Luteinizing hormone modulates cognition and amyloid-deposition in Alzheimer APP transgenic mice
Until recently, the study of hormonal influences in Alzheimer disease was limited to the role of sex steroids. Despite numerous epidemiological studies supporting a protective role for estrogen in Alzheimer disease, recent studies show that estrogen administration in elderly women increases the risk of disease. Reconciling these contradictory reports, we previously hypothesized that other hormones of the hypothalamic-pituitary-gonadal axis, such as luteinizing hormone, may be involved in the onset and development of the disease. In this regard, luteinizing hormone is elevated in Alzheimer disease and is known to modulate amyloidogenic processing of amyloid-beta protein precursor. Therefore, in this study, to evaluate the therapeutic potential of luteinizing hormone ablation, we administered a gonadotropin-releasing hormone analogue, leuprolide acetate, to an aged transgenic mouse model of Alzheimer disease (Tg 2576) and measured cognitive Y-maze performance and amyloid-beta deposition after 3 months of treatment. Our data indicate that luteinizing hormone ablation significantly attenuated cognitive decline and decreased amyloid-beta deposition as compared to placebo-treated animals. Importantly, leuprolide acetate-mediated reduction of amyloid-beta correlated with improved cognition. Since both cognitive loss and amyloid-beta deposition are features of Alzheimer disease, leuprolide acetate treatment may prove to be a useful therapeutic strategy for this disease.
Luteinizing hormone modulates cognition and amyloid-β deposition in
Alzheimer APP transgenic mice
, Kate M. Webber
, Craig S. Atwood
, Miguel A. Pappolla
, Richard L. Bowen
, Mark A. Smith
Institute of Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106, USA
School of Medicine, University of Wisconsin and William S. Middleton Memorial Veterans Administration, Madison, WI 53705, USA
Department of Neuroscience, LSU Health Sciences Center, School of Medicine, New Orleans, LA 70112, USA
Voyager Pharmaceutical Corporation, Raleigh, NC 27615, USA
Received 17 August 2005; received in revised form 17 January 2006; accepted 18 January 2006
Available online 13 February 2006
Until recently, the study of hormonal influences in Alzheimer disease was limited to the role of sex steroids. Despite numerous epidemiological
studies supporting a protective role for estrogen in Alzheimer disease, recent studies show that estrogen administration in elderly women increases
the risk of disease. Reconciling these contradictory reports, we previously hypothesized that other hormones of the hypothalamic–pituitary–
gonadal axis, such as luteinizing hormone, may be involved in the onset and development of the disease. In this regard, luteinizing hormone is
elevated in Alzheimer disease and is known to modulate amyloidogenic processing of amyloid-β protein precursor. Therefore, in this study, to
evaluate the therapeutic potential of luteinizing hormone ablation, we administered a gonadotropin-releasing hormone analogue, leuprolide
acetate, to an aged transgenic mouse model of Alzheimer disease (Tg 2576) and measured cognitive Y-maze performance and amyloid-β
deposition after 3 months of treatment. Our data indicate that luteinizing hormone ablation significantly attenuated cognitive decline and decreased
amyloid-β deposition as compared to placebo-treated animals. Importantly, leuprolide acetate-mediated reduction of amyloid-β correlated with
improved cognition. Since both cognitive loss and amyloid-β deposition are features of Alzheimer disease, leuprolide acetate treatment may prove
to be a useful therapeutic strategy for this disease.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Alzheimer disease; Amyloid-β; Cognition; Hippocampal function; Luteinizing hormone; Therapeutics
Various lines of evid ence suggest the involvement of
menopause a nd age-related testosterone d ecline-induced
changes in hypothalamic–pituitary–gonadal (HPG) axis hor-
mone levels in the etiology of Alzheimer disease (AD)
(reviewed in ). Declines in sex steroids have long been
associated with AD incidence and prevalence  and hormone
replacement therapy (HRT) linked to a decreased risk of
developing AD [3,4]. However, recent findings, reporting
negative cognitive effects following HRT in women at an AD-
vulnerable age [5–7]. Alternative theories also involve the role
of free testosterone and high sex hormone-binding globulin
(SHBG) levels in the disease such that reduced testosterone
levels, as those found in women, and increased levels of SHBG,
as those found in HRT takers, which result in reduced free
testosterone, may account for the higher incidence of disease in
women [8–10]. Nevertheless, changes in estrogen and testos-
terone do not account for why men with Down's syndrome have
a significantly higher risk for developing AD-type changes than
women since the levels of sex steroids in individuals with
Down's syndrome are comparable to those found in the general
population [11,12]. This phenomenon indicates that hormones
other than estrogen or testosterone per se may be important.
Interestingly, in Down's syndrome individuals have higher
levels of gonadotropins such as LH , and more importantly,
men have higher level s of luteinizing hormone (LH) than do
Biochimica et Biophysica Acta 1762 (2006) 447 – 452
Corresponding author. Tel.: +1 216 368 3670; fax: +1 216 368 8964.
E-mail address: email@example.com (M.A. Smith).
0925-4439/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
age-matched women , whereas the opposite is found in the
general population (i.e., higher levels of LH in women than in
men of the same age ). Therefore, LH represents the only
factor, thus far, that explains the gender predispositi on in the
incidence of AD as well as its reversal in Down's syndrome
. Supporting this view, individuals with AD have a two-fold
elevation in LH serum concentrations when compared to age-
matched controls [14,16]. However, two recent studies reported
no elevation of LH in AD [17,18]. This disparity in results likely
reflects the different ages and grouping techniques used across
the studies. This aspect aside, it is important to note that
gonadotropin (LH)-based etiopathogenic etiology of AD is
supported by several other pieces of evidence. First, questions
regarding a “critical period” of HRT-based protection against
dementia [19,20] suggest that members of the HPG axis other
than sex steroids could be involved. In this regard, the levels of
LH are highest during the peri-menopause and early meno-
pausal periods  and it is notable that HRT du ring this period,
which by increasing estrogen would lower LH levels, has been
observed to be most succ essful at preventing dementia.
Secondly, in the brain, likely due to the fact that LH can cross
the blood brain barrier freely [22,23], patients with AD show
increased levels of LH in topographical locations that mirror the
selective and vulnerable neuronal populations . This latter
finding likely relates to the fact that the highest density of LH
receptors in the brain are found within the hippocampus ,a
region that is particularly vulnerable in AD, and the
degeneration of which leads to early memory loss in the disease
([25,26] for review). In early studies, suggestive of a key role of
LH in AD pathogenesis, we showed that LH drives amyloid-β
protein precursor (AβPP) processing towards the amyloido-
genic pathway in vitro . These findings indicate that
gondadotropins such as LH may play an important role in the
onset and progression of AD. Therefore, the aim of this study
was to e xamine the capacity of a gonadotropin-re leasing
hormone agonist, leuprolide acetate, which abolishes the release
of LH [27,28], to modulate cognitive performance and amyloid-
β deposition in the brains of aged Tg2576 mice carrying the
Swedish APP mutation. Aged females as opposed to younger
females were used to attempt to mimic the capacity of this drug
to modulate advanced stages of AD.
2. Materials and methods
We used female 21 month-old non-cycling transgenic mice Tg2576 that
over-express the 695-amino-acid isoform of human AβPP containing a
Lys670 → Asn, Met671 → Leu mutation found in a Swedish family with
early onset AD. In these animals, levels of amyloid-β begin to rise in the brain
by 6 months of age and by 9–10 months they develop senile plaque-like
deposits of amyloid-β . All animals were genotyped using a standard PCR
protocol . All animals were group housed (n = 3/cage), provided ad libitum
access to food and water, and maintained on a 12 h light/dark cycle. The
experimental groups were chosen in a random fashion and received leuprolide
acetate [7.5 mg/kg, slow release (depot) formulation] (n = 8) or physiological
0.9% saline (n = 5) injected intra-muscularly (IM) twice monthly for a duration of
3 months and all measurements (behavioral/immunocytochemical) were carried
out by the observer blind to the treatment. All animals were weighed 3 times
during the study and showed no significant weight loss across groups or time. All
animals were in good health with no obvious signs of disease, however, four
animals died during the course of the study. Weight and death data are
summarized in Table 1. We have previously shown that this dose regimen
successfully abolishes LH levels in these mice .
2.2. LH measurements
Additional animals (n=4/group) of the same age, genotype, gender, and
handling to animals tested for behavior were used to determine treatment
efficacy of leuprolide acetate on LH serum levels. Blood samples were taken
upon sacrifice, centrifuged to collect serum, and LH levels were measured
by RIA analysis (Dr. Nett-ARBL-Endocrine Laboratory, Colorado State
2.3. Y-maze procedure
We used the Y-maze task (32 cm × 10 cm × 26 cm) to measure spontaneous
alternation behavior, a cognitive parameter  previously used by others to
measure effectiveness of potential treatments in animal models of AD [31–33]
as well as exploratory activity. Each animal was tested prior to treatment (age 21
months) and at the end (age 24 months). As previously described , animals
were placed in one of three arms of the maze chosen at random across trials, and
left to explore all arms of the maze for 5 min during which time the sequence and
number of arm choices were recorded. Spontaneous alterations were expressed
as a percentage and referred to as the proportion of arm choices that differed
from previous choices .
2.4. Amyloid-β immunohistochemistry and quantification
For amyloid-β immunohistochemistry, animals were sacrificed with a lethal
dose of pentobarbital and their brains removed and fixed in 4% paraformal-
dehyde. All brains were simultaneously sagitally sectioned (50 μm) across the
hippocampus to ensure accurate region sectioning and quantification .
Sections were stained free-floating; after a H
treatment and blocking serum,
sections were immunostained with a primary amyloid-β antibody (4G8, which
recognizes the sequence of amyloid-β in the 17–24 region) (0.164 mg/ml)
(1:5000 mouse monoclonal) for 24 h (at 4 °C), a goat anti-mouse secondary
antibody for 30 min at RT, and avidin–biotin–HRP complex (Vectastain Elite
ABC kit, Vector, Burlingame, CA) for 1 h RT. Sections were developed with
diaminobenzidine tetrahydrachloride (DAB) with H
and mounted on glass
Quantification of amyloid-β deposition was carried out using a Zeiss
Axiocam (Munchen-Hallbergmoss, Germany) and compatible image analysis
software, KS300 (Carl Zeiss Vision GmbH, Munchen-Hallbergmoss, Germany).
For each animal, every 6th section of a series through the dorsal hippocampus
(approximately 240 μm apart) was selected (approximately 11 sections/animal)
and quantified for amyloid-β deposition as previously described . Briefly,
using a 5× objective, a single field encompassing the entire hippocampus was
manually selected and positive staining was expressed as the percent area
stained across the area. The values obtained from all sections per animal were
2.5. Statistical analysis
Using appropriate software (Sigmastat, SPSS-Inc., Chicago, IL), a two-way
repeated measures ANOVA was used to determine differences in Y-maze
alternation behavior across treatment groups (leuprolide acetate vs. saline) and
Weight and animal death information
Number of animals Weight (g)
Baseline 3 months Baseline 3 months
Saline 8 5 32.1±3.7 28.8± 3.3
Leuprolide 9 8 27.8± 1.0 28.7 ± 1.7
448 G. Casadesus et al. / Biochimica et Biophysica Acta 1762 (2006) 447–452
time (Baseline and 3 months) as a repeated measures factor. The Student's t test
was used to determine differences in amyloid-β burden between leuprolide
acetate and saline-treated animals.
Leuprolide acetate treatment, in accord with previous data
[27,28], significantly lowered LH secretion in our model
(P b 0.02) (Fig. 1). Importantly, these declines led to sustained
cognitive ability as m easured by spontaneous alternation
behavior in the Y-maze task when compared to saline treatment
=14.745, P b 0.01) (Fig . 2). Specifically, while both
groups were comparable at baseline (t
= 0.944, P b 0.35),
leuprolide acetate-treated animals showed significantly lower
rate of decline when compared to saline treated animals at 3
months post-treatment (t
= 29.438, P b 0.02). Importantly,
sustained performance was present in the absence of differences
in locomotor activity across groups (total number of arms
entered/5 min trial) (F
= 0.670, Pb 0.430).
A Student's t test statistical analysis used to determine
differences in amyloid-β deposition in treated and non-treated
groups revealed that leuprolide acetate treatment significantly
decreased the levels of amyloid-β deposition in the hippocampi
of treated versus non-treated aged animals (t
P b 0.01) (Fig. 3). Notably, reductions in amyloid-β were
significantly correlated (Pearson's analysis) with improvements
in cognitive performance (r = −0.75, P b 0.05), such tha t
leuprolide acetate-mediated decreases in levels of amyloid-β
deposition were associated with improved performance in the
Our data reveal that treatments that target HPG axis
hormones such as LH can modulate cognitive behavior in
aged AβPP transgenic mice, and also decrease the extensive
deposition of amyloid-β. These results support our hypothesis
that HPG axis function, and in particular the changes that occur
later in life (i.e., elevations in LH following menopause/age-
related declines in testosterone ), may play an important role in
the pathogenesis of AD [1,15,37,38].
Coupled with the decreases in LH, leuprolide acetate
treatment leads to decreases in sex steroids such as estrogen
which have been associated with declines in cognitive output
[39–44]. Therefore, our data suggest that, at least in aged
AβPP transgenic mice, the positive effects of LH ablation
override any negative effects of estrogen depletion. Indeed, as
shown in this study, leuprolide acetate treatment maintains
alternation behavior in the Y-maze task, which has been
interpreted to reflect intact working memory. Alternation
behavior also depends on the animal's innate tendency/
preference to alternate, leading to the possibility that
treatment, rather than improving/sustaining memory, could
increase alternating preference. The fact that our data show
sustained rather than improved behavioral output in the
treated animals compared to controls and the fact that treated
animals did not show increases in overall arm entries nor any
directional biases, suggests that treatment did indeed sustain
short-term memory rather than potentiate their preference to
alternate. Such an assertion is in concert with data
demonstrating that the modulation estrogen in the AβPP/
PS-1 animal model of AD leads to improvements in cognitive
behavior but, and unlike our findings, no changes in
pathological features of AD . This slight discrepancy in
results can be explained by a differential LH status in the
animals of the two studies since while in our study we ablated
both estrogen and LH concurrently, ovariectomy  leads to
declines in estrogen but a rise in the levels of LH and
administration of estrogen (c.f. HRT) does not decrease LH
levels beyond baseline. Therefore, one possibility is that it is
only the decrease in estrogen when it is coupled with an
increase in LH that leads to behavioral impairments and it is
only the ablation of LH that leads to changes in amyloid-β
pathology in these mice. Additionally, such differences could
Fig. 1. Serum mouse LH levels (mLH pg/ml± SEM) measured by RIA for saline
and leuprolide acetate-treated animals (n = 4/group). *Indicates significance at
P b 0.02.
Fig. 2. Y-Maze performance in Tg2576 mice after leuprolide acetate (n =8) or
saline treatment (n = 5) at baseline and after 3 months. Figure illustrates the mean
% alternations expressed as % change from baseline. * Indicates significance at
P b 0.05.
449G. Casadesus et al. / Biochimica et Biophysica Acta 1762 (2006) 447–452
also arise from the differences in techniques used (silver stain
vs. 4G8, a non-human specific antibody). Clearly, future, more
in depth, studies targeting all of these varia bles should be
carried out to clarify the interactive role of these hormones on
cognition and AD-related pathology. That withstanding, the
data do provide compelling evidence that LH is an important
component in the pathophysiological processes of AD.
The precise mechanistic pathway by which LH is involved in
AD is still under investigation, nonetheless, that LH modulates
the processing of Aβ PP  may be important given the
proposed centr al role of amyloid- β in AD pathogenesis  or
its proposed indirect role as a surrogate marker of neuronal
health [47,48]. In this regard, the reduction of amyloid-β and
coincident sustainment of alternation behavior following LH
ablation, observed in this study, is in agreement with previous
studies showing a correlation between amyloid-β burden and
cognition [49,50]. Importantly, given the high density of LH
receptors in the hippocampal region  and the higher levels
of LH in the brains of patients with AD [14,16] it is likely that
LH plays a significant role in the pathogenesis of the disease
dependent, as well as independent, of amyloid-β-related
Based on the aforementioned notion that LH is a driving
pathogenic force in AD, leuprolide acetate, a gonadotropin-
releasing hormone agonist, which suppresses LH to undetect-
able levels by down-regulating pituitary gonadotropin-releasing
hormone receptors [27,28], might be an effective method of
treatment for patients with AD. A previous study in humans
demonstrated that treatment with leuprolide acetate led to initial
increases in serum concentrations of amyloid-β 1–40 ; this
led to the assumption that declines in sex steroids by chemical
castration could lead to an increase in AD pathogenesis.
However, it is important to note that leuprolide acetate is a
gonadotropin-releasing hormone agonist which initially
increases the levels of LH before the levels decline and
therefore, an initial increase in LH could account for the initial
increases in serum amyloid-β observed. On the other hand,
increased serum amyloid-β may also reflect increased efflux of
amyloid-β from brain, as has been documented in transgenic
AβPP mice after immunotherapy .
Importantly, to reflect a realistic therapeutic window as it
would apply to human patients, in our study we used aged mice
where cognitive decline and amyloid deposition are both
evident. In addition, and without attempting direct comparisons
across species, studying aged rather than young female mice
provided a similar hormonal environment to that of a post-
menopausal female (i.e., deregulation and responsiveness to
gonadotropins as well as low estrogen levels), which is typically
the population that develops AD . However, in order to
study the full spectrum of leuprolide acetate treatment effects,
future studies should include a young group as well as an
ovariectomized group. Certainly, the complex interaction
between all the HPG axis components suggests that it is
unlikely that any one hormone of this axis plays a single and
predominant role but rather it may be the balance or ratio of one
hormone to another, i.e., LH to estrogen or testosterone. In this
Fig. 3. Amyloid-β burden measured as % area stained in the entire hippocampus of 11 sections/brain/animal is significantly lower in animals treated with leuprolide
(n = 8) compared to saline-treated animals (n=5, *P b 0.05). Representative image of amyloid-β-burden in Tg2576 mice after saline (S) or leuprolide (L). Scale bar,
450 G. Casadesus et al. / Biochimica et Biophysica Acta 1762 (2006) 447–452
regard, individuals with higher LH to sex steroid ratios such as
women  and men  with lower endogenous sex steroid
levels (i.e., high LH to estrogen ratio) show higher rates of AD
and would likely benefit from leuprolide acetate treatment more
than women/men with higher endogenous levels of estrogen/
testosterone (lower LH:estrogen ratio), who have lower rates of
AD. Likewise, in men, treatment of leuprolide acetate could be
beneficial albeit masked by the effects of testosterone depletion
, in which case, testosterone replacement could be ideal.
Therefore, and considering that the totality of hormonal
influences cannot be ignored, our study demon strates that
targeting LH with leuprolide acetate, a product already safety-
approved for therapeutic use in prostate cancer, is as effective if
not more effective, as shown by its capacity to both positively
modulate cognition and reduce amyloid-β load, than estrogen
therapy alone. Likewise, the fact that leuprolide acetate was
capable of modulating cognitive behavior and reducing
advanced amyloid-β deposition in aged animals suggests that
this treatment may be an effective treatment strategy even at late
stages of disease. In this regard, a recently completed phase II
clinical trial (http://clinicaltrials.gov/ct/show/NCT00076440?
order=6) indicates that patients treated with high doses of
leuprolide acetate show a stabilization in cognitive decline
(ADAS-Cog, ADCS-CGIC) and activities of daily living
pp. 56–64), therefore our findings are in agreement with
those in human trials.
We would like to thank Dr. Bob Switzer (Neuroscience
Associates Inc.) for his invaluable help with the tissue
preparation and amyloid-β staining and Dr. Nett (Endocrine
Laboratory at ARBL-Colorado State University) for his help
and expert input on serum LH measurements. Work in authors'
laboratories is supported by Voyager Pharmaceutical Corpora-
tion, the Alzheimer's Association (MAS), Philip Morris USA
Inc., and Philip Morris International (GC). Drs. Atwood, Perry
and Smith are consultants to Voyager and own equity.
 G. Casadesus, X. Zhu, C.S. Atwood, K.M. Webber, G. Perry, R.L. Bowen,
M.A. Smith, Beyond estrogen: targeting gonadotropin hormones in the
treatment of Alzheimer's disease, Curr. Drug Targets CNS Neurol. Disord.
3 (2004) 281–285.
 J.J. Manly, C.A. Merchant, D.M. Jacobs, S.A. Small, K. Bell, M. Ferin, R.
Mayeux, Endogenous estrogen levels and Alzheimer's disease among
postmenopausal women, Neurology 54 (2000) 833–837.
 V.W. Henderson, A. Paganini-Hill, C.K. Emanuel, M.E. Dunn, J.G.
Buckwalter, Estrogen replacement therapy in older women. Comparisons
between Alzheimer's disease cases and nondemented control subjects,
Arch. Neurol. 51 (1994) 896–900.
 C. Kawas, S. Resnick, A. Morrison, R. Brookmeyer, M. Corrada, A.
Zonderman, C. Bacal, D.D. Lingle, E. Metter, A prospective study of
estrogen replacement therapy and the risk of developing Alzheimer's
disease: the Baltimore Longitudinal Study of Aging, Neurology 48 (1997)
 S.R. Rapp, M.A. Espeland, S.A. Shumaker, V.W. Henderson, R.L.
Brunner, J.E. Manson, M.L. Gass, M.L. Stefanick, D.S. Lane, J. Hays,
K.C. Johnson, L.H. Coker, M. Dailey, D. Bowen, Effect of estrogen plus
progestin on global cognitive function in postmenopausal women: the
Women's Health Initiative Memory Study: a randomized controlled trial,
JAMA 289 (2003) 2663–2672.
 V.W. Henderson, J.R. Guthrie, E.C. Dudley, H.G. Burger, L. Dennerstein,
Estrogen exposures and memory at midlife: a population-based study of
women, Neurology 60 (2003) 1369–1371.
 S.A. Shumaker, C. Legault, S.R. Rapp, L. Thal, R.B. Wallace, J.K.
Ockene, S.L. Hendrix, B.N. Jones III, A.R. Assaf, R.D. Jackson, J.M.
Kotchen, S. Wassertheil-Smoller, J. Wactawski-Wende, Estrogen plus
progestin and the incidence of dementia and mild cognitive impairment in
postmenopausal women: the Women's Health Initiative Memory Study: a
randomized controlled trial, JAMA 289 (2003) 2651–2662.
 V.W. Henderson, E. Hogervorst, Testosterone and Alzheimer disease: is it
men's turn now? Neurology 62 (2004) 170–171.
 A.M. Paoletti, S. Congia, S. Lello, D. Tedde, M. Orru, M. Pistis, M.
Pilloni, P. Zedda, A. Loddo, G.B. Melis, Low androgenization index in
elderly women and elderly men with Alzheimer's disease, Neurology 62
 J.E. Morley, Testosterone and behavior, Clin. Geriatr. Med. 19 (2003)
 N. Schupf, D. Kapell, B. Nightingale, A. Rodriguez, B. Tycko, R. Mayeux,
Earlier onset of Alzheimer's disease in men with Down syndrome,
Neurology 50 (1998) 991–995.
 J. Hasen, R.M. Boyar, L.R. Shapiro, Gonadal function in trisomy 21,
Horm. Res. 12 (1980) 345–350.
 Y.H. Hsiang, G.D. Berkovitz, G.L. Bland, C.J. Migeon, A.C. Warren,
Gonadal function in patients with Down syndrome, Am. J. Med. Genet. 27
 R.A. Short, R.L. Bowen, P.C. O'Brien, N.R. Graff-Radford, Elevated
gonadotropin levels in patients with Alzheimer disease, Mayo Clin. Proc.
76 (2001) 906–909.
 M.A. Smith, G. Perry, C.S. Atwood, R.L. Bowen, Estrogen replacement
and risk of Alzheimer disease, JAMA 289 (2003) 1100 (author reply
 R.L. Bowen, J.P. Isley, R.L. Atkinson, An association of elevated serum
gonadotropin concentrations and Alzheimer disease? J. Neuroendocrinol.
12 (2000) 351–354.
 E.K. Hoskin, M.X. Tang, J.J. Manly, R. Mayeux, Elevated sex-hormone
binding globulin in elderly women with Alzheimer's disease, Neurobiol.
Aging 25 (2004) 141–147.
 E. Hogervorst, J. Williams, M. Combrinck, A. David Smith, Measuring
serum oestradiol in women with Alzheimer's disease: the importance of
the sensitivity of the assay method, Eur. J. Endocrinol. 148 (2003)
 P.P. Zandi, M.C. Carlson, B.L. Plassman, K.A. Welsh-Bohmer, L.S.
Mayer, D.C. Steffens, J.C. Breitner, Hormone replacement therapy and
incidence of Alzheimer disease in older women: the Cache County Study,
JAMA 288 (2002) 2123–2129.
 R.B. Gibbs, R. Gabor, Estrogen and cognition: applying preclinical
findings to clinical perspectives, J. Neurosci. Res. 74 (2003) 637–643.
 H.G. Burger, The endocrinology of the menopause, Maturitas 23 (1996)
 H. Lukacs, E.S. Hiatt, Z.M. Lei, C.V. Rao, Peripheral and intracerebro-
ventricular administration of human chorionic gonadotropin alters several
hippocampus-associated behaviors in cycling female rats, Horm. Behav.
29 (1995) 42–58.
 Z.M. Lei, C.V. Rao, J.L. Kornyei, P. Licht, E.S. Hiatt, Novel expression of
human chorionic gonadotropin/luteinizing hormone receptor gene in brain,
Endocrinology 132 (1993) 2262–2270.
 R.L. Bowen, M.A. Smith, P.L. Harris, Z. Kubat, R.N. Martins, R.J.
Castellani, G. Perry, C.S. Atwood, Elevated luteinizing hormone
expression colocalizes with neurons vulnerable to Alzheimer's disease
pathology, J. Neurosci. Res. 70 (2002) 514–518.
 L. De Toledo-Morrell, I. Goncharova, B. Dickerson, R.S. Wilson, D.A.
Bennett, From healthy aging to early Alzheimer's disease: in vivo
detection of entorhinal cortex atrophy, Ann. N. Y. Acad. Sci. 911 (2000)
451G. Casadesus et al. / Biochimica et Biophysica Acta 1762 (2006) 447–452
 J.H. Morrison, P.R. Hof, Selective vulnerability of corticocortical and
hippocampal circuits in aging and Alzheimer's disease, Prog. Brain Res.
136 (2002) 467–486.
 R.L. Bowen, G. Verdile, T. Liu, A.F. Parlow, G. Perry, M.A. Smith, R.N.
Martins, C.S. Atwood, Luteinizing hormone, a reproductive regulator that
modulates the processing of amyloid-beta precursor protein and amyloid-
beta deposition, J. Biol. Chem. 279 (2004) 20539–20545.
 H. Okada, Y. Doken, Y. Ogawa, Persistent suppression of the pituitary–
gonadal system in female rats by three-month depot injectable micro-
spheres of leuprorelin acetate, J. Pharm. Sci. 85 (1996) 1044–1048.
 K. Hsiao, P. Chapman, S. Nilsen, C. Eckman, Y. Harigaya, S. Younkin, F.
Yang, G. Cole, Correlative memory deficits, Abeta elevation, and amyloid
plaques in transgenic mice, Science 274 (1996) 99–102.
 R. Lalonde, The neurobio logical basis of spontaneous alternation,
Neurosci. Biobehav. Rev. 26 (2002) 91–104.
 J.A. Joseph, N.A. Denisova, G. Arendash, M. Gordon, D. Diamond, B.
Shukitt-Hale, D. Morgan, Blueberry supplementation enhances signaling
and prevents behavioral deficits in an Alzheimer disease model, Nutr.
Neurosci. 6 (2003) 153 –162.
 L. Holcomb, M.N. Gordon, E. McGowan, X. Yu, S. Benkovic, P. Jantzen,
K. Wright, I. Saad, R. Mueller, D. Morgan, S. Sanders, C. Zehr, K.
O'Campo, J. Hardy, C.M. Prada, C. Eckman, S. Younkin, K. Hsiao, K.
Duff, Accelerated Alzheimer-type phenotype in transgenic mice carrying
both mutant amyloid precursor protein and presenilin 1 transgenes, Nat.
Med. 4 (1998) 97–100.
 D.M. Wilcock, A. Rojiani, A. Rosenthal, G. Levkowitz, S. Subbarao, J.
Alamed, D. Wilson, N. Wilson, M.J. Freeman, M.N. Gordon, D. Morgan,
Passive amyloid immunotherapy clears amyloid and transiently activates
microglia in a transgenic mouse model of amyloid deposition, J. Neurosci.
24 (2004) 6144–6151.
 H. Anisman, Dissociation of disinhibitory effects of scopolamine: strain
and task factors, Pharmacol. Biochem. Behav. 3 (1975) 613–618.
 M .J. West, L. Sl omianka, H.J. Gunders en, Unbiased stereological
estimation of the total number of neurons in the subdivisions of the rat
hippocampus using the optical fractionator, Anat. Rec. 231 (1991)
 A. Nunomura, G. Perry, G. Aliev, K. Hirai, A. Takeda, E.K. Balraj, P.K.
Jones, H. Ghanbari, T. Wataya, S. Shimohama, S. Chiba, C.S. Atwood, R.
B. Petersen, M.A. Smith, Oxidative damage is the earliest event in
Alzheimer disease, J. Neuropathol. Exp. Neurol. 60 (2001) 759–767.
 K.M. Webber, R. Bowen, G. Casadesus, G. Perry, C.S. Atwood, M.A.
Smith, Gonadotropins and Alzheimer's disease: the link between estrogen
replacement therapy and neuroprotection, Acta Neurobiol. Exp. (Wars) 64
 G. Casadesus, C.S. Atwood, X. Zhu, A.W. Hartzler, K.M. Webber, G.
Perry, R.L. Bowen, M.A. Smith, Evidence for the role of gonadotropin
hormones in the development of Alzheimer disease, Cell Mol. Life Sci. 62
 H.A. Bimonte, V.H. Denenberg, Estradiol facilitates performance as
working memory load increases, Psychoneuroendocrinology 24 (1999)
 J.M. Daniel, G.P. Dohanich, Acetylcholine mediates the estrogen-induced
increase in NMDA receptor binding in CA1 of the hippocampus and the
associated improvement in working memory, J. Neurosci. 21 (2001)
 J.M. Daniel, A.J. Fader, A.L. Spencer, G.P. Dohanich, Estrogen enhances
performance of female rats during acquisition of a radial arm maze, Horm.
Behav. 32 (1997) 217–225.
 A.J. Fader, P.E. Johnson, G.P. Dohanich, Estrogen improves working but
not reference memory and prevents amnestic effects of scopolamine of a
radial-arm maze, Pharmacol. Biochem. Behav. 62 (1999) 711–717.
 T. Heikkinen, J. Puolivali, L. Liu, A. Rissanen, H. Tanila, Effects of
ovariectomy and estr ogen treatment on learning and hippocampal
neurotransmitters in mice, Horm. Behav. 41 (2002) 22–32.
 M.M. Miller, S.M. Hyder, R. Assayag, S.R. Panarella, P. Tousignant,
K.B. Franklin, Estrogen modulates spontaneous alternation and the
cholinergic phenotype in the basal forebrain, Neuroscience 91 (1999)
 T. Heikkinen, J. Puolivali, H. Tanila, Effects of long-term ovariectomy and
estrogen treatment on maze learning in aged mice, Exp. Gerontol. 39
 D.J. Selkoe, Aging, amyloid, and Alzheimer's disease: a perspective in
honor of Carl Cotman, Neurochem. Res. 28 (2003) 1705–1713.
 M.A. Smith, G. Casadesus, J.A. Joseph, G. Perry, Amyloid-beta and tau
serve antioxidant functions in the aging and Alzheimer brain, Free Radic.
Biol. Med. 33 (2002) 1194
 J. Joseph, B. Shukitt-Hale, N.A. Denisova, A. Martin, G. Perry, M.A.
Smith, Copernicus revisited: amyloid beta in Alzheimer's disease,
Neurobiol. Aging 22 (2001) 131–146.
 G. Chen, K.S. Chen, J. Knox, J. Inglis, A. Bernard, S.J. Martin, A. Justice,
L. McConlogue, D. Games, S.B. Freedman, R.G. Morris, A learning
deficit related to age and beta-amyloid plaques in a mouse model of
Alzheimer's disease, Nature 408 (2000) 975–979.
 B.J. Cummings, E. Head, A.J. Afagh, N.W. Milgram, C.W. Cotman, Beta-
amyloid accumulation correlates with cognitive dysfunction in the aged
canine, Neurobiol. Learn Mem. 66 (1996) 11–23.
 S. Gandy, S. Petanceska, Regulation of Alzheimer beta-amyloid precursor
trafficking and metabolism, Adv. Exp. Med. Biol. 487 (2001) 85–100.
 R.B. DeMattos, K.R. Bales, D.J. Cummins, J.C. Dodart, S.M. Paul, D.M.
Holtzman, Peripheral anti-A beta antibody alters CNS and plasma A beta
clearance and decreases brain A beta burden in a mouse model of
Alzheimer's disease, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 8850–8855.
 C.E. Finch, L.S. Felicio, C.V. Mobbs, J.F. Nelson, Ovarian and steroidal
influences on neuroendocrine aging processes in female rodents, Endocr.
Rev. 5 (1984) 467–497.
 X. Hong, X. Zhang, H. Li, A case-control study of endogenous estrogen
and risk of Alzheimer's disease, Zhonghua Liu Xing Bing Xue Za Zhi 22
 E. Hogervorst, J. Williams, M. Budge, L. Barnetson, M. Combrinck, A.D.
Smith, Serum total testosterone is lower in men with Alzheimer's disease,
Neuro-endocrinol. Lett. 22 (2001) 163–168.
 H.J. Green, K.I. Pakenham, B.C. Headley, J. Yaxley, D.L. Nicol, P.N.
Mactaggart, C. Swanson, R.B. Watson, R.A. Gardiner, Altered cognitive
function in men treated for prostate cancer with luteinizing hormone-
releasing hormone analogues and cyproterone acetate: a randomized
controlled trial, BJU Int. 90 (2002) 427–432.
452 G. Casadesus et al. / Biochimica et Biophysica Acta 1762 (2006) 447–452