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Co
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Available online 1 April 2011
Keywords:
Nogo
CamkII
Aging
Hippocampus
ne occurs without frank neurodegeneration and is the most common cause of
Neurobiology of Disease 43 (2011) 201–212
Contents lists available at ScienceDirect
Neurobiology
.e lAge-related cognitive decline impacts a variety of brain functions,
and reduces quality of life for aging individuals by diminishing
healthspan and increasing dependence. Currently, an estimated 40%
of the otherwise healthy population over age 60 is affected by
cognitive decline (Small, 2002). Common age-related conditions such
as hypertension and heart disease are risk factors for cognitive
decline, and are associated with increased severity of cognitive
deficits with advancing age (Qiu et al., 2005; Dahle et al., 2009;
Okonkwo et al., 2010). Due to lifespan increases, demographic shifts,
and health care advances, the percentage of the population over age
60 is expected to increase to 20% by 2050 (Shrestha, 2006). This
Previous characterizations of the hippocampal proteome and
transcriptome with aging and cognitive decline (Poon et al., 2006;
Blalock et al., 2003, 2005; Rowe et al., 2007Butterfield et al., 2006;
Freeman et al., 2009b) have identified alterations in neurobiologi-
cally-relevant processes associated with advancing age, including
increased oxidative stress, decreased glucose utilization and bioener-
getic metabolism, and aberrant protein synthesis and trafficking.
Although these processes are important to healthy neuronal function,
a more immediate cause of cognitive decline is likely dysregulation of
neurotransmission and synaptic plasticity. Electrophysiological cor-
relates of hippocampal function are disrupted with aging and learningprevalence of aged individuals is unique in
incidence of age-related health condition
concomitantly with our increasing lifespan. A
⁎ Corresponding author at: Penn State College of Med
R130, Hershey, PA 17033, USA. Fax: +1 717 531 5013.
E-mail address: wfreeman@psu.edu (W.M. Freeman
Available online on ScienceDirect (www.scienced
0969-9961/$ – see front matter © 2011 Elsevier Inc. Al
doi:10.1016/j.nbd.2011.03.012are needed to understand, prevent, and treat age-related cognitive
decline.IntroductionSynapse
Learning and memorybasis of age-related cognitive decline remains unknown. While concerted efforts have led to the identification
of neurobiological changes with aging, few age-related alterations have been definitively correlated to
behavioral measures of cognitive decline. In this work, adult (12 months) and aged (28 months) rats were
categorized by Morris water maze performance as Adult cognitively Intact, Aged cognitively Intact or Aged
cognitively Impaired, and protein expression was examined in hippocampal synaptosome preparations.
Previously described differences in synaptic expression of neurotransmission-associated proteins (Dnm1,
Hpca, Stx1, Syn1, Syn2, Syp, SNAP25, VAMP2 and 14-3-3 eta, gamma, and zeta) were confirmed between
Adult and Aged rats, with no further dysregulation associated with cognitive impairment. Proteins related to
synaptic structural stability (MAP2, drebrin, Nogo-A) and activity-dependent signaling (PSD-95, 14-3-3θ,
CaMKIIα) were up- and down-regulated, respectively, with cognitive impairment but were not altered with
increasing age. Localization of MAP2, PSD-95, and CaMKIIα demonstrated protein expression alterations
throughout the hippocampus. The altered expression of activity- and structural stability-associated proteins
suggests that impaired synaptic plasticity is a distinct phenomenon that occurs with age-related cognitive
decline, and demonstrates that cognitive decline is not simply an exacerbation of the aging phenotype.
© 2011 Elsevier Inc. All rights reserved.human history, and the
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l rights reserved.ncreasing in prevalence. Nevertheless, the neurobiologicalReceived 28 February 2011
Accepted 18 March 2011memory impairment in aging individuals. With increasing longevity, cognitive deficits, especially in
hippocampus-dependent memory processes, are iArticle history: Age-related cognitive decliHippocampal dysregulation of synaptic p
age-related cognitive decline
Heather D. VanGuilder a, Julie A. Farley b, Han Yan b,
William E. Sonntag b, Willard M. Freeman a,⁎
a Penn State College of Medicine, Department of Pharmacology, Hershey Center for Applie
b Reynolds Oklahoma Center on Aging, University of Oklahoma Health Sciences Center, 97
a b s t r a c ta r t i c l e i n f o
j ourna l homepage: wwwsticity-associated proteins with
lleen A. Van Kirk a, Matthew Mitschelen b,
search, 500 University Drive, Hershey, PA, 17033, USA
10th Street, BRC-1303, Oklahoma City, OK, 73104, USA
of Disease
sev ie r.com/ locate /ynbd id are consistent with unstable encoding of spatial
(Barnes et al., 1997; Kumar et al., 2007; Norris et al.,
ig and Barnes, 2003; Wilson et al., 2003). This
ests in resistance to LTP induction, facilitation of LTD,
tiotemporal activation of ensemble networks. These
ay be related to atypical synapse morphology,
r synthesis and receptor signaling, and neuronal
expression (Liu et al., 2008; Shi et al., 2005; Burke

and Barnes, 2006). Additional work is required, however, to establish
a definitive link between these phenomena and impaired cognitive
function.
Using a rodent model of age-related cognitive decline that enables
segregation of aged rats into cognitively intact and impaired groups
based onMorris water maze performance, we have demonstrated two
distinct shifts in the hippocampal cytosolic proteomewith age-related
cognitive decline: one related to aging and a second specific to
cognitive function (Freeman et al., 2009b). Further, we recently
reported age-related dysregulation of components of the hippocam-
pal synaptoproteome with roles in initiation and modulation of
neurotransmission (VanGuilder et al., 2010). The goal of the current
study was to identify synaptic proteins regulated specifically with
Assessment and Accreditation of Laboratory Animal Care, and all
animal procedureswere approvedby the Institutional Animal Care and
Use Committee in compliance with the Public Health Service Policy on
Humane Care and Use of Laboratory Animals and the National
Research Council's Guide for the Care and Use of Laboratory Animals.
Morris water maze testing
Rats were acclimatized to the OUHSC Barrier Facility for twoweeks
prior to cognitive assessment conducted with a variation of theMorris
water maze using methods similar to those previously described
(Freeman et al., 2009b; Mitschelen et al., 2009). The water maze
consisted of a galvanized metal tank 1.7 m in diameter and 0.6 m in
ct
aire
ct
aire
202 H.D. VanGuilder et al. / Neurobiology of Disease 43 (2011) 201–212age-related cognitive decline and to differentiate this phenomenon
from the general effects of aging. This investigation focused on
proteins that mediate activity-responsive synaptic signaling and
structural remodeling, and that have well-characterized regulatory
functions in hippocampus-dependent spatial learning and memory
(Elgersma et al., 2004; Khuchua et al., 2003; Kojima and Shirao, 2007;
Migaud et al., 1998; Miller et al., 2002; Mizui et al., 2005; Zagrebelsky
et al., 2010). We observed increased expression of proteins vital to
synaptic structural stability (MAP2, drebrin, Nogo-A) and decreased
expression of synaptic activity-dependent signaling proteins (PSD-95,
14-3-3θ, CaMKIIα) that strongly correlate with declining cognitive
performance. These findings provide evidence for disrupted hippo-
campal synaptic plasticity specifically in cognitively impaired aged
animals whichmay act synergistically with age-related decrements of
neurotransmission efficacy.
Materials and methods
Animals
Three independent cohorts of male Fischer 344×Brown Norway
(F1) hybrid rats (see Table 1 for cohort information) were obtained
from the National Institute on Aging colony at Harlan Industries
(Indianapolis, IN). Synaptosome samples used in aging-only analyses
(set 1) were derived from Young-adult, Adult, and Aged rats
(VanGuilder et al., 2010). Rats were singly housed in laminar flow
cages (Polysulfone) in the OUHSC (University of Oklahoma Health
Sciences Center) specific pathogen-free Barrier Facilitywithwater and
food freely available (Purina Mills, Richmond, IN). Environmental
controls maintained a 12-hour light/dark cycle and constant temper-
ature and humidity. One week following completion of behavioral
testing, rats were sacrificed by decapitation without anesthesia, and
the hippocampi rapidly dissected for preparation of synaptosomes (set
2). Alternatively, rats were perfusion-fixed, and their brains collected
for immunohistochemical analysis (set 3). At necropsy, animals were
examined for exclusionary criteria including peripheral tumors, frank
kidney disease and cardiac hypertrophy as well as gross brain
neuropathology (e.g., cortical atrophy and pituitary tumors). The
OUHSC animal facilities are fully accredited by the Association for
Table 1
Animal data.
Animal cohort Symbol Cognitive assessment Age (months) Group
Set 1a ■ No 3 Young
12 Adult
26 Aged
Set 2 ● Yes 12 Adult
28 Aged Inta
28 Aged Imp
Set 3 ▲ Yes 13 Adult
26 Aged Inta
26 Aged Imp
a Previously described in (VanGuilder et al., 2010).height. Water made opaque with non-toxic water-based white food
coloring was added to a depth of 25 cm, and a retractable 12 cm
escape platform was fixed 2 cm below the water's surface. A curtain
with fixed-position visual cues, serving as reference cues for the
location of the escape platform, surrounded the maze pool. A center-
mounted camera located 1.6 m above the water's surface provided
image input to an automated tracking system (Noldus Ethovision XT,
Wageningen, Netherlands) that recorded water maze performance.
Task acquisition was conducted in four training blocks consisting of
five individual trials performed over two days, for a total of eight days
of task acquisition with the escape platform located in the same
position across all days of training. Rats were placed into the maze,
facing the wall of the pool in one of four locations with start positions
pseudo-randomized, and were given 60 s to locate the escape
platform based on surrounding spatial cues. Path length to find the
platform was the dependent measure, with shorter path lengths
indicating better performance. After completion of each acquisition
block (i.e., on days 2, 4, 6 and 8), a probe trial was performed with the
escape platform removed. Rats were placed into the maze and the
mean proximity to the platform location, duration in the annulus-40
(the area 40 cm around the platform location), cumulative distance,
and mean swim velocity were recorded during the first 30 s. To avoid
extinguishingmemory of the platform location, the platformwas then
replaced and rats were given an additional 60 s to locate it using the
surrounding cues. Probe trial data were used to segregate Aged
animals into cognitively Intact and Impaired groups relative to Adult
group performance, allowing retrospective analysis of acquisition
phase data by group. Mean proximity to the escape platform location
was used as the primary measure of cognitive performance on probe
trials based on demonstration of its superior sensitivity compared
to alternative measures (Maei et al., 2009). The number of cumulative
platform location crossings was used as a secondary measure of
cognitive performance (Terry, 2009). To ascertain successful task
acquisition, data were statistically analyzed across blocks by one-way
repeated measures ANOVA with Holm–Sidak post hoc testing.
Significance of group differences for individual acquisition blocks
and probe trials was assessed by one-way ANOVA with Student
Newman Keuls post hoc testing. Two days following conclusion
of water maze testing, visual performance was assessed over four
n Sample Type Biochemical assessment
10 Hippocampal synaptosomes Immunoblot analysis of protein targets
with aging10
10
5 Hippocampal synaptosomes Immunoblot/qPCR analysis of targets
with cognitive status and aging8
d 7
9 Fixed, frozen sections Immunohistochemical localization of
cognition-related protein targets10
d 5