Modulation of human neural stem cell differentiation
in Alzheimer (APP23) transgenic mice by phenserine
Amelia Marutle*†, Masao Ohmitsu*, Mats Nilbratt‡, Nigel H. Greig§, Agneta Nordberg‡, and Kiminobu Sugaya*
*Biomolecular Sciences Center, Burnett College of Biomedical Sciences, University of Central Florida, Orlando, FL 32816;‡Division of Alzheimer
Neurobiology, Department of Neurobiology, Care Sciences, and Society, Karolinska Institutet, Karolinska University Hospital Huddinge,
S-141 86 Stockholm, Sweden; and§Section on Drug Design and Delivery, Laboratory of Neurosciences, National Institute on Aging,
National Institutes of Health, Baltimore, MD 21224
Communicated by Tomas Ho ¨kfelt, Karolinska Institutet, Stockholm, Sweden, June 14, 2007 (received for review March 9, 2007)
exposed to high concentrations of secreted amyloid-precursor
protein (sAPP) in vitro differentiated into mainly astrocytes, sug-
gesting that pathological alterations in APP processing during
neurodegenerative conditions such as Alzheimer’s disease (AD)
may prevent neuronal differentiation of HNSCs. Thus, successful
neuroplacement therapy for AD may require regulating APP ex-
pression to favorable levels to enhance neuronal differentiation of
HNSCs. Phenserine, a recently developed cholinesterase inhibitor
(ChEI), has been reported to reduce APP levels in vitro and in vivo.
In this study, we found reductions of APP and glial fibrillary acidic
protein (GFAP) levels in the hippocampus of APP23 mice after 14
days treatment with (?)-phenserine (25 mg/kg) lacking ChEI ac-
tivity. No significant change in APP gene expression was detected,
suggesting that (?)-phenserine decreases APP levels and reactive
astrocytes by posttranscription regulation. HNSCs transplanted
into (?)-phenserine-treated APP23 mice followed by an additional
7 days of treatment with (?)-phenserine migrated and differenti-
ated into neurons in the hippocampus and cortex after 6 weeks.
Moreover, (?)-phenserine significantly increased neuronal differ-
entiation of implanted HNSCs in hippocampal and cortical regions
of APP23 mice and in the CA1 region of control mice. These results
indicate that (?)-phenserine reduces APP protein in vivo and
increases neuronal differentiation of HNSCs. Combination use of
HNSC transplantation and treatment with drugs such as (?)-
phenserine that modulate APP levels in the brain may be a useful
tool for understanding mechanisms regulating stem cell migration
and differentiation during neurodegenerative conditions in AD.
amyloid precursor protein ? transplantation ? immunohistochemistry ?
neurogenesis ? Alzheimer’s disease
onstrated that migration and differentiation of these cells is
regulated primarily by environmental cues (1–4). Pathological
changes that occur in neurodegenerative disorders such as
Alzheimer’s disease (AD) may profoundly affect the brain
microenvironment, which may in turn affect the fate of NSCs.
The amyloid hypothesis, which postulates that ?-amyloid (A?)
neurotoxicity plays a causative role in AD, has dominated much
of AD research (5) and the absence of a lethal phenotype in
amyloid-precursor protein (APP) knockout mice (6) has de-
tracted attention from the physiological functions of APP.
Several studies have shown that APP is involved in regulating
neurite outgrowth, cell proliferation, neuronal migration, and
differentiation (7–10). APP expression is also increased after
brain injury, and increased levels are observed in apoptotic cells
(11, 12). Other studies report that A? inhibits NSC migration by
increasing amyloid-associated cell death and by dysregulation of
not only A? but that also altered APP processing during the
course of AD may have effects on stem cell biology.
Previously, we showed that human NSCs (HNSCs) trans-
planted into aged rats differentiated into neural cells and could
ransplantation of neural stem cells (NSCs) to the developing
brain and in animal models of neurodegeneration has dem-
reverse age-associated cognitive impairment in these animals
(3). This study demonstrated that the aged rat brain was capable
of providing necessary environmental conditions for HNSCs to
retain their multipotency and provided some evidence for the
potential of stem cell replacement therapies to improve memory
and cognitive deficits in AD. However, we recently found
increased in vitro glial differentiation of HNSCs treated with
high doses of secreted APP or transfected with wild-type APP
would have reduced effectiveness in the AD brain, in which
impaired APP metabolism would prevent or reduce neuronal
differentiation of implanted cells. Therefore, we suggest that
regulation of APP levels in the brain is necessary for imple-
menting neuroplacement strategies.
(?)-Phenserine is a recently developed cholinesterase inhib-
itor (ChEI) currently in clinical trials for treatment of mild to
moderate AD. Recent studies have reported that besides its
ChEI activity, (?)-phenserine also lowers APP and A? levels in
neuronal cells in culture and in rodents by translational regula-
tion of APP protein synthesis (16–18). However, the doses at
which (?)-phenserine decreases APP production in vitro are
higher than those that elicit its ChEI activity in patients treated
with the experimental drug. Typically, ChEIs have dose limita-
tions and may cause undesirable side effects due to the excessive
amounts of acetylcholine produced after treatment. Chirally
pure (?)-phenserine lacks ChEI activity but has similar effects
on APP production as its (?)-enantiomer (16). In this study, we
APP protein levels in an AD transgenic mouse model (APP23
mice) at 4–7 months of age. We also investigated whether
(?)-phenserine-induced alterations of endogenous APP levels
in these mice, which in turn could influence the migration and
differentiation of transplanted HNSCs. Here we show a physi-
ological function of APP in regulating HNSC migration and
differentiation fate in vivo.
Reduced APP Protein Expression After (?)-Phenserine Treatment. To
investigate the effects of (?)-phenserine on full-length APP
protein expression, Western blot analysis was performed on
cortical and hippocampal tissues from APP23 mice treated with
either (?)-phenserine (25 mg/kg i.p. per day for 14 days) or
saline. APP23 mice showed significantly (P ? 0.05) higher levels
Author contributions: A.M. and K.S. designed research; A.M., M.O., and M.N. performed
research; N.H.G. and A.N. contributed new reagents/analytic tools; A.M., M.O., and M.N.
analyzed data; and A.M. and K.S. wrote the paper.
The authors declare no conflict of interest.
Abbreviations: A?, ?-amyloid; AD, Alzheimer’s disease; APP, amyloid precursor protein;
ChEI, cholinesterase inhibitor; GFAP, glial fibrillary acidic protein; HNSC, human neural
stem cell; sAPP, secreted APP.
†To whom correspondence should be sent at the ‡ address. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
July 24, 2007 ?
vol. 104 ?
of APP (77% increase) compared with that of controls (Fig. 1A).
After (?)-phenserine treatment, a significant decrease (38%) in
APP protein expression was observed in the hippocampus of
APP23 mice (P ? 0.01) compared with saline treated mice (Fig.
1A). No significant change in APP protein expression was
observed between (?)-phenserine treated and saline treated
wild type mice (Fig. 1A). APP protein expression was also
reduced in the cerebral cortices of APP23 and wild-type mice
after (?)-phenserine treatment, but these reductions did not
reach statistical significance (P ? 0.05, data not shown).
Reduced Glial Fibrillary Acidic Protein (GFAP) Protein Expression After
(?)-Phenserine Treatment. A previous study reported that APP
overexpression in APP23 mice is also associated with marked
gliogenesis in the brains of these mice (19). Therefore we also
whether treatment with (?)-phenserine could alter GFAP lev-
els. We observed that APP23 mice had significantly (P ? 0.05)
higher (109%) GFAP protein expressed in the hippocampus
compared with that of wild-type mice. After (?)-phenserine
treatment (25 mg/kg i.p. per day for 14 days), a significant (P ?
0.05) reduction (36%) in GFAP expression was measured in
APP23 mice (Fig. 1B). No significant changes were observed
between (?)-phenserine-treated and saline-treated wild-type
mice (Fig. 1B). GFAP protein expression in the cerebral cortices
of APP23 and wild-type mice was similar, and no significant
change was observed after (?)-phenserine treatment (data not
Effect of (?)-Phenserine on APP Gene Expression in APP23 Mice. To
investigate whether (?)-phenserine-induced reduction of APP
protein expression in APP23 mice was mediated at the tran-
scriptional level, quantitative real-time RT-PCR analysis was
performed on cortical and hippocampal tissues from treated
animals. However, no significant changes were observed in APP
gene expression after (?)-phenserine treatment in both APP23
and wild-type mice (data not shown), indicating that (?)-
Effects of (?)-Phenserine on Glial Differentiation of Transplanted
HNSCs in APP23 Mice. Six weeks after implantation, fluorescent
immunohistochemistry was used to identify cells derived from
transplanted HNSCs (BrdU-labeled) and to examine their dif-
ferentiation into neural and glial cells. We also sought to
determine whether the (?)-phenserine-induced effects on APP
could influence the differentiation fate of transplanted HNSCs.
Transplanted HNSCs survived in vivo, and an extensive number
of cells exhibiting characteristic astroglial morphologies, and
coexpressing BrdU with the astrocytic marker for human GFAP
(GFAP?/BrdU?) were observed in the molecular and granule
layers of the hippocampal CA1 region (Fig. 2 A–D). Typically,
APP23 mice showed more pronounced immunoreactivity for
GFAP?/BrdU?compared with controls. Cells expressing
GFAP?/BrdU?in hippocampal regions were counted, and the
results were expressed as the average number of GFAP?/BrdU?
cells per region for each treatment group (Fig. 3 A–C). APP23
mice showed significantly (P ? 0.01) more GFAP?/BrdU?
double immunopositive cells compared with that of wild-type
mice (Fig. 3 A–C). In addition, a significant correlation (P ?
0.05; linear regression r ? 0.47) between the number of GFAP?/
was demonstrated in the hippocampus of APP23 mice (Fig. 3D).
After (?)-phenserine treatment, a marked reduction (ranging
from 28% to 40%) in the number of GFAP?/BrdU?double
immunopositive cells was observed in hippocampal regions of
APP23 mice (Fig. 3 A–C), indicating that (?)-phenserine re-
duces glial differentiation caused by APP overexpression. No
significant differences in the number of GFAP?/BrdU?double
immunopositive cells were observed in the hippocampus of
wild-type mice after (?)-phenserine treatment (Fig. 3 A–C). To
eliminate the possibility that (?)-phenserine contributed to
increased cell death rather than decreased glial differentiation of
transplanted HNSCs, we measured caspase-3 immunoreactivity
in brain sections from saline and phenserine treated APP23
hippocampus of 6- to 8-month-old APP23 and nontransgenic mice that were
treated with either saline or (?)-phenserine (25 mg/kg), respectively, for 14
days.*, P ? 0.05 and**, P ? 0.01 indicates significantly different from
saline-treated (ANOVA). ‡, P ? 0.05 indicates significantly different within
saline-treated group (ANOVA). All values are expressed as mean ? SEM from
three to four independent experiments.
Relative protein levels of total sAPP (22C11) (A) and GFAP (B) in the
after treatment with either saline or (?)-phenserine (25 mg/kg). Representa-
of 6- to 7-month-old APP23 and nontransgenic mice 6 weeks after HNSCs
transplantation. Sections were double-immunofluorescence stained with
GFAP (green) and BrdU (red) markers for astroglia cells and donor cells,
(E–H). All nuclei were counterstained by DAPI (blue). (Scale bars: 20 ?m.)
Marutle et al.
July 24, 2007 ?
vol. 104 ?
no. 30 ?
mice. We detected a few apoptotic cells-derived from trans-
planted HNSCs in both the dentate gyrus and CA1 hippocampus
of APP23 mice (Fig. 3 E–H), yet no significant difference in the
number of apoptotic nuclei was detected in mice treated with
(?)-phenserine compared with those who received saline. These
results indicate that (?)-phenserine did not mediate any signif-
icant toxic effects on transplanted cells.
Effects of (?)-Phenserine on Neuronal Differentiation of Transplanted
HNSCs in APP23 Transgenic Mice. Examination of neuronal differ-
entiation of transplanted HNSCs was performed in brain sec-
tions from APP23 and wild-type mice treated with (?)-
phenserine or saline. The number of cells coexpressing BrdU
with the neuronal marker for human ?-III tubulin (?-III tubu-
lin?/BrdU?) were counted (per square millimeter) in the mo-
lecular and granule layers of the hippocampal CA1 and CA2 and
the dentate gyrus and in the pyramidal layers of the somato-
sensory and motor cortex. Transplanted cells that differentiated
into ?-III tubulin?/BrdU?cells within the CA1 region had large
pyramidal morphologies (Fig. 2 E–H), whereas those in the
dentate granule layer displayed a small ovoid appearance char-
acteristic for dentate granule neurons (data not shown). In the
somatosensory and motor cortical regions, ?-III tubulin?/
BrdU?cells exhibited both pyramidal and nonpyramidal mor-
phologies (data not shown). To exclude the possibility that
measured neuronal immunoreactivity was also detecting endog-
enous neurons in mouse that were not derived from the trans-
planted HNSCs, we stained, in parallel experiments, sections
together with an antibody that specifically labels human nuclei.
Similar results were obtained for human nuclei staining as with
?-III tubulin and BrdU, thus verifying that differentiated cells
were of human origin [supporting information (SI) Fig. 5]. We
anticipated a reduced neuronal differentiation of transplanted
HNSCs in saline-treated APP23 mice on the basis of earlier
findings in vitro in which more glial differentiation of HNSCs was
observed after treatment with secreted APP (sAPP) (23). How-
ever, no significant difference in the number of ?-III tubulin?/
BrdU?double immunopositive cells was observed in hippocam-
pal regions of APP23 mice compared with wild-type mice (Fig.
4 A, C, and D), even though fewer ?-III tubulin?/BrdU?double
immunopositive cells were detected in the motor and somato-
(Fig. 4 B and E). Interestingly, we observed a significant increase
(ranging from 32% to 112%) in the number of ?-III tubulin?/
after 6 weeks of differentiation in hippocampal regions of 6- to 7-month-old
APP23 and nontransgenic mice that were treated with either saline or (?)-
phenserine (25 mg/kg). All values are expressed as the mean ? SEM (n ? 6–7
within each group) and were obtained by averaging counts of immunoreac-
tive human-specific astroglial cells in the CA1 (A), CA2 (B), and dentate gyrus
(C), measured bilaterally on four to six alternate sections for each mouse. ‡‡,
P ? 0.01 indicates a significant difference within the saline-treated group
(ANOVA). (D) Correlation of APP protein levels with the number of GFAP?/
saline only. Each point corresponds to average APP protein levels and the
number of GFAP?/BrdU?cells in the CA1, CA2, and dentate gyrus regions of
each individual mouse. (Linear regression r ? 0.47; P ? 0.05). (E–H) Anti-
caspase-3 staining of apoptotic cells-derived from transplanted HNSCs in the
(green) (G and H). Nuclei are stained with DAPI (blue); small arrows indicate
apoptotic cell nuclei. (H) Colocalization of BrdU (red) and caspase-3 (green).
(Scale bar: E, 10 ?m; F, 100 ?m.)
Transplanted HNSC expressing immunoreactivity for GFAP and BrdU
and cortical regions of 6- to 7-month-old APP23 and nontransgenic mice that
were treated with either saline or (?)-phenserine (25 mg/kg). All values are
expressed as mean ? SEM. n ? 6–7 within each group and were obtained by
averaging counts of immunoreactive human-specific neuronal cells in the
measured bilaterally on four to six alternate sections for each mouse.*, P ?
0.05 and***, P ? 0.0001 indicates a significant difference from saline-treated
(ANOVA). ‡, P ? 0.05 indicates a significant difference within (?)-phenserine-
treated groups (ANOVA).
Transplanted HNSC expressing immunoreactivity for neuronal
www.pnas.org?cgi?doi?10.1073?pnas.0705346104Marutle et al.
BrdU?double immunopositive cells in the hippocampal CA1
and CA2 (P ? 0.0001) and in the motor and the somatosensory
cortex (P ? 0.05) of APP23 mice after (?)-phenserine treatment
compared with the number of cells found in APP23 mice treated
with saline (Fig. 4 A–C and E). A significant (P ? 0.05) increase
(40%) in the number of ?-III tubulin?/BrdU?cells was observed
only in the CA1 hippocampal region of wild-type mice treated
with (?)-phenserine (Fig. 4A).
regulate HNSC biology in normal or diseased brain is still in its
infancy. Several studies have shown that APP expression is
up-regulated during development of the CNS, coinciding with a
peak in neuronal differentiation (20, 21). Increased APP levels
are also observed after brain damage (22, 23). Both of these
events involve migration and differentiation of NSCs, suggesting
that APP may also play an important physiological function in
regulating stem cell biology. In a recent study, we demonstrated
that treatment with recombinant sAPP promoted migration and
differentiation of HNSCs in culture, and 22C11 antibody-
mediated neutralization of sAPP in media inhibited these effects
dose dependently (15). We also reported that HNSCs trans-
planted into APP knockout mice showed less migration and
differentiation compared with wild-type mice (15). On the basis
of these observations, we suggest that APP may be acting as a
signaling factor in migration and differentiation of HNSCs.
neurofibrillary tangles and extracellular A? deposits generated
from proteolytic cleavage of APP (24). In addition, a severe
impairment of cholinergic neurotransmission is observed in AD
patients because of a pronounced loss of basal forebrain cho-
linergic neurons projecting to hippocampal and cortical regions.
The resulting deficits in these regions correlate with the memory
and cognitive impairment manifested clinically (25, 26). To date,
the most effective treatment for AD is with ChEIs that stimulate
an increase in levels of the neurotransmitter acetylcholine (27).
Several of these drugs have been shown to affect APP processing
and to lower A? in cell culture through mechanism(s) that are
independent from their activities as ChEIs (28–30). The ChEI
(?)-phenserine is currently being tested in clinical trials for the
symptomatic treatment of mild to moderate AD, and its positive
enantiomeric form, (?)-phenserine, has been found to signifi-
cantly reduce APP and A? in both neuronal cell lines in culture
and in animals by regulating APP protein synthesis (16, 18). As
a consequence of its apparent lack of ChEI activity, (?)-
phenserine may be administered in vivo in relatively high doses
without adverse effects (31), and the compound is currently in
clinical trials for AD treatment.
In the present study, we examined the effects of (?)-
phenserine on APP protein expression, and the migration and
differentiation of transplanted HNSCs in APP23 transgenic
mice. To study the effects on APP and HNSC differentiation in
APP23 mice, (?)-phenserine treatment and subsequent trans-
plantation of HNSCs were performed in 3- to 4-month-old mice,
which is before the onset of AD-like pathology. APP23 mice can
express a 7-fold overexpression of mutated human APP751 in
the brain, with A? plaque-like deposits that begin to appear in
the hippocampus and neocortex from 6 months of age, and
increased deposition is observed with age (19). Here we showed
that (?)-phenserine significantly reduced APP as well as GFAP
protein expression in the hippocampus of APP23 transgenic
mice. (?)-Phenserine suppressed APP protein expression with-
out altering APP gene expression, indicating the involvement of
a posttranscriptional regulatory mechanism. Our findings are in
agreement with earlier studies that showed that ChEIs, such as
tacrine and (?)-phenserine, induced similar reductions in levels
32–33). A dramatic increase of APP in cholinergic projection
areas has been demonstrated in a study using rats with forebrain
cholinergic lesions (17). Further findings from this study showed
that phenserine could reverse this effect and additionally reduce
APP production in naı ¨ve animals (17). In our study, we also
found a reduced glial differentiation of transplanted HNSCs in
hippocampal regions of (?)-phenserine treated APP23 mice. In
regions such as the CA1 hippocampus, glial differentiation of
HNSCs was decreased by ?50% in the APP23 mice after
treatment with (?)-phenserine, which corresponded with a shift
from a 2:1 to 1:1 ratio in the number of transplanted cells
differentiating into a glial versus a neuronal lineage (SI Table 1).
The shift to increased neuronal differentiation after (?)-
phenserine treatment was most apparent in the CA2 region of
APP23 mice, in which glial differentiation decreased by 36% (SI
Table 2). However, (?)-phenserine treatment did not signifi-
cantly affect neural differentiation of transplanted HNSCs in the
dentate gyrus of either wild-type or APP23 mice. Adult neuro-
genesis typically occurs in the subventricular zone and the
dentate gyrus of the hippocampus (34). Endogenous neurore-
generation in the dentate gyrus may therefore depend mainly on
the stem cells that already reside in the subgranular zone of the
dentate granule cell layer of the hippocampus (35), whereas
endogenous stem cells residing in the subventricular zone may
not migrate into the dentate gyrus (36). Thus, it is possible that
exogenous HNSCs may not necessarily follow the same distri-
bution pattern as endogenous stem cells.
It has been proposed that (?)-phenserine mediates a specific
effect on human APP through translational regulation of protein
synthesis (16, 18, 37). We would therefore expect APP levels to
remain unaffected after (?)-phenserine treatment in the control
mice, because these mice do not carry the human form of APP.
However, we did observe an effect of (?)-phenserine on neu-
ronal differentiation of transplanted HNSCs in wild-type mice in
the present study, suggesting that other mechanisms exist.
Earlier studies have implicated that APP exerts its effects on cell
ERK signaling pathway (38). Accordingly, a recent study in our
group showed that APP is involved in promoting astrocytic
differentiation of NT2-/D1 neural precursor cells induced by
treatment with staurosporin, a protein kinase C inhibitor and
inducer of cell differentiation. Staurosporin treatment increased
sAPP in these cells, which led to activation of the Erk1/2
signaling pathway and increased astrocytic differentiation of the
NT2-/D1 cells (39). To confirm APP involvement, APP expres-
sion was suppressed in these cells by using RNA interference
methods, and this resulted in reduced GFAP expression (23). In
another study, we showed that treatment of HNSCs in culture
with sAPP was associated with an increased expression of genes
related to the Notch and JAK/STAT-signaling cascades (15).
These cascades are known to play a pivotal role in neuron–glia
differentiation (40), and we suggest that it is possible that the
reduction in glial differentiation of transplanted HNSCs in
APP23 mice observed herein could be a consequence of (?)-
phenserine-mediated inhibition of APP effect(s) on Notch and
JAK/STAT pathways. Only a few studies up to date have
investigated the cell fate of endogenous populations of stem cells
in the adult brain in regards to APP overexpression and A?
pathogenesis. One study demonstrated impaired neurogenesis in
the dentate gyrus of transgenic mice expressing the Swedish
double mutation (K595N, M596L) (14), whereas other studies
measured increased neurogenesis both in the AD human post-
mortem brain (41) and in the brains of transgenic mice express-
ing the Swedish and Indiana APP (PDGF-APPSw,Ind) muta-
tions (42). In the present investigation we have measured
increased neurogenesis in the hippocampus and cortex of APP23
mice and in the CA1 hippocampal region of wild-type mice after
(?)-phenserine treatment. It is possible that a discrepancy in the
Marutle et al.
July 24, 2007 ?
vol. 104 ?
no. 30 ?
findings of both decreased (14) and increased (42) neurogenesis
in AD transgenic mice and those presented here could be
attributed to cell-intrinsic differences between endogenous and
exogenous stem cells. Our current findings suggest that (?)-
phenserine may stimulate increased neuronal differentiation or
neurogenesis by a mechanism that may involve APP interac-
tion(s) with other factors. To confirm these results, additional
studies were performed in vitro on differentiating HNSCs
treated with (?)-phenserine. Similar to our in vivo findings, we
observed that (?)-phenserine suppressed APP and GFAP pro-
tein expression, and increased the number of neuronal cells in
differentiated cell populations of HNSCs in vitro (SI Fig. 6).
A recent study by Jin et al. (43) demonstrated that the ChEIs
tacrine, galanthamine and the NMDA receptor antagonist me-
mantine, promote increased neurogenesis both in isolated cul-
tures from cortical progenitor cells and in mice. The mechanisms
through which these disparate drugs increase neurogenes is still
unclear, yet the investigators suggested that a common mecha-
nism, mediated through muscarinic receptor-coupled phospho-
inositide signaling is involved (43). They reported that this effect
could also be due to activation of cholinergic receptors that are
expressed on neuronal progenitors and that these receptors in
turn may stimulate neurogenic factors (43–45). Because (?)-
phenserine does not possess ChEI activity, stimulation of neu-
rogenesis may likely not be mediated through cholinergic recep-
tors that may be expressed on the differentiated HNSCs. The
exact mechanisms, with regards to which signaling pathway(s)
are involved in mediating the (?)-phenserine-induced effects on
APP in regulating stem cell migration and differentiation in vivo,
are beyond the scope of our present study. Thus, future studies
will be crucial for investigating the specific molecular mecha-
nisms underlying this phenomena, as well as comparative studies
for determining the efficacy of various doses of (?)-phenserine.
In conclusion, our present findings suggest that altered APP
levels regulate NSC biology in the adult brain, and this may have
serious implications for the pathophysiology of AD and other
diseases involving dysregulation of APP metabolism such as
Down’s syndrome. High levels of APP in the brain may exhaust
stem cell populations as a result of premature or increased glial
differentiation. Further understanding of the mechanisms in-
volved in regulating stem cell biology during neurodegeneration
is needed, and a combination of augmentation of stem cell
populations by transplantation and a pharmacological approach
to regulate APP levels may aid future development of novel
strategies for therapeutical interventions of these diseases.
Materials and Methods
HNSC Culture. HNSCs originally isolated from 9-week-old fetal
cortical tissue were purchased from BioWhittaker (Walkersville,
MD), and the cells were expanded and passaged in serum-free
culture media, as described in ref. 46. Briefly, HNSCs were
cultured in DMEM/F12 (GIBCO, Burlington, ON, Canada)
supplemented with 20 ng/ml EGF and 20 ng/ml basic fibroblast
growth factor (bFGF) (R & D, Minneapolis, MN), B27 (1:50;
GIBCO), 5 ?g/ml heparin (Sigma, St. Louis, MO), and antibi-
otic-antimycotic mixture (1:100; GIBCO) in a humidified atmo-
sphere of 5% CO2at 37°C. Before transplantation, HNSCs were
incubated with 3 ?M BrdU (Sigma) for 48 h to label cell nuclei
to distinguish them from the host cells.
Animals. APP23 mice, expressing the 751-aa human APP
(hAPP751) with the Swedish double mutation (K670N, M671L)
(47) were received as a gift from NovartisPharma (Basel,
Switzerland) and were used to breed a colony of experimental
animals by backcrossing to C57BL/6 mice. Mice were housed in
standard cages with access to food and water ad libitum during
a 12/12 h light/dark cycle. Genotypes were confirmed by PCR
(48), and in all experiments wild-type littermates served as
compliance with National Institutes of Health Guidelines for
Care and Use of Laboratory Animals and were approved by the
Animal Research Committee (protocol 00-24) at the University
of Central Florida.
(?)-Phenserine Treatment. A total of 55 age- and sex-matched
APP23 (n ? 30) and wild-type (n ? 25) mice (ages ranged from
mg/kg per day i.p.) or 0.9% saline for 14 consecutive days.
Animals were subsequently divided into two groups that were
either killed after 14 days of treatment (n ? 17 APP23 and n ?
13 wild-type, respectively) or received HSNCs transplanted into
the lateral ventricle (n ? 13 APP23 mice and n ? 12 wild-type,
respectively). (?)-Phenserine or saline injections were contin-
ued once a day for 1 week after a 2-day recovery from surgery.
by an overdose of a 1:1 mixture of 100 mg/kg ketamine and 20
mg/kg xylazine, followed by transcardial perfusion with PBS.
Brains were removed and dissected into the hippocampus and
cortex, and tissue samples were stored at ?80°C until experi-
transcardially perfused with 4% paraformaldehyde (pH 7.4).
Brains were removed, postfixed for 12 h, and cryoprotected in
20% sucrose in PBS overnight. Twenty-micrometer coronal
brain sections were cut and processed for immunofluorescence.
Animal Surgery and Transplantation. Anesthetized animals were
mounted on a stereotaxic apparatus (ASI Instruments, Warren,
MI). HNSCs (?105cells) were suspended in 10 ?l of PBS and
slowly injected into the right lateral ventricle of each mouse.
Intraventricular injection minimizes disruption of brain tissue
and may leverage endogenous signals (e.g., chemokines released
by microglia in response to damage) that might affect stem cell
migration. No immunosuppressant was used, and animals were
monitored for body weight, swelling, and proper healing of the
Protein Isolation and Western Blot Analysis. Dissected cortical and
hippocampal tissues from (?)-phenserine and saline-treated
Nonidet P-40, 150 mM NaCl, 50 mM Tris (pH 8.0), and protease
inhibitor mixture (Roche, Indianapolis, IN). The homogenates
were centrifuged and washed twice at 12,000 ? g for 15 min at
4°C. Fifteen micrograms of protein was loaded per well, and
proteins were separated by SDS/PAGE and then blotted onto
PVDF membranes for 120 min at 30 V. For the detection of
full-length APP and GFAP protein, membranes were incubated
overnight with primary antibodies mouse monoclonal anti-
Alzheimer precursor protein A4 (22C11) (1:1,000; Chemicon,
Temecula, CA), rabbit anti-GFAP (1:1,000; Promega, Madison,
WI), and polyclonal rabbit anti-?-actin (1:1,000; Cell Signaling
Technology, Danvers, MA). After washing, membranes were
incubated with horseradish peroxidase-conjugated secondary
antibodies (anti-mouse IgG and anti-rabbit IgG; Jackson Immu-
noresearch, West Grove, PA) for 1–2 h. Signals were visualized
by incubation of membranes in ECL Plus reagents and exposure
sciences, Buckinghamshire, U.K.). Films were scanned, and the
optical density of each specific band relative to ?-actin was
analyzed by the public domain National Institutes of Health
Image J software.
Real-Time RT-PCR Analysis. Total RNA from hippocampal and
cortical tissues from treated animals was extracted with TRIzol
(Invitrogen) according to the manufacturer’s protocol. cDNA
www.pnas.org?cgi?doi?10.1073?pnas.0705346104 Marutle et al.
synthesis was performed with 1 ?g of total RNA and reagents Download full-text
from the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA)
according to kit instructions. Relative quantification with real-
time PCR was determined using the MyiQ Real-Time PCR
Detection System software (Bio-Rad), and reactions were per-
formed in a thermal iCycler by using the Bio-Rad MyIQ SYBR
Green Supermix as described in SI Table 3. The authenticity of
the PCR products was verified by a melt-curve analysis.
Fluorescent Immunohistochemistry. Free-floating coronal brain
sections (20 ?m) were denatured with 1 M HCl for 20 min and
neutralized with PBS for 30 min at room temperature (RT)
to increase the accessibility of the anti-BrdU antibody to the
BrdU incorporated in the cell nuclei. The sections were then
blocked in PBS containing 0.25% Triton X-100 and 3% normal
donkey serum for 1 h and incubated with sheep polyclonal
anti-BrdU (1:1,000; Abcam, Cambridge, MA), mouse anti-
human nuclei (1:100; Chemicon), mouse IgG2b anti-human
?-III tubulin, clone SDL3D10 (1:2,000; Sigma) and mouse
anti-NeuN (1:1,000; Abcam), or rabbit IgG anti-human GFAP
(1:500; Sigma) overnight at 4°C. For apoptosis measurements,
sections were incubated with rabbit anti-active caspase-3 anti-
body (1:125; Promega, Madison, WI). Sections were incubated
with corresponding secondary antibodies (1:500 to 1:1,000),
conjugated with fluorescein (FITC) or rhodamine (TRITC)
(Jackson Immunoresearch) for 2 h at RT. After final washes in
PBS-T, sections were mounted and cover slipped with Vectash-
ield with DAPI (Vector Laboratories, Burlingame, CA) for
fluorescent microscopic analysis.
Microscopy and Analysis of Differentiation. Cell migration and
differentiation in transplanted mice (n ? 6–7 mice in each
group) were quantified by unbiased bilateral counts of the
number of BrdU-positive cells expressing either the neuronal
marker, ?-III tubulin, or the glial marker, GFAP, in the molec-
ular and granule layers of hippocampal CA1, CA2, and dentate
gyrus, and motor and sensory regions of the cerebral cortex by
using a Leica (Deerfield, IL) DMRB fluorescent microscope at
?400 magnification. Microscopic images were taken with an
Axiocam digital camera (Zeiss, Oberkochen, Germany)
mounted on the DMRB and processed using the QImaging with
Q Capture software (QImaging, Burnaby, Canada). An average
of four to six sections were counted for each animal. The
numbers of transplanted cells counted in each section were
averaged for each side so that the final numbers represented the
mean neuron or astrocyte number per sampling area or per
Data and Statistical Analysis. Data are presented as mean ? SEM
of different experiments, and differences between groups were
analyzed with one-way ANOVA followed by Bonferroni/Dunn
and Scheffe ´ post hoc comparison testing. Correlations between
variables were determined by linear regression analysis (PRISM
3.0; GraphPad, San Diego, CA).
This work was supported by National Institutes of Health Grant AG
23472, a grant from BioFlorida, and Alzheimer Association Grant
IIRG-03-5577. M.N. was supported by a grant from the Erik and Edith
Fernstro ¨ms Foundation (Sweden) and Swedish Medical Research Coun-
cil Contract Grant 05817.
1. Sheen V, Macklis J (1995) J Neurosci 15:8378–8392.
2. Brustle O, McKay R (1996) Curr Opin Neurobiol 6:688–695.
3. Qu T, Brannen C, Kim H, Sugaya K (2001) NeuroReport 12:1127–1132.
4. Englund U, Fricker-Gates R, Lundberg C, Bjorklund A, Wictorin K (2002) Exp
5. Selkoe DJ (1991) Neuron 6:487–498.
6. Muller U, Cristina N, Li Z, Wolfer D, Lipp H, Rulicke T, Brandner S, Aguzzi
A, Weissmann C (1994) Cell 79:755–765.
7. Salinero O, Moreno-Flores M, Wandosell F (2000) J Neurosci Res 60:87–97.
8. Caille I, Allinquant B, Dupont E, Bouillot C, Langer A, Muller U, Prochiantz
A (2004) Development (Cambridge, UK) 131:2173–2181.
9. De Strooper B, Annaert W (2000) J Cell Sci 113:1857–1870.
10. Ando K, Oishi M, Takeda S, Iijima K, Isohara T, Nairn A, Kirino Y, Greengard
P, Suzuki T (1999) J Neurosci 19:4421–4427.
11. Koszyca B, Blumbergs P, Manavis J, Wainwright H, James R, Gilbert J, Jones
N, Reilly P (1998) J Neurotrauma 15:675–683.
12. Wang C, Wurtman R, Lee R (2000) Brain Res 865:157–167.
13. Bondolfi L, Calhoun M, Ermini F, Kuhn H, Wiederhold K, Walker L,
Staufenbiel M, Jucker M (2002) J Neurosci 22:515–522.
14. Haughey N, Nath A, Chan S, Borchard A, Rao M, Mattson M (2002)
J Neurochem 83:1509–1524.
15. Kwak YD, Brannen C, Qu T, Kim H, Dong X, Soba P, Majumdar A, Kaplan
A, Beyreuther K, Sugaya K (2006) Stem Cells Dev 15:381–389.
16. Shaw K, Utsuki T, Rogers J, Yu Q, Sambamurti K, Brossi A, Ge Y, Lahiri D,
Greig N (2001) Proc Natl Acad Sci USA 98:7605–7610.
17. Haroutunian V, Greig N, Pei X, Utsuki T, Gluck R, Acevedo L, Davis KL,
Wallace W (1997) Brain Res Mol Brain Res 46:161–168.
18. Utsuki T, Yu Q, Davidson D, Chen D, Holloway H, Brossi A, Sambamurti K,
Lahiri D, Greig N, Giordano T (2006) J Pharmacol Exp Ther 318:855–862.
19. Sturchler-Pierrat C, Staufenbiel M (2000) Ann NY Acad Sci 920:134–139.
20. Salbaum J, Ruddle F (1994) J Exp Zool 269:116–127.
21. Trapp B, Hauer P (1994) J Neurosci Res 37:538–550.
22. Kirazov E, Kirazov L, Bigl V, Schliebs R (2001) Int J Dev Neurosci 19:287–296.
23. Murakami N, Yamaki T, Iwamoto Y, Sakakibara T, Kobori N, Fushiki S, Ueda
S (1998) J Neurotrauma 15:993–1003.
24. Haass C, Hung A, Schlossmacher M, Oltersdorf T, Teplow D, Selkoe D (1993)
Ann NY Acad Sci 695:109–116.
25. Bierer L, Haroutunian V, Gabriel S, Knott P, Carlin L, Purohit D, Perl D,
Schmeidler J, Kanof P, Davis K (1995) J Neurochem 64:749–760.
26. Nordberg A (2001) Biol Psychiatry 49:200–210.
27. Doody R, Stevens J, Beck C, Dubinsky R, Kaye J, Gwyther L, Mohs RC, Thal
L, Whitehouse P, DeKosky S, Cummings J (2001) Neurology 56:1154–1166.
28. Lahiri D, Farlow M, Nurnberger J, Jr, Greig N (1997) Ann NY Acad Sci
29. Pakaski M, Kasa P (2003) Curr Drug Targets CNS Neurol Disord 2:163–171.
30. Racchi M, Mazzucchelli M, Lenzken S, Porrello E, Lanni C, Govoni S (2005)
Chem Biol Interact 157–158:335–338.
31. Greig N, Ruckle J, Comer P, Brownell L, Holloway H, Flanagan D, Jr, Canfield
C, Burford R (2005) Curr Alzheimer Res 2:483–492.
32. Lahiri D, Lewis S, Farlow M (1994) J Neurosci Res 37:777–787.
34. Gage FH (2000) Science 287:1433–1438.
35. van Praag H, Schinder A, Christie B, Toni N, Palmer T, Gage F (2002) Nature
36. Doetsch F, Alvarez-Buylla A (1996) Proc Natl Acad Sci USA 93:14895–14900.
37. Maloney B, Ge YW, Greig N, Lahiri D (2004) FASEB J 18:1288–1290.
38. Greenberg S, Koo E, Selkoe D, Qiu W, Kosik K (1994) Proc Natl Acad Sci USA
39. Kwak YD, Choumkina E, Sugaya K (2006) Biochem Biophys Res Commun
40. Fischer D, van Dijk R, Sluijs J, Nair S, Racchi M, Levelt CN, van Leeuwen F,
Hol E (2005) FASEB J 19:1451–1458.
41. Jin K, Peel A, Mao XO, Xie L, Cottrell B, Henshall D, Greenberg D (2004)
Proc Natl Acad Sci USA 101:343–347.
42. Jin K, Galvan V, Xie L, Mao X, Gorostiza O, Bredesen D, Greenberg D (2004)
Proc Natl Acad Sci USA 101:13363–13367.
43. Jin K, Xie L, Mao X, Greenberg D (2006) Brain Res 1085:183–188.
44. Atluri P, Fleck M, Shen Q, Mah S, Stadfelt D, Barnes W, Goderie S, Temple
S, Schneider A (2001) Dev Biol 240:143–156.
45. Ma W, Maric D, Li B, Hu Q, Andreadis J, Grant G, Liu Q, Shaffer K, Chang
Y, Zhang L, Pancrazio J, Pant H, Stenger D, Barker J (2000) Eur J Neurosci
46. Brannen C, Sugaya K (2000) NeuroReport 11:1123–1128.
47. Sturchler-Pierrat C, Abramowski D, Duke M, Wiederhold K, Mistl C, Roth-
acher S, Ledermann B, Burki K, Frey P, Paganetti P, et al. (1997) Proc Natl
Acad Sci USA 94:13287–13292.
48. Calhoun M, Burgermeister P, Phinney A, Stalder M, Tolnay M, Wiederhold K,
Abramowski D, Sturchler-Pierrat C, Sommer B, Staufenbiel M, Jucker M
(1999) Proc Natl Acad Sci USA 96:14088–14093.
Marutle et al.
July 24, 2007 ?
vol. 104 ?
no. 30 ?