Key Aging-Associated Alterations in Primary Microglia Response to Beta-Amyloid Stimulation

Abstract

Alzheimer’s disease (AD) is characterized by a progressive cognitive decline and believed to be driven by the self-aggregation of amyloid-β (Aβ) peptide into oligomers and fibrils that accumulate as senile plaques. It is widely accepted that microglia-mediated inflammation is a significant contributor to disease pathogenesis; however, different microglia phenotypes were identified along AD progression and excessive Aβ production was shown to dysregulate cell function. As so, the contribution of microglia to AD pathogenesis remains to be elucidated. In this study, we wondered if isolated microglia cultured for 16 days in vitro (DIV) would react differentially from the 2 DIV cells upon treatment with 1000 nM Aβ1–42 for 24 h. No changes in cell viability were observed and morphometric alterations associated to microglia activation, such as volume increase and process shortening, were obvious in 2 DIV microglia, but less evident in 16 DIV cells. These cells showed lower phagocytic, migration and autophagic properties after Aβ treatment than the 2 DIV cultured microglia. Reduced phagocytosis may derive from increased CD33 expression, reduced triggering receptor expressed on myeloid cells 2 (TREM2) and milk fat globule-EGF factor 8 protein (MFG-E8) levels, which were mainly observed in 16 DIV cells. Activation of inflammatory mediators, such as high mobility group box 1 (HMGB1) and pro-inflammatory cytokines, as well as increased expression of Toll-like receptor 2 (TLR2), TLR4 and fractalkine/CX3C chemokine receptor 1 (CX3CR1) cell surface receptors were prominent in 2 DIV microglia, while elevation of matrix metalloproteinase 9 (MMP9) was marked in 16 DIV cells. Increased senescence-associated β-galactosidase (SA-β-gal) and upregulated miR-146a expression that were observed in 16 DIV cells showed to increase by Aβ in 2 DIV microglia. Additionally, Aβ downregulated miR-155 and miR-124, and reduced the CD11b+ subpopulation in 2 DIV microglia, while increased the number of CD86+ cells in 16 DIV microglia. Simultaneous M1 and M2 markers were found after Aβ treatment, but at lower expression in the in vitro aged microglia. Data show key-aging associated responses by microglia when incubated with Aβ, with a loss of reactivity from the 2 DIV to the 16 DIV cells, which course with a reduced phagocytosis, migration and lower expression of inflammatory miRNAs. These findings help to improve our understanding on the heterogeneous responses that microglia can have along the progression of AD disease and imply that therapeutic approaches may differ from early to late stages.

Full text

ORIGINAL RESEARCH
published: 31 August 2017
doi: 10.3389/fnagi.2017.00277
Key Aging-Associated Alterations in
Primary Microglia Response to
Beta-Amyloid Stimulation
Cláudia Caldeira 1,Carolina Cunha 1,Ana R. Vaz1,2,Ana S. Falcão 2,
Andreia Barateiro 1,Elsa Seixas 3,Adelaide Fernandes 1,2 and Dora Brites 1,2*
1Neuron Glia Biology in Health and Disease, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy,
Universidade de Lisboa, Lisbon, Portugal, 2Department of Biochemistry and Human Biology, Faculty of Pharmacy,
Universidade de Lisboa, Lisbon, Portugal, 3Obesity Laboratory, Instituto Gulbenkian de Ciência, Oeiras, Portugal
Edited by:
Isidre Ferrer,
University of Barcelona, Spain
Reviewed by:
Abdu Adem,
United Arab Emirates University,
United Arab Emirates
Alberto Serrano-Pozo,
Massachusetts General Hospital,
United States
*Correspondence:
Dora Brites
dbrites@ff.ulisboa.pt
Received: 20 March 2017
Accepted: 03 August 2017
Published: 31 August 2017
Citation:
Caldeira C, Cunha C, Vaz AR,
Falcão AS, Barateiro A, Seixas E,
Fernandes A and Brites D (2017) Key
Aging-Associated Alterations in
Primary Microglia Response to
Beta-Amyloid Stimulation.
Front. Aging Neurosci. 9:277.
doi: 10.3389/fnagi.2017.00277
Alzheimer’s disease (AD) is characterized by a progressive cognitive decline and believed
to be driven by the self-aggregation of amyloid-β(Aβ) peptide into oligomers and
fibrils that accumulate as senile plaques. It is widely accepted that microglia-mediated
inflammation is a significant contributor to disease pathogenesis; however, different
microglia phenotypes were identified along AD progression and excessive Aβproduction
was shown to dysregulate cell function. As so, the contribution of microglia to AD
pathogenesis remains to be elucidated. In this study, we wondered if isolated microglia
cultured for 16 days in vitro (DIV) would react differentially from the 2 DIV cells upon
treatment with 1000 nM Aβ1–42 for 24 h. No changes in cell viability were observed and
morphometric alterations associated to microglia activation, such as volume increase
and process shortening, were obvious in 2 DIV microglia, but less evident in 16 DIV
cells. These cells showed lower phagocytic, migration and autophagic properties after
Aβtreatment than the 2 DIV cultured microglia. Reduced phagocytosis may derive from
increased CD33 expression, reduced triggering receptor expressed on myeloid cells 2
(TREM2) and milk fat globule-EGF factor 8 protein (MFG-E8) levels, which were mainly
observed in 16 DIV cells. Activation of inflammatory mediators, such as high mobility
group box 1 (HMGB1) and pro-inflammatory cytokines, as well as increased expression
of Toll-like receptor 2 (TLR2), TLR4 and fractalkine/CX3C chemokine receptor 1
(CX3CR1) cell surface receptors were prominent in 2 DIV microglia, while elevation of
matrix metalloproteinase 9 (MMP9) was marked in 16 DIV cells. Increased senescence-
associated β-galactosidase (SA-β-gal) and upregulated miR-146a expression that were
observed in 16 DIV cells showed to increase by Aβin 2 DIV microglia. Additionally, Aβ
downregulated miR-155 and miR-124, and reduced the CD11b+ subpopulation in 2 DIV
microglia, while increased the number of CD86+ cells in 16 DIV microglia. Simultaneous
M1 and M2 markers were found after Aβtreatment, but at lower expression in the
in vitro aged microglia. Data show key-aging associated responses by microglia when
incubated with Aβ, with a loss of reactivity from the 2 DIV to the 16 DIV cells, which
course with a reduced phagocytosis, migration and lower expression of inflammatory
miRNAs. These findings help to improve our understanding on the heterogeneous
responses that microglia can have along the progression of AD disease and imply that
therapeutic approaches may differ from early to late stages.
Keywords: Alzheimer’s disease, amyloid-βpeptide, neuroinflammation, aged-cultured microglia, inflammatory-
microRNAs, M1/M2 microglia subtypes, CD11b, CD86
Frontiers in Aging Neuroscience | www.frontiersin.org 1August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
INTRODUCTION
Alzheimer’s disease (AD) is the most common dementing
disorder in the elderly affecting around 35.6 million people
worldwide and is expected to duplicate in the years to come
(Prince et al., 2013). Amyloid precursor protein (APP) expression
is elevated in AD, and increased amyloidogenic cleavage has been
considered to cause the deposition of extracellular β-amyloid
(Aβ) plaques (Rubio-Perez and Morillas-Ruiz, 2012). Deposition
of Aβwas shown to trigger the activation of both astrocytes
and microglia leading to the production of pro-inflammatory
cytokines, such as interleukin (IL)-1βand tumor necrosis factor
(TNF)-α, among other inflammatory mediators (Wyss-Coray,
2006), thus generating neuroinflammation and contributing to
AD progression and severity (Heneka et al., 2015). Nevertheless,
treatment with nonsteroidal anti-inflammatory drugs (NSAIDs)
has consistently failed in efficacy in AD patients (von
Bernhardi, 2010), unless administered early in the disease (Varvel
et al., 2009). Lately, it was shown that fenamate NSAIDs
decreased microglia activation and IL-1βprocessing in rodent
models, which led the authors to suggest that they may be
repurposed as inhibitors of the NOD-like receptor family pyrin
domain containing 3 (NLRP3) inflammasome (Daniels et al.,
2016). Microglial malfunction has been associated with AD
pathogenesis. It has been indicated to contribute to changes
in cell microenvironment and to precede or facilitate AD
onset (Regen et al., 2017). However, the underlying molecular
mechanisms of microglia failure in AD pathogenesis remain to
be identified.
Microglia are the first line and the main immune defense
against disease and injury in the central nervous system (CNS).
When activated by stress stimuli the cells migrate and restrict the
damage by surrounding the site lesion and by clearing cellular
debris by phagocytosis. Microglia are activated by oligomeric
and fibrillar species of Aβ, as well as by molecules derived
from degenerating neurons (Mizuno, 2012), and have been
described to play a key role in removal of Aβfrom the brain
(Morgan, 2009). However, microglia is differently polarized
depending on the stimuli and the time of exposure, reason
why in some circumstances the phagocytic and inflammatory
phenotypes may alternate (Silva et al., 2010). It is well recognized
that microglia may comprise a family of cells with diverse
phenotypes exerting beneficial or destructive effects (Schwartz
et al., 2006). Other studies also propose that age-dependent
neuroinflammatory changes trigger decreased neurogenesis and
cognitive impairments in AD (Lynch et al., 2010; Varnum and
Ikezu, 2012). In addition, it has been claimed that these cells lose
their ability to phagocytose Aβwith age and disease progression,
and that in late disease stages inflammation no longer exists
(Floden and Combs, 2011; Wojtera et al., 2012). As so, there is an
urgent need to understand age-dependent changes in microglia
function and associated differences in responding to stimuli
to better recognize the diverse roles that these cells may have
in early and late-stages of AD. Recent evidences showed that
endogenous microRNAs (miRNAs), a subset of small noncoding
RNA molecules that play an important role in the regulation of
gene expression at the posttranscriptional level, are associated to
microglia activation (Guedes et al., 2013) and that miRNA(miR)-
155 can contribute to neuroinflammation in AD (Guedes et al.,
2014). Interestingly, upregulated miR-155 and miR-146a plus
downregulated miR-124 were recently observed in microglia
upon stimulation with lipopolysaccharide (LPS) (Cunha et al.,
2016). Actually, these miRNAs are considered to modulate the
inflammatory status (Olivieri et al., 2013b) and to be associated
to microglia activation and polarization (Ponomarev et al.,
2013).
Over the years several studies attempted the identification
of microglia activation subtypes in several in vitro and in vivo
models, as well as in AD brain autopsy specimens, trying
to fit them into the described polarization schemes (Walker
and Lue, 2015). Although the priming of microglia and the
polarization into the M1 phenotype have been suggested by
most of the works in AD (Heneka et al., 2015; Hoeijmakers
et al., 2016), others also indicate increased expression of
Arginase 1 (Colton et al., 2006) and co-expression of M1, M2a,
M2b and M2c markers (Wilcock, 2012; Sudduth et al., 2013).
Lately, five microglia morphological phenotypes (i.e., ramified,
hypertrophic, dystrophic, rod-shaped and amoeboid) were
identified in AD patient autopsied samples, together with an
increased prevalence of dystrophic microglia in cases of dementia
with Lewy bodies (Bachstetter et al., 2015). Contrasting results
obtained so far derive from the diversity of the experimental
models that are tentatively used to recapitulate the in vivo AD
condition.
Relatively to microglia, in vitro cell models, either microglial
cell lines, or primary microglia isolated from embryonic
(Gingras et al., 2007) or neonatal animals (Floden and Combs,
2007), though largely used (Moussaud and Draheim, 2010),
fail in mimicking adult behavior cells (Sierra et al., 2007).
Furthermore, primary cultures of microglia were shown to
change their activation profile according with the time in culture
(Cristóvão et al., 2010). All of these features contribute to data
inconsistency.
Since AD is considered an age-related disease, the use of
aged animal models have been proposed (Bachstetter et al.,
2015). However, a lot of problems must be considered. Actually,
the need to wait for 2–3 years for animals aging to assess
differences in cell function, and only in the survival population,
together with a high result variability (Birch et al., 2014), have
contributed to misunderstand many of the elderly processes and
to failure in obtaining successful innovative strategic approaches
to AD. Therefore, we hypothesized that our experimental model
of in vitro aging microglia (Caldeira et al., 2014) would add
additional information on the microglia phenotypes occurring
in AD onset and later along the disease progression, while
also allowing the work with aged microglia, once there are no
processes to isolate degenerating microglia for experimentation
(Njie et al., 2012).
In the present study we assumed that the recently isolated
microglia maintained for 2 days in vitro (DIV) and the 16 DIV
aged cultured microglia represent distinct cell populations
that should react differently to the Aβstressful stimulus.
These subtypes may underlie diverse vulnerabilities along AD
progression, from onset to late stages, and serve as models to
Frontiers in Aging Neuroscience | www.frontiersin.org 2August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
better understand changes associated to cell malfunction by
Aβaccumulation and by aging, not completely clarified so far.
We considered that the 2 DIV young microglia phenotype of
our previous study (Caldeira et al., 2014) mostly resemble the
activation of the cell in the subacute inflammation state, while the
16 DIV aged cells recapitulate cells unable to mount an efficient
response against a stressor stimulus. Hence, we aimed to examine
the behavior of these two in vitro cultured microglia phenotypes,
young/reactive (2 DIV) and aged/desensitized (16 DIV) cells,
when facing a non-toxic mixture of Aβ1–42 oligomeric and
fibrillar species at a concentration of 1000 nM. For that, we
decided to assess cell morphology, phagocytic ability, migration
capacity, autophagy and senescence markers, as well as a set
of inflammatory-associated miRNAs, inflammatory cytokines,
the alarmin high mobility group box 1 (HMGB1) protein,
key regulatory receptors and inflammasome complex, together
with matrix metalloproteinase 2 (MMP2) and MMP9 activation.
Further, we evaluated gene expression of microglia phenotype
M1 and M2 biomarkers and explored their subtype distribution.
Our results indicate that Aβ1–42, although prompting an
acute inflammatory reaction, promote the switch of the activated
microglia towards a miscellaneous polarized population, while
eliciting microglia senescence and impairing phagocytosis in the
2 DIV in vitro microglia. Data also highlight the presence of an
increased population of CD86+ microglia in the 16 DIV cells,
whose expression is associated to cell plasticity and multipolar
morphology. The number of CD86+ cells that increased in
the presence of Aβ1–42, further suggests the simultaneous
existence of pro- and anti-inflammatory phenotypes and a lower
ability to mount immune and neuroprotective responses by
the aged microglia. We believe that a better understanding
on the significance of these two activated/dysfunctional cell
states on AD pathogenesis will contribute to dissect microglial
mechanisms in AD. If microglial diversity is confirmed in
subsequent studies, different therapeutic approaches may be
required to ensure effectiveness in a disease where personalized
medicine and patient stratification are considered critical issues.
MATERIALS AND METHODS
Animals
Animal care followed the recommendations of the European
Convention for the Protection of Vertebrate Animals Used for
Experimental and Other Scientific Purposes (Council Directive
86/609/EEC) and National Law 1005/92 (rules for protection of
experimental animals). All animal procedures were approved by
the Institutional animal care and use committee. Every effort
was made to minimize the number of animals used and their
suffering.
Primary Culture of Microglia
Mixed glial cultures were prepared from 1 to 2 day-old CD1 mice,
as previously described (McCarthy and de Vellis, 1980), with
minor modifications (Gordo et al., 2006). Cells (4 ×105
cells/cm2) were plated on uncoated 12-well tissue culture plates
(with 18 mm coverslips) or 75-cm2culture flasks in culture
medium (DMEM-Ham’s F12 medium supplemented with 2 mM
L-glutamine, 1 mM sodium pyruvate, 1% nonessential amino
acids, 10% fetal bovine serum (FBS), and 1% antibiotic-
antimycotic solution), and maintained at 37◦C in a humidified
atmosphere of 5% CO2.
Microglia were isolated as previously described (Saura et al.,
2003). Briefly, after 21 days in culture, microglia were obtained
by mild trypsinization with a trypsin-EDTA solution diluted
1:3 in DMEM-Ham’s F12 for 45–60 min. The trypsinization
resulted in detachment of an upper layer of cells containing
astrocytes, whereas microglia remained attached to the bottom
of the well. The medium containing detached cells was removed
and the initial mixed glial-conditioned medium was added. The
use of this isolation procedure of mixed astrocyte-microglia
cultures for 21 DIV cells allows a maximal yield and purity of
microglia after trypsinization. In fact, astrocyte contamination
was less than 2%, as assessed by immunocytochemical staining
using a primary antibody against glial fibrillary acidic protein
(GFAP) and a species-specific fluorescent-labeled secondary
antibody. Neuronal contamination was 0%, as assessed by
immunocytochemical staining with a primary antibody against
microtubule-associated protein 2 (MAP2) followed by a species-
specific fluorescent-labeled secondary antibody (Silva et al.,
2010).
Treatment of Microglia with a Mixture of
Aβ1–42 Oligomers and Fibrils
Aβ1–42 peptide was diluted in DMEM-Ham’s F12 culture
medium to a stock concentration of 111 µM and allowed to
incubate for 24 h at 37◦C to preaggregate the peptides, as
formerly indicated (Hjorth et al., 2013). Cells were incubated
with 50 nM and 1000 nM Aβ1–42, during 24 h, at 37◦C, although
the lower concentration was later abandoned in favor of the
more consistent results obtained with the higher one. Cells
incubated in the absence of Aβ1–42 were used as controls. We
have previously observed that such Aβ1–42 solution was mainly
constituted by large oligomers and fibrils with a small proportion
of monomers and dimers, after a 24 h period of time (Falcão et al.,
2017).
Isolated microglia were differentially aged in culture for 2 and
16 DIV in order to obtain two diverse microglia phenotypes, in
accordance with our prior publication (Caldeira et al., 2014), and
to assess whether they differentially respond to the Aβstimulus.
Actually, the 2 DIV cells represent an activated microglia subtype
determined by the acute process of isolation, and the 16 DIV a
more unresponsive/dormant subclass.
Determination of Cell Death
To evaluate microglia cell death, we used phycoerythrin-
conjugated annexin V (V-PE) and 7-amino-actinomycin D
(7-AAD; Guava NexinrReagent, #4500-0450, Merck Millipore,
Billerica, MA, USA) to determine the percentage of viable, early-
apoptotic and late-apoptotic/necrotic cells by flow cytometry.
After incubation, plated microglia were trypsinized and added
to cells in the incubation media, which were then stained
with annexin V-PE and 7-AAD, following manufacturer’s
instructions, and analyzed on a Guava easyCyte 5HT flow
cytometer (Guava NexinrSoftware module, Millipore),
Frontiers in Aging Neuroscience | www.frontiersin.org 3August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
as previously described (Barateiro et al., 2012). The three
populations of cells that can be distinguished by this assay are
the viable cells (annexin V-PE and 7-AAD negative), the early
apoptotic cells (annexin V-PE positive and 7-AAD negative),
and the cells in late stages of apoptosis or dead cells (annexin
V-PE and 7-AAD positive).
Cell Morphological Analysis
For morphological analysis, cells were fixed for 20 min with
freshly prepared 4% (w/v) paraformaldehyde in phosphate buffer
saline (PBS), and stained with a primary antibody against
Ionized calcium binding adaptor molecule 1 (Iba1) (rabbit, 1:250;
#019-19741, Wako Pure Chemical Industries Ltd, Osaka, Japan),
and a secondary antibody Alexa Fluor 594 goat anti-rabbit
(1:1000; #R37117, Invitrogen Corporation, Carlsbad, CA, USA).
To identify the total number of cells, microglial nuclei were
stained with Hoechst 33258 dye (Sigma Chemical Co., St. Louis,
MO, USA). Fluorescence was visualized using an AxioCam
HRm camera adapted to an AxioSkopermicroscope (Zeiss,
Germany). Pairs of U.V. and red-fluorescence images of 10
random microscopic fields (original magnification: 400×) were
acquired per sample. To characterize microglia morphology we
used the particle measurement feature in ImageJ (1.47v, USA)
to automatically obtain the 2D area, perimeter, circularity, and
Feret’s diameter of single microglia. Circularity of microglia was
obtained by the formula: Circularity = 4π(area/perimeter2).
A circularity value of 1.0 indicates a perfectly circular cell,
and values near zero indicate elongated and ramified microglia.
Feret’s (maximum) diameter, a measure of cell length, is the
highest distance between any two points along the cell perimeter.
Evaluation of MMP2 and MMP9 Activities
Assessment of MMP2 and MMP9 activities in the extracellular
medium was based on their ability to degrade gelatin. For
that, 20 µl of incubation medium was resolved using a
SDS-PAGE zymography of 0.1% gelatin—10% acrylamide gel.
After electrophoresis, gels were washed for 1 h with 2.5%
TritonX-100 (in 50 nM CaCl2; 1 µM ZnCl2) to remove SDS and
renature MMP species in the gel. To allow gelatin degradation
by MMPs, gels were incubated overnight, at 37◦C, in the
developing buffer (50 mM Tris pH 7.4; 5 mM CaCl2; 1 µM
ZnCl2). For enzyme activity analysis, the gels were stained
with 0.5% Coomassie Brilliant Blue R-250 and distained in
30% ethanol/10% acetic acid/H2O. Gelatinase activity, detected
as a white band on a blue background, was measured by
computerized image analysis (Image LabTM Software 3.0,
Bio-Rad Laboratories Inc., Grand Junction, CO, USA) and
normalized to cellular protein content (Silva et al., 2010).
Activities of MMP2 and MMP9 were distinguished thanks to
their different relative molecular weight, i.e., MMP2 of 72 kDa
and MMP9 of 92 kDa (Frankowski et al., 2012).
Assessment of Microglia Autophagy
Autophagy was determined by immunocytochemistry based
on the punctate pattern of the microtubule-associated-protein-
light-chain-3 (LC3) and Western Blot detection of Beclin-1
bands, as previously described (Caldeira et al., 2014). For
immunocytochemistry, cells were fixed as above, and we
used rabbit anti-LC3 protein (1:500; #2775S, Cell Signaling
Technology Inc., Danvers, MA, USA) as a primary antibody,
and Alexa Fluor 488 goat anti-rabbit (1:1000; #A-11034,
Invitrogen Corporation) as the secondary one. Nuclei
were stained with Hoechst 33258 dye. Fluorescence was
visualized and images acquired as above mentioned. Increased
LC3 autophagosome puncta indicates induced autophagy. For
Western Blot, cell extracts were separated in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred to
a nitrocellulose membrane. Immunoblots were performed
as usual in our lab using as primary antibodies mouse anti-
Beclin-1 (1:500; #MABC34, MerckMillipore) and mouse
anti-β-actin (1:5000; #A5441, Sigma), followed by respective
secondary horseradish peroxidase-labeled antibodies. Results
were normalized to β-actin expression and expressed as fold vs.
2 DIV non-treated (control) cells.
Determination of Microglia Senescence
Microglial senescence was determined using the Cellular
senescence assay kit (Millipore) that evaluates the activity of
senescence-associated β-galactosidase (SA-β-gal), according
to manufacturer instructions. Microglial nuclei were
counterstained with hematoxylin. Light microscopy images
of 10 random microscopic fields (original magnification: 400×)
were acquired per sample using a Leica DC 100 camera (Leica,
Wetzlar, Germany) adapted to an Axioskop microscope (Zeiss).
Turquoise blue stained microglia were considered as senescent
cells and their percentage calculated relatively to the total
number of cells. Protein expression of ferritin was performed
by Western Blot as above, using the primary antibody rabbit
anti-FHT1 (1:500, #4393, Cell Signaling Technology Inc.,
Danvers, MA, USA).
Microglia Migration Assessment
Cell migration is often assessed with the classic Boyden Chamber
assay, where cells loaded in the upper well are allowed to migrate
through filter pores to the lower compartment of the chamber.
Assays were performed in a 48-well microchemotaxis Boyden
chamber (Neuro Probe, Gaithersburg, MD, USA), as previously
described (Miller and Stella, 2009), with minor modifications.
The bottom wells were filled with control medium (DMEM-
Ham’s F12) and Aβ(1000 nM) to evaluate the ability of microglia
to move towards Aβ. ATP (10 µM) applied in the lower well was
also used as a positive control for microglia migration, since it
is a known chemoattractant for microglia. The 8 µm diameter
polycarbonate membranes with polyvinylpyrrolidone (PVP)
surface treatment was equilibrated in control medium and after
chamber set up, 50 µl of cell suspension containing 2 ×104was
added to each top well. After 6 h incubation in a CO2incubator
at 37◦C to allow microglia migration, membrane was fixed with
ice-cold methanol and cells stained with 10% Giemsa (Sigma)
in PBS. Non-migrated cells on the upper side of the membrane
were wiped off with a filter wiper. The rate of migration was
determined by counting the cells on the lower membrane surface,
using 10 microscopic fields (original magnification: 100×).
Images were acquired with a Leica DFC490 camera adapted to an
Frontiers in Aging Neuroscience | www.frontiersin.org 4August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
AxioSkope HBO50 microscope. For each experiment, data from
at least three wells per condition were acquired.
Evaluation of Microglia Phagocytic Ability
The efficiency of the microglial phagocytosis was assessed by
counting the number of ingested beads per cell, considering the
total number of cells, to obtain the average amount of ingested
beads per cell, as well as by the percentage of cells phagocytosing
less than 5, 5–10, or more than 10 beads. The method consists
in incubating the primary microglial cultures, differentially aged
in culture with 0.0025% (w/w) of 1 µm fluorescent latex beads
(Sigma) for 75 min at 37◦C. Thereafter, cells were fixed with
freshly prepared 4% (w/v) paraformaldehyde in PBS. Microglia
were stained for Iba1 and nuclei counterstained with Hoechst
33258 dye. Fluorescence was visualized and acquired as above
mentioned.
Determination of Aβin Cells and Lysates
Aβin cell lysates was determined by Western Blot using
anti-Aβclone W0-2 (1:500, #MABN10, MerkMillipore) as the
primary antibody. Extracellular deposition of Aβwas observed
by immunocytochemistry using antibodies against Iba1 to detect
microglia cell body and Aβclone W0-2 for amyloid deposits.
Nuclei were counterstained with Hoechst 33258 dye.
Detection of Specific miRNA Expression
Changes
To evaluate changes in miRNAs with a crucial role in
microglia function/dysfunction and polarization, we assessed the
expression of miR-124, miR-155 and miR-146a by quantitative
realtime PCR (qRT-PCR). Total RNA was extracted from
primary microglia cultures using the miRCURYTM Isolation
Kit-Cells (Exiqon, Denmark), according to the manufacturer’s
recommendations for cultured cells. Briefly, after cell lysis, the
total RNA was adsorbed to a silica matrix, washed with the
recommended buffers and eluted with 35 µl RNase-free water by
centrifugation. After RNA quantification, conversion to cDNA
was performed using the universal cDNA Synthesis Kit (Exiqon)
and 20 ng total RNA according to the following protocol:
60 min at 42◦C followed by heat-inactivation of the reverse
transcriptase for 5 min at 95◦C. qRT-PCR was performed in an
7300 Real time PCR System (Applied Biosystems, Madrid, Spain)
using 96-well plates. For miRNA quantification the miRCURY
LNATM Universal RT microRNA PCR system (Exiqon) was
used in combination with pre-designed primers (Exiqon), which
are represented in Supplementary Table S1 (Supplementary
Material), using SNORD110 as reference gene. The reaction
conditions consisted of polymerase activation/denaturation and
well-factor determination at 95◦C for 10 min, followed by
50 amplification cycles at 95◦C for 10 s and 60◦C for 1 min
(ramp-rate 1.6◦/s). The miRNA fold increase/decrease with
respect to control samples was determined by the Pfaffl method,
taking into consideration different amplification efficiencies of
miRNAs in all experiments. The amplification efficiency for each
target was determined according to the formula: E= 10(−1/S)−1,
where S is the slope of the obtained standard curve.
Gene Expression Profiling
qRT-PCR was performed for mRNA expression, as usual in our
laboratory (Barateiro et al., 2013). Total RNA was extracted from
microglia using TRIzolr(Life Technologies, Inc., Grand Islands,
USA), according to manufacturer’s instructions. Total RNA
was quantified using Nanodrop ND-100 Spectrophotometer
(NanoDrop Technologies, Wilmington, DE, USA). Aliquots of
0.5 µg of total RNA were treated with DNase I and then
reverse transcribed to produce cDNA using oligo-dT primers and
SuperScript II Reverse Transcriptase under the recommended
conditions. qRT-PCR was performed on a 7300 Real time
PCR System (Applied Biosystems) using a SYBR Green qPCR
Master Mix (Fermentas, Ontario, Canada), and β-actin as an
endogenous control to normalize the expression level of the
different genes. Primer sequences that were used are indicated
in Supplementary Table S2 (Supplementary Material). PCR
was performed in 96 well plates and triplicate analysis was
accomplished for each sample. No-template control was included
for each amplificate. Cycling conditions were 94◦C for 3 min
followed by 40 cycles at 94◦C for 0.15 min, 62◦C for 0.2 min
and 72◦C for 0.15 min. A melt-curve analysis was used to
verify the specificity of the amplification, immediately after
the amplification protocol. Non-specific products of PCR were
not found in any case. Relative mRNA concentrations were
calculated using the Pfaffl modification of the ∆∆CT equation
(CT, cycle number at which fluorescence passes the threshold
level of detection), considering the efficiencies of individual
genes. The results were normalized to β-actin in the same sample,
and the initial amount of the template of each sample determined
as relative expression by the formula 2-∆∆CT. ∆CT in each
sample derives from the difference between the mean CT value
of each gene and the mean CT value of β-actin. ∆∆CT of
one sample is the difference between its ∆CT value and ∆CT
of the selected reference, in our case the 2 DIV non-treated
(control) cells.
Identification of CD11b and CD86 Microglia
Populations by Flow Cytometry
Cells were resuspended in PBS and kept in the flow buffer (PBS
plus 2% FBS and 0.02% sodium azide). To prevent non-specific
binding, cells were incubated for 20 min with CD16/CD32
(1:100) to block Fc receptors, at 4◦C. Afterwards, cell suspension
was incubated with the fluorescent labeled antibodies (CD11b
PerCp-Cy5, CD45 PE and CD86 Bio-SAV PE) for 30 min,
at 4◦C (1:100). Following the incubation, cells were washed
with the flow buffer, incubated with streptavidin (1:100) for
the CD86 Bio-SAV PE antibody during 30 min, and then
resuspended in the flow buffer. Expression of surface antigens
was evaluated using the BD FACSCalibur flow cytometer (Becton
Dickinson, San Jose, CA, USA) and data analyzed using the
FlowJo software.
Statistical Analysis
Results of at least four different experiments are expressed as
mean ±SEM. Significant differences between control and Aβ
treated groups were determined by t-test. To compare the effects
of Aβtreatment and microglia differentially aged in culture,
Frontiers in Aging Neuroscience | www.frontiersin.org 5August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
two-way analysis of variance (ANOVA) was performed using
GraphPad Prismr5.0 (GraphPad Software, San Diego, CA,
USA). Statistical significance was considered for p<0.05.
RESULTS
Microglia Treated with Aβdo Not Show
Age-Dependent Changes in Cell Viability
In the present work we used our established model of reactive
and aged-like microglia phenotypes, in which the microglial
cells are separated from mixed cultures with astrocytes and
maintained for 2 DIV and 16 DIV in culture, respectively
(Caldeira et al., 2014). Our main interest was to see whether
activated/young cells would respond differently from the aged
cells to Aβ. For that, we decided to incubate the cells with
a mixture of Aβoligomers and fibrils, as previously used in
the N9 microglial cell line (Falcão et al., 2017), to recapitulate
the neuropathology of AD associated to the different activation
processes of microglia by such species (Sondag et al., 2009).
Although we have tested 50 nM and 1000 nM Aβconcentrations,
as in our earlier study (Falcão et al., 2017), the effects obtained
were more reliable for the highest level, reason why we decided
to only proceed with Aβ1000 nM for a 24 h treatment
period.
As a first step, and in order to guarantee that the viability of
the aged-cultured microglia (16 DIV) was equivalent to that of
the acutely (2 DIV) isolated cells, we assessed the percentage of
viable, early-apoptotic and late-apoptotic/necrotic cells by flow
cytometry in both adherent and detached cells, as described
in ‘‘Materials and Methods’’ Section. As depicted in Table 1,
although a slight increase was observed in the number of cells
showing late-apoptosis/necrosis upon treatment with Aβ, namely
in the 16 DIV microglia, the lack of significance of such effects
point out no direct influence of cell viability differences on the
events presented in the following sections.
Young and Aged Cultured Microglia Show
Soma Enlargement by Aβtreatment
Characterization of microglia morphometric features has been
associated to cell polarization and activation state (Torres-Platas
et al., 2014). While the ramified morphology relates with the
surveilling cell, the amoeboid microglia are associated with
activation and believed to favor phagocytosis and mobility (Lull
and Block, 2010).
The cells cultured for 2 DIV (young/reactive) showed a
predominant amoeboid morphology, resulting from the acutely
isolation protocol that causes the activation of microglia
(Figure 1A), as previously demonstrated (Caldeira et al.,
2014). When aged in culture for 16 DIV, the cells exhibited
polarized and ramified populations, including rod-like and
bipolar morphologies, determining increased cell perimeter and
Feret’s maximum diameter (1.6-fold, p<0.05; Figures 1B,C).
Curiously, while Aβtreatment of 2 DIV microglia promoted
a prevalent ovoid shape with an enlarged cell area (1.9-fold,
p<0.05; Figure 1D), that of 16 DIV led to heterogeneous
morphologies, with polarized microglia bearing one and two
large processes, or determined a large lamellipodia with a
thin process. In this case the cells showed a reduction in
cell perimeter (0.7-fold, p<0.05) and in Feret’s maximum
diameter (interaction between DIV and Aβtreatment of F(4.74)
and F(5.27), respectively, p<0.05), as well as an increased
circularity (1.2-fold, p<0.05; interaction between DIV and Aβ
treatment of F(5.14),p<0.05; Figure 1E). These morphometric
alterations suggest that both young and aged cells suffer an
increase in soma volume, although the process shortening after
Aβtreatment was less notorious in aged cells. To what extent
these changes represent an equally activated microglia or a
different functional state will be examined in the following
sections.
AβDiversely Activates MMP2 and MMP9 in
Reactive and Aged Cultured Microglia
MMPs were shown to be important for Aβdegradation (Qiu
et al., 1997), and MMP3, MMP12 and MMP13 to be activated
by Aβ(Ito et al., 2007). Intriguingly, MMP2 and MMP9 revealed
to be differently activated in diverse experimental and animal
models, as well as in AD patients, and to be related with the
aggravation of AD disease (Brkic et al., 2015). We observed
that Aβtriggered an increased activation of both MMP2 and
MMP9 in the aged cells (2.4- and 1.5-fold, p<0.01 and p<0.05,
respectively; interaction between DIV and Aβtreatment for
MMP2 F(4.39),p<0.05), while only stimulated MMP9 in the
young reactive microglia (1.7-fold, p<0.05; Figure 2). Data
suggest that aged microglia may use these MMPs to degrade Aβ
and inhibit its accumulation. However, the dual roles of MMPs
complicate the understanding of the significance of such results
relatively to their potential beneficial or harmful effects in AD
(Wang et al., 2014).
TABLE 1 | Microglia viability is not altered by amyloid-β(Aβ) treatment.
2 DIV 16 DIV
Control AβControl Aβ
Viable 73.6 (±9.1) 65.6 (±6.7) 70.7 (±7.1) 66.5 (±5.7)
Early-apoptosis 18.8 (±8.6) 17.8 (±4.8) 21.7 (±8.1) 21.6 (±5.3)
Late-apoptosis/necrosis 2.9 (±1.4) 3.7 (±1.4) 5.8 (±1.0) 9.1 (±2.4)
Results are expressed as percentage of total number of cells. Values are mean ±SEM from at least four independent experiments. Microglial cells were kept in culture
for 2 days in vitro (DIV) and 16 DIV and treated with Aβat 1000 nM for 24 h. The percentage of viable microglia and microglia in early- and late-apoptosis/necrosis was
determined by flow cytometer with phycoerythrin-conjugated annexin V (annexin V-PE) and 7-amino-actinomycin D (7-AAD). The three populations were distinguished
as follows: viable cells (annexin V-PE and 7-AAD negative), early apoptotic cells (annexin V-PE positive and 7-AAD negative), and late stages of apoptosis or dead cells
(annexin V-PE and 7-AAD positive).
Frontiers in Aging Neuroscience | www.frontiersin.org 6August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
FIGURE 1 | Reactive and aged cultured microglia display soma enlargement and amoeboid morphology after treatment with amyloid-β(Aβ) peptide. Microglia that
were kept in culture for 2 and 16 days in vitro (DIV) were treated with 1000 nM Aβfor 24 h. Cells were immunostained for Iba1 and characterized for their
morphometric features. (A) Representative images show increased ramification by age, which was counteracted by Aβexposure. Microglia perimeter (B), Feret’s
diameter (C), area (D) and circularity values (E) were measured using ImageJ software and expressed in graph bars as mean ±SEM. Cultures, n= 4 per group.
Two-way analysis of variance (ANOVA; Post hoc Bonferroni test): ∗p<0.05 and ∗∗p<0.01 vs. respective non-treated Control; †p<0.05 vs. 2 DIV cells; (B)
DIV ×Aβinteraction F(4.74),p<0.05; (C) DIV ×Aβinteraction F(5.27),p<0.05; (E) DIV ×Aβinteraction F(5.14),p<0.05. Scale bar equals 50 µm.
AβInduces Age-Dependent Changes in
Autophagy-Related Beclin-l Gene and
LC3 Puncta
Autophagy, or cellular self-digestion, is a highly regulated
and evolutionarily conserved process that was shown to be
impaired in AD (Zare-Shahabadi et al., 2015). We first evaluated
microglia autophagic capacity by assessing the expression of
Beclin-1, a protein known to be recruited to phagosomal
membranes, and to participate in the early stages of autophagy
and LC3-associated phagocytosis (Chifenti et al., 2013). We
Frontiers in Aging Neuroscience | www.frontiersin.org 7August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
FIGURE 2 | Release of matrix metalloproteinase 2 (MMP2) and MMP9 is
differently induced by amyloid-β(Aβ) peptide in reactive and aged cultured
microglia. Microglia that were kept in culture for 2 and 16 days in vitro (DIV)
were treated with 1000 nM Aβfor 24 h. Activities of MMP2 and MMP9 were
evaluated by gelatin zymography. (A) Representative images of zymography
gels. (B) Results are expressed in graph bars as mean ±SEM. Cultures,
n= 4 per group. Two-way ANOVA (Post hoc Bonferroni test): ∗p<0.05 and
∗∗p<0.01 vs. respective non-treated Control; ††p<0.01 vs. 2 DIV;
(B) DIV ×Aβinteraction F(4.39),p<0.05.
observed that Beclin-1 was upregulated by Aβin young/activated
2 DIV cells (1.6-fold, p<0.05, Figures 3A,B), while was
downregulated in aged 16 DIV cells and only suffered a
slight and not significant increase upon Aβstimulation. Based
on the importance of LC3 processing for autophagosome
formation, we next determined LC3-positive puncta indicative
of such formation/accumulation (Klionsky et al., 2016). As
shown Figures 3C,D, Aβonly slightly increased the number
of 2 DIV cells presenting LC3-positive puncta. Conversely,
reduced autophagy in aged cells was upregulated under Aβ
treatment to values closely resembling those of 2 DIV cells.
Together, these results suggest that Aβpromotes the formation
of autophagosomes, which turnover may be impaired in aged
microglia and contribute to Aβaccumulation within the cells.
In addition, Aβswitches the reactive 2 DIV microglia towards
a senescent-like cell phenotype, perhaps compromising the
response to stressors.
AβUpregulates Senescence-Associated
Biomarkers in 2 DIV Microglia Towards
Values of 16 DIV Cells
Since Beclin-1 levels were shown to decline by aging (Shibata
et al., 2006), we next assessed the cells that positively stained for
SA-β-gal, a biomarker of cellular senescence (Sikora et al., 2011).
As shown in Figures 4A,B, treatment of young/reactive microglia
with Aβincreased the number of SA-β-gal positive cells (2.1-fold,
p<0.05) to values near to those of 16 DIV aged cells (interaction
between DIV and Aβtreatment F(28.1),p<0.01), independently
of being treated or not with Aβ.
Ferritin was found most related with proliferative microglia
in AD hippocampus from patients (Grundke-Iqbal et al., 1990).
However, a subpopulation of dystrophic microglia were also
shown to be positive for ferritin (Lopes et al., 2008). Indeed,
we have observed that both 2 DIV and 16 DIV Aβ-treated
microglia showed increased levels of ferritin, although the
expression was less predominant in the aged cells (data not
shown). Other studies also observed a decrease in ferritin
accumulation with age in substantia nigra (Walker et al., 2016),
which was referred to compromise cell resistance to reactive
oxygen species (ROS) (Yang et al., 2013). To further assess
if Aβinduces a senescent-like response in 2 DIV microglial
cells, we decided to evaluate miR-146a expression in the
differently in vitro aged microglial cells. Actually, besides its
numerous described functions and targets (Cardoso et al., 2016),
miR-146a was reported to contribute to age-related dysfunction
of macrophages (Jiang et al., 2012), and to loss of mitochondrial
integrity and function in aged cells (Rippo et al., 2014). As
anticipated, miR-146a increased expression was observed in
the 2 DIV microglia treated with Aβ, although not reaching
the values of 16 DIV cells (Figure 4C). Overall, Aβswitches
the reactive 2 DIV microglia towards a senescent-like cell
phenotype with potential negative consequences to a stress
response.
AβImpairs Microglia Migration Ability in
the Aged Cultured Cells
Microglia important functions in the CNS include migration
dynamics, synaptic pruning and phagocytosis of neuronal cells
and their debris (Xavier et al., 2014; Zhang et al., 2016).
Cell migration can be triggered by several chemoattractants,
including ATP that when released by damaged neurons acts
on P2Y12 and P2X4receptors in microglia stimulating their
migration (Miller and Stella, 2009). Our data showed that
young microglia exhibited higher migration ability than the
older cells (Figure 5) and that, in contrast with those, positively
respond to Aβ(1.7-fold, p<0.01) and ATP chemotactic
signals (2.0-fold, p<0.01). These findings besides indicating
that aged cells are in a dormancy-like state relatively to their
capacity of migration, when compared with the 2 DIV cells,
also demonstrate their unresponsiveness to chemoattractants,
Frontiers in Aging Neuroscience | www.frontiersin.org 8August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
FIGURE 3 | Autophagy is differently promoted by amyloid-β(Aβ) peptide in
reactive and aged cultured microglia. Microglia that were kept in culture for
2 and 16 days in vitro (DIV) were treated with 1000 nM Aβfor 24 h. Total cell
lysates were analyzed for the presence of Beclin 1. (A) Representative images
of Beclin 1 protein expression. (B) Results of densitometric analysis of Beclin
1 blots are expressed in graph bars as mean ±SEM. Microtubule-associated-
protein-light-chain-3 (LC3)-positive puncta cells were detected by
immunostaining for LC3. (C) Representative images of immunocytochemistry
for LC3 (green) and nuclei staining (blue). Scale bar equals 50 µm.
(D) Percentage of cells showing LC3-positive puncta are expressed in graph
bars as mean ±SEM. Cultures, n= 4 per group. Two-way ANOVA (Post hoc
Bonferroni test): ∗p<0.05 vs. respective non-treated Control; †p<0.05 and
††p<0.01 vs. 2 DIV.
including Aβ, that may further compromise the phagocytic
activity of 16 DIV microglia.
AβImpairs Microglia Phagocytic Ability
Mainly in the Reactive Cultured Microglia
Microglia phagocytosis is an important protective role for the
efficient elimination of apoptotic cells and for neuronal circuitry
reshape (Xavier et al., 2014). Our results corroborate previous
data demonstrating a reduced phagocytosis by in vitro aged
microglia (Caldeira et al., 2014). Here, we showed that 24 h
incubation of reactive microglia with Aβled to 2-fold reduction
(p<0.01) in the number of phagocytosed beads per cell,
approaching the values obtained for 16 DIV cells, treated or
not with Aβ, as depicted in Figures 6A,B. In addition, the
number of 2 DIV cells able to engulf more than 10 beads was
dramatically decreased upon Aβinteraction (10-fold reduction,
p<0.01, Figure 6C). Interestingly, the diminished number of
16 DIV cells phagocytosing 5–10 beads decreased even more
upon Aβtreatment. These results suggest that Aβreduces 2 DIV
microglia phagocytosis towards the levels of aged/unresponsive
microglia. We may conclude that despite the ability to migrate
to sites of Aβdeposition the young/activated microglia lose
their phagocytic capacity when facing Aβ, at least in primary
cultures.
Phagocytosis may be mediated by several pathways. We next
decided to explore milk fat globule factor-EGF factor 8 protein
(MFG-E8) expression, a key factor involved in the phagocytosis
of apoptotic cells, such as neurons (Fuller and Van Eldik, 2008;
Liu et al., 2013). Following the same protocol as above, we
tested whether Aβinterfered with the expression of MFG-E8 by
2 DIV and 16 DIV microglia. Interestingly, we observed that
the levels of MFG-E8 suffered more than a 2-fold reduction
(p<0.01) in the 2 DIV microglia (Figure 6D). As before, the aged
cells showed downregulated expression of this phagocytic-related
protein, which was sustained in the presence of Aβ(significant
interaction between DIV and Aβtreatment F(13.92),p<0.01),
thus reinforcing their unresponsive nature. To next assess the
expression of cell membrane surface receptors associated to
phagocytosis, we evaluated the triggering receptor expressed
on myeloid cells 2 (TREM2) and the type 1 transmembrane
protein CD33. Both are members of the sialic acid-binding
immunoglobulin-like lectins (Siglecs) and are expressed by
immune cells. TREM2 is involved in the phagocytosis of
damaged cells and showed to reduce the inflammatory response
(Walter, 2016). Most attractively, heterozygous rare variants
in TREM2 have been associated with a significant increase
in the risk of AD emergence (Guerreiro et al., 2013), while
TREM2 deficiency in an AD animal model, the 5xFAD, increased
cerebral Aβaccumulation (Wang Y. et al., 2015). Expression
of TREM2 mRNA was markedly increased in 2 DIV microglia
upon Aβexposure (5.2-fold, p<0.05), as depicted in Figure 6D.
However, unchanged values upon Aβtreatment were observed in
16 DIV microglia.
CD33 gene is considered a risk factor for AD and increased
number of CD33-immunoreactive microglia were shown to
correlate with insoluble Aβ42 levels and plaque burden in
AD brain (Griciuc et al., 2013). Actually, increased expression
Frontiers in Aging Neuroscience | www.frontiersin.org 9August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
FIGURE 4 | Amyloid-β(Aβ) peptide promotes cell senescence in reactive
cultured microglia. Microglia that were kept in culture for 2 and 16 days in vitro
(DIV) were treated with 1000 nM Aβfor 24 h. Activity of
senescence-associated β-galactosidase (SA-β-gal) was determined using a
commercial kit, and SA-β-gal-positive cells were counted. (A) Representative
images of cells showing blue turquoise SA-β-gal staining. Scale bar equals
20 µm. (B) Percentage of cells showing SA-β-gal-positive staining are
expressed in graph bars as mean ±SEM. (C) MicroRNA (miR)-146a
expression was evaluated by Real-Time PCR. Results are expressed in graph
bars as mean ±SEM. Cultures, n= 4 per group. Two-way ANOVA (Post hoc
Bonferroni test): ∗p<0.05 and ∗∗p<0.01 vs. respective non-treated Control;
††p<0.01 vs. 2 DIV cells; (B) DIV ×Aβinteraction F(28.1),p<0.01.
of CD33 determines an impairment in microglia-mediated
clearance of Aβ(Jiang et al., 2014). Our data revealed an
increased expression of CD33 in 2 DIV microglia (1.7-fold,
p<0.05), which was intensified in 16 DIV cells (2.8-fold,
p<0.01; Figure 6D), thus reinforcing previous results showing
a decreased microglial phagocytic ability towards Aβspecies.
To further explore differences in Aβphagocytosis
between young/reactive and aged/unresponsive microglia,
we evaluated Aβspecies in cell lysates. As depicted in
Supplementary Figure S1A, the 2 DIV microglia revealed a
higher content of Aβ, namely of dimers and monomers, as
compared to 16 DIV microglia that seem to contain increased
oligomers. Aβ-immunostaining (Supplementary Figure S1B)
confirmed the increased uptake of this peptide by young
microglia. Although also detected in 16 DIV/aged microglia,
the majority of deposits revealed to be localized outside
the cells. Overall, our results indicate that young/reactive
microglia attempt to phagocytose Aβ, based on the elevation of
TREM2 expression, but that increased CD33 expression may
counteract this feature. Deposits of Aβsurrounding the 16 DIV
cells and marked expression of CD33 in 16 DIV microglia
confirm the low ability of these cells to phagocytose Aβ.
AβReduces the Expression of
Inflammatory-Related miR-155 and
miR-124 in 2 DIV Microglia
Recent studies indicate that miR-155 and miR-124a regulate
T-cell functions during inflammation (Heyn et al., 2016). Both
miRNAs are directly involved in microglia polarization,
where miR-124 is considered to be associated with an
anti-inflammatory M2 phenotype, and miR-155 as having
a determinant role in microglia activation toward the
M1 phenotype (Ponomarev et al., 2013). To gain insight
into the Aβ-induced alterations in microglia polarization we
assessed the expression of these inflammation-related miRNAs
in the 2 DIV and 16 DIV microglia (Figure 7). Interestingly,
while we obtained a downregulated expression of both miR-155
and miR-124 by Aβtreatment in the 2 DIV microglial cells
(0.7-fold and 0.6-fold, respectively, p<0.05), no changes were
observed in the aged cultured microglia, which basal levels were
already inferior to the 2 DIV control cells (∼0.5-fold, p<0.01)
attesting a dormancy-like behavior of such cells. These results
suggest that Aβcounteracts either M2 (low miR-124) or M1
(low miR-155) polarization in the 2 DIV microglia already
activated by the isolation procedure. Thus, presence of mixed
subpopulations and less responsive microglia subtypes following
Aβinteraction should be envisaged as a consequence of this
noxious stimulus.
16 DIV Cells Only React to AβStimulus by
Increasing the Expression of TNF-αand
IL-1β, while the 2 DIV Microglia Show a
Larger and More Intense Spectrum of
Activation
To determine whether 2 DIV and 16 DIV microglia, despite the
downregulation of miR-155 and miR-124 expression, were still
able to mount an inflammatory response upon Aβinsult, we
assessed common biomarkers of microglia activation. We started
Frontiers in Aging Neuroscience | www.frontiersin.org 10 August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
FIGURE 5 | Reactive cultured microglia show increased ability to migrate towards amyloid-β(Aβ) peptide and ATP, while aged microglia are immotile and
unresponsive to such chemoattractants. Microglia were kept in culture for 2 and 16 days in vitro (DIV) and cellular chemotactic migration to 1000 nM Aβand 10 µM
ATP (positive chemotactic control) was evaluated after 6 h incubation using the Boyden chamber method. (A) Representative images of cells that have migrated to
Aβand ATP. Scale bar equals 50 µm. (B) Number of migrated cells was counted and results are expressed in graph bars as mean ±SEM. Cultures, n= 4 per group.
Two-way ANOVA (Post hoc Bonferroni test): ∗∗p<0.01 vs. respective non-treated Control; ††p<0.01 vs. 2 DIV cells; (B) DIV ×Aβinteraction F(4.36),p<0.05.
by evaluating the gene expression of the first line cytokines
TNF-α, IL-1βand IL-6. We obtained a clear upregulation of all
these pro-inflammatory cytokines in the young cultured 2 DIV
cells (Figure 8A). The 16 DIV cells showed a 10-fold reduction
in the mRNA expression of TNF-α, IL-1βand IL-6, as compared
with the 2 DIV control cells (p<0.01). These cells, although
less markedly than the 2 DIV cells, reacted to Aβexposure
by significantly increasing TNF-αand IL-1βgene expression,
but not that of IL-6. Since we and others previously showed
that HMGB1 is released by LPS-treated N9 microglia (Cunha
et al., 2016) and promote the synthesis of pro-IL-1βand pro-
IL-18 (Jiang et al., 2012), as well as the activation of NLRP3-
inflammasome (Chi et al., 2015), we additionally explored these
signaling pathways in our differentially aged microglia model
treated with Aβ. In accordance with the previous results on
cytokines, we observed a net elevation of HMGB1 and IL-18 gene
expression (1.6- and 2.1-fold, respectively, p<0.05) in 2 DIV
microglia exposed to Aβ. However, no changes on NLRP3 were
observed. Again, significantly decreased levels were obtained for
all biomarkers in 16 DIV microglia (p<0.01), as compared with
2 DIV cells, which revealed to be almost unreactive to the Aβ
stimulus (Figure 8B).
Surface Toll-like receptors (TLRs) are abundantly expressed
in microglia and recruitment of TLR2 and TLR4 by HMGB1 and
IL-1βwas shown to amplify inflammation (Park et al., 2004;
Facci et al., 2014). In agreement with previous results, Aβ
enhanced the expression of both TLR2 and TLR4 in 2 DIV cells
(2.4- and 2.0-fold, p<0.05, respectively), but not in 16 DIV
Frontiers in Aging Neuroscience | www.frontiersin.org 11 August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
FIGURE 6 | Ability of microglia to phagocytose is reduced by amyloid-β(Aβ) peptide mainly in the reactive cultured cells. Microglia that were kept in culture for 2 and
16 days in vitro (DIV) were treated with 1000 nM Aβfor 24 h. Phagocytic capacity was assessed after 75 min incubation with fluorescent latex beads.
(A) Representative images of microglia immunostained for Iba1 (red) and stained with Hoechst for nuclei staining (blue) containing phagocytosed fluorescent latex
beads (green). Scale bar equals 50 µm. (B) Number of phagocytosed beads per cell and (C) number of microglial cells phagocytosing less than 5, 5–10 and more
than 10 beads was counted. (D) Expression of milk fat globule-EGF factor 8 protein (MFG-E8), triggering receptor expressed on myeloid cells 2 (TREM2) and CD33,
associated to microglial phagocytosis, was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ±SEM. Cultures, n= 4 per group. Two-way
ANOVA (Post hoc Bonferroni test): ∗p<0.05 and ∗∗p<0.01 vs. respective non-treated Control; †p<0.05 and ††p<0.01 vs. 2 DIV; (D) MFG-E8: DIV ×Aβ
interaction F(13.9),p<0.01.
microglia (interaction between DIV and Aβtreatment for
TLR2 F(5.29),p<0.05, Figures 9A,B), which again revealed
an already suppressed basal expression of these receptors. The
fractalkine/CX3C chemokine receptor 1 (CX3CR1) signaling
pathway was also previously demonstrated to modulate
microglial activation (Limatola and Ransohoff, 2014). As
documented for TLR2 and TLR4, a similar profile was
obtained for the CX3XR1 expression in 2 DIV (2.6-fold,
p<0.05) and 16 DIV aged microglia (Figure 9C). Data
reinforce the ability of 2 DIV cells to develop an effective
inflammatory response upon Aβexposure, and confirm the low
responsiveness of 16 DIV microglia in conformity with more
Frontiers in Aging Neuroscience | www.frontiersin.org 12 August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
FIGURE 7 | Amyloid-β(Aβ) peptide decreases the expression of miRNA
(miR)-155 and miR-124 in the reactive cultured microglia toward that of
16 days in vitro (DIV) cells. Microglia that were kept in culture for 2 and 16 DIV
were treated with 1000 nM Aβfor 24 h. (A) Expression of
M1/pro-inflammatory-related miR-155 and of (B) M2/anti-inflammatory-related
miR-124 was evaluated by Real-Time PCR. Results are expressed in graph
bars as mean ±SEM. Cultures, n= 4 per group. Two-way ANOVA (Post hoc
Bonferroni test); ∗p<0.05 vs. respective non-treated Control; †p<0.05 and
††p<0.01 vs. 2 DIV cells.
unresponsive/dormant and senescent-like phenotype of these
cells.
Imbalance of M1 and M2 Phenotypes in
Aβ-Treated 2 DIV and 16 DIV Microglia
Suggests the Formation of Different Cell
Subsets
From previous data, the elevation of IL-1βexpression,
mainly in 2 DIV microglia, indicates that the cell assumes
preferentially the M1 phenotype upon Aβexposure. However,
the CX3CR1 increased expression also suggests microglia
subclasses with the M2a polarization (Chhor et al., 2013).
Therefore, we decided to further characterize the microglia
phenotypes in 2 DIV and 16 DIV cells exposed to Aβby
evaluating additional M1 and M2 markers. For that we evaluated
the gene expression of inducible nitric oxide synthase (iNOS) and
of major histocompatibility (MHC) class II, which are considered
M1/pro-inflammatory microglia markers, although MHC class II
has also been attributed to M2b polarized macrophages (Roszer,
2015). As shown in Figure 10A, while iNOS was highly induced
by Aβin both young and aged microglia (4.7- and 5.4-fold for
2 and 16 DIV, p<0.05 and p<0.01, respectively), MHC class II
was particularly high in young cultured cells (11.4-fold, p<0.01,
interaction between DIV and AβF(4.53),p<0.05), indicating
a preferential M1 polarization in 2 DIV cells and a mixture
of phenotypes in 16 DIV microglia. Then, we characterized
M2/anti-inflammatory microglia markers, such as Arginase 1
(prevalent in M2a activation state) and transforming growth
factor β(TGFβ) (suggested to be increased in the M2a/M2c/M2d
subtypes (Roszer, 2015)). As observed in Figure 10B, Arginase
1, considered to be a repair/regenerative gene (Chhor et al.,
2013), was only increased by Aβin young cultured cells (2.6-fold,
p<0.05), with levels that, although slightly elevated upon Aβ,
represented in the 16 DIV cells less than 40% (p<0.01) of
those in 2 DIV cells. In what concerns TGFβexpression, with
neuroprotective and pro-survival properties (Dobolyi et al., 2012;
Ryu et al., 2012), both young and aged Aβ-treated cells showed an
increased expression (2.4- and 2.3-fold, respectively, p<0.05).
Overall, these results suggest that M1 and M2 subpopulations
are present upon Aβtreatment. However, while 2 DIV cells
mainly express M1 markers, a phenotypic dysregulation with
overlapping of microglial M1 and M2 markers is present in aged
microglia. These cells additionally showed a decreased ability
to mount an adequate inflammatory response when stressed
with Aβ.
Proportion of CD11b and CD86 Positive
Microglia Differs between 2 DIV and 16 DIV
Cells after Incubation with Aβ
To further understand whether the lower reactivity of 16 DIV
microglia towards Aβwas associated with an increased
expression of CD86, which was previously indicated to be
age-related (Kohman et al., 2013), we evaluated changes in
the proportion of the M1 markers CD11b+ (co-stimulatory
ligand) and CD86+ (integrin αM) cells, in our microglia aged
model of 2 DIV and 16 DIV after Aβstimulus, by flow
cytometry. As depicted in Figure 11A, the naïve aged microglia
showed a decreased number of CD11b+ cells when compared
to young/activated 2 DIV cells. In addition, these aged cells
had a more elevated number of CD11b−/CD86−cells (∼50%),
together with elevated proportions of mixed CD11b−/CD86+
and CD11b+/CD86+ populations, than those showed by 2 DIV
microglia, corroborating the aging-like profile status of 16 DIV
cells (Figures 11B,C, Supplementary Table S3). When treated
with Aβboth 2 DIV and 16 DIV cells showed a decreased
population of CD11b+ cells. While 2 DIV microglia shifted from
medium to high density in terms of CD11b−/CD86−cells,
the number of CD11b−/CD86+ in 16 DIV microglia increased
Frontiers in Aging Neuroscience | www.frontiersin.org 13 August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
FIGURE 8 | Increased expression of inflammatory mediators in microglia treated with amyloid-β(Aβ) peptide is more evident in the reactive cultured cells. Microglia
that were kept in culture for 2 and 16 days in vitro (DIV) were treated with 1000 nM Aβfor 24 h. (A) Expression of inflammatory cytokines [e.g., tumor necrosis
factor-α(TNF-α), interleukin (IL)-1βand IL-6] and of (B) inflammasome-related proteins [e.g., high-mobility group protein B1 (HMGB1), IL-18 and NOD-like receptor
family pyrin domain containing 3 (NLRP3)] were evaluated by Real-Time PCR. Results are expressed in graph bars as mean ±SEM. Cultures, n= 4 per group.
Two-way ANOVA (Post hoc Bonferroni test): ∗p<0.05 vs. respective non-treated Control; ††p<0.01 vs. 2 DIV cells.
4-fold (∼25%) upon Aβtreatment and represented a 24-fold
increase relatively to their 2 DIV counterparts (p<0.01). No
relevant changes were noticed when we assessed the expression
of CD45 (data not shown). These results highlight that in vitro
aging reduces CD11b+ microglia reactivity to Aβ. Increased
CD86 signaling, namely in the presence of Aβ, further suggests
the existence of M2b microglia with pro- and anti-inflammatory
functions and a gain of function for co-stimulating other immune
cells.
DISCUSSION
In the present manuscript we assessed whether in vitro
aged microglia (Caldeira et al., 2014) and 2 DIV activated
microglia differently reacted to Aβstimulation, to better realize
the complexity of microglia activation and cell dysfunctional
processes in AD, as well as the relevance of immunosenescence
to AD emergence. Actually, since AD pathophysiology is overlaid
by the aging effects on the CNS, and microglia were shown to be
dysfunctional in aging and AD (Mosher and Wyss-Coray, 2014;
Cykowski et al., 2016), we aimed to recognize the relevance that
microglia diverse phenotypes may have along the progression of
the disease, and the role of subacute neuroinflammation in AD
pathogenesis.
We observed that several neuroprotective functions, namely
phagocytosis and migration abilities, as well as autophagy, were
impaired by in vitro aging, contributing to Aβdeposition. Aged
16 DIV cells showed lower ability to mount an Aβ-induced
inflammatory response with compromised expression of
inflammation-related miRNAs and CD11b marker, but
enhanced expression of the co-stimulatory CD86 molecule.
In addition, our data pointed toward Aβas a stressor-inducer
molecule of microglia senescence. This is not without precedent
since senescent astrocytes were shown to increase in human
Frontiers in Aging Neuroscience | www.frontiersin.org 14 August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
FIGURE 9 | Amyloid-β(Aβ) peptide upregulates the expression of Toll-like
receptor 2 (TLR2), TLR4 and fractalkine/CX3C chemokine receptor 1
(CX3CR1) in the reactive cultured microglia, but not in aged cells. Microglia
that were kept in culture for 2 and 16 days in vitro (DIV) were treated with
1000 nM Aβfor 24 h. Expression of TLR2 (A), TLR4 (B) and CX3CR1 (C) was
evaluated by Real-Time PCR. Results are expressed in graph bars as
mean ±SEM. Cultures, n= 4 per group. Two-way ANOVA (Post hoc
Bonferroni test): ∗p<0.05 vs. respective non-treated Control; ††p<0.01 vs.
2 DIV cells; (A) DIV ×Aβinteraction F(5.29),p<0.05.
brain during aging and AD (Bhat et al., 2012), and dystrophic
microglia was found in AD brain specimens (Streit et al.,
2009). Findings support that aged microglia have compromised
function and that Aβreduces microglia ability to fully develop
neuroprotective and inflammatory reaction against this noxious
stimulus.
We have previously shown that impaired cell function by
in vitro aging is not associated with loss of cell viability (Caldeira
et al., 2014). Similarly, we did not observe age-dependent changes
in cell death, either in the absence or in the presence of Aβ
treatment. These results further validate our in vitro differently
aged microglia model to evaluate perturbing effects by aging
and Aβ. Changes in microglia morphology are associated with
different functional states, where activation relates with larger
somata, shorter processes and amoeboid morphology (Harry,
2013). Aged microglia also revealed to be smaller, less branched
and less effective in mounting a normal response to injury.
These cells with dystrophic appearance and less capacity to
phagocytose and migrate, probably due to intracellular oxidative
stress, were reported to be senescent (Streit et al., 2008), and to
show increased ferritin immunoreactivity (Lopes et al., 2008).
We previously demonstrated that cells acutely isolated and
maintained for 2 DIV in culture behave as activated microglia,
while if maintained in culture for 16 DIV exhibit a more bipolar
shape and shorter large processes (Caldeira et al., 2014). Here,
aged cells showed a thin and elongated shape with altered nuclei
morphology. This type of cells, commonly called as rod cells, have
been associated to chronically inflamed cerebral cortex (Hof and
Mobbs, 2009) and acutely dementing processes (Graeber, 2010).
As expected, when microglia were exposed to Aβ, in particular
the 2 DIV cells, they acquired an amoeboid morphology, which
is a morphometric characteristic of reactive microglia (Nakajima
and Kohsaka, 2004). To note, however, that aged 16 DIV cells
additionally showed distinct microglia morphological subclasses,
as recently observed in the hippocampus of AD patients
(Bachstetter et al., 2015). The elevated ferritin levels we observed
in 2 DIV microglia reinforce activation by Aβand suggest a
putative defense mechanism against oxidative stress (Grundke-
Iqbal et al., 1990; Yang et al., 2013). We also identified moderated
accumulation of ferritin in aged cells, which is in accordance with
dystrophic (senescent) microglia (Lopes et al., 2008) and may
determine low resistance to ROS (Yang et al., 2013).
MMPs are important inflammatory components and their
activation was shown to be implicated in AD pathogenesis
(Wang et al., 2014). Besides their multiple roles in AD they are
considered important for Aβdegradation (Miners et al., 2011).
While 2 DIV cells secrete MMP9, but not MMP2, upon Aβ
treatment, aged cells release both. MMP2 is considered the major
protective gelatinase in AD and is overexpressed by astrocytes
surrounding senile plaques, whereas MMP9 expression has a
potential neurotoxic side and is described as a characteristic
feature of AD (Wang et al., 2014). Indeed, activation of MMP9,
but not of MMP2, was reported in serum and brain samples
of patients with mild cognitive impairment and with AD
(Lorenzl et al., 2008; Bruno et al., 2009). Increased release of
MMP2 by 16 DIV cells may imply enhanced ability to cleave
Aβ(Konnecke and Bechmann, 2013). Activation of MMP9 in
Frontiers in Aging Neuroscience | www.frontiersin.org 15 August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
FIGURE 10 | Mixed representation of M1/pro-inflammatory and M2/anti-inflammatory polarization markers in 2 and 16 days in vitro (DIV) microglia treated with
amyloid-β(Aβ) peptide suggests the presence of different cell subsets. Microglia that were kept in culture for 2 and 16 DIV were treated with 1000 nM Aβfor 24 h.
(A) Expression of M1/pro-inflammatory [e.g., inducible nitric oxide synthase (iNOS) and major histocompatibility (MHC) class II] and of (B) M2/anti-inflammatory [e.g.,
Arginase and transforming growth factor β(TGFβ)] was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ±SEM. Cultures, n= 4 per
group. Two-way ANOVA (Post hoc Bonferroni test): ∗p<0.05 and ∗∗p<0.01 vs. respective non-treated Control; †p<0.05 and ††p<0.01 vs. 2 DIV cells; (A) MHC
class II: DIV ×Aβinteraction F(4.53),p<0.05.
both differentially aged cells may also disturb blood-brain barrier
dynamic properties (Turner and Sharp, 2016), critically affecting
brain Aβhomeostasis and its trans-endothelial transport and
clearance (Provias and Jeynes, 2014).
Microglia migration is essential for many pathophysiological
processes and a feature of the activated cell (Kettenmann et al.,
2011). As in our previous study (Caldeira et al., 2014), aged
microglia was unresponsive to ATP-induced chemotactic signals.
In ex vivo retinal explants from aged mice (18–24 months
of age), microglia process motility was reduced relatively to
young adult animal cells (2–3 months of age; Damani et al.,
2011). Interestingly, intranasally and intravenously administered
microglia to mice migrate to brain in young and aged
recipients, if derived from young, but not from aged donors
(Leovsky et al., 2015). Such findings sustain microglia migration
impairment with age. Again, young microglia, but not aged
cells, were shown to migrate towards Aβ, following stimulation
of ATP release by fibrillar and oligomeric Aβ1–42 species (Kim
et al., 2012). Actually, microglial-mediated clearance of tissue
debris was demonstrated to decay with aging (Neumann et al.,
2009), to be compromised in older AD animal models (Njie
et al., 2012), and to be associated with immunosenescence
(Li, 2013). Functional impairment of microglia leading to
phagocytic capacity decline was shown to coincide with Aβ
deposition in a mice model of AD (Krabbe et al., 2013).
In our in vitro aging microglia model the phagocytic ability
of 2 DIV cells was decreased by Aβ, either in the number
of beads per cell or maximum amount in each cell. Values
Frontiers in Aging Neuroscience | www.frontiersin.org 16 August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
FIGURE 11 | Cell aging and treatment with amyloid-β(Aβ) peptide decrease the population of microglia CD11b+ cells, while increase the number of CD86+ cells.
Microglia that were kept in culture for 2 and 16 days in vitro (DIV) were treated with 1000 nM Aβfor 24 h. The population of CD11b+ and CD86+ cells was detected
by flow cytometry. (A) Analysis of microglia expressing CD11b. Results are expressed in graph bars as mean ±SEM. Cultures, n= 4 per group. Two-way ANOVA
(Post hoc Bonferroni test): ∗p<0.05 and ∗∗p<0.01 vs. respective non-treated Control; †p<0.05 vs. 2 DIV cells. (B) Representative flow cytogram of CD11b+ and
CD86+ cells in reactive (2 DIV) and aged (16 DIV) cultured microglia. (C) Results are expressed in 2D pie graphs as mean. Cultures, n= 4 per group. Two-way
ANOVA (Post hoc Bonferroni test): ∗p<0.05 and ∗∗p<0.01 vs. respective non-treated Control; †p<0.05 and ††p<0.01 vs. 2 DIV cells.
obtained were at the same level of those presented by 16 DIV
cells, whose phagocytic dysfunction was not modified by Aβ.
Microglia phagocytosis is also related with the recognition of
phosphatidylserine receptors following docking of the MFG-E8
molecule (Li, 2012). Neuroprotective effects of MFG-E8 against
oligomeric Aβtoxicity were previously shown (Li et al., 2012),
although phagocytosis of viable neurons may also occur,
which is a disadvantage (Fricker et al., 2012). We were the
first demonstrating MFG-E8 downregulation due to age and
Aβin primary cultures of microglia, though others have
detected a reduced expression in AD patients (Boddaert et al.,
2007).
AD risk is modulated by genetic factors that influence
microglial activation. Most attractively, mutations in the Siglecs
TREM2 and CD33 have been distinctly associated with the
development of AD, and shown to act in opposing directions
relatively to microglial activation and AD risk; alleles that
inhibit TREM2 function increase AD risk, whereas alleles that
inhibit CD33 function reduce such risk (Malik et al., 2013).
TREM2 was indicated to support microgliosis (Zheng et al.,
2017) and its deficiency to attenuate microglia phagocytic activity
(Kawabori et al., 2015). Therefore, our results indicating an
elevated expression of TREM2 in Aβ-treated 2 DIV microglia,
but not in 16 DIV microglia, again point to a gain-of-function of
Frontiers in Aging Neuroscience | www.frontiersin.org 17 August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
the young cell relatively to the aged one. Furthermore, elevated
expression of CD33 was observed in microglia treated with
Aβ, mostly if aged. CD33 expression was reported to inhibit
uptake and clearance of Aβ1–42 in microglial cell cultures and
microglia immunoreactive for CD33 were shown to correlate
with insoluble Aβlevels and plaque burden in the AD brain
(Griciuc et al., 2013). In this sense, we observed that young cells
expressing elevated TREM2 had enhanced internalization of Aβ,
while aged cells with low TREM2 and upregulated CD33 showed
reduced intracellular Aβand increased number of extracellular
deposits, further corroborating their inability to clear Aβ.
Upregulation of TLR2, TLR4 and CX3CR1 in 2 DIV cells
upon treatment with Aβ, but not in 16 DIV cells, are in
line with higher neuroprotection by young cells than by older
ones. Increased expression of TLR2 and TLR4 was found in
AD human brains and suggested to require stimulation by Aβ
fibrils (Reed-Geaghan et al., 2009). Interestingly, TLR-signaling
was shown to link the autophagy pathway to phagocytosis
(Sanjuan et al., 2007) and to be involved in the clearance of
Aβdeposits (Trudler et al., 2010). Role of CX3CR1 signaling
in AD is still controversial. The ablation of CX3CR1 gene in a
rodent AD model increased cytokine levels and Tau pathology,
while also increased protofibrillar Aβphagocytosis (Merino
et al., 2016). However, CX3CR1 deficiency was associated
with aberrant microglial activation and AD-related cognitive
deficits (Cho et al., 2011). In another study, a protracted
reduction of CX3CR1 expression in aged microglia was observed
after lipopolysaccharide injection, together with incomplete
resolution of inflammation and delayed recovery from sickness
behavior (Wynne et al., 2010). Thus, the importance of CX3CR1
in AD is controversial and needs further clarification.
Altered TLRs/ligands interaction may derive from key
signaling modulator miRNAs, particularly the trio miR-155,
miR-21 ad miR-146a, during age-related changes of immune
system functions (Olivieri et al., 2013a). Studies support the
pivotal role of miRNAs in the regulation of microglial phenotype
by promoting microglial quiescence (miR-124), or by driving
microglial inflammatory and immune responses (miR-155 and
miR-146a) (Ponomarev et al., 2013). While miR-124 was shown
to be downregulated in hippocampal brain samples of AD
patients from early to severe disease stages (Lukiw, 2007),
miR-155 was reported to be overexpressed in circulating fluids
and cells of AD individuals (Alexandrov et al., 2012; Guedes
et al., 2016), as well as in 3xTg-AD mice brain (Guedes
et al., 2014). However, in other works, miR-155 expression
was found significantly reduced in old individuals (Noren
Hooten et al., 2010). Our results indicate that both miR-124
and miR-155 are decreased in 16 DIV cells, and that their
downregulation in 2 DIV microglia comes from Aβinteraction.
Such reduction may have important consequences in AD
progression since miR-155 and miR-124 were recognized as
critical modulators of immunological responses and to possibly
act as anti-inflammatory factors (Li et al., 2016; Qin et al., 2016).
TNF-α, IL-1β, IL-6, HMGB1 and IL-18, but not NLRP3, were
increased in 2 DIV cells upon Aβtreatment, while only the first
two were enhanced in the 16 DIV cells. A better understanding
of the pro-inflammatory signaling pathways associated to AD
is crucial to define their beneficial or harmful consequences,
and if their targeting by NSAIDs is advantageous. Based on our
data, we postulate that NSAIDs therapy should be envisaged as a
stage-dependent disease strategy with potential benefits in early
inflammatory states of AD disease, as suggested by others (Cole
and Frautschy, 2010; Imbimbo et al., 2010; Wang J. et al., 2015).
TNF-αand IL-1βincrease is consensual in AD pathogenesis
(Wang W. Y. et al., 2015) and was here observed in 2 DIV and
16 DIV microglia, which may then be considered as targets for
selective tuning. We observed that Aβwas unable to stimulate
the production of other inflammatory mediators in aged cells.
Relationship between IL-6 concentration and aging is not clearly
established, and although suggested to increase, implicated cell
is not recognized and conflicting results have been published
(Maggio et al., 2006). We have established that Aβand LPS
trigger the release of HMGB1 from microglia (Cunha et al.,
2016; Falcão et al., 2017), and we observed its upregulation in
the 2 DIV Aβ-treated microglia. HMGB1 is a nuclear protein
acting as a co-factor for gene transcription. However, when in the
extracellular fluid, it acts as an alarmin and a pro-inflammatory
cytokine that signals through TLR2/TLR4 (Park et al., 2004),
which were increased in the reactive microglia. HMGB1 is
involved in AD pathology by inducing neurite degeneration
(Fujita et al., 2016). Our results do not sustain NLRP3 activation,
although clearly show upregulated IL-18, again more notoriously
in 2 DIV than in 16 DIV cells. Although IL-18 has been
indicated to be produced downstream of NLRP3 (Zaki et al.,
2010), it was recently associated to NLRP1 inflammasome, as
well (Murphy et al., 2016). In a recent article both inflammasome
components were indicated to be activated in AD, but their direct
association with microglia was not investigated (Saresella et al.,
2016). Most interesting, increased expression of pro-IL-18 with
defective NLRP3 activation was observed in dendritic cells from
elderly mice during influenza infection, highlighting that IL-18
upregulation may occur in the absence of NLRP3 activation
(Stout-Delgado et al., 2012).
The increase in pro-inflammatory cytokines, as well as
in iNOS and MHC class II, indicates that Aβtriggers the
polarization of microglia into the M1 phenotype (Wang W. Y.
et al., 2015; Cunha et al., 2016), namely in the 2 DIV
cultured microglia. Nevertheless, increased expression of TGFβ
and Arginase 1, and in some cases also of MHC class II,
suggests the presence of M2 subclasses in both differentially
aged cells (Chhor et al., 2013; Roszer, 2015). Increased
microglial iNOS and TGFβsignaling by aging and AD was
observed in experimental models and patients (Dheen et al.,
2005; Doyle et al., 2010; Mosher and Wyss-Coray, 2014; von
Bernhardi et al., 2015). Actually, M1 and M2 phenotypes
are the extreme subtypes of microglia polarization, and
the existence of different heterogeneous activation states
reflect the plastic nature of microglia (Bachstetter et al.,
2015; Grabert et al., 2016). Heterogeneous populations of
microglia in our model result from the co-existence of four
separated CD11b−/CD86−, CD11b−/CD86+, CD11b+/CD86−
and CD11b+/CD86+ subtypes in 2 DIV microglia, but more
extensively in 16 DIV microglia. These distinct subsets may
derive from differentiation dissimilarities, contributing to
Frontiers in Aging Neuroscience | www.frontiersin.org 18 August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
morphological and functional diversities. Major differences
induced by Aβincluded a decrease in the number of CD11b+
cells in 2 DIV cells and an increase in CD86+ cells in 16 DIV
microglia. This aging-associated diversity is in line with previous
studies showing that aged mice (22 months) have a greater
proportion of CD86+ microglia in hippocampus than adult
animals (4 months) (Kohman et al., 2013). CD86 was shown
to have co-stimulatory effects on T cells activation (Tambuyzer
et al., 2009) and Monsonego et al. (2003) demonstrated that
Aβ-reactive T cell activation was CD86 microglia-dependent.
Therefore, we hypothesize that aging and Aβmay potentiate
interactions between microglia and infiltrating T cells, thus
concurring for immune dysfunction. M2b polarized microglia
with high MHC class II and CD86 expression, pro-/anti-
inflammatory properties, and enhanced T cell recruitment
capacity may be a subset of Aβ-treated 16 DIV cells. Loss of
CD11b+ cells, containing the αMβ2 integrin receptor, in the Aβ-
treated 2 DIV microglia can account to reduced phagocytic and
migration abilities, since αMβ2 was reported to be implicated
in phagocytosis, cell-mediated killing, chemotaxis and cellular
activation (Cougoule et al., 2004; Chen et al., 2008).
In this study we used highly enriched primary cultures of
microglia isolated from the cortex of mice pups, as described
(Saura et al., 2003). A mild state of microglia activation was
observed at 2 DIV cultures after the isolation procedure (Caldeira
et al., 2014). Indeed, the calming state requires time in culture
(Cristóvão et al., 2010). We used 2 DIV cells and treatment
with 1000 nM Aβto mimic activation of mild microglia-
associated neuroinflammation, a risk factor for developing AD
(Eikelenboom et al., 2012; Wang W. Y. et al., 2015). Considering
that cellular senescence is interconnected with AD pathogenesis
(Boccardi et al., 2015), and that microglial degeneration and loss
of neuroprotection by the dystrophic/senescent microglia, rather
than activated microglia, contributes to AD (Streit et al., 2009;
Mosher and Wyss-Coray, 2014), we used in vitro 16 DIV aged
microglia (Caldeira et al., 2014) and incubation with Aβto also
test this alternative hypothesis.
Data show that microglia activation by Aβdepends on the
polarization state of the cell. If already activated, microglia
react with increased migration and expression of all major
inflammatory biomarkers (except NLRP3), but also showing
dysfunctional consequences as low phagocytic ability, increased
senescence-like behavior, decreased CD11b immunoreactivity
and reduced inflammatory miR-155 and miR-124 expression.
Changes are much less notorious in the mature/aged microglia
that only respond by activation of MMP2 and MMP9, increased
LC3-punta and CD86 immunostaining, together with elevated
iNOS, TGFβand TNF-αgene expression. Distribution of
M1 and M2 polarized markers indicates that 2 DIV cells
assume a predominant M1 phenotype in the presence of Aβ,
while 16 DIV cells comprise diverse microglia subtypes that
include M2 subclasses. Altogether, we hypothesize that diverse
microglia polarized cells distinctly contribute to AD initiation
and progression. However, given the complexity of AD and
the involvement of multiple cell types, our results should be
interpreted with caution and their translation to humans will
require further studies.
New insights may be obtained with microglia isolated from
human post-mortem brain tissue (Mizee et al., 2017), or
derived from induced pluripotent stem cells generated from
AD patients (Abud et al., 2017). Moreover, because neuron-
astrocyte-microglia communication plays a crucial role in
AD pathogenesis, and microglia activation triggers astrocyte
neurotoxicity (Liddelow et al., 2017), additional studies using
3D culture models that allow cell-to-cell interplay and best
recapitulate AD (Kim et al., 2015; Choi et al., 2016; Lee
et al., 2016) should be used to corroborate or complement our
findings.
AUTHOR CONTRIBUTIONS
DB conceived the project. AF and DB planned and designed the
experiments. AF, ARV and ASF performed microglia cultures.
CCaldeira performed the experiments. CCunha evaluated
microRNA profiling. ARV assessed autophagy. AB and ES
performed flow cytometry measurements and AF analyzed the
results. CCaldeira, AF and DB interpreted experiments and wrote
the manuscript. DB edited the final version. The manuscript has
been read and approved by all named authors.
ACKNOWLEDGMENTS
This work was supported by FEDER (COMPETE Programme)
and by National funds (Fundação para a Ciência e a Tecnologia,
FCT) Project FCT-PEst-OE/SAU/UI4013 to iMed.ULisboa
and Project FCT-EXPL/NEU-NMC/1003/2013 to AF, and, in
part, by the EU Joint Programme—Neurodegenerative Disease
Research (JPND) MADGIC project JPco-fuND/0003/2015 to
DB, as well as by the prize Edgar Cruz e Silva 2012 from
Grupo de Estudo do Envelhecimento Cerebral e Demência
to DB. ASF holds a post-doctoral research position (C2007-
FFUL/UBMBE/02/2011), ARV together with AB are recipients of
postdoctoral research fellowships (SFRH/BPD/76590/2011 and
SFRH/BPD/96794/2013, respectively), and CCunha of a PhD
fellowship (SFRH/BD/91316/2012), all from FCT. The funding
organization had no role in study design, data collection
and analysis, decision to publish, or preparation of the
manuscript.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online
at: http://journal.frontiersin.org/article/10.3389/fnagi.2017.002
77/full#supplementary-material
FIGURE S1 | Reactive cultured microglia phagocytose higher amount of
monomeric and dimeric species than the aged cells, which otherwise exhibit
increased number of extracellular amyloid-β(Aβ) deposits. Microglia that were
kept in culture for 2 and 16 days in vitro (DIV) were treated with 1000 nM Aβ
for 24 h. (A) Representative images of Aβimmunoblots in cell lysates using the
anti-Aβclone W0–2 antibody. (B) Representative images of microglia
immunostained for Iba1 (red) and Aβ(green) with nuclei staining (blue). Scale
bar equals 50 µm.
Frontiers in Aging Neuroscience | www.frontiersin.org 19 August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
REFERENCES
Abud, E. M., Ramirez, R. N., Martinez, E. S., Healy, L. M., Nguyen, C. H. H.,
Newman, S. A., et al. (2017). iPSC-derived human microglia-like cells to study
neurological diseases. Neuron 94, 278–293.e9. doi: 10.1016/j.neuron.2017.
03.042
Alexandrov, P. N., Dua, P., Hill, J. M., Bhattacharjee, S., Zhao, Y., and
Lukiw, W. J. (2012). microRNA (miRNA) speciation in Alzheimer’s disease
(AD) cerebrospinal fluid (CSF) and extracellular fluid (ECF). Int. J. Biochem.
Mol. Biol. 3, 365–373.
Bachstetter, A. D., Van Eldik, L. J., Schmitt, F. A., Neltner, J. H., Ighodaro, E. T.,
Webster, S. J., et al. (2015). Disease-related microglia heterogeneity in
the hippocampus of Alzheimer’s disease, dementia with Lewy bodies,
and hippocampal sclerosis of aging. Acta Neuropathol. Commun. 3:32.
doi: 10.1186/s40478-015-0209-z
Barateiro, A., Miron, V. E., Santos, S. D., Relvas, J. B., Fernandes, A., Ffrench-
Constant, C., et al. (2013). Unconjugated bilirubin restricts oligodendrocyte
differentiation and axonal myelination. Mol. Neurobiol. 47, 632–644.
doi: 10.1007/s12035-012-8364-8
Barateiro, A., Vaz, A. R., Silva, S. L., Fernandes, A., and Brites, D.
(2012). ER stress, mitochondrial dysfunction and calpain/JNK activation are
involved in oligodendrocyte precursor cell death by unconjugated bilirubin.
Neuromolecular Med. 14, 285–302. doi: 10.1007/s12017-012-8187-9
Bhat, R., Crowe, E. P., Bitto, A., Moh, M., Katsetos, C. D., Garcia, F. U., et al.
(2012). Astrocyte senescence as a component of Alzheimer’s disease. PLoS One
7:e45069. doi: 10.1371/journal.pone.0045069
Birch, A. M., Katsouri, L., and Sastre, M. (2014). Modulation of inflammation
in transgenic models of Alzheimer’s disease. J. Neuroinflammation 11:25.
doi: 10.1186/1742-2094-11-25
Boccardi, V., Pelini, L., Ercolani, S., Ruggiero, C., and Mecocci, P. (2015). From
cellular senescence to Alzheimer’s disease: the role of telomere shortening.
Ageing Res. Rev. 22, 1–8. doi: 10.1016/j.arr.2015.04.003
Boddaert, J., Kinugawa, K., Lambert, J. C., Boukhtouche, F., Zoll, J., Merval, R.,
et al. (2007). Evidence of a role for lactadherin in Alzheimer’s disease. Am.
J. Pathol. 170, 921–929. doi: 10.2353/ajpath.2007.060664
Brkic, M., Balusu, S., Libert, C., and Vandenbroucke, R. E. (2015). Friends or foes:
matrix metalloproteinases and their multifaceted roles in neurodegenerative
diseases. Mediators Inflamm. 2015:620581. doi: 10.1155/2015/620581
Bruno, M. A., Mufson, E. J., Wuu, J., and Cuello, A. C. (2009). Increased matrix
metalloproteinase 9 activity in mild cognitive impairment. J. Neuropathol. Exp.
Neurol. 68, 1309–1318. doi: 10.1097/nen.0b013e3181c22569
Caldeira, C., Oliveira, A. F., Cunha, C., Vaz, A. R., Falcão, A. S., Fernandes, A.,
et al. (2014). Microglia change from a reactive to an age-like phenotype with
the time in culture. Front. Cell. Neurosci. 8:152. doi: 10.3389/fncel.2014.00152
Cardoso, A. L., Guedes, J. R., and de Lima, M. C. (2016). Role of microRNAs in the
regulation of innate immune cells under neuroinflammatory conditions. Curr.
Opin. Pharmacol. 26, 1–9. doi: 10.1016/j.coph.2015.09.001
Chen, J., Namiki, S., Toma-Hirano, M., Miyatake, S., Ishida, K., Shibata, Y., et al.
(2008). The role of CD11b in phagocytosis and dendritic cell development.
Immunol. Lett. 120, 42–48. doi: 10.1016/j.imlet.2008.06.010
Chhor, V., Le Charpentier, T., Lebon, S., Oré, M. V., Celador, I. L., Josserand, J.,
et al. (2013). Characterization of phenotype markers and neuronotoxic
potential of polarised primary microglia in vitro.Brain Behav. Immun. 32,
70–85. doi: 10.1016/j.bbi.2013.02.005
Chi, W., Chen, H., Li, F., Zhu, Y., Yin, W., and Zhuo, Y. (2015). HMGB1 promotes
the activation of NLRP3 and caspase-8 inflammasomes via NF-κB pathway in
acute glaucoma. J. Neuroinflammation 12:137. doi: 10.1186/s12974-015-0360-2
Chifenti, B., Locci, M. T., Lazzeri, G., Guagnozzi, M., Dinucci, D., Chiellini, F.,
et al. (2013). Autophagy-related protein LC3 and Beclin-1 in the first trimester
of pregnancy. Clin. Exp. Reprod. Med. 40, 33–37. doi: 10.5653/cerm.2013.
40.1.33
Cho, S. H., Sun, B., Zhou, Y., Kauppinen, T. M., Halabisky, B., Wes, P., et al. (2011).
CX3CR1 protein signaling modulates microglial activation and protects against
plaque-independent cognitive deficits in a mouse model of Alzheimer disease.
J. Biol. Chem. 286, 32713–32722. doi: 10.1074/jbc.m111.254268
Choi, S. H., Kim, Y. H., Quinti, L., Tanzi, R. E., and Kim, D. Y. (2016). 3D
culture models of Alzheimer’s disease: a road map to a ‘‘cure-in-a-dish’’. Mol.
Neurodegener. 11:75. doi: 10.1186/s13024-016-0139-7
Cole, G. M., and Frautschy, S. A. (2010). Mechanisms of action of non-steroidal
anti-inflammatory drugs for the prevention of Alzheimer’s disease. CNS
Neurol. Disord. Drug Targets 9, 140–148. doi: 10.2174/187152710791011991
Colton, C. A., Mott, R. T., Sharpe, H., Xu, Q., Van Nostrand, W. E., and
Vitek, M. P. (2006). Expression profiles for macrophage alternative activation
genes in AD and in mouse models of AD. J. Neuroinflammation 3:27.
doi: 10.1186/1742-2094-3-27
Cougoule, C., Wiedemann, A., Lim, J., and Caron, E. (2004). Phagocytosis, an
alternative model system for the study of cell adhesion. Semin. Cell Dev. Biol.
15, 679–689. doi: 10.1016/s1084-9521(04)00087-4
Cristóvão, A. C., Saavedra, A., Fonseca, C. P., Campos, F., Duarte, E. P., and
Baltazar, G. (2010). Microglia of rat ventral midbrain recovers its resting state
over time in vitro: let microglia rest before work. J. Neurosci. Res. 88, 552–562.
doi: 10.1002/jnr.22219
Cunha, C., Gomes, C., Vaz, A. R., and Brites, D. (2016). Exploring new
inflammatory biomarkers and pathways during LPS-Induced M1 polarization.
Mediators Inflamm. 2016:6986175. doi: 10.1155/2016/6986175
Cykowski, M. D., Takei, H., Van Eldik, L. J., Schmitt, F. A., Jicha, G. A.,
Powell, S. Z., et al. (2016). Hippocampal sclerosis but not normal aging
or Alzheimer disease is associated with TDP-43 pathology in the basal
forebrain of aged persons. J. Neuropathol. Exp. Neurol. 75, 397–407.
doi: 10.1093/jnen/nlw014
Damani, M. R., Zhao, L., Fontainhas, A. M., Amaral, J., Fariss, R. N., and
Wong, W. T. (2011). Age-related alterations in the dynamic behavior of
microglia. Aging Cell 10, 263–276. doi: 10.1111/j.1474-9726.2010.00660.x
Daniels, M. J., Rivers-Auty, J., Schilling, T., Spencer, N. G., Watremez, W.,
Fasolino, V., et al. (2016). Fenamate NSAIDs inhibit the NLRP3 inflammasome
and protect against Alzheimer’s disease in rodent models. Nat. Commun.
7:12504. doi: 10.1038/ncomms12504
Dheen, S. T., Jun, Y., Yan, Z., Tay, S. S., and Ling, E. A. (2005). Retinoic acid
inhibits expression of TNF-αand iNOS in activated rat microglia. Glia 50,
21–31. doi: 10.1002/glia.20153
Dobolyi, A., Vincze, C., Pal, G., and Lovas, G. (2012). The neuroprotective
functions of transforming growth factor beta proteins. Int. J. Mol. Sci. 13,
8219–8258. doi: 10.3390/ijms13078219
Doyle, K. P., Cekanaviciute, E., Mamer, L. E., and Buckwalter, M. S. (2010).
TGFβsignaling in the brain increases with aging and signals to astrocytes
and innate immune cells in the weeks after stroke. J. Neuroinflammation 7:62.
doi: 10.1186/1742-2094-7-62
Eikelenboom, P., Hoozemans, J. J., Veerhuis, R., van Exel, E., Rozemuller, A. J.,
and van Gool, W. A. (2012). Whether, when and how chronic inflammation
increases the risk of developing late-onset Alzheimer’s disease. Alzheimers Res.
Ther. 4:15. doi: 10.1186/alzrt118
Facci, L., Barbierato, M., Marinelli, C., Argentini, C., Skaper, S. D., and Giusti, P.
(2014). Toll-like receptors 2, -3 and -4 prime microglia but not astrocytes across
central nervous system regions for ATP-dependent interleukin-1βrelease. Sci.
Rep. 4:6824. doi: 10.1038/srep06824
Falcão, A. S., Carvalho, L. A., Lidónio, G., Vaz, A. R., Lucas, S. D.,
Moreira, R., et al. (2017). Dipeptidyl vinyl sulfone as a novel chemical
tool to inhibit HMGB1/NLRP3-Inflammasome and Inflamma-miRs in
Aβ-mediated microglial inflammation. ACS Chem. Neurosci. 8, 89–99.
doi: 10.1021/acschemneuro.6b00250
Floden, A. M., and Combs, C. K. (2007). Microglia repetitively isolated from
in vitro mixed glial cultures retain their initial phenotype. J. Neurosci. Methods
164, 218–224. doi: 10.1016/j.jneumeth.2007.04.018
Floden, A. M., and Combs, C. K. (2011). Microglia demonstrate age-dependent
interaction with amyloid-βfibrils. J. Alzheimers Dis. 25, 279–293.
doi: 10.3233/JAD-2011-101014
Frankowski, H., Gu, Y. H., Heo, J. H., Milner, R., and Del Zoppo, G. J. (2012). Use
of gel zymography to examine matrix metalloproteinase (gelatinase) expression
in brain tissue or in primary glial cultures. Methods Mol. Biol. 814, 221–233.
doi: 10.1007/978-1-61779-452-0_15
Fricker, M., Neher, J. J., Zhao, J. W., Thery, C., Tolkovsky, A. M., and Brown, G. C.
(2012). MFG-E8 mediates primary phagocytosis of viable neurons during
neuroinflammation. J. Neurosci. 32, 2657–2666. doi: 10.1523/JNEUROSCI.
4837-11.2012
Fujita, K., Motoki, K., Tagawa, K., Chen, X., Hama, H., Nakajima, K., et al.
(2016). HMGB1, a pathogenic molecule that induces neurite degeneration via
Frontiers in Aging Neuroscience | www.frontiersin.org 20 August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
TLR4-MARCKS, is a potential therapeutic target for Alzheimer’s disease. Sci.
Rep. 6:31895. doi: 10.1038/srep31895
Fuller, A. D., and Van Eldik, L. J. (2008). MFG-E8 regulates microglial
phagocytosis of apoptotic neurons. J. Neuroimmune Pharmacol. 3, 246–256.
doi: 10.1007/s11481-008-9118-2
Gingras, M., Gagnon, V., Minotti, S., Durham, H. D., and Berthod, F. (2007).
Optimized protocols for isolation of primary motor neurons, astrocytes and
microglia from embryonic mouse spinal cord. J. Neurosci. Methods 163,
111–118. doi: 10.1016/j.jneumeth.2007.02.024
Gordo, A. C., Falcão, A. S., Fernandes, A., Brito, M. A., Silva, R. F. M., and Brites, D.
(2006). Unconjugated bilirubin activates and damages microglia. J. Neurosci.
Res. 84, 194–201. doi: 10.1002/jnr.20857
Grabert, K., Michoel, T., Karavolos, M. H., Clohisey, S., Baillie, J. K., Stevens, M. P.,
et al. (2016). Microglial brain region-dependent diversity and selective regional
sensitivities to aging. Nat. Neurosci. 19, 504–516. doi: 10.1038/nn.4222
Graeber, M. B. (2010). Changing face of microglia. Science 330, 783–788.
doi: 10.1126/science.1190929
Griciuc, A., Serrano-Pozo, A., Parrado, A. R., Lesinski, A. N., Asselin, C. N.,
Mullin, K., et al. (2013). Alzheimer’s disease risk gene CD33 inhibits microglial
uptake of amyloid β.Neuron 78, 631–643. doi: 10.1016/j.neuron.2013.04.014
Grundke-Iqbal, I., Fleming, J., Tung, Y.-C., Lassmann, H., Iqbal, K., and Joshi, J. G.
(1990). Ferritin is a component of the neuritic (senile) plaque in Alzheimer
dementia. Acta Neuropathol. 81, 105–110. doi: 10.1007/bf00334497
Guedes, J., Cardoso, A. L., and Pedroso De Lima, M. C. (2013). Involvement
of microRNA in microglia-mediated immune response. Clin. Dev. Immunol.
2013:186872. doi: 10.1155/2013/186872
Guedes, J. R., Custódia, C. M., Silva, R. J., de Almeida, L. P., Pedroso de
Lima, M. C., and Cardoso, A. L. (2014). Early miR-155 upregulation contributes
to neuroinflammation in Alzheimer’s disease triple transgenic mouse model.
Hum. Mol. Genet. 23, 6286–6301. doi: 10.1093/hmg/ddu348
Guedes, J. R., Santana, I., Cunha, C., Duro, D., Almeida, M. R., Cardoso, A. M.,
et al. (2016). MicroRNA deregulation and chemotaxis and phagocytosis
impairment in Alzheimer’s disease. Alzheimers Dement. (Amst) 3, 7–17.
doi: 10.1016/j.dadm.2015.11.004
Guerreiro, R., Wojtas, A., Bras, J., Carrasquillo, M., Rogaeva, E., Majounie, E., et al.
(2013). TREM2 variants in Alzheimer’s disease. N Engl J. Med. 368, 117–127.
doi: 10.1056/NEJMoa1211851
Harry, G. J. (2013). Microglia during development and aging. Pharmacol. Ther.
139, 313–326. doi: 10.1016/j.pharmthera.2013.04.013
Heneka, M. T., Carson, M. J., El Khoury, J., Landreth, G. E., Brosseron, F.,
Feinstein, D. L., et al. (2015). Neuroinflammation in Alzheimer’s
disease. Lancet Neurol. 14, 388–405. doi: 10.1016/S1474-4422(15)
70016-5
Heyn, J., Luchting, B., Hinske, L. C., Hübner, M., Azad, S. C., and Kreth, S. (2016).
miR-124a and miR-155 enhance differentiation of regulatory T cells in patients
with neuropathic pain. J. Neuroinflammation 13:248. doi: 10.1186/s12974-016-
0712-6
Hjorth, E., Zhu, M., Toro, V. C., Vedin, I., Palmblad, J., Cederholm, T., et al.
(2013). Omega-3 fatty acids enhance phagocytosis of Alzheimer’s disease-
related amyloid-β42 by human microglia and decrease inflammatory markers.
J. Alzheimers Dis. 35, 697–713. doi: 10.3233/JAD-130131
Hoeijmakers, L., Heinen, Y., Van Dam, A. M., Lucassen, P. J., and Korosi, A.
(2016). Microglial priming and Alzheimer’s disease: a possible role for
(early) immune challenges and epigenetics? Front. Hum. Neurosci. 10:398.
doi: 10.3389/fnhum.2016.00398
Hof, P. R., and Mobbs, C. V. (2009). Handbook of the Neuroscience of Aging.
Amsterdam: Academic Press.
Imbimbo, B. P., Solfrizzi, V., and Panza, F. (2010). Are NSAIDs useful to treat
Alzheimer’s disease or mild cognitive impairment? Front. Aging Neurosci. 2:19.
doi: 10.3389/fnagi.2010.00019
Ito, S., Kimura, K., Haneda, M., Ishida, Y., Sawada, M., and Isobe, K.
(2007). Induction of matrix metalloproteinases (MMP3, MMP12 and
MMP13) expression in the microglia by amyloid-βstimulation via the
PI3K/Akt pathway. Exp. Gerontol. 42, 532–537. doi: 10.1016/j.exger.2006.
11.012
Jiang, M., Xiang, Y., Wang, D., Gao, J., Liu, D., Liu, Y., et al. (2012). Dysregulated
expression of miR-146a contributes to age-related dysfunction of macrophages.
Aging Cell 11, 29–40. doi: 10.1111/j.1474-9726.2011.00757.x
Jiang, T., Yu, J.-T., Hu, N., Tan, M.-S., Zhu, X.-C., and Tan, L. (2014). CD33 in
Alzheimer’s disease. Mol. Neurobiol. 49, 529–535. doi: 10.1007/s12035-013-
8536-1
Kawabori, M., Kacimi, R., Kauppinen, T., Calosing, C., Kim, J. Y., Hsieh, C. L., et al.
(2015). Triggering receptor expressed on myeloid cells 2 (TREM2) deficiency
attenuates phagocytic activities of microglia and exacerbates ischemic damage
in experimental stroke. J. Neurosci. 35, 3384–3396. doi: 10.1523/JNEUROSCI.
2620-14.2015
Kettenmann, H., Hanisch, U. K., Noda, M., and Verkhratsky, A. (2011). Physiology
of microglia. Physiol. Rev. 91, 461–553. doi: 10.1152/physrev.00011.2010
Kim, H. J., Ajit, D., Peterson, T. S., Wang, Y., Camden, J. M., Gibson Wood, W.,
et al. (2012). Nucleotides released from Aβ1−42-treated microglial cells
increase cell migration and Aβ1−42 uptake through P2Y2receptor activation.
J. Neurochem. 121, 228–238. doi: 10.1111/j.1471-4159.2012.07700.x
Kim, Y. H., Choi, S. H., D’Avanzo, C., Hebisch, M., Sliwinski, C., Bylykbashi, E.,
et al. (2015). A 3D human neural cell culture system for modeling Alzheimer’s
disease. Nat. Protoc. 10, 985–1006. doi: 10.1038/nprot.2015.065
Klionsky, D. J., Abdelmohsen, K., Abe, A., Abedin, M. J., Abeliovich, H.,
Acevedo Arozena, A., et al. (2016). Guidelines for the use and interpretation
of assays for monitoring autophagy (3rd edition). Autophagy 12, 1–222.
doi: 10.1080/15548627.2015.1100356
Kohman, R. A., Bhattacharya, T. K., Wojcik, E., and Rhodes, J. S. (2013). Exercise
reduces activation of microglia isolated from hippocampus and brain of aged
mice. J. Neuroinflammation 10:114. doi: 10.1186/1742-2094-10-114
Konnecke, H., and Bechmann, I. (2013). The role of microglia and matrix
metalloproteinases involvement in neuroinflammation and gliomas. Clin. Dev.
Immunol. 2013:914104. doi: 10.1155/2013/914104
Krabbe, G., Halle, A., Matyash, V., Rinnenthal, J. L., Eom, G. D., Bernhardt, U.,
et al. (2013). Functional impairment of microglia coincides with beta-
amyloid deposition in mice with Alzheimer-like pathology. PLoS One 8:e60921.
doi: 10.1371/journal.pone.0060921
Lee, H. K., Velazquez Sanchez, C., Chen, M., Morin, P. J., Wells, J. M.,
Hanlon, E. B., et al. (2016). Three dimensional human neuro-spheroid model
of Alzheimer’s disease based on differentiated induced pluripotent stem cells.
PLoS One 11:e0163072. doi: 10.1371/journal.pone.0163072
Leovsky, C., Fabian, C., Naaldijk, Y., Jäger, C., Jang, H. J., Böhme, J., et al.
(2015). Biodistribution of in vitro-derived microglia applied intranasally
and intravenously to mice: effects of aging. Cytotherapy 17, 1617–1626.
doi: 10.1016/j.jcyt.2015.07.019
Li, W. (2012). Eat-me signals: keys to molecular phagocyte biology and ‘‘appetite’’
control. J. Cell. Physiol. 227, 1291–1297. doi: 10.1002/jcp.22815
Li, W. (2013). Phagocyte dysfunction, tissue aging and degeneration. Ageing Res.
Rev. 12, 1005–1012. doi: 10.1016/j.arr.2013.05.006
Li, X., Kong, D., Chen, H., Liu, S., Hu, H., Wu, T., et al. (2016). miR-155 acts as
an anti-inflammatory factor in atherosclerosis-associated foam cell formation
by repressing calcium-regulated heat stable protein 1. Sci. Rep. 6:21789.
doi: 10.1038/srep21789
Li, E., Noda, M., Doi, Y., Parajuli, B., Kawanokuchi, J., Sonobe, Y., et al. (2012).
The neuroprotective effects of milk fat globule-EGF factor 8 against oligomeric
amyloid βtoxicity. J. Neuroinflammation 9:148. doi: 10.1186/1742-2094-9-148
Liddelow, S. A., Guttenplan, K. A., Clarke, L. E., Bennett, F. C., Bohlen, C. J.,
Schirmer, L., et al. (2017). Neurotoxic reactive astrocytes are induced
by activated microglia. Nature 541, 481–487. doi: 10.1038/nature
21029
Limatola, C., and Ransohoff, R. M. (2014). Modulating neurotoxicity through
CX3CL1/CX3CR1 signaling. Front. Cell. Neurosci. 8:229. doi: 10.3389/fncel.
2014.00229
Liu, Y., Yang, X., Guo, C., Nie, P., Liu, Y., and Ma, J. (2013). Essential role
of MFG-E8 for phagocytic properties of microglial cells. PLoS One 8:e55754.
doi: 10.1371/journal.pone.0055754
Lopes, K. O., Sparks, D. L., and Streit, W. J. (2008). Microglial dystrophy
in the aged and Alzheimer’s disease brain is associated with ferritin
immunoreactivity. Glia 56, 1048–1060. doi: 10.1002/glia.20678
Lorenzl, S., Buerger, K., Hampel, H., and Beal, M. F. (2008). Profiles of matrix
metalloproteinases and their inhibitors in plasma of patients with dementia.
Int. Psychogeriatr. 20, 67–76. doi: 10.1017/s1041610207005790
Lukiw, W. J. (2007). Micro-RNA speciation in fetal, adult and Alzheimer’s disease
hippocampus. Neuroreport 18, 297–300. doi: 10.1097/WNR.0b013e3280148e8b
Frontiers in Aging Neuroscience | www.frontiersin.org 21 August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
Lull, M. E., and Block, M. L. (2010). Microglial activation and chronic
neurodegeneration. Neurotherapeutics 7, 354–365. doi: 10.1016/j.nurt.2010.
05.014
Lynch, A. M., Murphy, K. J., Deighan, B. F., O’Reilly, J. A., Gun’ko, Y. K.,
Cowley, T. R., et al. (2010). The impact of glial activation in the aging brain.
Aging Dis. 1, 262–278.
Maggio, M., Guralnik, J. M., Longo, D. L., and Ferrucci, L. (2006). Interleukin-6 in
aging and chronic disease: a magnificent pathway. J. Gerontol. A Biol. Sci. Med.
Sci. 61, 575–584. doi: 10.1093/gerona/61.6.575
Malik, M., Simpson, J. F., Parikh, I., Wilfred, B. R., Fardo, D. W., Nelson, P. T.,
et al. (2013). CD33 Alzheimer’s risk-altering polymorphism, CD33 expression,
and exon 2 splicing. J. Neurosci. 33, 13320–13325. doi: 10.1523/JNEUROSCI.
1224-13.2013
McCarthy, K. D., and de Vellis, J. (1980). Preparation of separate astroglial and
oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85, 890–902.
doi: 10.1083/jcb.85.3.890
Merino, J. J., Muñetón-Gómez, V., Alvárez, M. I., and Toledano-Díaz, A.
(2016). Effects of CX3CR1 and fractalkine chemokines in amyloid βclearance
and p-Tau accumulation in Alzheimer’s disease (AD) rodent models: is
fractalkine a systemic biomarker for AD? Curr. Alzheimer Res. 13, 403–412.
doi: 10.2174/1567205013666151116125714
Miller, A. M., and Stella, N. (2009). Microglial cell migration stimulated by ATP
and C5a involve distinct molecular mechanisms: quantification of migration by
a novel near-infrared method. Glia 57, 875–883. doi: 10.1002/glia.20813
Miners, J. S., Barua, N., Kehoe, P. G., Gill, S., and Love, S. (2011). Aβ-degrading
enzymes: potential for treatment of Alzheimer disease. J. Neuropathol. Exp.
Neurol. 70, 944–959. doi: 10.1097/NEN.0b013e3182345e46
Mizee, M. R., Miedema, S. S. M., van der Poel, M., Adelia, Schuurman, K. G.,
van Strien, M. E., et al. (2017). Isolation of primary microglia from the
human post-mortem brain: effects of ante- and post-mortem variables. Acta
Neuropathol. Commun. 5:16. doi: 10.1186/s40478-017-0418-8
Mizuno, T. (2012). The biphasic role of microglia in Alzheimer’s disease. Int.
J. Alzheimers Dis. 2012:737846. doi: 10.1155/2012/737846
Monsonego, A., Imitola, J., Zota, V., Oida, T., and Weiner, H. L. (2003). Microglia-
mediated nitric oxide cytotoxicity of T cells following amyloid β-peptide
presentation to Th1 cells. J. Immunol. 171, 2216–2224. doi: 10.4049/jimmunol.
172.1.717-b
Morgan, D. (2009). The role of microglia in antibody-mediated clearance
of amyloid-βfrom the brain. CNS Neurol. Disord. Drug Targets 8, 7–15.
doi: 10.2174/187152709787601821
Mosher, K. I., and Wyss-Coray, T. (2014). Microglial dysfunction in brain aging
and Alzheimer’s disease. Biochem. Pharmacol. 88, 594–604. doi: 10.1016/j.bcp.
2014.01.008
Moussaud, S., and Draheim, H. J. (2010). A new method to isolate microglia
from adult mice and culture them for an extended period of time. J. Neurosci.
Methods 187, 243–253. doi: 10.1016/j.jneumeth.2010.01.017
Murphy, A. J., Kraakman, M. J., Kammoun, H. L., Dragoljevic, D., Lee, M. K.,
Lawlor, K. E., et al. (2016). IL-18 production from the NLRP1 inflammasome
prevents obesity and metabolic syndrome. Cell Metab. 23, 155–164.
doi: 10.1016/j.cmet.2015.09.024
Nakajima, K., and Kohsaka, S. (2004). Microglia: neuroprotective and
neurotrophic cells in the central nervous system. Curr. Drug Targets
Cardiovasc. Hematol. Disord. 4, 65–84. doi: 10.2174/15680060434
81284
Neumann, H., Kotter, M. R., and Franklin, R. J. (2009). Debris clearance by
microglia: an essential link between degeneration and regeneration. Brain 132,
288–295. doi: 10.1093/brain/awn109
Njie, E. G., Boelen, E., Stassen, F. R., Steinbusch, H. W., Borchelt, D. R., and
Streit, W. J. (2012). Ex vivo cultures of microglia from young and aged rodent
brain reveal age-related changes in microglial function. Neurobiol. Aging 33,
195.e1–195.e12. doi: 10.1016/j.neurobiolaging.2010.05.008
Noren Hooten, N., Abdelmohsen, K., Gorospe, M., Ejiogu, N., Zonderman, A. B.,
and Evans, M. K. (2010). microRNA expression patterns reveal differential
expression of target genes with age. PLoS One 5:e10724. doi: 10.1371/journal.
pone.0010724
Olivieri, F., Rippo, M. R., Prattichizzo, F., Babini, L., Graciotti, L., Recchioni, R.,
et al. (2013a). Toll like receptor signaling in ‘‘inflammaging’’: microRNA as new
players. Immun. Ageing 10:11. doi: 10.1186/1742-4933-10-11
Olivieri, F., Rippo, M. R., Procopio, A. D., and Fazioli, F. (2013b). Circulating
inflamma-miRs in aging and age-related diseases. Front. Genet. 4:121.
doi: 10.3389/fgene.2013.00121
Park, J. S., Svetkauskaite, D., He, Q., Kim, J. Y., Strassheim, D., Ishizaka, A., et al.
(2004). Involvement of toll-like receptors 2 and 4 in cellular activation by high
mobility group box 1 protein. J. Biol. Chem. 279, 7370–7377. doi: 10.1074/jbc.
M306793200
Ponomarev, E. D., Veremeyko, T., and Weiner, H. L. (2013). MicroRNAs
are universal regulators of differentiation, activation, and polarization of
microglia and macrophages in normal and diseased CNS. Glia 61, 91–103.
doi: 10.1002/glia.22363
Prince, M., Bryce, R., Albanese, E., Wimo, A., Ribeiro, W., and Ferri, C. P. (2013).
The global prevalence of dementia: a systematic review and metaanalysis.
Alzheimers Dement. 9, 63.e2–75.e2. doi: 10.1016/j.jalz.2012.11.007
Provias, J., and Jeynes, B. (2014). The role of the blood-brain barrier in the
pathogenesis of senile plaques in Alzheimer’s disease. Int. J. Alzheimers Dis.
2014:191863. doi: 10.1155/2014/191863
Qin, Z., Wang, P. Y., Su, D. F., and Liu, X. (2016). miRNA-124 in immune system
and immune disorders. Front. Immunol. 7:406. doi: 10.3389/fimmu.2016.00406
Qiu, W. Q., Ye, Z., Kholodenko, D., Seubert, P., and Selkoe, D. J. (1997).
Degradation of amyloid β-protein by a metalloprotease secreted by microglia
and other neural and non-neural cells. J. Biol. Chem. 272, 6641–6646.
doi: 10.1074/jbc.272.10.6641
Reed-Geaghan, E. G., Savage, J. C., Hise, A. G., and Landreth, G. E. (2009).
CD14 and toll-like receptors 2 and 4 are required for fibrillar Aβ-stimulated
microglial activation. J. Neurosci. 29, 11982–11992. doi: 10.1523/JNEUROSCI.
3158-09.2009
Regen, F., Hellmann-Regen, J., Costantini, E., and Reale, M. (2017).
Neuroinflammation and Alzheimer’s disease: implications for microglial
activation. Curr. Alzheimer Res. doi: 10.2174/1567205014666170203141717
[Epub ahead of print].
Rippo, M. R., Olivieri, F., Monsurrò, V., Prattichizzo, F., Albertini, M. C., and
Procopio, A. D. (2014). MitomiRs in human inflamm-aging: a hypothesis
involving miR-181a, miR-34a and miR-146a. Exp. Gerontol. 56, 154–163.
doi: 10.1016/j.exger.2014.03.002
Roszer, T. (2015). Understanding the mysterious M2 macrophage through
activation markers and effector mechanisms. Mediators Inflamm. 2015:816460.
doi: 10.1155/2015/816460
Rubio-Perez, J. M., and Morillas-Ruiz, J. M. (2012). A review: inflammatory
process in Alzheimer’s disease, role of cytokines. ScientificWorldJournal
2012:756357. doi: 10.1100/2012/756357
Ryu, K. Y., Cho, G. S., Piao, H. Z., and Kim, W. K. (2012). Role of TGF-βin survival
of phagocytizing microglia: autocrine suppression of TNF-αproduction and
oxidative stress. Exp. Neurobiol. 21, 151–157. doi: 10.5607/en.2012.21.4.151
Sanjuan, M. A., Dillon, C. P., Tait, S. W., Moshiach, S., Dorsey, F., Connell, S.,
et al. (2007). Toll-like receptor signalling in macrophages links the autophagy
pathway to phagocytosis. Nature 450, 1253–1257. doi: 10.1038/nature06421
Saresella, M., La Rosa, F., Piancone, F., Zoppis, M., Marventano, I., Calabrese, E.,
et al. (2016). The NLRP3 and NLRP1 inflammasomes are activated in
Alzheimer’s disease. Mol. Neurodegener. 11:23. doi: 10.1186/s13024-016-
0088-1
Saura, J., Tusell, J. M., and Serratosa, J. (2003). High-yield isolation of murine
microglia by mild trypsinization. Glia 44, 183–189. doi: 10.1002/glia.10274
Schwartz, M., Butovsky, O., Brück, W., and Hanisch, U. K. (2006). Microglial
phenotype: is the commitment reversible? Trends Neurosci. 29, 68–74.
doi: 10.1016/j.tins.2005.12.005
Shibata, M., Lu, T., Furuya, T., Degterev, A., Mizushima, N., Yoshimori, T.,
et al. (2006). Regulation of intracellular accumulation of mutant Huntingtin
by Beclin 1. J. Biol. Chem. 281, 14474–14485. doi: 10.1074/jbc.M600364200
Sierra, A., Gottfried-Blackmore, A. C., McEwen, B. S., and Bulloch, K. (2007).
Microglia derived from aging mice exhibit an altered inflammatory profile. Glia
55, 412–424. doi: 10.1002/glia.20468
Sikora, E., Arendt, T., Bennett, M., and Narita, M. (2011). Impact of cellular
senescence signature on ageing research. Ageing Res. Rev. 10, 146–152.
doi: 10.1016/j.arr.2010.10.002
Silva, S. L., Vaz, A. R., Barateiro, A., Falcão, A. S., Fernandes, A., Brito, M. A., et al.
(2010). Features of bilirubin-induced reactive microglia: from phagocytosis to
inflammation. Neurobiol. Dis. 40, 663–675. doi: 10.1016/j.nbd.2010.08.010
Frontiers in Aging Neuroscience | www.frontiersin.org 22 August 2017 | Volume 9 | Article 277

Caldeira et al. Microglia Phenotype Governs Reactivity to Aβ
Sondag, C. M., Dhawan, G., and Combs, C. K. (2009). Beta amyloid
oligomers and fibrils stimulate differential activation of primary microglia.
J. Neuroinflammation 6:1. doi: 10.1186/1742-2094-6-1
Stout-Delgado, H. W., Vaughan, S. E., Shirali, A. C., Jaramillo, R. J., and
Harrod, K. S. (2012). Impaired NLRP3 inflammasome function in elderly mice
during influenza infection is rescued by treatment with nigericin. J. Immunol.
188, 2815–2824. doi: 10.4049/jimmunol.1103051
Streit, W. J., Braak, H., Xue, Q. S., and Bechmann, I. (2009). Dystrophic (senescent)
rather than activated microglial cells are associated with tau pathology and
likely precede neurodegeneration in Alzheimer’s disease. Acta Neuropathol.
118, 475–485. doi: 10.1007/s00401-009-0556-6
Streit, W. J., Miller, K. R., Lopes, K. O., and Njie, E. (2008). Microglial degeneration
in the aging brain-bad news for neurons? Front. Biosci. 13, 3423–3438.
doi: 10.2741/2937
Sudduth, T. L., Schmitt, F. A., Nelson, P. T., and Wilcock, D. M. (2013).
Neuroinflammatory phenotype in early Alzheimer’s disease. Neurobiol. Aging
34, 1051–1059. doi: 10.1016/j.neurobiolaging.2012.09.012
Tambuyzer, B. R., Ponsaerts, P., and Nouwen, E. J. (2009). Microglia: gatekeepers
of central nervous system immunology. J. Leukoc. Biol. 85, 352–370.
doi: 10.1189/jlb.0608385
Torres-Platas, S. G., Comeau, S., Rachalski, A., Bo, G. D., Cruceanu, C.,
Turecki, G., et al. (2014). Morphometric characterization of microglial
phenotypes in human cerebral cortex. J. Neuroinflammation 11:12.
doi: 10.1186/1742-2094-11-12
Trudler, D., Farfara, D., and Frenkel, D. (2010). Toll-like receptors
expression and signaling in glia cells in neuro-amyloidogenic diseases:
towards future therapeutic application. Mediators Inflamm. 2010:497987.
doi: 10.1155/2010/497987
Turner, R. J., and Sharp, F. R. (2016). Implications of MMP9 for blood brain
barrier disruption and hemorrhagic transformation following ischemic stroke.
Front. Cell. Neurosci. 10:56. doi: 10.3389/fncel.2016.00056
Varnum, M. M., and Ikezu, T. (2012). The classification of microglial activation
phenotypes on neurodegeneration and regeneration in Alzheimer’s disease
brain. Arch. Immunol. Ther. Exp. (Warsz) 60, 251–266. doi: 10.1007/s00005-
012-0181-2
Varvel, N. H., Bhaskar, K., Kounnas, M. Z., Wagner, S. L., Yang, Y., Lamb, B. T.,
et al. (2009). NSAIDs prevent, but do not reverse, neuronal cell cycle reentry
in a mouse model of Alzheimer disease. J. Clin. Invest. 119, 3692–3702.
doi: 10.1172/JCI39716
von Bernhardi, R. (2010). Immunotherapy in Alzheimer’s disease: where do we
stand? Where should we go? J. Alzheimers Dis. 19, 405–421. doi: 10.3233/JAD-
2010-1248
von Bernhardi, R., Eugenín-Von Bernhardi, L., and Eugenín, J. (2015). Microglial
cell dysregulation in brain aging and neurodegeneration. Front. Aging Neurosci.
7:124. doi: 10.3389/fnagi.2015.00124
Walter, J. (2016). The triggering receptor expressed on myeloid cells 2: a molecular
link of neuroinflammation and neurodegenerative diseases. J. Biol. Chem. 291,
4334–4341. doi: 10.1074/jbc.R115.704981
Walker, D. G., and Lue, L. F. (2015). Immune phenotypes of microglia in human
neurodegenerative disease: challenges to detecting microglial polarization
in human brains. Alzheimers Res. Ther. 7:56. doi: 10.1186/s13195-015-
0139-9
Walker, T., Michaelides, C., Ekonomou, A., Geraki, K., Parkes, H. G.,
Suessmilch, M., et al. (2016). Dissociation between iron accumulation
and ferritin upregulation in the aged substantia nigra: attenuation by
dietary restriction. Aging (Albany NY) 8, 2488–2508. doi: 10.18632/aging.
101069
Wang, Y., Cella, M., Mallinson, K., Ulrich, J. D., Young, K. L., Robinette, M. L.,
et al. (2015). TREM2 lipid sensing sustains the microglial response in an
Alzheimer’s disease model. Cell 160, 1061–1071. doi: 10.1016/j.cell.2015.
01.049
Wang, J., Tan, L., Wang, H. F., Tan, C. C., Meng, X. F., Wang, C., et al. (2015). Anti-
inflammatory drugs and risk of Alzheimer’s disease: an updated systematic
review and meta-analysis. J. Alzheimers Dis. 44, 385–396. doi: 10.3233/JAD-
141506
Wang, X. X., Tan, M. S., Yu, J. T., and Tan, L. (2014). Matrix metalloproteinases
and their multiple roles in Alzheimer’s disease. Biomed Res. Int. 2014:908636.
doi: 10.1155/2014/908636
Wang, W. Y., Tan, M. S., Yu, J. T., and Tan, L. (2015). Role of pro-inflammatory
cytokines released from microglia in Alzheimer’s disease. Ann. Transl. Med.
3:136. doi: 10.3978/j.issn.2305-5839.2015.03.49
Wilcock, D. M. (2012). A changing perspective on the role of
neuroinflammation in Alzheimer’s disease. Int. J. Alzheimers Dis. 2012:495243.
doi: 10.1155/2012/495243
Wojtera, M., Sobow, T., Kloszewska, I., Liberski, P. P., Brown, D. R., and
Sikorska, B. (2012). Expression of immunohistochemical markers on microglia
in Creutzfeldt-Jakob disease and Alzheimer’s disease: morphometric study and
review of the literature. Folia Neuropathol. 50, 74–84.
Wynne, A. M., Henry, C. J., Huang, Y., Cleland, A., and Godbout, J. P.
(2010). Protracted downregulation of CX3CR1 on microglia of aged mice
after lipopolysaccharide challenge. Brain Behav. Immun. 24, 1190–1201.
doi: 10.1016/j.bbi.2010.05.011
Wyss-Coray, T. (2006). Inflammation in Alzheimer disease: driving
force, bystander or beneficial response? Nat. Med. 12, 1005–1015.
doi: 10.1038/nm1484
Xavier, A. L., Menezes, J. R., Goldman, S. A., and Nedergaard, M. (2014). Fine-
tuning the central nervous system: microglial modelling of cells and synapses.
Philos. Trans. R. Soc. Lond. B Biol. Sci. 369:20130593. doi: 10.1098/rstb.20
13.0593
Yang, H., Yang, M., Guan, H., Liu, Z., Zhao, S., Takeuchi, S., et al. (2013).
Mitochondrial ferritin in neurodegenerative diseases. Neurosci. Res. 77, 1–7.
doi: 10.1016/j.neures.2013.07.005
Zaki, M. H., Vogel, P., Body-Malapel, M., Lamkanfi, M., and Kanneganti, T. D.
(2010). IL-18 production downstream of the Nlrp3 inflammasome confers
protection against colorectal tumor formation. J. Immunol. 185, 4912–4920.
doi: 10.4049/jimmunol.1002046
Zare-Shahabadi, A., Masliah, E., Johnson, G. V., and Rezaei, N. (2015). Autophagy
in Alzheimer’s disease. Rev. Neurosci. 26, 385–395. doi: 10.1515/revneuro-
2014-0076
Zhang, F., Nance, E., Alnasser, Y., Kannan, R., and Kannan, S. (2016).
Microglial migration and interactions with dendrimer nanoparticles are
altered in the presence of neuroinflammation. J. Neuroinflammation 13:65.
doi: 10.1186/s12974-016-0529-3
Zheng, H., Jia, L., Liu, C. C., Rong, Z., Zhong, L., Yang, L., et al. (2017).
TREM2 promotes microglial survival by activating Wnt/β-catenin pathway.
J. Neurosci. 37, 1772–1784. doi: 10.1523/JNEUROSCI.2459-16.2017
Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2017 Caldeira, Cunha, Vaz, Falcão, Barateiro, Seixas, Fernandes and
Brites. This is an open-access article distributed under the terms of the Creative
Commons Attribution License (CC BY). The use, distribution or reproduction in
other forums is permitted, provided the original author(s) or licensor are credited
and that the original publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution or reproduction is permitted which does not
comply with these terms.
Frontiers in Aging Neuroscience | www.frontiersin.org 23 August 2017 | Volume 9 | Article 277