576 mice fed curcumin (Tg+Cur; C) at 16 months of
age. Neuronal fibers in Tg2576 mice had a defect in myelination and a vacuole developed within the myelination sheath, resulting in ring-like
shapes in their transverse sections (inset in B). Administration of curcumin partially suppressed the demyelination and vacuolization (C). Insets
are high magnification. Scale bars in Fig. 9C; 50 µm. D-I) Electron micrographs of transverse sections of the sciatic nerves of non-transgenic
control mice (D, G), Tg2576 mice (E, H) and Tg2576 mice fed curcumin (F, I) at 16 months of age. Low (D-F) and high (G-I) magnifications
are shown. Scale bars are indicated (F, I).
tion), whereas in the spinal cord of Tg2576 mice, vis-
ible plaques were not deposited, but neuronal loss oc-
curred. Our analysis suggests that the level of Aβ1−42
in the spinal cord of Tg2576 mice at 16 monthsof age
is similar to the level detected in the brain of Tg2576
mice at 10 months of age and approximately 10% of
that in the brain of Tg2576 mice at 14.5 monthsof age
([10] unpublished observation). The lipid peroxidation
level in the spinal cord was similar to that in the brain
of Tg2576 mice at 14.5 months of age (this study) [10].
Because soluble or oligomeric Aβis pathogenic [31–
33], we speculate that Aβitself, or ROS produced by
Aβ, might cause neuronal injury in the spinal cord of
Tg2576 mice. It is also possible that antioxidant sys-
tems in the spinal cord are not as powerfully fortified
as in the brain of Tg2576 mice at 14.5 months of age,
and so neuronal loss occurs in the spinal cord but is not
in the brain in the similar condition. In addition, the
spinal cord of Tg2576 mice at 16 months has deficits
in mitochondrial function (Fig. 5). Mitochondria are a
major source of free radical production and mitochon-
drial metabolism impairment is a prominent feature of
neurodegenerative diseases [34]. Recent studies sug-
gest that Aβpeptides cause mitochondrial respiratory
dysfunctions [35,36]. Together, these results suggest
that accumulated Aβin the spinal cord of Tg2576 mice
might cause mitochondrial dysfunctions and neurotox-
icity.
It may be worth noting that both neuronal loss and
sciatic nerve degeneration of Tg2576mice are reminis-
cent of the pathology displayed by mouse models of
amyotrophic lateral sclerosis (ALS) in several respects.
ALS shows progressive neurodegeneration of motor
neurons in the primary motor cortex, brainstem, and
spinal cord and produces motor function defects [37].
Mutations in the gene for SOD1 can cause familial
ALS [38,39]. Moreover, transgenic G93A-SOD1 mice
overexpressing a mutant form of human SOD1 show
J.-S. Seo et al. / Spinal Cord Phenotypes of Tg2576 Mice 275
spinal cord phenotypes, including high ROS levels and
neural loss in the spinal cord, such as in Tg2576 mice.
Age-dependentsevereneuronalloss occursinthe spinal
cord of Tg2576 mice andG93A-SOD1 mice. These re-
sults support the notion that high ROS levels may cause
to neuronal loss in the spinal cords of both Tg2576and
Tg-G93A-SOD1 mice.
Our results suggest that the severities of motor func-
tion defects of Tg2576 mice, including hindlimb ex-
tension reflex impairment, body trembling, hindlimb
tremors, and abnormal gaits, were not linearly propor-
tional to the decrease in the number of ChAT-positive
neuronsor cresyl violet-stainedcells intheventral horn.
Non-transgenicmice showed adetectabledecrease over
ages in the numbers of ChAT-positive neurons in the
ventral horn at 16 months of age comparedwith those
at 6 months of age, but did not show severe phenotypes
at 16 months of age (Figs 1 and 2). In addition, our
results showed that motor function defects of Tg2576
mice appear to be expressed at a significant level when
the numbers of cresyl violet-stained cells in the ventral
horn were declined below a certain level (Figs 1 and
2). Moreover, the nerve fibers in the ventral root had
a defect in myelination and were reduced in number
(Supplemental Fig. 3), suggesting the possibility that
muscle denervation might occur in Tg2576 mice. It
may be worthy to also note that the behavioral data we
analyzed were about overtly expressed motor function
phenotypes, rather than healthy state of motor func-
tion or motor function competence. We do not exclude
the possibility that motor function defects of Tg2576
mice are produced in part by sciatic nerve degenera-
tion, although the present study does not explore the
time course of its progression.
Administration of curcumin ameliorates the neuro-
logical phenotypes of Tg2576 mice. The therapeutic
effects of curcumin were so strong that both the neu-
ronal loss and the behavioral abnormalities appeared to
be rescued by treatment with this natural compound, as
it suppressed plaque pathology in the brain of Tg2576
mice [22]. While the therapeutic effects of curcumin
on neuronal loss and behavioral deficits were dramat-
ic for the early stages of pathological expressions, the
effects were detectably diminished at 15–16 months of
age. EM study revealed that the sciatic nerveswere im-
paired in Tg2576 mice. Though the present study did
not provide the mechanismunderlying the sciatic nerve
degeneration, curcumin appearsto be beneficial by de-
laying the pathogenesis of nerve degeneration, possi-
bly due to its antioxidant effect [40] or its capability
to reduce Aβlevels [18,41]. However, our EM study
of the sciatic nerves showed that the therapeutic effects
of curcumin practically vanished in Tg2576 mice at 16
monthsof age,though thevisible cell bodiesin the ven-
tralhorn of thelumbar cordremained detectably higher
thanin untreatedTg2576mice. Wespeculate thatcatas-
trophic pressures in Tg2576 mice at 15–16 months of
age, which might be caused by multiple mechanisms,
override the therapeutic effects of curcumin. Consider-
ing these results and the fact that Tg2576 mice at 10–
13 months of age showed severe body trembling and
hindlimb tremors (Fig. 1D), it is possible that defects in
axonalfibers, includingdemyelination,in Tg2576 mice
are responsible, at least in part, for the pathological
expression.
ACKNOWLEDGMENTS
This work was supported by a grant (KRF-2008-
313-E00483) from Korea Research Foundation and a
grant (M103KV010014-08K2201-01410) from Brain
ResearchCenter ofThe 21stCentury FrontierResearch
Program, of the Ministry of Education, Science and
Technology, Republic of Korea.
Authors’ disclosures available online (http://www.j-
alz.com/disclosures/view.php?id=364).
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