Neurochemical Research

https://doi.org/10.1007/s11064-021-03456-1

REVIEW

Microglia at the Centre of Brain Research: Accomplishments

and Challenges for the Future
Nuno L. Soares1   · Helena L. A. Vieira1,2,3

Received: 11 August 2021 / Revised: 17 September 2021 / Accepted: 20 September 2021

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
Microglia are the immune guardians of the central nervous system (CNS), with critical functions in development, main-
tenance of homeostatic tissue balance, injury and repair. For a long time considered a forgotten ‘third element’ with basic
phagocytic functions, a recent surge in interest, accompanied by technological progress, has demonstrated that these distinct
myeloid cells have a wide-ranging importance for brain function. This review reports microglial origins, development, and
function in the healthy brain. Moreover, it also targets microglia dysfunction and how it contributes to the progression of
several neurological disorders, focusing on particular molecular mechanisms and whether these may present themselves as
opportunities for novel, microglia-targeted therapeutic approaches, an ever-enticing prospect. Finally, as it has been recently
celebrated 100 years of microglia research, the review highlights key landmarks from the past century and looked into the
future. Many challenging problems have arisen, thus it points out some of the most pressing questions and experimental
challenges for the ensuing century.

Glial Cells and Historical Perspective
of Microglia
The 19th century neuroscientists Camillo Golgi, Santiago
Ramón y Cajal and Rudolf Virchow were seminal in intro-
ducing and characterizing the concept of a new cell type in
the central nervous system (CNS): glial cells, the ‘glue of
the brain’ (glia meaning ‘glue’ in Greek) [1–3]. This his-
torical presumption of glia as a connective neural tissue has
evolved. We know today that these cells display a wide range
of paramount supportive mechanisms in the brain and spinal
cord [4].
* Nuno L. Soares
nuno.soares@nms.unl.pt
1 ‘element’ would be revealed to be, in fact, composed of two
Chronic Diseases Research Center (CEDOC) ‑ Faculdade
de Ciências Médicas/NOVA Medical School, Universidade
Nova de Lisboa, Campo dos Mártires da Pátria 130,
1169‑056 Lisboa, Portugal
2
Department of Chemistry, UCIBIO, Applied Molecular
Biosciences Unit, NOVA School of Science and Technology,
Universidade Nova de Lisboa, Lisboa, Portugal
3
Associate Laboratory i4HB - Institute for Health
and Bioeconomy, NOVA School of Science and Technology,
NOVA University Lisbon, Lisboa, Portugal
Glia can be subdivided into macroglia (which include
Astrocytes, Oligodendrocytes and Schwann cells, among
others) and microglia (Fig. 1).
These cells are active in the maintenance of appropriate
biological and chemical CNS homeostasis: ion regulation,
metabolic support, extracellular matrix formation, neuro-
transmitter recycling and modulation of synaptic transmis-
sion [5,6]. Astrocytes also play a role in vasoconstriction/
vasodilation and form part of the blood–brain barrier (BBB)
[5]. Their size and complex star-shape allow them to be in
close contact with multiple neurons at same time.
Microglia are the first line of defence for the CNS and
make up for about 5–10% of total glia in the human brain
[7]. Initially described by Cajal in the early 20th century as
non-neuron and non-astrocyte parenchymal cells which he
would christen as a ‘third element’ [2]. It was not until Rio-
Hortega’s ground-breaking brain staining methods that this
cells types—‘interfascicular glia’ (later oligodendrocytes),
and a population of a small population of ‘mesodermal ori-
gin and more related to leukocytes’, introducing the term
‘microglia’ [8]. These breakthrough findings would help in
characterizing microglial cells as active phagocytes of debris
and neuronal material, plastic in their interaction with other
populations and migratory [3,9].

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Fig. 1  Glial cell populations and interaction with neurons. Neurons
co-exist with other cells in the nervous system—Glia, which have
several important housekeeping functions. Oligodendrocytes insu-
late axons, which is crucial for proper action potential formation;
Astrocytes provide structural and metabolic support and are required
for synaptic and neurovascular function. Microglia are the ‘resident
immune cells’, clearing tissue and initiating an inflammatory response
While the origin of this cell type remained a hot topic
for some years, interest around microglia would eventually
die down until the 1960s, when novel techniques of histo-
logical analysis and methods for culture systems relaunched
[10–12]. In vitro studies allowed to further understand how
these cells respond to different stimuli and for further char-
acterization of microglial antigen-presentation, cytokine
producing and electrophysiology properties [13, 14]. Imag-
ing techniques in live settings, on the other hand, has had a
major role for understanding the role of these cells in brain
development and homeostasis, as well as how dysfunction
occurs and affects pathology of multiple brain disorders [15,
16].
The last two decades in particular really saw a boom
in microglia interest. Due to this, we know today those
Fig. 2  Microglial origins.
Microglia originate from hemat-
opoietic precursors (a) that exit
yolk sac’s blood islands into the
circulating system (b) and colo-
nize the neuroepithelium at E
9.5. This population proliferate
and differentiate locally when in
contact with specific environ-
mental and molecular cues,
growing to colonize the CNS as
a whole, self-sustaining locally
into adulthood. Migration of
circulation immune popula-
tions onto the brain parenchyma
occurs during inflammatory
events
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microglia are transcriptomically and functionally heterog-
enous, with functions that include innate immunity, phago-
cytosis, synaptic regulation, neuronal activity, and BBB
integrity.
Microglia’s Origins
One ever elusive topic has been the origin of microglial
progeny. Rio-Hortega initially hypothesized that these cells
originated from a mesodermal precursor [3], but several
alternative theories claimed that microglia was of neuro-
ectodermal origin [17] or even that they originated from
circulating monocytes which colonized the brain [18]. Dif-
ferent theories continued emerging into the early 2000s, with
several studies indicating that microglia shared ancestry with
astrocytes [17], and other hypothesis claiming that origi-
nated from pericytes [19].
Rio-Hortega’s mesodermal theory gained traction, with
studies revealing homology in terms of morphological fea-
tures and specific markers. However, it was only in 2010
that Ginhoux and colleagues produced a watershed moment
for the field [20]. Ginhoux’s team revealed that microglia
originate from primitive myeloid progenitors from yolk sac,
which enter circulation and colonize the brain parenchyma,
all this occurring before BBB development [20] (Fig. 2).
This embryonic-derived population differentiate locally in
the brain, with Colony Stimulating Factor-1 (CSF-1) having
a key role in driving microglial population [20, 21]. Single-
cell technology has unveiled that transcription factors such
as Pu.1, Irf8 and Sall1 are also relevant for myeloid cell
commitment to the microglial route [22, 23].
Unlike initially speculated, microglia appear to be able
to self-sustain throughout adulthood once it colonizes the
brain, and not be dependent on blood myeloid cells, which
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are mostly isolated from the parenchyma [20]. In fact, in an
adult mice model where microglia were genetically ablated,
there was a replenishment of a steady-state population within
1 week of depletion via proliferation of clusters of surviving
cells, in a interleukin-1 (IL-1) dependent mechanism [24].
In the homeostatic brain, microglial turnover appears
to depend on a delicate balance between proliferation and
apoptosis, with Réu and colleagues reporting that microglial
cells are renewed approximately every 4 years, with prolif-
eration occurring in random brain regions and that there
is no evidence of quiescent, long-lived populations in the
human brain [25]. In contrast, under pathological condi-
tions, microglia proliferation becomes very plastic, with Tay
describing that injury (facial nerve axotomy, FNA) results
in region specific selective expansion of microgliotic clones
(high ­MHCII+ ­ki67+ cells) [26]. Conversely, as mice recov-
ered from injury, microglia migrated out of the site of injury
and underwent selective apoptosis, with microglial network
being re-established [26].
Still, particularly with the onset of single-cell tech-
nologies, there is major progress to be made in regard to
understanding microglial identity and functionality at the
individual cell level, both in the context of physiology and
pathology.
Microglia—Function and Characterization
Microglia, the immunocompetent cells of the CNS, have a
small soma with a distinct ramified morphology for actively
patrolling the brain parenchyma, scavenging for dead cells,
debris, infectious and other potential noxious agents [7], as
well as establishing contacts at the synapse level, with other
glial cells or with the BBB [7]. Adult microglia are armed
with several surface molecules, including pattern-recogni-
tion receptors (PRRs), cytokine and chemokine receptors,
purinergic receptors, adhesion molecules, Fc receptors, ion
channels, and others that allow recognition and response
to homeostatic imbalances [7]. Phenotypically, microglia
express several markers also present in macrophages, such
as Iba-1, CD11b, MHCII or F4/80. CD45 [7, 27] expression
has been used to distinguish between macrophages (­ CD45hi)
and microglia (­ CD45lo), but this readout is often seen as
unreliable [27]. Recently, the transmembrane protein 119
(TMEM119) has been described as a microglia-specific
marker which is not expressed in infiltrating myeloid cells
[28].
When encountering noxious stimuli, microglia undergo
physiological and morphological alterations, namely: (i)
adopting an ‘amoeboid’ shape, (ii) overexpressing several
surface molecules (eg. MHCII, CD86, CD32, FcγRs), (iii)
producing and secreting a wide range of proteases, reac-
tive oxygen/nitrogen species (ROS/RNS), chemokines (eg.
CCL1, CCL5, CCL12) and cytokines (eg. Tumour necro-
sis factor-α, IL-1β, IL-6, IL-18), (iv) priming other glial
cells and (v) recruiting peripheral immune populations [7,
29–31]. This robust response allows for higher motility, the
scavenging and phagocytic removal of pathogenic agents
(bacteria, viruses, misfolded proteins), cellular and myelin
debris and induce cell death in compromised neural tissue
[7], returning the tissue to homeostasis. Microglia is also
crucial for the priming of the adaptive immune system, act-
ing as antigen-presenting cells for infiltrating leukocytes in
auto-immune disorders such as multiple sclerosis [32].
During the inflammatory response, microglia phenotype
is generally referred as ‘activated’ or M1 profile. In con-
trast with microglial populations transitioning to inflamma-
tory resolution or in homeostasis, which are referred as M2
phenotype, or ‘alternatively activated’ [27], and have cru-
cial roles in tissue healing or phagocytic resolution [7, 27]
(Fig. 3). M2 profile is characterized by presenting surface
markers CD206, CD163 and TREM-2 (triggering receptor
expressed on myeloid cells 2) [33], and by the secretion of
lectin Ym1 [34] and differential cytokine secretion pattern
(suppression of inflammatory cytokine release, high levels of
IL-10, IL-4, Transforming growth factor-β or Brain-derived
neurotrophic factor) [33]. Arginase-1 (Arg-1) is the ‘classi-
cal’ M2 marker. In fact, as this enzyme competes with induc-
ible nitic oxide synthase (iNOS), overexpressed in inflamma-
tion, for the substrate L-Arginine, yielding L-ornithine and
urea, and not the inflammatory mediator nitric oxide [35].
While currently also applied to microglia, initially the M1/
M2 paradigm originated from the need to contextualize the
polarization of macrophage populations, akin to what had
previously been done with characterization of T ­ h1 and T
­ h2
populations, which were functionally characterized by the
profile of cytokine secretion [36]. Yet, both microglial cells
and macrophages are very plastic and have region-specific
characteristics. The M1 vs M2 dichotomy is a ‘black-and-
white’ answer, which tells us more about how we tend to
box things for the sake of simplicity and not about how these
cells really operate. As Richard Ransohoff wrote a few years
ago, it is a matter of semantics [37]. The shortcomings of
the M1 vs M2 framework has been further put under the
microscope by the increasing reporting of new microglia ‘M
subphenotypes’, such as M2a, M2b and M2c [38]. Whether
this means that we should see the M1/M2 dichotomy as a
spectrum that has these two states as opposites of a gradi-
ent or whether something more heterogenous and multipolar
should be posited remains to be answered.
In reality, the fact that microglia have constant functional
relevancy, as well as region-specific characteristic, could be
a bookmark for the future. For instance, in the mouse brain,
microglia have higher levels of reactive markers CD11b,
F4/80 and FcγRI in the hippocampus [39], whereas in the
frontal cortex CD11b is lower and CX3CR1 (fractalkine
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Fig. 3  Microglial ‘classical’ vs ‘alternative’ states. Upon damage or
inflammatory stimuli (LPS, IFN-γ), microglia respond by expressing
surface reactive markers and receptors, over-express iNOS, adopt a
smaller, rounder ‘amoeboid shape’ and secrete inflammatory media-
tors (cytokines, ROS, glutamate, proteases). In this state, microglia
also tend to be more proliferative and motile, accumulating around
the injury site. Conversely, signals such as IL-10 or TGF-β lead to
receptor) is particularly high [39]. In the substantia nigra,
microglial density is locally very high and CX3CR1 and
CD200R are both elevated [40, 41]. In terms of number,
other microglia-rich regions include the basal ganglia and
olfactory telencephalon, whereas the brainstem and the cer-
ebellum are more sparsely populated [41, 42].
Microglia in the Homeostatic Brain
Microglia play important roles in non-immune surveillance
mechanisms. These cells are involved in homeostatic pro-
cesses in brain development, trophic support, myelination
and synaptic plasticity [43–45](Fig. 4).
In early brain development, microglia is a key cell type in
the regulation of cellular population number, as they selec-
tively phagocyte dying neuronal precursor cells, while also
inducing apoptosis via secretion of ROS and inflammatory
factors and subsequently engulfing cells in specific brain
regions [46–48]. Several of these steps also occur during
adult hippocampal neurogenesis, as microglia control neural
precursor stem cell (NPSC) number and remove apoptotic
neuroblasts [49, 50]. Interestingly, the secretome of phago-
cytic microglia also contributes to the regulation of the pro-
liferation/differentiation balance in neurogenic niches [51].
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a more restorative profile, with a more ramified morphology, down-
regulation of inflammatory mediator expression, increased levels of
receptors such as CD206, as well as enzyme Arginase-1. Anti-inflam-
matory cytokines, neurotrophins and growing factors see increased
secretion. In this state, microglia become more active in phagocytosis
of debris and promote myelination
Conversely, microglia also produce several factors, such
as BDNF and thrombospondin, which act as survival cues
[52–57]. Neural fate in the layer V of the neocortex, for
example, is actively dependent on Insulin-like growth factor
1 (IGF-1) locally produced by a microglial subpopulation
[58]. Additionally, during brain development, breakthrough
studies indicate the existence of an intimate connection
between immunity and neuron wiring, as microglia orches-
trate mechanisms of axonal outgrowth and the stimulation
of cortical interneuron migration [59–61].
These myeloid cells are also active synaptic modulators
[61–63]. For example, in post-natal period, microglia selec-
tively eliminate ‘weak’ or deficient pre-synapses in spe-
cific regions, namely in the dorsolateral geniculate nucleus
(dLGN) of the thalamus, hippocampal CA1 region and spi-
nal cord [62, 64]. Its activity in synaptic pruning is actively
regulated by neuron-astrocyte-microglia tripartite communi-
cation, as astrocytic derived IL-33 has recently been shown
to drive microglia synapse removal [64]. Similarly, specific
neuron-microglia communication pathways are required
for microglia synaptic refinement. Examples of this are the
CX3CL1-CX3CR1 pair and the immune surface molecule
TREM-2 [65]. Disruption of these signalling mechanisms
lead to long-term consequences in brain network connectiv-
ity and signal transmission [46, 58]. Inversely, microglia can
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Fig. 4  Microglia functions during brain development and homeostatic
support. Microglia are involved in pruning of weak, immature, or
superfluous synapses (a), removal of apoptotic neurons and induction
of apoptosis signalling in early brain development (b), as well as neu-
also induce synaptogenesis and spine formation [44, 66].
Identified microglia synaptogenic factors include soluble
BDNF and IL-10 [67–70]. Additionally, cargo from extra-
cellular vesicles (EVs) can also regulate synaptic activity,
such as (i) microvesicles, which increase excitatory trans-
mission via enhancement of neuron sphingosine metabo-
lism [71]; (ii) endocannabinoids from EVs, which activate
type-1 receptors in neuron hippocampal cultures and inhibit
GABA release [72]; or (iii) EVs containing microglia spe-
cific microRNA 146a-5P which suppresses excitatory syn-
apses [73].
More recently, Anne Schaefer’s group revealed a new,
important new link between innate immunity and neural
function. It was shown that microglia actively regulate neu-
ronal activity by sensing and catabolizing neuron-derived
extracellular ATP, inhibiting neuron activity via pre-synap-
tic ­A1R negative feedback mechanism [74]. This prevents
excessive neurostimulation, regulating synchrony and firing
frequency of striatal neurons in vivo [74].
Microglia also appear to play a role in function and dif-
ferentiation of other glial cells. Microglia locally promotes
oligodendrogenesis in the early Postnatal Subventricular
Zone via release of pro-inflammatory cytokines, such as
IL-1β, IL-6 and TNF-α [57]. Similarly, microglia can induce
in vitro astrocyte differentiation from neural stem cells [75].
ronal process formation and synaptogenesis (c). Additionally, micro-
glia are also involved in trophic support signalling (d), preservation
of the microvasculature and BBB integrity (e), as well as regulation
of microglia excitability via purinergic communication (f)
Other data have also revealed, using in vivo models, that
microglial activity results in enhancing of myelination [76].
Microglia can also play a role in modelling in vivo vascu-
larization by paracrine release of TGF-β1 [77]. Even though
microglial inflammation can disrupt BBB integrity, vessel-
associated microglia is important for preserving BBB per-
meability, interacting closely with endothelial cells and pro-
ducing tight-junction protein Claudin-5 [78]. In summary,
microglial function is very heterogeneous, playing a central
role not only in CNS immunity, but also development and
overall homeostasis.
Microglial Dysfunction and Disease
Both acute and low-grade, chronic inflammatory mecha-
nisms perturb the CNS equilibrium and can irreversibly
damage brain tissue [79–81]. At a molecular level, several
mediators can be neurotoxic and cause cell death, namely
ROS and RNS, which damage cellular components, gluta-
mate, activating N-methyl-D-aspartate (NMDA) receptors
cytokines such as TNF-α, which is a cell-death ligand (cas-
pase-8 dependent mechanism or necroptosis) and a wide
range of proteases [79, 82–85]. BBB integrity can also be
eroded, which causes a consequent leakage of peripheral
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immune populations and other circulating molecules into
the brain parenchyma [86], further amplifying the noxious
nature of the response. Aberrant phagocytosis can occur as
a consequence of this and lower levels of nourishing factors
is also a common cause of local damage [87–90].
CNS inflammation is a predominant feature of neurologi-
cal diseases, both chronic (Parkinson’s Disease, Alzheimer’s
Disease, Multiple Sclerosis, Schizophrenia) [29, 79, 91, 92]
and acute (Brain ischaemia, Traumatic brain injury) [80,93].
While these disorders are very heterogeneous in nature, they
all present similar local inflammatory features and target-
ing neuroinflammation can contribute to limit pathological
severity [94–96].
In AD, systemic inflammation is a pathological driving
force [81, 96]. In brains of both human patients and AD
mouse models, microglial cells proliferate, migrate and accu-
mulate around Amyloid β (Aβ) deposits, acting as physical
barrier between compromised and non-compromised tissue
[81]. Moreover, microglia recognize, bind to and degrade
soluble forms of Aβ via micropinocytosis [97, 98]. These
cells also engulf aggregates via phagocytosis mediators like
scavenger and toll-like receptor (TLR) families [99, 100].
Yet, data have implied that microglial cells, which engulf Aβ
are unable to efficiently degrade fibrils, and that this chronic
deficient clearance mechanism contributes significantly to
inflammation and progression of disease [101]. Some have
also argued that the pre-existence of a low-grade inflamma-
tory state leads not only to functional damage to neurons but
also contribute to limit plaque clearance [102, 103].
The evidence that loss of function variations in TREM-
2, which is strictly expressed by microglia in the brain, can
be a risk factor for late-onset AD development, is another
indicator of microglia’s central role in AD pathogenic-
ity [104]. In fact, in TREM-2 deficient mice, it has been
reported that microglia present altered proliferation and
surveying functions, and do not migrate towards injured
regions, nor accumulate around plaques [105–107]. Moreo-
ver, animals lacking TREM-2 have also displayed higher
number of dystrophic neurons, compared to non-mutants
[105, 106]. At the cell level, microglia populations carry-
ing TREM-2 mutations display altered activation signatures,
which could indicate that the cells fail to evolve towards a
pro-inflammatory state in tissue [108]. In other chronic neu-
rological disorders, there are similar hallmarks of microglia
dysregulation: in tissue samples from Parkinson’s Disease
(PD) human brains, microglia overexpress reactive markers
[109]. Additionally, phagocytic dysregulation also appears
to be involved in pathogenicity, as α-synuclein (α-syn) can
disrupt engulfment [110, 111].
In a prevalent acute pathology like brain ischaemia,
microglia migrate to the injury site, driven by damage-
associated molecular pattern molecules and locally engulf
and degrade compromised cells, in order to return the tissue
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to homeostasis [112]. However, sustained inflammatory
response carried by these cells can have an opposite effect
and act as a secondary damage inducer in the penumbra
[80, 113]. Prolonged inflammation can lead to synaptic
dysfunction, neuronal atrophy, and eventual cell death, by
excitotoxicity, oxidative stress, or cytokine-driven apoptosis
[113], as well as oligodendrocyte loss, resulting in improper
myelination [114]. Also, microglia response will activate
astrocytic cells, which further amplifies inflammation, per-
turb glutamate buffering and release of neurotrophic fac-
tors [115, 116]. All these events contribute to an increase in
microvasculature permeability by overexpression of adhe-
sion molecules and detachment of astrocytic components
and pericytes [117–119], and subsequent inflow of circulat-
ing immune populations [113].
Microglia (dys)function during ageing is also an object
of great interest. Several studies in aged brains of humans
and other mammals show that microglia have considerable
physiological alterations and present a constant ‘primed
state’, with higher expression of several surface receptors
and pro-inflammatory mediators [120–124]. Morphologi-
cal dynamics are also altered, as microglia present reduced
processes, similar to the inflammatory ‘amoeboid’ pheno-
type, become less motile and phagocytic [125–127]. These
cells are more responsive to stimuli and elicit an exaggerated
and prolonged immune response. This hyper reactive milieu
correlates with and accompanies increasing tissue cell death
and cognitive decline [128, 129]. Understanding the mecha-
nisms of microglia senescence can be a steppingstone to bet-
ter comprehend the biological significance of its dysfunction
in the context of neurodegeneration.
Current literature highlights how microglial function is a
double edged-sword under a constant and tight control net-
work [130]. Such control network is comprised by the other
brain populations, microglia itself and the environmental
milieu [130]. Loss of this equilibrium can lead to exacer-
bated microglial activation, which can turn its function from
damage response to a damage perpetuation [131].
Signalling Factors in Microglia Cell‑to‑Cell
Communication and Inflammatory
Modulation
As priorly stated, microglia shift between a surveillance
‘resting’ mode and a ‘reactive’ state, which initiates robust
innate immune functions and is pivotal for CNS homeosta-
sis. However, this balance is very delicate and needs con-
stant and tight regulation. Loss of equilibrium results in the
creation of a sustained inflammatory milieu with a damaging
nature.
For regulating neuroinflammation, CNS innate immune
system is exposed to ubiquitous microenvironmental cues
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[130, 132–137]. Microglia cells are in constant commu-
nication with themselves, other glial cells and neurons.
Production of anti-inflammatory factors (such as IL-10,
IL-4, IL-13 and TGF-β), of neurotransmitter and neuro-
peptides (like Vasoactive intestinal peptide—VIP and Pitu-
itary adenylate cyclase-activating polypeptide—PACAP),
circulating EVs and of specific microRNAs is key in the
resolution of inflammation [138–145]. Likewise, sev-
eral neuron-microglia specific pathways are relevant for
microglial activity fine-tuning, including CD200-CD200R,
CD45-CD45L, CX3CL1-CX3CR1, CD172a-CD47 and
TREM-2 [146–151].
CD200-CD200R is a pair of membrane Immunoglob-
ulin superfamily glycoproteins [152, 153]. Microglia
express CD200R whose direct interaction with neuronal
CD200 acts as a ‘calming’ input on inflammatory activ-
ity [154, 155]. Interruption of this pathway leads to an
enhanced microglia response to activation stimuli [149,
156]. Low levels of the CD200-CD200R pair has been
registered in brains of Alzheimer’s Disease (AD) patients,
particularly in the hippocampus [157]. Moreover, CD200-
CD200R disruption in an in vivo model of experimental
autoimmune encephalomyelitis (EAE) increases tissue
inflammation and disease severity [158]. This is reversed
by overexpressing CD200 on neurons [158]. Additionally,
recent data show that CD200-CD200R pathway could have
a significant impact on microglia phagocytosis as well
[159, 160].
Similarly, CX3CL1 expression in neurons can pro-
vide a ‘calming cue’ to microglia populations, regulating
engulfment of synaptic material [161, 162]. Ransohoff
and colleagues published that CX3CL1 depletion results
in exacerbated microglia inflammatory function in vivo
[163]. Still, other data show conflicting information: this
communication pair is overexpressed in samples from
EAE rodents and its effect is even more ambiguous in AD
models [164, 165]. It could be argued that this heterogene-
ity is related to the fact that neuronal CX3CL1 has both a
membrane bound form and a soluble one, which may have
varying regulatory consequences on the receptor.
TREM-2, another membrane microglial Ig superfam-
ily receptor, is an additional inhibitory ‘check-point’ for
microglial inflammatory response [150, 166]. Addition-
ally, TREM-2 has a crucial role in phagocytosis, as its
depletion causes an impairment of apoptotic cell clearance
[167].
Overall, microglial cells are in constant crosstalk with
a dynamic regulatory milieu, which actively regulates
several of microglial functions [130]. Understanding and
describing these inter-cellular communication pathways
is an important step for comprehension of the brain and
offers potential therapeutic tools and approaches in the
future.
Phagocytosis at the Center of Microglial
Function
Phagocytosis is one of the key cellular functions of micro-
glia. It is a complex, multi-step process by which cells rec-
ognize, bind, engulf and digest large particles (≥ 0.5 µm),
which can be pathogens, cellular debris, senescent cells or
foreign bodies of a varied nature. It is a fundamental part
of innate immunity and key in the initiation of adaptative
response [168, 169]. Likewise, phagocytosis is also cru-
cial for tissue homeostasis by eliminating apoptotic cells
[170]. Cells with capacity to phagocyte are also referred
to as ‘professional phagocytes’ and include, macrophages,
neutrophils, monocytes and dendritic cells [171].
In the brain, microglia are the major professional phago-
cyte cell type [16]. These cells are equipped with most of
the surface receptors expressed by other tissue phagocytes
and have similar physiological functions [16] (Fig. 5). Par-
ticle recognition (pathogens, aggregates, myelin debris) is
mediated by an array of receptors: Foreign bodies express
surface pathogen-associated molecular patterns (PAMPs),
which are recognized by PRRs such as TLRs, and oth-
ers like Dectin-1 and scavenger receptor A [7, 172–174].
Potential pathogens can also be recognized by circulating
antibodies, which can be subsequently bound to a cascade
of complement system components [7]. Phagocytes can
then bind, through specific surface receptors, to the Fc
region of Igs that coat the foreign body or to specific com-
plement regions, and initiate phagocytosis [175–177]. At
the level of the synapse, complement proteins—receptor
pairs (C3-C3R) are crucial for interaction [178].
Microglia have specific molecular mechanisms for the
detection and engulfment of dying cells. Cells undergoing
cell death release soluble ‘find me’ signals, such as nucleo-
tides and lysophosphatidylcholine (LPC), that facilitate
recruitment [179–181]. Also, apoptotic cells express sur-
face ‘eat me’ signals (phosphatidylserine, oxidized LDL,
Intercellular adhesion molecule 3) [182–185], which are
in turn recognized by phagocyte surface receptors, as are
MerTK (MER proto-oncogene, tyrosine kinase), α vβ 3-
integrin (vitronectin), CD36 or brain-specific angiogenesis
inhibitor 1 (BAI1) [183, 186–188], either directly or via
adaptor molecules. Inversely, healthy cells express surface
receptors, like CD31 and CD47, which have been identi-
fied as ‘don’t eat me’ signals [189, 190].
As stated previously, microglia detect and degrade for-
eign bodies, remove dying or superfluous cells and act
as synaptic and neurite ‘strippers’ [16, 48, 61, 62, 112].
In addition to removal of dying or dead cells, microglia
phagocytosis plays an important part in regulating the
number of infiltrating immune cells, such as neutrophiles
and leukocytes [191, 192]. This house-cleaning function
13

Fig. 5  Microglia phagocytosis and implications in the CNS. Micro-
glial cells are professional phagocytes of the brain, clearing the tissue
by removing dying cells and cellular debris (a), misfolded proteins
(b), and potential pathogens, such as bacteria and viruses (c). Addi-
tionally, microglia are involved in the engulfment of synapses and
neuronal processes (d), as well as targeted removal of neuronal pre-
cursors (e), key mechanisms during brain development. These cells
highlights that these myeloid cells not only can initiate
immunity in situ, but also act as a quality control mecha-
nism, which is crucial for tissue homeostasis and the main-
tenance of immune-privileged nature of the CNS, elimi-
nating supernumerary immune populations that infiltrate
the brain upon lesion [191, 192]. In fact, blockade of this
microglial phagocytosis of infiltrating neutrophils, leads
to loss of neuronal survival in organotypic slices [191].
Also, microglia can engulf and kill other live, non-neu-
ronal cells, such as glioma cells, in an in vitro co-culture
system [193]. This mechanism has described to be depend-
ent on recognition via microglia surface receptors Siglec-
h/DAP12 [193].
Phagocytosis of apoptotic cells has been connected with
an autocrine regulation of the phagocyte’s inflammatory
state [194, 195]. In microglia, this has been partially cor-
roborated by some literature, where engulfment of apoptotic
neuronal and T cells results in the inhibition of pro-inflam-
matory cytokine secretion in  vitro [196, 197]. Whether
there is a specific requirement at the molecular level that
triggers microglial anti-inflammatory priming is still to be
understood.
The role of phagocytosis in the framework of the M1/M2
dichotomy has also been a source of doubt and appears to
be very much context dependent, as both phenotypes appear
13
Neurochemical Research
can also remove other populations, such as invading neutrophils and
other granulocytes (f), through cell-specific signalling. In pathology,
stressed neurons (exposed to oxidative stress, inflammation or other
sublethal stimulus) can express low levels of membrane phosphatidyl-
serine (PtdSer), which microglia will recognize, engulfing live cells
and causing cell death (g)
to be active in engulfing and digesting different targets [198,
199].
Microglial Phagocytosis and Pathology
In 2014, Brown and colleagues have described ‘phagoptosis’
as a novel cell death form: death by phagocytosis of other-
wise viable cells [200]. Also known as ‘primary phagocyto-
sis’, it differs from ‘regular’ phagocytosis, where engulfment
and removal are a consequence of target cell apoptosis and
not the reason. Phagoptosis mediates turnover of erythro-
cytes, tumours, and other populations [46, 47, 201, 202],
and thus is quantitatively one of the main forms of cell death
in the body. In the brain, phagocytosis of live neurons has
also been referred to as ‘neurophagy’, and is a key event to
several brain physiological processes [203].
Phagoptosis can have, however, a pathological role [200].
In the brain, several sub-lethal insults can lead to activation
of microglia, which engulf viable neurons and cause sub-
sequent pathological neuronal loss [89]. Sustained micro-
glial production of ROS and RNS causes adjacent viable
neurons to expose PtdSer at their membrane surface, trig-
gering ‘eat-me’ signalling and initiating engulfment and
consequent neuronal death [204, 205]. In vivo injection of
Neurochemical Research
lipopolysaccharides (LPS) into rat brains caused neuroin-
flammation, which in turn promoted microglia engulfment
and neuronal loss [90]. These events were partially reverted
by MFG-E8 (Milk Fat Globule-Epidermal Growth Factor-
Factor 8) genetic ablation, as well as vitronectin chemical
inhibition [90]. Phagoptosis is also involved in damage
propagation in stroke, particularly in the penumbra area,
where stressed neurons reversibly expose phosphatidylser-
ine [206]. Again, microglia overexpression of phagocytosis
receptors can directly influence the extent of damage in this
key region. In fact, MerTK deficient and MFG-E8 knockout
mice experienced reduced neuronal loss and motor deficit
in the weeks after the ischaemic event [206]. Similar results
have been registered in PD animal models, where knockout
of complement component 3 (C3) can limit loss of dopamin-
ergic neurons in the substantia nigra [207].
Therefore, phagoptosis is an extremely dynamic and
delicate process, where the target cell ceases to exist, and as
such, cannot be analysed. Modulation of these mechanisms
currently seem like an enticing prospect, but one needs to
better understand the molecular players and interactions that
govern phagoptosis, as the mechanisms that lead to dele-
terious phagoptosis are the same involved in mechanisms
relevant for tissue homeostasis, such as removal of protein
aggregates and turnover of particular cell populations.
Approaches to Regulate Microglial (dys)
Function
As it became clear that neuroinflammation is a driving force
in the pathophysiology of several CNS-related disorders,
new approaches targeting microglial function have been
designed. These include classical pharmacological agents,
but also cytokine-targeting mechanisms, metabolic messen-
gers, and microRNAs, all of which can regulate the plasticity
of the immune patrollers of the brain.
Classical pharmacological approaches, such as usage of
non-steroidal anti-inflammatory drugs (NSAIDS), which
target cyclooxygenases, have been used to alleviate LPS-
induced brain inflammation in neonatal rats, reducing acti-
vated microglia, as well as inflammatory cytokine secre-
tion [208]. Melatonin and minocycline are other examples
of compounds used to alleviate brain inflammation, with
minocycline decreasing microglial activation and demy-
elination in a EAE model [209]. Novel strategies, such
as using EV-based approaches, are becoming burgeoning
topics in CNS therapeutics. Their capacity to penetrate
barriers, low immunogenicity and bioavailability have
made EVs prime candidates as modern vehicles for drug
delivery. In fact, EVs from human bone marrow mesen-
chymal stem cells have been administered intranasally and
shown to be uptaken by microglia in the forebrain of rats
[210]. Other than being used as delivery systems, EVs are
now being seen as potential biomarkers with diagnostic
and therapeutic potential, such as in cancer [211], or in
neurodegenerative disorders, where cerebrospinal fluid
vesicle content can be profiled.
Recently, new selective target approaches have also been
emerging. These include manipulation of specific neuron-
microglia cross-talk pathways, such as TREM-2 [167],
CD200-CD200R [154], and other pairs. As aforemen-
tioned, CD200 overexpression limits disease progression in
a EAE model [158], and CD200Fc administration to micro-
glia in vitro inhibited reactive macrophage phagocytosis
of oligodendrocyte progenitors [160]. Similarly, blockade
of inflammatory cytokines, such as TNF-α inhibitors have
been used to regulate inflammatory disorders, as rheuma-
toid arthritis and Crohn’s disease [212], but results in brain
in vivo models have been polarizing.
Targeting of surface receptors, like histamine [213], endo-
cannabinoid [214] and acetylcholine receptors [215] have
also garnered anti-inflammatory responses in microglia.
Conversely, neurotrophins (NGF) [216] and cytokines (IL-
10, IL-4) [88, 217] involved in inflammatory resolution also
favour microglial anti-inflammatory polarization.
Blockade of potassium channels, which are crucial for
microglial respiratory burst during inflammation, has also
been used as a recent approach to inhibit sustained micro-
glial activation in in vivo models that include spinal cord
[218] and ischemia/reperfusion injuries [219].
One novel approach that only recently started being taken
advantage of is the usage of microRNAs [220]. These short,
non-coding RNA molecules are important endogenous post-
transcriptional regulators, thus offering great control of cel-
lular mechanisms, making them ideal tools for microglial
manipulation. miRNA-124 inhibits inflammatory genes (IL-
6, TNF-α) [221] and drives resolution when administered
in liposomes in a EAE model [222]. miRNA-124 silences
CCAAT/enhancer‐binding protein C/EBP‐α, an important
inflammatory transcription factor. Recently, other microR-
NAs, such as miRNA-200b, and -155 both by direct delivery
and viral delivery [223, 224]. Another strategy for regula-
tion of microglial polarization is metabolic reprogramming.
Increasing evidence show that microglial inflammatory
polarization is coupled with a metabolic switch towards
glycolysis [225]. Glucose transporters have been reported
to be upregulated upon LPS and IFN-γ triggered activa-
tion [226], enhancing glucose uptake, and enzymes such as
hexokinase (HK) [227] and 6-phosphofructo-2-kinase/fruc-
tose-2,6-biphosphatase 3 (PFKFB3) have also been reported
to be upregulated [228]. In fact, blockade of HK2 leads to a
dampening in microglial reactivity in a stroke model [227],
while 2-deoxy-D-glucose (2-DG), which competitively
inhibits glucose-6-phosphate production, reduces microglial
activation and production of NO [229].
13

Recently, Motterlini and colleagues showed that in vitro
administration of carbon monoxide (CO) leads to an attenu-
ation of this glycolytic switch in LPS-treated BV2 microglia,
which limits inflammatory output [230]. Other more spe-
cific metabolic targets have also been explored in order to
regulate neuroinflammation, such as fatty acid metabolism,
which microglia use as alternative sources of energy in the
brain [231].
Concluding Remarks and Future Challenges
The recent centenary of microglia’s discovery has served as
a symbolic hallmark which helps putting in perspective how
research about these cells has progressed to be at the centre
of neurobiology. Several important steps have been taken,
allowing for a better characterization of: (i) microglial yolk
sac origin and (ii) function, ranging from inflammation to
synaptic regulation and neuronal excitatory control as well
as interactome with other cells and the environment.
Modern findings have been powered by ever-evolving
technology, as sophisticated imaging and physiology meth-
ods emerged and animal cell culture procedures became
of widespread use [10–12, 15]. In vivo models, such as
zebrafish and rodent models, as well as the usage of induced
pluripotent stem cells (iPSCs) are powerful present and
future tools to translate knowledge into real-life impact on
human lives. The emergence of single-cell transcriptomics
aims to fill big knowledge gaps at the cell level, as ques-
tions related to the multiple activation states (and respec-
tive functions) continue to multiply. Moreover, this type of
technology will aid in better characterizing the patterns of
microglial differentiation once these cells populate the early
parenchyma, while potentially allowing for the better char-
acterization region and sex-specific differences of particular
populations in the brain, which may be a steppingstone to
better understand new microglial functions. Sex differences
in particular have been shown to lead to different outcomes
in animal models of neuropathic pain or anxiety [232, 233].
Genome-wide association studies (GWAS), which have
been increasingly used, can help in elucidating the involve-
ment of microglial dysfunction and risk genes in disease.
This is particularly useful in complex, multifactorial disor-
ders like neurodegenerative diseases, where microglial dys-
function and overall neuroinflammation clearly contributes
to the progression of disease, but questions still remain about
its involvement in the etiology of pathology, per se.
Another challenge will be to characterize the mecha-
nisms and pathways of microglia cell-to-cell commu-
nication, both with itself, other glia, and neurons. This
topic has become a bigger focus, as neurobiology moved
away from its very neurocentric roots, with the discov-
ery of multiple neuroimmune receptor-ligand pairs which
13
Neurochemical Research
regulate inflammation [130, 132]. Still, this is a particu-
larly difficult topic with a vast number of open questions
at the molecular level, such as what the true role of frac-
talkine is within the context of microglial activation or
what are the mechanisms through which astrocyte-micro-
glia-neuron triad communication at the synapse level.
Microglia are hematopoietic cells that populate the
CNS with multiple housekeeping functions and immune-
surveillance properties, all of which crucial for tissue
homeostasis [7]. One century of microglia has brought
much needed knowledge regarding these cells, but also
enticing tools, dilemmas, and new questions for the ensu-
ing century.
Funding  This work is  financed by national funds from FCT—
Fundação para a Ciência e a Tecnologia, I.P., in the scope of the
project UIDP/04378/2020 and UIDB/04378/2020 of the Research
Unit on Applied Molecular Biosciences—UCIBIO  and the pro-
ject LA/P/0140/2020 of the Associate Laboratory Institute for Health
and Bioeconomy—i4HB, and in the framework of FCT-ANR/NEU-
NMC/0022/2012 Grant and PTDC/MEC-NEU/28750/2017 Grant; and
FCT provided individual financial support NLS (PD/BD/127819/2016).
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