A selective role for ARMS/Kidins220 scaffold protein in spatial memory and trophic support of entorhinal and frontal cortical neurons.

Abstract

Progressive cortical pathology is common to several neurodegenerative and psychiatric disorders. The entorhinal cortex (EC) and frontal cortex (FC) are particularly vulnerable, and neurotrophins have been implicated because they appear to be protective. A downstream signal transducer of neurotrophins, the ankyrin repeat-rich membrane spanning scaffold protein/Kidins 220 (ARMS) is expressed in the cortex, where it could play an important role in trophic support. To test this hypothesis, we evaluated mice with a heterozygous deletion of ARMS (ARMS(+/-) mice). Remarkably, the EC and FC were the regions that demonstrated the greatest defects. Many EC and FC neurons became pyknotic in ARMS(+/-) mice, so that large areas of the EC and FC were affected by 12 months of age. Areas with pyknosis in the EC and FC of ARMS(+/-) mice were also characterized by a loss of immunoreactivity to a neuronal antigen, NeuN, which has been reported after insult or injury to cortical neurons. Electron microscopy showed that there were defects in mitochondria, myelination, and multilamellar bodies in the EC and FC of ARMS(+/-) mice. Although primarily restricted to the EC and FC, pathology appeared to be sufficient to cause functional impairments, because ARMS(+/-) mice performed worse than wild-type on the Morris water maze. Comparisons of males and females showed that female mice were the affected sex in all comparisons. Taken together, the results suggest that the expression of a prominent neurotrophin receptor substrate normally protects the EC and FC, and that ARMS may be particularly important in females.

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A selective role for ARMS/Kidins220 scaffold protein in spatial memory and trophic
support of entorhinal and frontal cortical neurons
Aine M. Duffya,⁎, Michael J. Schanera, Synphen H. Wub, Agnieszka Staniszewskif, Asok Kumara,
Juan Carlos Arévalog, Ottavio Aranciof, Moses V. Chaob, Helen E. Scharfmana,c,d,e
aThe Nathan Kline Institute for Psychiatric Research, Center for Dementia Research, Orangeburg, New York, NY 10962, USA
bMolecular Neurobiology Program, Skirball Institute of Biomolecular Medicine, Departments of Cell Biology, Physiology & Neuroscience and Psychiatry,
New York University Langone Medical Center, New York, NY 10016, USA
cDepartment of Child & Adolescent Psychiatry, New York University Langone Medical Center, New York, NY 10016, USA
dDepartment of Psychiatry, New York University Langone Medical Center, New York, NY 10016, USA
eDepartment of Physiology & Neuroscience, New York University Langone Medical Center, New York, NY 10016, USA
fDepartment of Pathology & Cell Biology and Taub Institute for Research on Alzheimer's Disease & the Aging Brain, Columbia University Medical Center New York, NY 10032, USA
gDepartment of Cell Biology and Pathology, Instituto de Neurociencias Castilla y Léon (INCyL), Universidad de Salamanca, Salamanca 37007, Spain
a b s t r a c ta r t i c l ei n f o
Article history:
Received 11 October 2010
Revised 1 March 2011
Accepted 4 March 2011
Available online 16 March 2011
Keywords:
BDNF
Neurotrophin
Sex differences
Spatial memory
Myelin
Oxidative stress
Progressive cortical pathology is common to several neurodegenerative and psychiatric disorders. The
entorhinal cortex (EC) and frontal cortex (FC) are particularly vulnerable, and neurotrophins have been
implicated because they appear to be protective. A downstream signal transducer of neurotrophins, the
ankyrin repeat-rich membrane spanning scaffold protein/Kidins 220 (ARMS) is expressed in the cortex,
where it could play an important role in trophic support. To test this hypothesis, we evaluated mice with a
heterozygous deletion of ARMS (ARMS+/−mice). Remarkably, the EC and FC were the regions that
demonstrated the greatest defects. Many EC and FC neurons became pyknotic in ARMS+/−mice, so that large
areas of the EC and FC were affected by 12 months of age. Areas with pyknosis in the EC and FC of ARMS+/−
mice were also characterized by a loss of immunoreactivity to a neuronal antigen, NeuN, which has been
reported after insult or injury to cortical neurons. Electron microscopy showed that there were defects in
mitochondria, myelination, and multilamellar bodies in the EC and FC of ARMS+/−mice. Although primarily
restricted to the EC and FC, pathology appeared to be sufficient to cause functional impairments, because
ARMS+/−mice performed worse than wild-type on the Morris water maze. Comparisons of males and
females showed that female mice were the affected sex in all comparisons. Taken together, the results suggest
that the expression of a prominent neurotrophin receptor substrate normally protects the EC and FC, and that
ARMS may be particularly important in females.
Published by Elsevier Inc.
Introduction
Neurons in the entorhinal cortex (EC) and frontal cortex (FC) are
vulnerable in many neurological and psychiatric disorders. Abnormal
cytoarchitecture and neuron density have been observed in the EC of
patients with schizophrenia (Arnold et al., 1995; Krimer et al., 1997;
Arnold, 2000), temporal lobe epilepsy (Du et al., 1993; Scharfman,
2000), Alzheimer's disease (AD) and even in normal aging (Gomez-
Isla et al., 1996; de Toledo-Morrell et al., 2000; Kordower et al., 2001).
In the FC, it has been reported that pyramidal neurons show signs of
atrophy in patients with schizophrenia (Lewis et al., 2003), bipolar
disorder (Rajkowska, 2002), depression (Drevets, 2000; Shah et al.,
2002), aging and AD (Holland et al., 2009; McDonald et al., 2009;
Schroeter et al., 2009).
Neurotrophins may normally protect the EC and FC, because
reduced expression of neurotrophins or altered neurotrophin recep-
tors have been identified in the EC and FC in several diseases. For
example, decreased levels of brain-derived neurotrophic factor
(BDNF) and neurotrophin-3 (NT-3) have been found in the FC of
patients with schizophrenia and drug-resistant depression (Durany et
al., 2001; Weickert et al., 2003; Shoval and Weizman, 2005; Weickert
et al., 2005). In addition, BDNF and its primary receptor, tropomyosin
receptor kinase B (TrkB), are decreased in the cortex during aging and
AD, based on both animal models of the disease, and clinical research
(Lindvall et al., 1994; Knusel and Gao, 1996; Chao et al., 2006; Tapia-
Arancibia et al., 2008; Nagahara et al., 2009; Peng et al., 2009).
The reasons why neurotrophins are protective in the EC and FC are
unclear. Newly described signaling mechanisms suggest reasons why
Experimental Neurology 229 (2011) 409–420
⁎ Corresponding author at: The Nathan Kline Institute for Psychiatric Research,
Center for Dementia Research, 140 Old Orangeburg Rd., Bldg. 35, Orangeburg, New
York, NY 10962, USA.
E-mail addresses: aduffy@nki.rfmh.org (A.M. Duffy), hscharfman@nki.rfmh.org
(H.E. Scharfman).
0014-4886/$ – see front matter. Published by Elsevier Inc.
doi:10.1016/j.expneurol.2011.03.008
Contents lists available at ScienceDirect
Experimental Neurology
journal homepage: www.elsevier.com/locate/yexnr

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neurotrophins are protective, because the new pathways appear to
mediate protection. One example is tyrosine phosphorylation of the
ankyrin repeat-rich membrane spanning protein (ARMS), also termed
Kidins220 (Iglesias et al., 2000). ARMS/Kidins220 (hereafter referred
to as ARMS) is a transmembrane scaffold protein downstream of Trk
(Kong et al., 2001). ARMS also is downstream of ephrin receptors, and
therefore may transmit signals involved in ephrin function, such as
changes in morphology and synaptic plasticity (Murai and Pasquale,
2004; Lai and Ip, 2009).
Phosphorylation of ARMS promotes cell survival in culture by
activating the mitogen-activated protein kinase (MAPK) and nuclear
factor κB (NFκB) (Arévalo et al., 2004, 2006; Sniderhan et al., 2008).
Knockdown of ARMS in culture results in increased neuronal death
(Lopez-Menendez et al., 2009), suggesting that ARMS is a candidate
mechanism linking neurotrophins, and possibly ephrins, to the
support of cortical neurons. Therefore, we hypothesized that
decreasing levels of ARMS would result in cortical pathology. To test
this hypothesis we used transgenic mice with reduced ARMS
expression (heterozygous mice; ARMS+/−). Histochemistry, immu-
nocytochemistry, and electron microscopy (EM) were used to
evaluate the EC and FC, as well as other cortical areas. The Morris
water maze was used to evaluate animals behaviorally because the EC
and FC have been shown to contribute to this task (Kolb et al., 1983;
Hebert and Dash, 2002; Spowart-Manning and van der Staay, 2005;
Nakazawa, 2006; Jo et al., 2007; Leon et al., 2010). The results suggest
that ARMS plays a critical role in the maintenance of structure in the
EC and FC, behaviors associated with these areas, and that sex is an
important variable in the vulnerability of these regions to decreased
levels of ARMS.
Materials and methods
Animals
The experimental procedures were carried out in accordance with
NIH guidelines, and were approved by the Institutional Animal Care
and Use Committees (IACUC) at The Nathan Kline Institute and New
York University (NYU) Langone Medical Center. All wild-type (WT)
and ARMS+/−mice were bred at NYU. The targeting construct for
ARMS−/−mice has been described elsewhere (Wu et al., 2009).
Mice were housed 3–4/cage, in standard mouse cages with corn
cob bedding and a 12-hour light–dark cycle (lights on, 7:00 am). Food
(Purina5001 chow;W.F.Fisher,Somerville,NJ,U.S.A.)andwaterwere
available ad libitum. For anatomical studies, females were perfused at
the same time of day (9:00–11:00 am) and on the same stage of the
estrous cycle (metestrus), to minimize variability. For the Morris
water maze, the cycle stage was monitored to ensure estrous cyclicity
was maintained for the duration of the testing period. To promote
estrous cyclicity, males were housed in cages next to females
(Whitten et al., 1968). Procedures used to establish cycle stage by
vaginal cytology are described elsewhere (Scharfman et al., 2003,
2009).
Fixation and tissue preparation
WT and ARMS+/−mice were deeply anesthetized by inhalation
(Isoflurane; Baxter Healthcare Corporation, Deerfield, IL, U.S.A.)
followed by urethane (2.5 g/kg i.p.; Sigma-Aldrich Chemical Co., St.
Louis, MI, U.S.A.; all chemicals were from Sigma-Aldrich unless stated
otherwise), prior to fixation by perfusion through the left ventricle
using a peristaltic pump (Minipuls 1, Gilson, Middleton, WI, U.S.A).
This approach allowed rapid (b5 min) and sequential delivery of two
solutions: normal saline (40 ml of 0.9% NaCl in distilled water; dH2O),
and fixative (40 ml of 4% paraformaldehyde in 0.1 M phosphate
buffer; PB; pH 7.4). Animals used for EM were perfused with 0.9%
NaCl, followed by 2% paraformaldehyde and 2.5% glutaraldehyde
(Electron Microscopy Sciences, Hartfield, PA, U.S.A.) in 0.1 M PB. The
brains were removed immediately, post-fixed for 24 h in 4%
paraformaldehyde in 0.1 M phosphate PB at 4 °C, and horizontal
sections (50 μm-thick) were cut using a vibratome (TPI 3000,
Vibratome Co., St. Louis, MO, U.S.A.), and collected sequentially.
Regions specified in the Results follow standard terminology (Paxinos
and Franklin, 2001).
Cresyl violet stain
After the entire brain was sectioned, sections (150 μm apart) were
mounted sequentially on subbed slides. After allowing slides to dry
overnight, slides were dehydrated in a graded series of ethanols
(Pharmaco-Aaper)dilutedin distilledH2O(dH2O; 70%, 1×3 min; 95%,
1×3 min; and 100%, 2×5 min). Following rehydration, slides were
stained with 0.1% (w/v) cresyl violet in dH2O for 1 min. Acetic acid
(0.01%; Fisher Chemical Co., Fairlawn, NJ, U.S.A.) was used to destain if
sections were too dark (i.e., cells were stained, but so were areas
between cells). Following dehydration, slides were cleared in xylene
(2×5 min; Fisher), and cover-slipped in Permount (Fisher).
NeuN immunohistochemistry
A monoclonal antibody against neuronal nuclei (NeuN; Clone A60,
MAB 377, Chemicon, Temecula, CA, U.S.A.) was purified from mouse
brain, and its specificity for mature neurons has been demonstrated
(Mullen et al., 1992). Notably, several recent studies have identified
thatneuronsare notalwaysimmunoreactiveusingtheNeuNantibody
if they are evaluated after insult or injury, when NeuN is phosphor-
ylated (Lind et al., 2005). Examples of conditions that lead to a loss of
NeuN-immunoreactivity (NeuN-ir) include metabolic or oxidative
stress, such as ischemia, aging, irradiation, and organophosphate
toxicity(Portiansky etal.,2006;Buckingham etal.,2008;Hayakawa et
al., 2008; Kadriu et al., 2009; Matsuda et al., 2009; Won et al., 2009).
Sections were chosen for NeuN-immunohistochemistry were
adjacent to those that were used for cresyl violet. Sections were
processed for NeuN-immunohistochemistry as previously published
(Scharfman et al., 2002; Winawer et al., 2007), analyzed using a
brightfield microscope (BX61, Olympus, Center Valley, PA, U.S.A.), and
photographed using a digital camera (RET 2000R-F-CLR-12, Q
Imaging, Surrey, BC, Canada). Some NeuN-labeled sections were
counterstained with cresyl violet. For counterstaining, sections were
first processed with the antibody to NeuN and then mounted on
subbed slides. They were allowed to dry overnight, and then stained
for cresyl violet using the protocol described above.
Image analysis (BioquantImage Analysis Corporation, Nashville,TN,
U.S.A.) was conducted as follows: first, the edges of each section were
demarcated at 2× magnification. The areas displaying weak cortical
NeuN-ir were then evaluated by computerized thresholding. The
thresholdwasset sothat areassuchas whitematter, almostexclusively
without neuronal nuclei, were below threshold, and regions that had
many neuronal nuclei were above the threshold. A complete 3-D
rendering of each brain was compiled based on the z-coordinates for
each section. Using a surface-rendering tool (Topographer Plug-in,
BioquantImageAnalysisCorporation)avirtualskinwasrenderedusing
a mesh modeling algorithm for the whole brain and each region with
weak NeuN-ir. This procedure led to a 3-D quantitative image of the
brain (see Fig. 3), from which volumes of areas with weak NeuN were
calculated.
Electron microscopy
Using tissue from animals that were fixed with paraformaldehyde/
glutaraldehyde (described above), sections were processed for NeuN
immunoperoxidase labeling, and adjacent sections were postfixed in
2% osmium tetroxide in 0.1 M PB for 1 h. They were processed for EM
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analysis as previously described (Duffy et al., 2009). Ultrathin sections
were collected on 300-mesh copper grids, counterstained with 0.5%
uranyl acetate (Electron Microscopy Sciences) and lead citrate (1.33 g
lead nitrate and 1.76 g trisodium citrate in 30 ml dH2O) and examined
by EM (Model CM10, Phillips, Eindoven, Netherlands). Micrographs
were captured using a digital camera (model C4742-95, Hamamatsu,
Shizuoka, Japan) and Image Capture Engine software (version
5.42.443a, Advanced Microscopy Techniques, Danvers, MA, U.S.A.).
The classification of cellular and subcellular elements was based on
the descriptions by Peters et al. (1991).
Morris water maze
The Morris water maze was used as described elsewhere
(Trinchese et al., 2004; Puzzo et al., 2009). Briefly, mice were trained
to learn the location of a hidden platform. A white tank was filled with
water (20–22 °C), made opaque with the addition of non-toxic white
paint, and a white platform was submerged under the surface of the
water. Single-housed mice were trained to locate a hidden platform in
2 daily sessions, 4 h apart for 3 days. Each session consisted of 3×60 s
trials, 30 s apart. The time taken to reach the platform was recorded
for each trial and the path was monitored with video tracking to
record velocity (HVS 2020; HVS Image, Hampton, UK). On the 4th day,
the mice were tested for long-term retention of the platform location
using 4 consecutive 60 s probe trials during which the platform was
removed. The tank was divided into 4 quadrants and video tracking
was used to chart the percent of time the animal spent in the target
quadrant containing the platform (quadrant 4). Following the
completion of the probe trials, the mice were tested on a visual task
to determine if there were differences that could be attributed to
vision or swimming velocity. The task consisted of 2 daily sessions for
2 days, 4 h apart. Each session contained 3×60 s trials, 30 s apart. For
the visual task, the platform was placed 1 cm above the water, and a
small silver feeding bowl was placed in the center of the platform. The
platform was moved for each trial. The time to reach the platform was
recorded for each trial and video tracking was used to calculate
swimming velocity.
Data analysis
For the analysis of weak NeuN-ir, volumes corresponding to
regions of weak NeuN-ir are presented as a percentage of the 3-D
Fig. 1. PyknoticneuronsandweakNeuN-immunoreactivityintheentorhinalcortex(EC)ofARMS+/−mice.(A)AhorizontalsectionshowingthelateralEC(LEC)fromaWTmouse,stained
with cresyl violet. MEC = medial EC; CA1 = area CA1; DG = dentate gyrus; A = anterior; M = medial. Inset: Layer II/III of the LEC shown at higher power. Arrows point to cresyl violet
stainedneurons withnormalmorphology. (B) A cresylviolet-stained section froman ARMS+/−mouse,froma similar dorso-ventral level to (A).Inset:Arrowspoint to layerII/IIIneurons
fromtheLECthatappeartobepyknoticbecausetheyaredarklystained,andsomataareshrunken,withangularedges.(C)AsectionlabeledusinganantibodytoNeuNthatwasadjacentto
the one in A shows normal NeuN-immunoreactivity (ir). Inset: Layer II/III of the LEC illustrates normal NeuN-ir labeling. (D) A NeuN-labeled section that was adjacent to the section in B
shows weak NeuN-ir, primarily in the superficial layers of the LEC (arrows). Inset: Layer II/III of the LEC is shown at higher power. Many cells show weak or no NeuN-ir (arrowheads) in
contrast to some cells with normalNeuN-ir (arrows). Calibration forA–D (shown inB)=100 μm; calibrationfor insets inA–B (shownininset for B)=25 μm,and calibration for insets in
C–D (shown in inset for D)=25 μm.
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estimate of total brain volume. For ventricular volume, the outlines
of ventriclesweretraced digitallyandusedtobasea 3-Destimate of
total ventricular volume using Bioquant software. For EM, quanti-
fication of abnormal mitochondria and myelin defects was
performed manually using micrographs. Data are presented as
the number of mitochondria per 100 μm2of tissue, without
correcting for shrinkage, because there was no evidence of
differential shrinkage between genotypes (based on observation
and calculated ventricular volumes; Table 1). Abnormal mitochon-
dria were defined as previously described (Kong and Xu, 1998;
Yazdani et al., 2006; Martin et al., 2007; Boland et al., 2008) using
these characteristics: 1) large size, 2) irregular cristae, and 3)
irregular or broken outer mitochondrial membranes. Defects in
myelination were defined by 1) breaks in the myelin sheath, and 2)
redundant whorls of myelin.
Data are presented as mean±standard error of the mean (SEM)
and Pb0.05 was considered significant. Statistical comparisons were
made using either a two-tailed Student's t-test or two-way analysis of
variance (ANOVA; Microsoft Excel 2007, Microsoft Corporation,
Redmond, WA, U.S.A.). Prior to ANOVA, Bartlett's test (Snedecor and
Cochran, 1989) was used to test for homoscedasticity of variance.
Wheresignificant departurefromhomoscedasticity wasdetected, this
was corrected by log transformation of the data, prior to ANOVA. To
analyze the latencies recorded in the hidden trials and probe trial in
the water maze, repeated measures analysis of variance (RMANOVA)
or ANOVA and Bonferroni/Dunn post-hoc test (Statview v. 5.0.1, SAS
Institute Inc., Cary, NC, U.S.A.) were used.
Results
Subjects
ARMS−/−mice that were previously generated by Cre-mediated
recombination displayed early embryonic lethality (Wu et al., 2009).
Therefore, ARMS+/−mice were used. ARMS+/−mice were viable and
fertile and displayed a 30–40% decrease in ARMS protein (Wu et al.,
2009).
ARMS+/−mice display abnormal cellular morphology in the EC and FC
Cresyl violet staining was used to screen adult ARMS+/−mice
(12-months-old) for detectable defects. There was no evidence of
abnormalities except for select cortical regions, notably the EC and
FC, and only at high magnification (Figs. 1–3). The cells in the EC of
ARMS+/−mice appeared to be pyknotic, i.e., the neurons stained
darker than normal, and had a shrunken or deformed appearance
(Figs. 1A and B, insets). The pyknotic neurons appeared to be
primarily in the superficial layers (Figs. 1A and B). In adjacent areas
of the ARMS+/−mice, and throughout the brain of WT mice,
neurons appeared to be relatively normal, i.e., cresyl violet stain
was lighter in staining intensity, and the somata did not appear to
be shrunken (Fig. 1A). There were no changes in total volume or
ventricular volume (Table 1), despite the presence of pyknotic
neurons.
Loss of NeuN immunoreactivity is observed in ARMS+/−mice
When areas of pyknotic cells, stained by cresyl violet, were
compared to adjacent sections stained with NeuN, the areas where
pyknotic cells were evident in one section corresponded to areas with
weak NeuN-ir in the adjacent section (Figs. 1B and D; 2). When NeuN
labeling was weak, cells were either very lightly labeled by the NeuN
antibody, or there was no detectable immunoreactivity, despite the
fact that in the same sections there was robust NeuN-ir in adjacent
regions (Figs. 1D, 2 and 3C). Therefore, weak NeuN-ir appeared to be a
surrogate marker for pyknotic cells.
NeuN-ir was evaluated using computerized thresholding to
quantify areas of robust vs. weak NeuN-ir (Figs. 3A and B; for
quantification see Fig. 4). Remarkably, there was no evidence of
pyknosis or weak NeuN-ir in the hippocampal pyramidal cell layers or
the cell layers of the dentate gyrus (Figs. 3A and B). Instead, weak
NeuN-ir was found within the adjacent EC, although the location was
not always in the exact same subregion of the EC, and sometimes the
adjacent areas (e.g., perirhinal cortex) were also affected. In addition,
weak NeuN-ir was also present in the FC (FC; see Figs. 3C and D).
Fig. 2. Weak NeuN immunoreactivity as a surrogate marker of pyknotic cells. (A) A horizontal section showing the EC and surrounding areas from a WT mouse, stained with cresyl
violet. MEC=medial EC; A=anterior; M=medial. Inset: Layers II/III of the LEC shown at higher power. Arrows point to cresyl violet-stained neurons in layers II/III with normal
morphology. Calibration for A–F (shown in C)=200 μm; calibration for insets in A–F (shown in inset for F)=20 μm. (B) NeuN-ir in a section adjacent to the one in part A shows
normal NeuN-ir. Inset: Cells in layer II/III of the LEC exhibit robust NeuN-ir (arrows). (C) A section adjacent to the one in part B labeled with NeuN and counterstained with cresyl
violet. Inset: Cells in layer II/III of the LEC are labeled with NeuN and stained with cresyl violet (arrows). Cells that are stained with cresyl violet only and have small irregular profiles
are presumably glia (arrowheads). (D) A cresyl violet-stained section from an ARMS+/−mouse at a similar dorso-ventral level as the WT mouse (A–C). Inset: Arrows point to cells in
layers II/III of the LEC that appear to be pyknotic because their somata are shrunken and have angular edges. (E) A NeuN-labeled section that was 150 μm ventral to the section in D
shows weak NeuN-ir the EC. Inset: Cells in layers II/III in the LEC are shown at higher power. Many cells with weak or no detectable NeuN-ir (arrows) are present, but some cells
exhibit strong NeuN-ir (arrowhead). (F) A section adjacent to the one in D labeled with NeuN and counterstained with cresyl violet. Inset: Arrows point to layer II/III cells of the LEC
that appear pyknotic and lack NeuN-ir. Some cells are labeled by cresyl violet and have robust NeuN-ir (arrowhead).
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Remarkably, the EC and FC were the only two areas that were affected
acrossall animals(Figs.3 and4). EMwasused toconfirm thatthecells
with weak NeuN labeling were in fact present, but abnormal (Figs. 3E
and F).
Quantification of weak NeuN-ir showed that the total volume of
weak NeuN-ir in the EC was greater in 12-month-old female ARMS+/−
mice compared to age-matched female WT mice (WT: 0.31±0.05%,
n=6; ARMS+/−: 0.69±0.12%, n=5; Student's t-test, P=0.0266;
Fig. 4B). However, there were no differences in the extent of weak
NeuN-ir in the EC of 12-month-old male ARMS+/−mice compared to
age-matched male WT mice (WT: 0.49±0.22%, n=4; ARMS+/−:
0.56±0.15%, n=4; Student's t-test, P=0.833), suggesting that the
female EC was affected by ARMS reduction, but not the male EC.
Within the FC, two regions exhibited weak NeuN-ir: 1) an area
close to the medial pial membrane (referred to here as the MFC), and
2)anareanearthelateraledgeoftheFC(forthepurposesofthisstudy,
werefertothisareaastheLFC;Figs.3CandD).Becauseeachpartofthe
MFCandLFCwithweakNeuN-irwasalwaysseparatedby a broadarea
of robust NeuN-ir, the areas we refer to as MFC and LFC were readily
differentiated,andthereforewerecomparedquantitatively.TheLFCof
female 12-month-old ARMS+/−mice exhibited a significantly greater
volume of weak NeuN-ir compared to age-matched female WT mice
(WT: 0.07±0.03%, n=6; ARMS+/−: 0.32±0.11%, n=5; Student's
t-test, P=0.0383; Fig. 4B), but the MFC did not (WT: 0.16±0.07%,
n=10; ARMS+/−: 0.26±0.09%, n=9; Student's t-test, P=0.251;
Fig. 4B). In male mice at the same age, there were no differences in the
volume of weak NeuN-ir, for both the LFC (WT: 0.0.20±0.17%, n=4;
ARMS+/−: 0.51±0.19%, n=4; Student's t-test, P=0.117) and MFC
(WT: 0.03±0.02%, n=4; ARMS+/−: 0.19±0.16%, n=4; Student's
t-test, P=0.193), suggesting that the female FC was vulnerable to
ARMS reduction but not the male FC.
We also evaluated NeuN-ir at a younger age (1 month) in females
(Fig. 4B). ANOVA revealed a significant effect of genotype
(F1,13=8.855, p=0.011) and of age (F1,13=8.644 p=0.012)
Fig. 3. QuantificationofNeuN-irinWTandARMS+/–mice.(AandB)RepresentativeexamplesofWT(A)andARMS+/−(B)miceareshown.Allsectionsfromtheanimalsthatwereusedfor
Figs.1and2weremergedtocreate3-Dimagesoftheentirebrainandareshownwithdifferentorientations(1–3)asindicatedbytheaxestotherightofeachreconstruction.Areasofweak
NeuN-ir,detected bycomputerizedthresholding, are shown incolor (green = LFC; blue= MFC; pink= ECand adjacentareas).Areas of robustNeuN-ir are showningray. A = anterior;
P=posterior,V=ventral,D=dorsal.Calibrationsin1–3=1 mm.(C)ANeuN-labeledhorizontalsectionshowsnormalNeuN-iroftheFCofaWTmouse(left)andweakNeuN-irintheFC
ofanARMS+/−mouse(right).LFC=lateralFC;MFC=medialFC.Inset:Theareathatisoutlinedbyaboxisshownathigherpower.AcellwithstrongNeuN-irismarkedbyanarrow;cells
withweakNeuN-iraremarkedbyarrowheads.CalibrationforC=100 μm;calibrationforinsetsinC=25 μm.(D)AnelectronmicrographofacellwithstrongNeuN-ir(left;NeuN-positive
orNeuN(+))exhibits normalultrastructure,whereasa cellwith weak NeuN-ir(right;NeuN-negative orNeuN (−))exhibitsabnormalcharacteristics. Thenucleus (Nu) isshrunkenand
there is clumping of nuclear chromatin (white arrowhead). Lf = lipofuscin bodies. Calibration=500 nm.
Table 1
Lack of brain and ventricular volume reduction in ARMS+/−mice.
Total volume (mm3)Ventricle volume (mm3)
Male
WT (n=4)
ARMS+/−(n=4)
P
255.78±12.34
225.73±14.18
0.162
5.94±0.35
5.09±0.73
0.351
Female
WT (n=6)
ARMS+/−(n=5)
P
186.02±23.24
225.75±20.21
0.229
6.20±1.40
6.65±0.70
0.780
Pooled
WT (n=10)
ARMS+/−(n=9)
P
213.93±18.17
225.74±12.12
0.601
6.09±0.82
5.96±0.55
0.891
Legend: Total brain volume and ventricular volume of WT and ARMS+/–mice is shown.
Data were generated from 3-D reconstructions of WT and ARMS+/−mice. There were no
statistically-significant differences in total brain or ventricular volumes (Student's t-tests).
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Therefore,weak NeuN-irwas greaterin the ARMS+/−mice than in the
WT mice, and the area of weak NeuN-ir was greatest at 12 months of
age. However, there was no age×genotype interaction (F1,13=2.558
p=0.134) so there is no evidence from these data that the increase in
weak NeuN-ir with age is genotype-dependent.
Ultrastructural analysis reveals abnormalities in mitochondria and
myelination in ARMS+/−mice
Mitochondria
We used EM to investigate the ultrastructural correlates of pyknosis
and weak NeuN-ir. In tissue from the EC and FC of ARMS+/−mice,
mitochondria appeared to be atypical. Typical mitochondria have a
smooth outer membrane enclosing a highly folded inner membrane,
forming cristae, and these were observed in the EC and FC of WT mice
(Fig.5A).IntheARMS+/−mice,manymitochondriaintheECandFChad
disorganized cristae and irregular outer membranes (Figs. 5B and C).
MLBs,consistingofa seriesof concentricallyarrangedmembraneswere
also observed (Fig. 5B), were evident in all subcellular compartments,
and were not observed in WT mice. Notably, abnormal mitochondria
and MLBs have been reported after oxidative stress (Kowaltowski and
Vercesi, 1999; Lin and Beal, 2006), retrograde degeneration of nerve
cells (Bogolepov, 1971), neurotoxic insults (Yazdani et al., 2006), in a
mouse model of amyotrophic lateral sclerosis (Kong and Xu, 1998) and
after impaired clearance of autophagosomes (Boland et al., 2008).
To quantify the ultrastructural changes, areas of weak NeuN-ir and
strong NeuN-ir in the superficial layers of the lateral EC (LEC) were
compared using 12-month-old female WT and ARMS+/−mice (n=5/
group; Figs. 4 and 5). The LEC was used for quantification because the
LECwasthemostcommonsiteof weakNeuN-irwithintheEC. Anarea
of the LEC that was 2380±499.73 μm2was examined per animal.
Quantification of the number of abnormal mitochondria (for criteria,
see Materials and methods) showed that there were greater numbers
in areas of weak NeuN-ir in ARMS+/−mice compared to areas of weak
NeuN-ir in WT mice (WT: 0.304±0.220 per 100 μm2, n=4 mice,
ARMS+/−: 2.468±0.705 per 100 μm2, n=5 mice; Student's t-test,
P=0.0402, Fig. 5D). The difference in the number of abnormal
mitochondria was also significant when areas of weak NeuN-ir
were compared to areas of strong NeuN-ir in ARMS+/−mice (strong
NeuN-ir: 0.1364±0.014 per 100 μm2, n=4 mice, weak NeuN-ir:
2.468±0.705 per 100 μm2, n=5 mice; Student's t-test, P=0.0401,
Fig. 5D), and areas of strong NeuN-ir in WT mice (WT: 0.105±0.075
per 100 μm2, n=3 mice, ARMS+/−: 2.468±0.705 per 100 μm2, n=5
mice; Student's t-test, P=0.0342, Fig. 5D). These results indicate that
in ARMS+/−mice, abnormal mitochondria were more frequently
detected compared to WT mice, and in areas with weak NeuN-ir, they
were more frequent compared to areas with robust NeuN-ir. Thus,
areas with abnormal mitochondria corresponded closely to the areas
of weak NeuN-ir and pyknotic neurons.
Myelin
In addition to abnormalities of mitochondria, myelin appeared
to be defective in ARMS+/−mice (Fig. 6). Specifically, the myelin
sheath appeared to contain breaks and redundant whorls of
myelin (Figs. 6B and C); these characteristics were used to define
abnormal myelin for quantification. Notably, abnormal myelin was
located in many areas, in and outside the area of weak NeuN-ir
(e.g., regions adjacent to the site of weak NeuN-ir in the EC or FC,
including all cortical layers, striatum, hippocampus and the
underlying white matter). This was a contrast to the location of
abnormal mitochondria, which appeared to be specific for areas of
weak NeuN-ir, as explained above. Thus, there were numerous
defective myelin profiles in areas of strong and weak NeuN-ir in
ARMS+/−mice, and they were not significantly different when
quantified per unit area (strong NeuN-ir: 0.807±0.381 per
100 μm2, n=4 mice; weak NeuN-ir: 1.854±0.627 per 100 μm2,
n=5 mice; Student's t-test, P=0.101, Fig. 6D). Taken together,
the EM analyses show that the mitochondrial defects exhibited
specificity for the areas that had weak NeuN-ir, but abnormal
myelin appeared to lack this specificity.
Notably, there were some defects in myelin that were found in 12-
month-old WT mice. These occurred in areas with weak or robust
NeuN-ir, but were substantially less frequent than in ARMS+/–mice.
Thus, the number of these abnormal myelin profiles was less than the
number in ARMS+/−mice when areas with weak NeuN-ir were
compared (WT: 0.163±0.087 per 100 μm2, n=3 mice, ARMS+/−:
1.854±0.627 per 100 μm2, n=5 mice; Student's t-test, P=0.0271,
Fig. 6D), or when areas with robust NeuN-ir was compared (WT:
0.137±0.068 per 100 μm2, n=3 mice, ARMS+/−: 1.854±0.627 per
100 μm2, n=5 mice; Student's t-test, P=0.0260, Fig. 6D).
Female ARMS+/−mice exhibit deficits in spatial memory
To determine if the anatomical abnormalities in ARMS+/−mice
were associated with functional defects, behavioral tests were
performed. Female ARMS+/−mice (n=19) were significantly slower
to learn the location of the hidden platform during training, compared
to female WT mice (n=20). RMANOVA revealed a significant effect of
genotype (F1,5=2.835, P=0.015) and Bonferroni/Dunn post hoc
Fig. 4. Quantification of weak NeuN-ir in ARMS+/−mice compared to WT mice. (A) The
total volumes of weak NeuN-ir in the EC and FC are shown for 12-month-old ARMS+/−
mice (black) and WT controls (white). Single asterisk indicates Pb0.05; double asterisk
indicates Pb0.01(for statistics, see text). (B) Comparisons are shown for the EC, LFC and
MFC of female ARMS+/−mice and female WT controls. There is a greater volume of
weak NeuN-ir in the EC and LFC of ARMS+/−mice compared to WT. Two-way ANOVA
showed an increase in the volume of weak NeuN-ir in both 1-month and 12-month-old
ARMS+/−mice (Pb0.05) and the 12-month-old mice had a larger extent of weak
NeuN-ir (Pb0.05).
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analysis revealed differences in the 2nd–5th sessions (Pb0.05;
Fig. 7B). However, there was no difference in time to find the hidden
platform between 12-month-old male ARMS+/−mice (n=14) and
male WT mice (n=14; Fig. 7A).
We also assessed spatial memory with the probe trial (Schenk and
Morris, 1985). Female ARMS+/−mice were impaired compared to
female WT mice (ANOVA F1,144=5.496, P=0.0207; Fig. 7D). How-
ever, there was no difference in the amount of time spent in the target
quadrant between male ARMS+/−mice and WT mice (Fig. 7C). The
visible platform trial, performed after the probe trial, did not reveal
any differences in time to reach the platform (males, F1,3=1.143,
P=0.333; females, F1,3=2.099, P=0.101) or swimming velocity
(males, F1,3=1.242, P=0.295; females, F1,3=0.637, P=0.593).
Discussion
Summary
The results demonstrated that ARMS is important to the EC and
FC in several ways. First, neurons in the EC and FC were
compromised by a decrease in ARMS levels. Second, animals
with a reduction in ARMS exhibited deficits in spatial learning and
memory, which depend on the EC and FC. Third, the results
indicate that females are affected but not males. Taken together,
these observations provide support for the hypothesis that
neurotrophin signaling is important to the maintenance of normal
cortical function, and supports the hypothesis that disruption of
neurotrophin signaling contributes to impairments in disorders
where areas like the EC and FC are implicated, and women often
exhibit a greater incidence or more affective symptoms compared
to men (Wizemann and Pardue, 2001).
NeuN-ir is not always a marker of neurons
The results support previous observations that the absence of
NeuN-ir does not always indicate neuronal loss. In some circum-
stances, loss of NeuN immunoreactivity occurs when NeuN is
phosphorylated (Igarashi et al., 2001; Davoli et al., 2002; Lind et al.,
2005; Portiansky et al., 2006; Buckingham et al., 2008; Matsuda et al.,
2009; Won et al., 2009; Wu et al., 2010). The results also show that
weak NeuN-ir appears to be a surrogate marker for pyknosis.
Cellular and subcellular abnormalities in the EC and FC of ARMS+/−mice
The cause of pyknosis and weak NeuN-ir in ARMS+/−mice could
be related to a type of oxidative stress, given what is known about
pyknosis, NeuN-ir, and ARMS. One reason to suggest this is that
pyknosis and weak NeuN-ir are often found when there is oxidative
stress (Lemaire et al., 2000; Ünal-Çevik et al., 2004; Portiansky et
al., 2006; Buckingham et al., 2008). In addition, our evaluation of
ARMS+/−mice showed that cells with weak NeuN-ir had irregular
nuclear morphology and mitochondria with abnormal internal
membranes. Similar observations have been reported in other
studies where oxidative stress occurs (Yakes and Van Houten,
1997; Kong and Xu, 1998; Terni et al., 2010), such as traumatic
brain injury (Singh et al., 2006) and neurotoxic insults (Kowal-
towski and Vercesi, 1999; Zhu et al., 2007; Rajeswari and Sabesan,
2008). It is interesting that the signs of oxidative stress in ARMS+/−
Fig. 5. Ultrastructural evidence for mitochondrial abnormalities in ARMS+/−mice with weak NeuN-ir. (A) An area of the EC of a WT mouse with normal NeuN-ir (NeuN (+)) shows
normal mitochondria (m; arrows). Synapses that appear normal are marked by curved arrows. (B and C) The nuclear cytoplasm from a cell in the EC of an ARMS+/−mouse with
weak NeuN-ir (NeuN (−)), illustrates abnormal mitochondria (arrowheads) and MLBs (arrow). ER = endoplasmic reticulum. (D) There were more abnormal mitochondria in NeuN
(−) areas of ARMS+/−mice compared to WT mice. Asterisks indicate statistical significance (Pb0.05; for statistics see text). Calibration=500 nm.
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mice were found primarily in the EC and FC, because oxidative
damage appears to occur in these areas in neurodegenerative
diseases and psychiatric illness. For example, proteins such as
mitochondrial adenosine triphosphate-synthase are lipoxidized in
the EC during the first stages of AD pathology (Terni et al., 2010). In
addition, signs of mitochondrial dysfunction and oxidative stress
are also observed in the FC of patients with psychiatric disorders
(Michel et al., 2007; Wang et al., 2009; Andreazza et al., 2010;
Gawryluk et al., 2010). Therefore, our findings are consistent with a
vulnerability of the EC and FC, and the results of this study suggest
that a reduction in ARMS plays a role.
The presence of abnormal mitochondria and MLBs is also
reminiscent of impaired clearance of damaged organelles by autop-
hagy (Mizushima et al., 2002; Rideout et al., 2004). Autophagy could
be impaired in the EC in ARMS+/−mice preferentially because the EC
is a metabolically active area, based on studies of cytochrome oxidase
(Hevner and Wong-Riley, 1992; Mutisya et al., 1994). A high
metabolic rate may lead to a high basal requirement for autophagy,
which could place the EC at risk if other insults or injury occur, such as
reduced ARMS levels. The importance of autophagy for the mainte-
nance of neuronal integrity has been suggested in the context of other
types of insults and injury such as aging and AD (Nixon et al., 2005;
Boland and Nixon, 2006; Boland et al., 2008; Finn and Dice, 2008).
Another potential reason for the defects in ARMS+/−mice is
related to the myelination defects, because oligodendrocytes are
normally dependent on BDNF and TrkB [in spinal cord, (McTigue et
al., 1998; Dougherty et al., 2000); in basal forebrain, (Van't Veer et
al., 2009) and in retinal ganglion cells, (Cellerino et al., 1997)].
Therefore, the abnormal myelination in ARMS+/−mice may be
due to the disruption of BDNF and TrkB signaling. It is possible that
myelination is also vulnerable because of the high metabolic
activity of oligodendrocytes (McTigue and Tripathi, 2008).
Behavioral impairments in ARMS+/−mice
It was not surprising that there was a defect in the Morris water
maze task in female mice, given that this task involves both the EC
(Hebert and Dash, 2002; Spowart-Manning and van der Staay, 2005;
Nakazawa, 2006) and the FC (Kolb et al., 1983; Jo et al., 2007; Silachev
etal.,2009;Leonet al.,2010),andthestructuraldefectswerein theEC
and FC of female mice. Based on the demonstration that extracellular
signal regulated kinase (ERK) in the EC and FC is critical to
performance in the Morris water maze (Hebert and Dash, 2002;
Leon et al., 2010), and the evidence that ERK is activated by ARMS
(Arévalo et al., 2006), reduced ERK activation may also play a role.
However, we cannot exclude that other mechanisms that are not ERK-
Fig. 6. Ultrastructural evidence for abnormalities of myelination and synaptic densities in ARMS+/−mice. (A) In the EC of a WT mouse with normal NeuN-ir, a normal myelinated
axon (Ax) and mitochondria (m) are marked by arrows. In addition, there is a synapse (curved arrow) that appears to be a normal asymmetric synapse, because there is a thickened
postsynaptic density and the axon terminal contains spherical vesicles (Peters et al., 1991). (B) A longitudinal section of a myelinated axon (Ax; arrow) from a NeuN (+) area of an
ARMS+/−mouse. An arrowhead marks a myelin whorl. (C) In an area of weak NeuN-ir (NeuN (−)) from an ARMS+/−mouse, a myelinated axon appears to have breaks in myelin
and a myelin whorl (arrowheads). In the same area, asymmetric postsynaptic densities (curved arrows) appear to be abnormal because they are disconnected from a plasma
membrane. In contrast, asymmetric postsynaptic densities in WT mice were connected to plasma membranes (see curved arrow in part A). (D) There were more abnormal myelin
profiles in ARMS+/−mice compared to WT mice, whether NeuN (−) areas or NeuN (+) areas were examined. Asterisks indicate statistical significance (Pb0.05; for statistics, see
text). Calibration=500 nm.
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dependent could have contributed, and more experiments will be
required to prove that the defects in the EC and FC caused impaired
performance. For example, the myelin defects, which were not
specific to the EC and FC, but would be likely to affect their functional
integration with other brain areas, could have played a role.
Vulnerability of the EC in ARMS+/−mice
The results of this study support the idea that the EC plays a central
role in spatial memory normally (Steffenach et al., 2005), that EC
pathology develops with aging (Braak and Braak, 1990; Barnes et al.,
2000), and that the EC is one of the earliest areas to be affected in AD
(de Leon et al., 2007; de Toledo-Morrell et al., 2007). Interestingly,
when the EC was compromised in ARMS+/−mice, the location was
primarily the superficial layers. The superficial layers are important
because they contain the cells of origin of the EC projection to the
hippocampus, the perforant path (Steward and Scoville, 1976; Witter
et al., 1989; Dolorfo and Amaral, 1998). In addition, there were myelin
defects in ARMS+/−mice which suggests that perforant path axons
would be impaired. Thisisinterestingbecausethedefectsrelatedtothe
EC that were observed in clinical studies of patients with AD were
related to a deficit in the perforant path axons (de Leon et al., 2007;
de Toledo-Morrell et al., 2007), and it was suggested that impairments
in the perforant path lead to a “disconnection” of the EC from
hippocampus that underlies early cognitive impairment in AD (de
Toledo-Morrell et al., 2007).
Sexual dimorphism in ARMS+/−mice
The fact that female mice were affected rather than male mice is
interesting because there is evidence for sex differences in spatial
memory. Thus, previous studies have documented worse perfor-
mance by females on spatial memory tasks, not only in rodents (Bucci
et al., 1995; La Buda et al., 2002; Gresack and Frick, 2003; Sutcliffe et
al., 2007), but also in humans (Perrot-Sinal et al., 1996; Astur et al.,
1998, 2004; Postma et al., 1999, 2004), although not all results agree
(Bucci et al., 1995). There also are sex differences that have been
identified in the perforant path projection, where females appear to
exhibit less synaptic plasticity than males (LTP, Maren et al., 1994).
Therefore, sex differences that we identified may be due to a specific
sexual dimorphism in the normal EC and FC circuits which is revealed
in theARMS+/−mouse.Indeed,theCoolidge effect,whererecognition
of females by males is critical, is blocked by lesions to the perirhinal
cortex/EC, but not lesions to hippocampus (Petrulis and Eichenbaum,
2003). Social odor discrimination in rats appears to depend on EC also
(Mayeaux and Johnston, 2004).
Why a reduction in ARMS would increase the vulnerability of EC
and FC neurons in females may be related to the normal effects of
estrogen in the brain, which increase neurotrophin synthesis (for
reviews, see Scharfman and MacLusky, 2005, 2006a). In the EC and FC,
estrogen treatment specifically increases neurotrophin levels in
females (Bimonte-Nelson et al., 2004). As a result, females may be
more dependent on neurotrophins than males, making them more
vulnerable to a reduction in ARMS.
Another explanation for the sex difference in ARMS+/−mice is based
on the idea that estrogen is not always protective. In transgenic mice that
simulate AD pathology, for example, females are more severely affected
than males of the same ages (Callahan et al., 2001; Wang et al., 2003;
Schuessel et al., 2005; Schafer et al., 2007; Hirata-Fukae et al., 2008). One
explanation is based on the idea that estrogen is not protective—and may
actually exacerbate damage—when oxidative stress develops (Brinton,
2008; Irwin et al., 2008; Henderson and Brinton, 2010). This is consistent
withthefindingsinthefemaleARMS+/−mice,wheretheeffectinfemales
was modest before puberty (at 1 month of age) but robust after puberty
(at 12 months of age). The normal protective effects of estrogen are also
disruptedduringaging(SohrabjiandBake,2006;SelvamaniandSohrabji,
2008, 2010). An additional contributing factor could be that estrogen
increases neuronal activity in the EC (Scharfman and MacLusky, 2005,
2006b; Skucas et al., 2009), which could lead to a greater metabolic
demand in the female EC, increasing vulnerability.
Significance
There are many types of neurological and psychiatric disorders
that have been related to a deficit in neurotrophins or their receptors
(Chaoet al., 2006). As a result, it has been suggested that neurotrophic
support is necessary to maintainnormal CNSfunction. However,there
are few successful treatment strategies based on neurotrophin
signaling at the present time. One reason for the lack of progress in
neurotrophin-based therapeutics is that few molecules have been
identified that would be specific enough and also have no side effects.
For example, BDNF itself can be protective, but has also been
associated with seizures (Binder, 2004; Koyama and Ikegaya, 2005;
Scharfman, 2005; Tongiorgi et al., 2006; McNamara and Scharfman,
2011). Another reason that therapeutics based on neurotrophins like
BDNF have not been implemented is that neurotrophins are large
molecules that do not diffuse readily (Thoenen and Sendtner, 2002).
Although direct infusion of cells that produce BDNF into areas like the
EC has shown great promise (Tuszynski et al., 2005; Nagahara et al.,
2009), BDNF itself may not be the best therapeutic molecule. Other
neurotrophin based strategies need to be developed. The regulation of
ARMS could be one approach to specifically target neurotrophin
signaling pathways in areas of the brain that are vulnerable to disease.
Acknowledgments
This study was supported by NIH NS-37562 and MH-084215 to
H.E.S., NIH NS-21072 and HD-23315 to M.V.C., NIH AG-034248 to O.A.,
the New York University Langone Medical Center CoE Seed Grant
Program, and the New York State Office of Mental Health. We thank
Fig. 7. Impaired Morris water maze performance in female ARMS+/−mice. (A) The mean
latencytofindthehiddenplatformisshownforalldaysoftraining.FemaleARMS+/−mice
showed impairments during training compared to female WT mice. Male ARMS+/−mice
(black) showed no significant impairments compared to male WT mice (white). Single
asterisks reflect Pb0.05(for statistics, see text). (B) After training,animals were tested for
memory of the location of the hidden platform (quadrant 4). Female ARMS+/−mice
showed impaired performance compared to WT mice (i.e. female ARMS+/−mice spent
less time inquadrant 4; asterisk reflects Pb0.05). There was no impairment inmemory in
male mice (i.e., all animals spent more time in quadrant 4).
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Dr. Ralph Nixon, Dr. Neil Maclusky, Dr. Daniel Wesson, and Patrice
Pearce for their suggestions.
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