1. health and fitness
2. disease

doi:10.1093/brain/aws195
Brain 2012: 135; 2826–2837
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BRAIN
A JOURNAL OF NEUROLOGY
Immunoglobulin G Fc receptor deficiency prevents
Alzheimer-like pathology and cognitive
impairment in mice
Paula Fernandez-Vizarra,1,* Oscar Lopez-Franco,1 Ben˜at Mallavia,1 Alejandro Higuera-Matas,3
Virginia Lopez-Parra,1 Guadalupe Ortiz-Mun˜oz,1 Emilio Ambrosio,3 Jesus Egido,1
Osborne F. X. Almeida2 and Carmen Gomez-Guerrero1
1 Renal and Vascular Inflammation, Nephrology Department, IIS-Fundacion Jimenez Diaz, Autonoma University, Avda. Reyes Catolicos 2,
28040 Madrid, Spain
2 NeuroAdaptations Group, Max Planck Institute for Psychiatry, Kraepelinstr. 2-10, 80804 Munich, Germany
3 Department of Psychobiology, School of Psychology, UNED, C/Juan del Rosal 10, 28040 Madrid, Spain
*Present address: NeuroAdaptations Group, Max Planck Institute of Psychiatry, Kraepelinstr. 2-10, 80804 Munich, Germany
Correspondence may also be addressed to: Paula Fernandez-Vizarra, PhD, NeuroAdaptations Group, Max Planck Institute of Psychiatry, Kraepelinstr.
2-10, 80804 Munich, Germany. E-mail: paula_fernandez@mpipsykl.mpg.de
Alzheimer’s disease is a severely debilitating disease of high and growing proportions. Hypercholesterolaemia is a key risk factor
in sporadic Alzheimer’s disease that links metabolic disorders (diabetes, obesity and atherosclerosis) with this pathology.
Hypercholesterolaemia is associated with increased levels of immunoglobulin G against oxidized lipoproteins. Patients with
Alzheimer’s disease produce autoantibodies against non-brain antigens and specific receptors for the constant Fc region of
immunoglobulin G have been found in vulnerable neuronal subpopulations. Here, we focused on the potential role of Fc
receptors as pathological players driving hypercholesterolaemia to Alzheimer’s disease. In a well-established model of hypercholesterolaemia, the apolipoprotein E knockout mouse, we report increased brain levels of immunoglobulin G and upregulation of
activating Fc receptors, predominantly of type IV, in neurons susceptible to amyloid b accumulation. In these mice, gene deletion
of -chain, the common subunit of activating Fc receptors, prevents learning and memory impairments without influencing
cholesterolaemia and brain and serum immunoglobulin G levels. These cognition-protective effects were associated with a
reduction in synapse loss, tau hyperphosphorylation and intracellular amyloid b accumulation both in cortical and hippocampal
pyramidal neurons. In vitro, activating Fc receptor engagement caused synapse loss, tau hyperphosphorylation and amyloid b
deposition in primary neurons by a mechanism involving mitogen-activated protein kinases and b-site amyloid precursor protein
cleaving enzyme 1. Our results represent the first demonstration that immunoglobulin G Fc receptors contribute to the development of hypercholesterolaemia-associated features of Alzheimer’s disease and suggest a new potential target for slowing or
preventing Alzheimer’s disease in hypercholesterolaemic patients.
Keywords: Alzheimer’s disease; hypercholesterolaemia; apolipoprotein E; immunoglobulin receptors; cognitive dysfunction
Received June 8, 2011. Revised June 8, 2012. Accepted June 10, 2012
ß The Author (2012). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: journals.permissions@oup.com
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Correspondence to: Carmen Gomez-Guerrero, PhD,
Renal and Vascular Inflammation
IIS-Fundacion Jimenez Diaz
Avda. Reyes Catolicos 2,
28040 Madrid, Spain
E-mail: cgomez@fjd.es or c.gomez@uam.es
FcR in sporadic Alzheimer-like disease
Brain 2012: 135; 2826–2837
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Abbreviations: apoE = apolipoprotein E; BACE1 = b-site amyloid precursor protein cleaving enzyme 1; JNK = c-Jun N-terminal
kinase; DKO = double knockout; ERK = extracellular regulated mitogen-activated protein kinase; GFAP = glial fibrillary acidic protein;
IgG = immunoglobulin G; MCP1 = monocyte chemoattractant protein 1; FcR = receptor for the constant Fc region of immunoglobulin G; TNF = tumour necrosis factor Introduction
Materials and methods
Reagents
Soluble IgG immune complexes (Oreskes and Mandel, 1983) were obtained by heat aggregation (30 min at 63 C) of monomeric mouse IgG
(Cappel, MP Biomedicals) and subsequent centrifugation to eliminate
insoluble immune complexes as described (Hernandez-Vargas et al.,
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The pre-dementia phase of Alzheimer’s disease can be modelled in
transgenic mice carrying human mutations (Ashe and Zahs, 2010).
Such models are considered useful in the development of therapeutics aimed at slowing the transition from asymptomatic
Alzheimer’s disease—in which the neuropathology is already
initiated—to full-blown Alzheimer’s disease. On the other hand,
the amplifying effects of secondary changes are now thought to
confound therapeutic interventions and given that Alzheimer’s disease develops over decades, it is clearly important to improve
understanding of the mechanisms that initiate this pathology.
Animal models burdened with risks associated with the development of Alzheimer’s disease serve this goal better than those
involving overexpression of molecules that are causally associated
with Alzheimer’s disease.
The neuropathological characteristics of Alzheimer’s disease
include amyloid b depositions and accumulations of abnormally
hyperphosphorylated tau protein in selected brain regions; in addition, Alzheimer’s disease brains show abundant signs of microvascular damage and pronounced inflammation (Bertram et al., 2010).
Current evidence suggests that amyloid b initiates a disease process
that progresses to cognitive impairment and that tau mediates cognitive dysfunction (Ashe and Zahs, 2010). Although the pathways
contributing to increased amyloid b levels appear to differ according
to predisposing risk factors, it is reasonable to assume that these
pathways converge at some point before triggering unbalanced
amyloid b metabolism; identification of such an upstream hub
would facilitate research and therapeutic developments.
Patients suffering from sporadic Alzheimer’s disease show elevated levels of immunoglobulin G (IgG) autoantibodies against
non-brain antigens (Ounanian et al., 1990), and express receptors
for the constant Fc region of IgG (FcR) (Bouras et al., 2005) in
neuronal populations that are vulnerable to the disease (Morrison
and Hof, 2002). Importantly, increased circulating and brain IgG
levels are found in individuals at risk of developing Alzheimer’s disease (Ballard et al., 2011), including advanced age (Listı` et al., 2006),
stress (Nakata et al., 2000) and metabolic disorders such as diabetes,
obesity and atherosclerosis (Lu et al., 2001; Turk et al., 2001; Haroun
and El-Sayed, 2007; Okamatsu et al., 2009; Winer et al., 2011).
Abnormal modifications of proteins, such as oxidation and
non-enzymatic glycosylation, are also observed during early stages
of metabolic disorders (Haroun and El-Sayed, 2007; Winer et al.,
2011); these result in an adaptive immune response with subsequently sustained high levels of circulating autoantibodies (mainly
IgG isotype) (Doyle and Mamula, 2001) and the corresponding
immune complexes formed by antibody–antigen interaction.
Antibody penetrance into the brain is severely limited in physiological conditions; however, weakening of the blood–brain barrier
allows entry of immunoglobulins and immune complexes, as has
been observed in individuals with metabolic syndrome, and risk for
Alzheimer’s disease, and also in experimental models of Alzheimer’s
disease-related pathologies (Methia et al., 2001; Kuang et al., 2004;
Hafezi-Moghadam et al., 2007; Bake et al., 2009; Diamond et al.,
2009). Thus, we hypothesize that brain FcR activation may be a
major convergence point for sporadic Alzheimer’s disease triggered
by the occurrence of two pathological phenomena: IgG overproduction and blood–brain barrier leakage.
In the mouse, four FcR classes that differ in affinity, specificity
and function have been described. Activating FcRs (I, III and IV)
associate with the common -chain which harbours the
immunoreceptor tyrosine-based activation motif and elicit an
immune response upon ligand (IgG or immune complexes) binding; in contrast, the inhibitory FcRIIb contains the immunoreceptor tyrosine-based inhibition motif and nullifies cell activation
(Nimmerjahn et al., 2005; Nimmerjahn and Ravetch, 2008).
FcRs have been localized in neurons, microglia, astroglia and
oligodendrocytes (Bouras et al., 2005) and have been shown to
play an important role in myelination (Nakahara et al., 2003).
Hypercholesterolaemia, a common feature in metabolic disorders,
is characterized by autoimmune responses triggered by most forms
of modified low density lipoproteins (Burut et al., 2010).
Interestingly, hypercholesterolaemic mice display several neuropathological hallmarks of Alzheimer’s disease, including increased
levels of amyloid precursor protein and amyloid b, abnormally hyperphosphorylated tau and inflammation, together with cognitive impairments (Oitzl et al., 1997; Veinbergs et al., 1999; Crisby et al.,
2004; Rahman et al., 2005; Bjelik et al., 2006).
In this study, we investigated the potential role of FcR in the
aetiopathogenesis of Alzheimer’s disease. Given that a multiplicity
of risk factors may be represented in hypercholesterolaemia and
the fact that Alzheimer’s disease is associated with IgG overproduction and blood–brain barrier leakage, studies were performed
in apolipoprotein E (apoE) knockout mice, which display overt
hypercholesterolaemia (Zhang et al., 1992). Therefore, the features characteristic of Alzheimer’s disease were comparatively studied between single apoE knockout (apoE / ) mice and double
knockout (DKO) mice (Hernandez-Vargas et al., 2006), which are
gene deficient in both apoE and -chain, the common subunit
necessary for assembly, cell-surface localization and functionality
of activating FcRs (Nimmerjahn and Ravetch, 2008).
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2006). Culture media, supplements and transfection reagent were
purchased from Lonza and Life Technologies; SP600125 and U0126
from Stressgen Bioreagents Corp.; small interfering RNA from Santa
Cruz Biotechnologies. The 9E9 FcR blocking antibody was generously
provided by Dr J.V. Ravetch (The Rockefeller University, NY)
(Nimmerjahn et al., 2005). Primary antibodies used were: synaptophysin,
microtubule associated protein 2, and tau phosphorylated at Ser199/202
(Millipore); tau doubly phosphorylated at Ser202/Thr205, Ser199/202
and Ser205/208 (AT8 antibody) (Pierce Biotechnology Inc.); glial
fibrillary acidic protein (GFAP; Sigma-Aldrich); a neoepitope in amyloid
b, to exclude cross-reactions with its precursor amyloid precursor protein
(IBL); FcRIV (Santa Cruz Biotechnologies); mouse IgG (Amersham);
b-site amyloid precursor protein cleaving enzyme 1 (BACE1; ProSci
Inc.); insulin degrading enzyme (Abcam); CD11b (Abcam); -tubulin
(Sigma-Aldrich); and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH, Millipore). Assays for real-time PCR were from Life
Technologies.
Mice
Behavioural tests
Mice were housed three to five per cage and allowed to habituate to
the cage environment for 2 weeks before behavioural testing. Housing
conditions were kept constant until the end of behavioural testing. All
behavioural tests were carried out under dim light in a sound-proof
room by the same researcher and were conducted in the following
order: elevated plus maze, object location test.
Elevated plus maze
Mice were allowed to freely explore the maze for 8 min. Anxiety index
was calculated as the time spent in open arms relative to the total time
spent in closed and open arms and in the centre. Locomotor activity
index was calculated as frequency of entry into closed arms.
Object location test
One day after the elevated plus maze test, mice were habituated to
the empty arena placed in a room with extra-maze cues for 15 min
during 5 days before training. In the training phase, mice were
exposed to two identical objects for 5 min. After a delay of 14 min,
the time spent exploring the objects in a new (novel) and in the old
(familiar) locations was recorded during 3 min (test phase). To analyse
cognitive performance, a location index (or relative exploration time)
was calculated as previously described (Murai et al., 2007) with a
slight modification: to/Te, where to = time spent exploring either the
displaced or the non-displaced object and Te = total time spent exploring both objects.
Biochemistry
Total serum cholesterol concentrations were measured using standard
enzymatic methods (Invitrogen). Total serum immunoglobulins were
measured by sandwich ELISA using specific antibodies recognizing
mouse IgG1, IgG2a/c and IgG3 (BD Biosciences).
Primary neuronal cultures
Mouse cortical neurons from prenatal embryonic Day 17 were mechanically dissociated and plated on poly-L-lysine-coated coverslips or
plates. Cell suspension (106 cells/ml) was seeded in Dulbecco’s modified Eagle medium containing 5% foetal bovine serum, 0.05%
GlutaMAXTM and 1% penicillin/streptomycin, and then cultured in
NeurobasalÕ medium supplemented with 2% B27, 1% GlutaMAXTM
and 0.1% penicillin/streptomycin. On Day 5 in vitro, 10 mM cytosine
arabinoside was added. Contaminating glial cells in neuronal cultures
accounted for 52% (astroglia and microglia combined) on the day of
use. Cultures at Day 12 were stimulated with IgG immune complexes
(150 mg/ml). In some experiments, cells were pretreated for 1 h with
mitogen-activated protein kinases inhibitors [c-Jun N-terminal kinase
(JNK) inhibitor, SP600125, 5 10 5 M; extracellular regulated
mitogen-activated protein kinase (ERK) inhibitor, U0126, 10 5 M] or
FcRIV blocking antibody (9E9 or irrelevant hamster IgG, 1 mg/106
cells; Nimmerjahn et al., 2005) before stimulation. For knock-down
experiments, cells were transfected with 60 pM of small interfering
RNA (FcRIV or irrelevant) and 8 ml of LipofectamineTM 2000 in culture medium. Small interfering RNA knocking-down efficiency checked
by real-time PCR was 72.1 0.3% at 24 h after transfection (n = 3).
Immunocytochemistry
Brains were fixed in 4% paraformaldehyde for 4 h. Cryostat sections
(10 mm) on slice were used for immunohistochemistry. For antigenretrieval in amyloid b immunostaining, sections were pretreated with
88% formic acid for 5 min. Non-specific binding sites were blocked by
incubating sections in 1% bovine serum albumin and 5% pre-immune
serum diluted in 0.5% Tween-20 in PBS for 1 h at room temperature in
slight orbital agitation. Primary antibody was incubated in 0.5%
Tween-20 (FcRIV 1:50, amyloid b 1:100, GFAP 1:2000; CD11b
1:20) for three overnights at 4 C in slight orbital agitation.
For immunocytochemistry, fixation in 2% paraformaldehyde
(10 min, room temperature) was performed before incubation of primary antibodies in PBS overnight at 4 C in slight agitation (FcRIV
1:50, microtubule associated protein 2 1:100, amyloid b 1:100, GFAP
1:2000, CD11b 1:20). For both in vivo and in vitro studies, fluorescent secondary antibodies (1:100 in PBS) were incubated at room
temperature for 1 h with orbital agitation. Incubation with 40 6-diamidino-2-phenylindole (DAPI; 1:10 000 in PBS) was then performed (10 min, room temperature). Confocal images were obtained
by sequential scan.
Western blot
Cytosolic proteins were extracted from cells (Ortiz-Munoz et al., 2010)
and the cerebral cortex from mice (Rodrigo et al., 2004) and resolved
on SDS–PAGE, transferred onto polyvinylidene fluoride membranes
and immunoblotted with specific antibodies (Ortiz-Munoz et al.,
2010). Optical densities of individual bands were normalized to loading controls (GAPDH or -tubulin) and n-fold changes were obtained
by normalization against results obtained under basal conditions (untreated cells) or control (wild-type) mice, as appropriate.
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All animal studies conformed to Directive 2010/63/EU of the European
Parliament and were approved by the Institutional Animal Care and
Use Committee. Wild-type and apoE knockout mice were purchased
from Jackson Laboratory; the DKO mice (gene deficient in both apoE
and -chain) were generated in our laboratory as previously described
(Hernandez-Vargas et al., 2006). Wild-type, apoE / and DKO mice
were fed a standard mouse lab chow diet (Panlab) and allowed to age.
Mice from each group were sacrificed at 12 months (wild-type, n = 5;
apoE / , n = 8; DKO, n = 7) and 21 months (wild-type, n = 5;
apoE / , n = 12; DKO, n = 11). Mice brains were obtained after
deep anaesthesia with a mixture (0.01 ml/g body weight, intraperitoneally) of ketamine (10 mg/ml) and xylazine (1 mg/ml) and perfusion
with saline.
P. Fernandez-Vizarra et al.
FcR in sporadic Alzheimer-like disease
Brain 2012: 135; 2826–2837
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Results
FcR deficiency restores cognitive and
synaptic status and attenuates
Alzheimer-like pathology in
hypercholesterolaemic mice
hypercholesterolaemic and control mice at middle-age
(12-month-old). (A) Western blot of IgG in the cerebral cortex
of wild-type (WT), apoE / and DKO mice. Representative
blots from each group (wild-type, n = 5; apoE / , n = 8;
DKO, n = 7) are shown. Summary of densitometric analysis is
expressed in arbitrary units (a.u.). (B) Immunoglobulin isotype
distribution in mouse sera measured by ELISA. Values are expressed as absorbance units at = 450 nm. Values are
mean SEM of five animals per group. (C) Real-time PCR
analysis of activating and inhibitory FcRs in the hippocampus
and cerebral cortex of mice. Values are mean SEM of studied
animals per group (wild-type, n = 5; apoE / , n = 8; DKO,
n = 7). *P 5 0.05 and **P 5 0.01 versus wild-type; #P 5 0.05
versus apoE / .
Messenger RNA expression
Total RNA from cells and tissues was extracted and retro-transcribed
as previously described (Ortiz-Munoz et al., 2010). Gene expression
was analysed in duplicate by real-time PCR on a TaqManÕ ABI 7500
sequence detection system (Applied Biosystems) and normalized to
housekeeping 18S transcripts. Results are given as n-fold changes,
relative to control groups. The relative expression levels of activating
and inhibitory FcR were calculated according to the formula
(FcRI + FcRIII + FcRIV)/FcRIIb and expressed as a percentage of
control values.
Statistics
Statistical significance was tested by unpaired Student’s t-test and
one-way ANOVA followed by an appropriate post hoc least significant
difference comparison test (GraphPad Prism software). A value of
P 5 0.05 was considered to be statistically significant.
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Figure 1 Levels of IgG and expression of FcR isoforms in
Western blot studies in brain lysates demonstrated very low levels
of IgG in middle-aged (12-month-old) wild-type mice and high
levels in apoE / and DKO (deficient in apoE and -chain
genes) mice, with both knockout strains showing a similar content
(Fig. 1A). ApoE / and DKO groups showed comparable serum
cholesterol levels (460 27 mg/dl versus 475 20 mg/dl,
P 4 0.05). Furthermore, serum antibody levels confirmed
increased brain IgG levels in apoE / and DKO mice, as compared to wild-type mice. The two strains of knockout mice
(apoE / and DKO) did not differ in their IgG subclass (IgG1,
IgG2 and IgG3) expression profiles (Fig. 1B), i.e. they shared a
similar immune imprint.
Given that high levels of IgG reportedly upregulate FcR
expression in different inflammatory disease models (Nimmerjahn
and Ravetch, 2008), we next examined whether experimental
hypercholesterolaemia altered FcR gene expression in the brain.
Consistent with the increase in IgG levels, we found an increased
expression of activating and inhibitory FcRs, mainly of the
FcRIV isoform, in brains from apoE / animals, when compared
with wild-type mice (Fig. 1C); this resulted in a net activating
profile (activating:inhibitory ratio, percentage versus wild-type
mice: cortex, 6.06; hippocampus, 13.99). In contrast, apart from
changes in the expression of FcRIII, hypercholesterolaemic
DKO mice did not show any difference in FcR expression in
the cortex and hippocampus, compared to wild-type animals
(Fig. 1C).
Middle-aged hypercholesterolaemic apoE / mice showed intracellular amyloid b immunostaining in pyramidal neurons of the
hippocampus and temporal cortex (Fig. 2A and C), consistent with
the early neuroanatomical and cellular distribution of intracellular
amyloid b deposition found in patients with Alzheimer’s disease
(Gouras et al., 2000; Gyure et al., 2001; Fernandez-Vizarra et al.,
2004). Immunohistochemical localization of amyloid b was similar to
that of FcRIV (Fig. 2D and E), the major receptor expressed in
hypercholesterolaemic mice brains; FcRIV staining was observed
in pyramidal neurons of the hippocampus, temporal cortex, but
also of the cingulate cortex (data not shown) of apoE / mice,
but not in other types of neuron or glial cells. ApoE / mice also
showed a significant increase in tau hyperphosphorylation in the
cerebral cortex, as compared to age-matched wild-type controls
(Fig. 2H). Importantly, deletion of activating FcR in DKO mice attenuated amyloid b immunostaining (Fig. 2A and C) and tau hyperphosphorylation (Fig. 2H) in all neuroanatomical areas studied.
Interestingly, FcR deficiency was also protective against amyloid b
deposition in aged (21-month-old) mice (Fig. 2B). Furthermore, lack
of functional FcRs significantly reduced the gene expression of the
inflammatory cytokine tumour necrosis factor (TNF) and the
monocyte chemoattractant protein 1 (MCP1, also known as Ccl2;
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P. Fernandez-Vizarra et al.
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Figure 2 Functional deficiency in activating FcRs protects against amyloid b deposition, tau phosphorylation, astrogliosis and cytokine
expression in hypercholesterolaemic mice. Role of FcRIV. (A and B) Representative confocal images (scale bar = 20 mm) and magnification details showing amyloid b (Ab) immunostaining (green) and nuclei (blue) taken from the hippocampus of apoE / and DKO mice
aged 12 and 21 months, as indicated. (C) Amyloid b immunostaining in the cerebral cortex from middle-aged (12-month-old) mice. Note
the increase in both intracellular and extracellular amyloid b immunostaining in apoE / brains. (D and E) Representative FcRIV
immunofluorescence in hippocampus (D, scale bar = 10 mm) and cerebral cortex (E, scale bar = 20 mm) from middle-aged mice, showing
FcRIV in green and cell nuclei in blue. Note the similar cellular distribution of amyloid b and FcRIV and the absence of FcRIV in glial
cells. (F and G) Representative confocal images of astrogliosis (F, red) and microgliosis (G, green) in the hippocampus from middle-aged
mice (nuclear staining in blue). (H and I) Western blot analysis of tau phosphorylation (ptau, H) and astrogliosis (GFAP marker, I) in the
cerebral cortex of middle-aged mice. Representative blots from each group are shown. Summary of densitometric analysis is expressed as
fold increases. (J) Cytokine gene expression in the hippocampus and cerebral cortex of middle-aged mice measured by real-time PCR.
IL-6 = interleukin 6; IFN- = interferon . Values are mean SEM of studied animals per group [for 12-month-old mice: wild-type (WT),
n = 5; apoE / , n = 8, DKO, n = 7; for 21-month-old mice: **P 5 0.01, n = 5; apoE / , n = 12; DKO, n = 11]. *P 5 0.05 and
**P 5 0.01 versus wild-type; #P 5 0.05 versus apoE / .
Fig. 2J) as well as the astroglial marker GFAP (Fig. 2I) in cerebral
cortex of hypercholesterolaemic mice. Immunohistochemical studies
revealed that astrogliosis was restricted to the hippocampus (Fig. 2F),
the region most affected by amyloid b deposition. By contrast, microgliosis, detected in the hippocampus (Fig. 2G) and across several
cortical areas (data not shown), did not correlate with the pattern
of amyloid b deposition or FcRIV overexpression and was not
affected by FcR deficiency.
A robust loss of synapses was found in apoE / mice (Fig. 3A),
as judged by the expression of synaptophysin; currently, synaptic
FcR in sporadic Alzheimer-like disease
Brain 2012: 135; 2826–2837
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Figure 3 Synapse loss and cognitive dysfunction are prevented by FcR deletion. (A) Representative immunoblots and quantification of
synaptophysin (syn) expression in the cerebral cortex of middle-aged mice. Values are mean SEM of the total of studied animals per
group: wild-type (WT), n = 5; apoE / , n = 8; DKO, n = 7. **P 5 0.01 versus wild-type; #P 5 0.05 versus apoE / . (B) Spatial learning
and memory measured using the object location test index (exploration time of a given location relative to total object exploration time).
(C) Anxiety levels and locomotor activity measured with the elevated plus maze test. Anxiety index was calculated as the relative time in
open arms, and locomotor activity as frequency of entry into closed arms. All behavioural studies were performed in middle-aged mice
(wild-type, n = 9; apoE / , n = 9; DKO, n = 10). **P 5 0.01 versus familiar location.
Engagement of neuronal FcR in
primary cultures induces Alzheimer-like
pathology
The role of neuronal FcR in hypercholesterolaemia-associated
Alzheimer-like pathology was further studied in primary neuronal
cultures from wild-type, apoE / and DKO mice. In wild-type and
apoE / neurons, IgG immune complexes induced a large increase in the expression of genes for all activating FcR isoforms,
mainly FcRIV (IV 4 I III); smaller effects were observed with
regard to the inhibitory FcRIIb isoform, resulting in a net
activating profile (activating/inhibitory ratio, n-fold versus basal:
wild-type, 2.58; apoE / , 1.57; Fig. 4A). Immmunofluorescence
confirmed the induction of the activating FcRIV isoform by IgG
immune complexes (Fig. 4B). In contrast, immune complexes failed
to induce activating FcR expression in neurons from DKO mice;
these neurons showed an upregulation of inhibitory FcRIIb only
(Fig. 4A and B). In parallel, amyloid b accumulation was observed
after FcR engagement in wild-type pyramidal neurons (Fig. 5A).
Immune complexes also induced tau hyperphosphorylation at epitopes that are considered important in Alzheimer’s disease (Fig. 5B
and C). Furthermore, significant synaptic loss was found in
immune complex-stimulated primary neurons from wild-type and
apoE / mice (Fig. 5D). Importantly, FcR deficiency attenuated
amyloid b deposition and tau hyperphosphorylation (Fig. 5A–C),
protected primary neurons against synapse loss (Fig. 5D) and significantly blocked the expression of inflammation-related genes
after stimulation with immune complexes (Fig. 5F).
To directly assess the involvement of FcRIV, the most inducible
activating FcR found in vivo and in vitro, inhibition experiments
were performed with small interfering RNA and neutralizing antibody. FcRIV small interfering RNA efficiently abrogated intracellular amyloid b accumulation (Fig. 5E), tau phosphorylation
(Fig. 5C) and synaptic loss (Fig. 5D) in response to immune complexes. However, FcRIV small interfering RNA partially reduced
MCP1 gene expression without significant effects on TNF
(Fig. 5F and G). Similarly, blocking FcRIV with a highly specific
antibody prevented tau phosphorylation and synaptic loss and
attenuated MCP1 protein expression (Fig. 5C, D and G).
Furthermore, and consistently with our in vivo results, confocal
microscopic studies using specific markers of astro- and microglial
cells (GFAP and CD11b, respectively) failed to reveal FcRIV
immunostaining in either astroglia or microglia (data not shown,
note that cultures contained 52% of glial cells), suggesting that
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loss appears to be the best correlate of cognitive dysfunction in
Alzheimer’s disease (Masliah et al., 2001; Reddy et al., 2005;
Gomez et al., 2010). Importantly, FcR deletion completely rescued synaptophysin protein levels in hypercholesterolaemic mice
(Fig. 3A). This recovery was shown to be functionally relevant as
shown by performance in the object location test, a measure of
hippocampus-medial temporal lobe related learning and memory
(Eichenbaum et al., 2007). In contrast to apoE / mice, DKO
mice and wild-type mice discriminated between novel and familiar
locations to similar extents, indicating intact cognitive function in
the DKO mice (Fig. 3B). Specifically, differences observed in spatial
learning and memory were not a function of total object exploration times in the training (wild-type, 15.7 6.1 s; apoE / ,
17.3 5.9 s; DKO, 5.3 1.2 s, n = 4–10) and test phases
(wild-type, 10.3 3.2 s; apoE / , 7.5 2.1 s; DKO, 4.0 1.6 s,
n = 4–10) or of different relative exploration times of the two
objects during the acquisition phase of the task (P 4 0.05 in all
cases). Furthermore, the observed differences in cognitive performance could not be explained by increased anxiety or
decreased locomotor activity, as revealed in the elevated plus
maze test (Fig. 3C).
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P. Fernandez-Vizarra et al.
Figure 4 Immune complexes induce FcR overexpression in
primary neurons. (A) The expression profile of activating FcRs
(I, III and IV) and inhibitory FcRIIb in primary neuronal cultures
from wild-type (WT), apoE / , and DKO mice was measured
by real-time PCR after stimulation with immune complexes (IC).
Basal is represented as a dashed line. Data are mean SEM of
three to six experiments. *P 5 0.05 and **P 5 0.01 versus
basal; #P 5 0.05 versus stimulated wild-type cells.
(B) Immunofluorescence of FcRIV (red) and the neuronal
marker microtubule associated protein 2 (MAP-2, green) in
wild-type and DKO neuronal cultures under basal conditions and
after 24-h treatment with IC. Representative of three independent experiments. Scale bar = 20 mm.
neurons are responsible for the observed responses following
treatment with immune complexes.
Role of BACE1, JNK and ERK pathways
in the neuroprotective effects of FcR
deficiency
Both increased production and decreased clearance of amyloid b
have been implicated in sporadic Alzheimer’s disease in humans
Discussion
This study used an animal model of hypercholesterolaemia to investigate the upstream pathways that lead to neuropathological
features characteristic of Alzheimer’s disease, i.e. those preceding
inappropriate increases in amyloid b. Targeting early pathological
mechanisms before the onset of accumulation of amyloid b and
associated amplifying events, such as neuroinflammation and vascular changes, can be expected to facilitate therapeutic interventions. While genetic and molecular data indicate the important role
of amyloid b in the initiation of Alzheimer’s disease (Selkoe, 2001;
Hardy, 2006), a number of recent clinical trials, aimed at reducing
amyloid b burden in mild-to-moderate stages of dementia, have
failed (Shepardson et al., 2011b). Accordingly, the importance of
focusing on factors that induce amyloid b accumulation and in
advance of secondary changes has become even more evident.
It is now recognized that hypercholesterolaemia, often found in
diabetes, obesity and atherosclerosis, is a key risk factor in sporadic
Alzheimer’s disease (Ballard et al., 2011; Shepardson et al. 2011a).
In support of this, several studies have shown that the molecular,
neuroanatomical and cognitive changes found in Alzheimer’s disease are recapitulated in hypercholesterolaemic mice (Oitzl et al.,
1997; Veinbergs et al., 1999; Crisby et al., 2004; Rahman et al.,
2005; Bjelik et al., 2006). The work presented here represents the
first to suggest a pivotal role for brain FcR in the pathogenesis of
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(Yang et al., 2003; Li et al., 2004; Mawuenyega et al., 2010).
Accordingly, we investigated whether the enzymes BACE1 and
insulin degrading enzyme were modulated by hypercholesterolaemia and FcR functional deletion. Western blot analysis for
BACE1 in the cerebral cortex from apoE / mice revealed a significant increase in the 140-kDa band, with a parallel decrease in
the 70-kDa band (Fig. 6A), suggestive of an increase in homodimeric BACE1 activity in hypercholesterolaemia (Schmechel et al.,
2004; Jin et al., 2010). Interestingly, the effects of hypercholesterolaemia on BACE1 were abolished when the FcR gene was
deleted (Fig. 6A). On the other hand, insulin degrading enzyme
levels remained unchanged under hypercholesterolaemic conditions and were unaffected by FcR deletion, as shown by western
blot analysis (Fig. 6B).
Activation of JNK and ERK pathways have been implicated in
synaptic plasticity (Curtis and Finkbeiner, 1999), amyloid b production and tau phosphorylation (Sato et al., 2002;
Colucci-D’Amato et al., 2003) and are known to be involved in
FcR signalling (Song et al., 2002; Luo et al., 2010). In our in vivo
model, hypercholesterolaemia was associated with activation of
JNK and ERK pathways; in contrast, JNK and ERK activation
levels were similar in wild-type and FcR-deficient mice
(Fig. 7A). In vitro, immune complexes induced a sustained activation of both JNK and ERK; these changes were seen before and
during expression of Alzheimer’s disease markers and synaptic loss
in wild-type cultures, and were precluded by FcR deficiency
(Fig. 7B). Moreover, pretreatment of neurons with specific inhibitors of JNK and ERK significantly attenuated the tau hyperphosphorylation and synaptic loss induced by immune complex
treatment (Fig. 7C and D).
FcR in sporadic Alzheimer-like disease
Brain 2012: 135; 2826–2837
| 2833
Representative immunofluorescence images from three independent experiments showing amyloid b (A) and phosphorylated tau (ptau, B)
in red combined with the neuronal marker microtubule associated protein 2 (MAP-2, green) and cell nuclei (blue), both in wild-type (WT)
and DKO neurons under basal conditions or following a 48-h exposure to immune complexes (IC). Arrow indicates typical intracellular
amyloid b deposit in a pyramidal neuron resembling a senile plaque. Arrowheads indicate basal and apical dendrites from pyramidal
neurons stained for AT8 in stimulated cells. Scale bars = 10 mm. (C and D) Western blot analysis of phosphorylated tau (ptau, C) and
synaptophysisn (syn, D) in primary neurons after a 48-h treatment with immune complexes. The effect of FcRIV inhibition in wild-type
neurons was assessed with small interfering RNA (siRNA) and blocking antibody (Block. Ab). Representative immunoblots of three to six
independent experiments are shown. (E) Photomicrographs of triple-colour immunofluorescence (red, amyloid b; green, microtubule
associated protein 2; and blue, nuclei) from wild-type neurons transfected with FcRIV small interfering RNA before stimulation with
immune complexes. Arrowhead denotes neuronal debris (irregular morphology without nuclear staining) strongly stained for amyloid b,
suggestive of a senile plaque derived from intracellular deposits. Scale bar = 20 mm; representative of three independent experiments. (F)
Time-dependent induction of TNF and MCP1 by immune complexes in primary cultures from WT, apoE / and DKO mice, measured by
real-time PCR. The involvement of FcRIV was assessed with small interfering RNA. (G) MCP1 protein production in wild-type neurons
was measured by ELISA. Dashed lines represent basal conditions. Data are mean SEM of three to six experiments per group. *P 5 0.05
and **P 5 0.01 versus basal; #P 5 0.05 and ##P 5 0.01 versus stimulated wild-type cells.
sporadic Alzheimer’s disease. We show that hypercholesterolaemia
results in a significant increase in brain IgG levels with a parallel
increase in activating FcR gene expression. Furthermore,
hypercholesterolaemic mice exhibited increased protein expression
of FcRIV isoform in pyramidal neurons vulnerable to intraneuronal amyloid b deposition in the hippocampus and temporal
cortex, regions similarly affected in the brains of patients with
Alzheimer’s disease (Gouras et al., 2000; Gyure et al., 2001;
Fernandez-Vizarra et al., 2004). Notably, we show that FcRIV
is more widely expressed than amyloid b; besides the hippocampus and temporal cortex, we observed FcRIV staining in the cingulate cortex, an area that is affected in early-stage Alzheimer’s
disease (Barnes et al., 2007; Pengas et al., 2010). These findings
are complemented by the observation that cross-linking of FcR
with IgG immune complexes leads to amyloid b accumulation,
hyperphosphorylation of tau at sites associated with Alzheimer’s
disease and synaptic loss in primary neuronal cultures. An important finding to emerge from this study is that deletion of FcR
function in hypercholesterolaemic mice prevents learning and
memory impairments. These cognition-protective effects were
associated with a reduction in amyloid b deposition, abnormal
tau hyperphosphorylation and synapse loss. Interestingly, the
above mentioned behavioural and neuronal consequences of
FcR deletion appear to occur independently of brain IgG and
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Figure 5 Over-activation of neuronal FcRs, and particularly the FcRIV isoform, induces Alzheimer-like pathology in vitro.
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| Brain 2012: 135; 2826–2837
P. Fernandez-Vizarra et al.
Figure 6 FcR deletion precludes the apparent increase in active BACE1 induced by hypercholesterolaemia. Western blot analysis of
BACE1 (A) and insulin degrading enzyme (IDE, B) protein levels in the cerebral cortex from middle-aged mice. Representative blots from
each group are shown. Values are mean SEM (wild-type, n = 5; apoE / , n = 8; DKO, n = 7). *P 5 0.05 versus wild-type; ##P 5 0.01
versus apoE / .
study in mice showed that disruption of the blood–brain barrier,
and subsequent deposition of IgG in the brain, causes synaptic loss
and memory impairment (Bell et al., 2010). Loss of integrity of the
blood–brain barrier, as well as IgG deposits and/or FcR expression, are also associated with Alzheimer’s disease risk factors other
than ageing, namely atherosclerosis, obesity and diabetes (Methia
et al., 2001; Kuang et al., 2004; Hafezi-Moghadam et al., 2007;
Bake et al., 2009; Diamond et al., 2009). Moreover, the brain is
among the most commonly affected organs in patients with
immune-mediated diseases, such as systemic lupus erythematosus,
subacute endocarditis and hepatitis C infection (Harris and Cobbs,
1996; Lister and Hickey, 2006; Carvalho-Filho et al., 2012), in
which neuronal dysfunction may result from direct immune effects
(autoantibody binding to cell surface, circulating immune complex
deposition and inflammation) on brain resident cells via FcR and
complement activation. Although these immune conditions might
influence the likelihood of developing Alzheimer’s disease and
other dementias, further research in this field is clearly worthwhile.
In light of strong evidence that amyloid b production is
increased (Yang et al., 2003; Li et al., 2004) and cleared with
reduced efficiency (Mawuenyega et al., 2010) in sporadic
Alzheimer’s disease, we here explored the mechanisms through
which FcR activation may lead to increased levels of cerebral
amyloid b. We focused on BACE1, whose homodimerization and
association with amyloid precursor protein leads to the initial
cleavage of amyloid precursor protein (Schmechel et al., 2004;
Jin et al., 2010) and whose expression and activity is upregulated
in patients with sporadic Alzheimer’s disease (Yang et al., 2003; Li
et al., 2004). We observed increased amounts of homodimeric
BACE1 in hypercholesterolaemic mice, an effect that was abolished when the FcR gene was deleted. Further, in line with previous work that reported that BACE1 homodimerization depends
on its phosphorylation at specific serine/threonine residues (Walter
et al., 2001), we observed sustained activation of JNK and ERK in
the brains of hypercholesterolaemic apoE / mice, and in cultured
neurons that were exposed to immune complexes; activated JNK
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blood cholesterol levels since neither of these parameters are
altered in FcR null mutant mice.
Support for the view that the FcRIV isoform is responsible for
mediating the detrimental effects of immune complexes on neurons
stems from experiments in which selective FcRIV inhibition (small
interfering RNA silencing and blocking antibody) rescued immune
complex-induced synaptic loss and the accumulation of amyloid b
and hyperphosphorylated tau. It is important to note that our analysis revealed that FcRIV expression is restricted to neurons both
in vivo and in vitro, as no signal was detected in astro- and microglia.
The results, obtained in primary neuronal cultures, indicate that
MCP1 and TNF are unlikely to exert substantial influence over the
development of amyloid b accumulation, tau pathology and synaptic
loss in primary neurons: although FcRIV knock-down in neurons did
not preclude cytokine (MCP1 and TNF) induction by immune complexes, this inflammatory response was not followed by the aforementioned neuropathological features. Nevertheless, a potentially
protective role of these cytokines cannot be ruled out at present.
In this context, it is worth mentioning that MCP1, a cytokine
involved in the recruitment, proliferation and activation of glial
cells, may trigger gliosis in the hippocampus; since astro- and microglia do not express the only hypercholesterolaemia-responsive FcR
isoform (FcRIV), gliosis in the hippocampus cannot be attributed to
activation of FcR. If early inflammatory responses serve to retard
hypercholesterolaemia-associated Alzheimer’s disease pathology,
interesting therapeutic opportunities could be explored by exploiting
the differential regulation of inflammation and Alzheimer-related
pathology by FcR isoforms; importantly, these would not necessarily interfere with the potentially protective functions of glial cells.
Our hypothesis that IgG is involved in the onset of certain sporadic forms of Alzheimer’s disease is consistent with recent data
suggesting that factors that induce weakening of the blood–brain
barrier may be causally related to neurodegenerative disease; since
this barrier gets leaky during normal ageing (Hafezi-Moghadam
et al., 2007), hypercholesterolaemia would add to or multiply
the risk of developing Alzheimer’s disease. Indeed, a recent
FcR in sporadic Alzheimer-like disease
Brain 2012: 135; 2826–2837
| 2835
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Figure 7 JNK and ERK activation are involved in the neuroprotective actions of FcR deletion. (A) JNK and ERK activity was measured by
western blot analysis of phosphorylated proteins (p-proteins: pJNK and pERK) in the cerebral cortex from middle-aged mice. (B) JNK and
ERK activation in primary neurons under basal conditions (B) or following 48-h stimulation with immune complexes (IC). (C and D)
Western blot analysis of tau phosphorylation (ptau, C) and synaptophysin levels (syn, D) in wild-type neurons pretreated with inhibitors of
JNK (SP600125, SP) and ERK (U0126, U) before stimulation. Basal condition is represented as a dashed line. Summary of densitometric
analysis is expressed as fold increases. Representative blots from in each group are shown. Values are mean SEM of studied animals per
group (wild-type, n = 5; apoE / , n = 8; DKO, n = 7; A) and of three to six independent experiments (B–D). *P 5 0.05 versus wild-type
(WT) mice or basal cells; #P 5 0.05 versus apoE / mice or stimulated wild-type cells.
and ERK have been previously implicated in the generation of
amyloid b and the abnormal hyperphosphorylation of tau (Sato
et al., 2002; Colucci-D’Amato et al., 2003).
In summary, our study describes a novel mechanism that
may trigger certain sporadic forms of Alzheimer’s disease. The
results point to FcR as a potential target for preventative intervention, at least in cases where hypercholesterolaemia is a risk
factor.
Although preventive approaches in those patients may primarily
include reduction of hypercholesterolaemia, recent evidence indicates that lipid-lowering drugs are inefficacious treatments for
Alzheimer’s disease (Shepardson et al., 2011b). Alternatively, strategies targeting antibody production and FcR balance and
activities could determine the net functional effect and hence
retard disease onset and progression. In future, it will be also important to determine whether levels of IgG immune complexes
can serve as early biomarkers of sporadic Alzheimer’s disease
and to assess the potential contribution of FcR signalling to
forms of Alzheimer’s disease that are not directly associated with
hypercholesterolaemia.
Acknowledgements
The authors thank Dr J. V. Ravetch (The Rockefeller University,
New York) for generously providing FcRIV blocking antibody, Dr
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| Brain 2012: 135; 2826–2837
M.P. Sanchez and A.M. Garcia-Cabrero (Laboratory of Neurology,
IIS-FJD,
Madrid)
and
Drs
C. Lopez-Menendez
and
L. Sanchez-Ruiloba (IIB Alberto Sols, CSIC-UAM) for antibody
samples and skilled advice with mice and primary cultures.
Funding
Spanish Ministry of Science (SAF2009/11794), Fondo de
Investigaciones Sanitarias (FIS PI10/00072, RECAVA RD06/
0014/0035), Fundacion Renal In˜igo Alvarez de Toledo, Spanish
Society of Nephrology and Lilly Foundation. P.F.-V. was supported
by postdoctoral fellowships from FIS (Sara Borrell program) and
Caja Madrid Foundation.
References
and PHF in clinically evaluated cases of Alzheimer’s disease. Histol
Histopathol 2004; 19: 823–44.
Gomez RM, Rosso OA, Berretta R, Moscato P. Uncovering molecular
biomarkers that correlate cognitive decline with the changes of hippocampus’ gene expression profiles in Alzheimer’s disease. PLoS One
2010; 5: e10153.
Gordon I, Genis I, Grauer E, Sehayek E, Michaelson DM. Biochemical and
cognitive studies of apolipoprotein-E-deficient mice. Mol Chem
Neuropathol 1996; 28: 97–103.
Gouras GK, Tsai J, Naslund J, Vincent B, Edgar M, Checler F, et al.
Intraneuronal Abeta42 accumulation in human brain. Am J Pathol
2000; 156: 15–20.
Gyure KA, Durham R, Stewart WF, Smialek JE, Troncoso JC. Intraneuronal
abeta-amyloid precedes development of amyloid plaques in Down syndrome. Arch Pathol Lab Med 2001; 125: 489–92.
Hafezi-Moghadam A, Thomas KL, Wagner DD. ApoE deficiency leads to
a progressive age dependent blood-brain barrier leakage. Am J Physiol
Cell Physiol 2007; 292: C1256–62.
Hardy J. Has the amyloid cascade hypothesis for Alzheimer’s disease
been proved? Curr Alzheimer Res 2006; 3: 71–3.
Haroun M, El-Sayed M. Measurement of IgG levels can serve as a biomarker in newly diagnosed diabetic children. J Clin Biochem Nutr
2007; 40: 56–61.
Harris PS, Cobbs CG. Cardiac, cerebral, and vascular complications of
infective endocarditis. Cardiol Clin 1996; 14: 437–50.
Hernandez-Vargas P, Ortiz-Mun˜oz G, Lopez-Franco O, Suzuki Y,
Gallego-Delgado J, Sanjuan G, et al. Fcgamma receptor deficiency
confers protection against atherosclerosis in apolipoprotein E knockout
mice. Circ Res 2006; 99: 1188–96.
Jin S, Agerman K, Kolmodin K, Gustafsson E, Dahlqvist C, Jureus A, et al.
Evidence for dimeric BACE-mediated APP processing. Biochem Biophys
Res Commun 2010; 393: 21–7.
Kuang F, Wang BR, Zhang P, Fei LL, Jia Y, Duan XL, et al. Extravasation
of blood-borne immunoglobulin G through blood-brain barrier during
adrenaline-induced transient hypertension in the rat. Int J Neurosci
2004; 114: 575–91.
Li R, Lindholm K, Yang LB, Yue X, Citron M, Yan R, et al. Amyloid beta
peptide load is correlated with increased betasecretase activity in sporadic Alzheimer’s disease patients. Proc Natl Acad Sci USA 2004; 101:
3632–7.
Lister KJ, Hickey MJ. Immune complexes alter cerebral microvessel permeability: roles of complement and leukocyte adhesion. Am J Physiol
Heart Circ Physiol 2006; 291: H694–704.
Listı` F, Candore G, Modica MA, Russo M, Di Lorenzo G, EspositoPellitteri M, et al. A study of serum immunoglobulin levels in elderly
persons that provides new insights into B cell immunosenescence. Ann
N Y Acad Sci 2006; 1089: 487–95.
Lu J, Moochhala S, Kaur C, Ling EA. Cellular inflammatory response
associated with breakdown of the blood-brain barrier after closed
head injury in rats. J Neurotrauma 2001; 18: 399–408.
Luo Y, Pollard JW, Casadevall A. Fcgamma receptor cross-linking stimulates cell proliferation of macrophages via the ERK pathway. J Biol
Chem 2010; 285: 4232–42.
Masliah E, Mallory M, Alford M, DeTeresa R, Hansen LA, McKeel DW Jr,
et al. Altered expression of synaptic proteins occurs early during progression of Alzheimer’s disease. Neurology 2001; 56: 127–9.
Mawuenyega KG, Sigurdson W, Ovod V, Munsell L, Kasten T, Morris JC,
et al. Decreased clearance of CNS beta-amyloid in Alzheimer’s disease.
Science 2010; 330: 1774.
Methia N, Andre P, Hafezi-Moghadam A, Economopoulos M,
Thomas KL, Wagner DD. ApoE deficiency compromises the blood
brain barrier especially after injury. Mol Med 2001; 7: 810–5.
Morrison JH, Hof PR. Selective vulnerability of corticocortical and hippocampal circuits in aging and Alzheimer’s disease. Prog Brain Res 2002;
136: 467–86.
Murai T, Okuda S, Tanaka T, Ohta H. Characteristics of object location
memory in mice: Behavioral and pharmacological studies. Physiol
Behav 2007; 90: 116–24.
Downloaded from by guest on October 21, 2014
Ashe KH, Zahs KR. Probing the biology of Alzheimer’s disease in mice.
Neuron 2010; 66: 631–45.
Bake S, Friedman JA, Sohrabji F. Reproductive age-related changes in the
blood brain barrier: expression of IgG and tight junction proteins.
Microvasc Res 2009; 78: 413–24.
Ballard C, Gauthier S, Corbett A, Brayne C, Aarsland D, Jones E.
Alzheimer’s disease. Lancet 2011; 377: 1019–31.
Barnes J, Godbolt AK, Frost C, Boyes RG, Jones BF, Scahill RI, et al.
Atrophy rates of the cingulate gyrus and hippocampus in
Alzheimer’s disease and FTLD. Neurobiol Aging 2007; 28: 20–28.
Bell RD, Winkler EA, Sagare AP, Singh I, LaRue B, Deane R, et al.
Pericytes control key neurovascular functions and neuronal phenotype
in the adult brain and during brain aging. Neuron 2010; 68: 409–27.
Bertram L, Lill CM, Tanzi RE. The genetics of Alzheimer disease: back to
the future. Neuron 2010; 68: 270–81.
Bjelik A, Bereczki E, Gonda S, Juha´sz A, Rimano´czy A, Zana M, et al.
Human apoB overexpression and a high-cholesterol diet differently
modify the brain APP metabolism in the transgenic mouse model of
atherosclerosis. Neurochem Int 2006; 49: 393–400.
Bouras C, Riederer BM, Kovari E, Hof PR, Giannakopoulos P. Humoral
immunity in brain aging and Alzheimer’s disease. Brain Res Brain Res
Rev 2005; 48: 477–87.
Burut DF, Karim Y, Ferns GA. The role of immune complexes in atherogenesis. Angiology 2010; 61: 679–89.
Carvalho-Filho RJ, Narciso-Schiavon JL, Tolentino LH, Schiavon LL,
Ferraz ML, Silva AE. Central nervous system vasculitis and polyneuropathy as first manifestations of hepatitis C. World J Gastroenterol
2012; 18: 188–91.
Colucci-D’Amato L, Perrone-Capano C, di PU. Chronic activation of ERK
and neurodegenerative diseases. Bioessays 2003; 25: 1085–95.
Crisby M, Rahman SM, Sylven C, Winblad B, Schultzberg M. Effects of
high cholesterol diet on gliosis in apolipoprotein E knockout mice.
Implications for Alzheimer’s disease and stroke. Neurosci Lett 2004;
369: 87–92.
Curtis J, Finkbeiner S. Sending signals from the synapse to the nucleus:
possible roles for CaMK, Ras/ERK, and SAPK pathways in the regulation of synaptic plasticity and neuronal growth. J Neurosci Res 1999;
58: 88–95.
Diamond B, Huerta PT, Mina-Osorio P, Kowal C, Volpe BT. Losing your
nerves? Maybe it’s the antibodies. Nat Rev Immunol 2009; 9: 449–56.
Doyle HA, Mamula MJ. Post-translational protein modifications in
antigen recognition and autoimmunity. Trends Immunol 2001; 22:
443–9.
Eichenbaum H, Yonelinas AP, Ranganath C. The medial temporal lobe
and recognition memory. Annu Rev Neurosci 2007; 30: 123–52.
Ferna´ndez-Vizarra P, Ferna´ndez AP, Castro-Blanco S, Serrano J,
Bentura ML, Martı´nez-Murillo R, et al. Intra- and extracellular Abeta
P. Fernandez-Vizarra et al.
FcR in sporadic Alzheimer-like disease
| 2837
Sato S, Tatebayashi Y, Akagi T, Chui DH, Murayama M, Miyasaka T,
et al. Aberrant tau phosphorylation by glycogen synthase kinase-3beta
and JNK3 induces oligomeric tau fibrils in COS-7 cells. J Biol Chem
2002; 277: 42060–5.
Schmechel A, Strauss M, Schlicksupp A, Pipkorn R, Haass C, Bayer TA,
et al. Human BACE forms dimers and colocalizes with APP. J Biol
Chem 2004; 279: 39710–7.
Selkoe DJ. Alzheimer’s disease: genes, proteins, and therapy. Physiol Rev
2001; 81: 741–66.
Shepardson NE, Shankar GM, Selkoe DJ. Cholesterol level and statin use
in Alzheimer disease: I. Review of epidemiological and preclinical studies. Arch Neurol 2011a; 68: 1239–44.
Shepardson NE, Shankar GM, Selkoe DJ. Cholesterol level and statin use
in Alzheimer Disease: II. Review of human trials and recommendations.
Arch Neurol 2011b; 68: 1385–92.
Song X, Shapiro S, Goldman DL, Casadevall A, Scharff M, Lee SC.
Fcgamma receptor I- and III-mediated macrophage inflammatory protein 1alpha induction in primary human and murine microglia. Infect
Immun 2002; 70: 5177–84.
Turk Z, Ljubic S, Turk N, Benko B. Detection of autoantibodies against
advanced glycation endproducts and AGE-immune complexes in
serum of patients with diabetes mellitus. Clin Chim Acta 2001; 303:
105–15.
Veinbergs I, Mante M, Jung MW, Van UE, Masliah E. Synaptotagmin
and synaptic transmission alterations in apolipoprotein E-deficient
mice. Prog Neuropsychopharmacol Biol Psychiatry 1999; 23: 519–31.
Walter J, Fluhrer R, Hartung B, Willem M, Kaether C, Capell A, et al.
Phosphorylation regulates intracellular trafficking of betasecretase. J
Biol Chem 2001; 276: 14634–41.
Winer DA, Winer S, Shen L, Wadia PP, Yantha J, Paltser G, et al. B cells
promote insulin resistance through modulation of T cells and production of pathogenic IgG antibodies. Nat Med 2011; 17: 610–17.
Yang LB, Lindholm K, Yan R, Citron M, Xia W, Yang XL, et al. Elevated
beta-secretase expression and enzymatic activity detected in sporadic
Alzheimer disease. Nat Med 2003; 9: 3–4.
Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E.
Science 1992; 258: 468–71.
Downloaded from by guest on October 21, 2014
Nakahara J, Tan-Takeuchi K, Seiwa C, Gotoh M, Kaifu T, Ujike A, et al.
Signaling via immunoglobulin Fc receptors induces oligodendrocyte
precursor cell differentiation. Dev Cell 2003; 4: 841–52.
Nakata A, Araki S, Tanigawa T, Miki A, Sakurai S, Kawakami N, et al.
Decrease of suppressor-inducer (CD4 + CD45RA) T lymphocytes and
increase of serum immunoglobulin G due to perceived job stress in
Japanese nuclear electric power plant workers. J Occup Environ Med
2000; 42: 143–50.
Nimmerjahn F, Bruhns P, Horiuchi K, Ravetch JV. FcgammaRIV: a novel
FcR with distinct IgG subclass specificity. Immunity 2005; 23: 41–51.
Nimmerjahn F, Ravetch JV. Fcgamma receptors as regulators of immune
responses. Nat Rev Immunol 2008; 8: 34–47.
Oitzl MS, Mulder M, Lucassen PJ, Havekes LM, Grootendorst J, de
Kloet ER. Severe learning deficits in apolipoprotein E-knockout mice
in a water maze task. Brain Res 1997; 752: 189–96.
Okamatsu Y, Matsuda K, Hiramoto I, Tani H, Kimura K, Yada Y, et al.
Ghrelin and leptin modulate immunity and liver function in overweight
children. Pediatr Int 2009; 51: 9–13.
Oreskes I, Mandel D. Size fractionation of thermal aggregates of immunoglobulin G. Anal Biochem 1983; 134: 199–204.
Ortiz-Mun˜oz G, Lopez-Parra V, Lopez-Franco O, Fernandez-Vizarra P,
Mallavia B, Flores C, et al. Suppressors of cytokine signalling abrogate
diabetic nephropathy. J Am Soc Nephrol 2010; 21: 763–72.
Ounanian A, Guilbert B, Renversez JC, Seigneurin JM, Avrameas S.
Antibodies to viral antigens, xenoantigens, and autoantigens in
Alzheimer’s disease. J Clin Lab Anal 1990; 4: 367–75.
Pengas G, Hodges JR, Watson P, Nestor PJ. Focal posterior cingulate
atrophy in incipient Alzheimer’s disease. Neurobiol Aging 2010; 31:
25–33.
Rahman A, Akterin S, Flores-Morales A, Crisby M, Kivipelto M,
Schultzberg M, et al. High cholesterol diet induces tau hyperphosphorylation in apolipoprotein E deficient mice. FEBS Lett 2005; 579:
6411–16.
Reddy PH, Mani G, Park BS, Jacques J, Murdoch G, Whetsell W Jr, et al.
Differential loss of synaptic proteins in Alzheimer’s disease: implications
for synaptic dysfunction. J Alzheimers Dis 2005; 7: 103–17.
Rodrigo J, Ferna´ndez-Vizarra P, Castro-Blanco S, Bentura ML, Nieto M,
Go´mez-Isla T, et al. Nitric oxide in the cerebral cortex of
amyloid-precursor
protein
(SW)
Tg2576
transgenic
mice.
Neuroscience 2004; 128: 73–89.
Brain 2012: 135; 2826–2837