1. Introduction
Microglia are brain-dwelling parenchymal macrophages [
1] that are distinct from other brain-dwelling non-parenchymal macrophagic populations and tissue macrophages [
2,
3]. Under healthy and normal conditions, microglia are self-maintained with no considerable contribution of peripheral myeloid cells [
4]. In addition, ~5% of the total cells of the neocortex are normally microglial [
5]. These macrophages have a uniform distribution throughout the central nervous system (CNS) parenchyma, with a population of approximately 10% of the total cells in the CNS. In addition, they can form a cellular grid with their ramified and highly motile process [
6]. During the developmental phase of the brain, microglia help shape neural circuits by modulating the strength of synaptic transmissions and sculpting neuronal synapses. When sensing any CNS injury, they become phagocytic and eliminate microbes, cell debris, protein aggregates or any form of CNS insult. However, microglial activation has been reported during several neurological conditions [
7,
8], in which they have evidently been playing both beneficial and detrimental roles, depending on disease progression.
The correct functioning of microglia is necessary for neural circuit remodelling and synaptic function, but their involvement in the pathogenesis of neurological diseases raises the debate of whether microglial activation is beneficial or detrimental. In response to CNS insults, microglia proliferate rapidly [
9], but their proliferation and turnover rates under homeostatic conditions have not been well documented. Filling this research gap could differentiate them from disease-causing agents [
10]. Several studies have shown interesting but conflicting results, either very low or very high microglial turnover in mice and the post-mortem brain [
11,
12,
13], although the validity of these techniques has been arguable and limited [
14,
15]. However, it is clear that understanding the regulation of microglial populations in the brain is crucial to understanding their functioning at different phases of age and age-related diseases. In one case, the mouse model of AD showed that early priming of individual microglia can induce long-lasting functional changes in life, whereas microglial senescence may have a role in age-related neurodegeneration [
10]. For example, stimulation of the brain’s immune system during development can induce long-lasting changes in microglial immune responses [
16,
17], and ageing and senescence of microglia contribute to neurodegenerative disorders [
18].
However, changes in microglial homeostasis and functioning during age and age-related neurodegenerative diseases could be due to alterations in the brain’s microenvironment or to the longevity of microglia, possibilities that remain unclear. Therefore, this article will discuss in detail microglia’s physiological role and changes due to exposure to different insults in individual age-related neurodegeneration.
2. Molecular and Functional Background of Microglia
Microglia are the resident immune cells of the brain and cover almost 5–12% of CNS cells [
19]. In addition, microglia are involved in the homeostasis of host defence against pathogens and consequent neurological disorders [
20,
21]. These cells are mesenchymal, originating in the yolk sac, and do not require hematopoietic stem cells for renewal [
13,
22]. Their survival and maintenance depend on cytokines, including CSF1 and interleukin (IL)-34 [
23], and on transcription factors such as IRF8 [
22]. However, microglia can be simply defined as innate immune cells of the CNS that originate from myeloid cells and express several genes, including
Cx3cr1,
CD11b,
Iba1 and
F4/80 [
21]. Depending on comprehensive knowledge of microglial gene expression and relevant functions [
21,
24], this study attempted to determine microglial functions in accordance with their gene expressions. There are three basic functions of microglia—sensing their environment, maintaining physiological homeostasis and protecting against self-modified and exogenous injurious agents. Furthermore, these normal functions are important regulators from a human being’s embryonic stages through to old age.
Sensing is the primary requirement for microglia to function in housekeeping and defending their host from injury (
Figure 1). Microglia form a network that spans throughout the CNS. Their thin processes are dynamic and in constant motion, allowing them to scan the area surrounding their cell body every few hours and rapidly polarise toward focal injury [
8,
19]. This network includes approximately 100 or more genes that consistently scan surrounding cell bodies and sense any changes in their microenvironment [
24,
25,
26]. The sensome mRNA is expressed uniformly in microglia of different regions of the brain, indicating that all microglia are capable of sensing functions.
The second and most important part of microglial function is physiological homeostasis. This function includes synaptic remodelling such as CNS development and maintenance, neurodegeneration [
27,
28], phagocytosis of dead or malfunctioning neuronal cells or cell debris [
29,
30], or myelin homeostasis [
31] (
Figure 2). In addition, microglia activate several inflammatory pathways that cause neuroinflammation and possibly neurodegeneration. In this process, microglia interact with astrocytes, and their interaction is also important in regulating homeostasis. Several chemokine and chemoattractant housekeeping genes are involved in phagocytosis (
Trem2), synaptic pruning and remodelling (
C1q and
Cx3cr1) [
24], and anomalies in these housekeeping genes may lead to neurodegeneration.
As carriers of innate immunity, microglia defend their host against pathogens (
Figure 1), injurious proteins including Aβ, aggregated α-synuclein, mutant huntingtin (
mHtt), mutant prion (mPrP
Sc) and oxidised superoxide dismutase (SOD). Microglia activate several receptors to incite host defences, such as expressing Fc receptors, Toll-like receptors (TLRs), and several antimicrobial peptides including
Camp and
Ngp [
24]. Therefore, microglia approach the neuroinflammatory threshold by producing peripheral inflammatory cytokines such as TNF-α and IL-1β [
33,
34]. In addition, this process involves chemokines (e.g., Ccl2) that recruit additional cells and work together to clear pathogens and normalise the brain in a homeostatic condition [
35]. However, consistent microglia-induced neuroinflammation leads to neurotoxicity and, eventually, neurodegeneration. In contrast to the anomalies in this microglial functioning, healthy neural microglia are always actively functioning through sensing, housekeeping and protecting their host.
3. Microglia and Ageing
Ageing is inevitable, and several structural and functional changes occur in the brain with advanced ageing. For instance, the brain loses a total mass of about 2 to 3% per decade after the age of 50. This loss of mass with age specifically affects the volume of grey and white matter in the prefrontal, parietal and temporal areas [
36,
37,
38]. Therefore, an individual gradually loses complex learning abilities and declines in cognitive function [
38,
39]. Several cellular-level changes also occur in ageing brains, such as genomic instability, shortening of telomeres and activation of tumour suppressor genes, protein mutation and accumulation, oxidative stress, reduced autophagy and mild to chronic inflammation. It is imperative to maintain the balance between pro- and anti-inflammatory cytokines. However, a brain undergoing advanced ageing shows an imbalance between these cytokine levels in response to chronic exposure to physical, chemical or biological agents, such as ionic radiation, pollutants and pathogens [
40,
41]. Studies have shown that chronic exposure to endogenous or exogenous pathogens decreases the anti-inflammatory cytokine IL-10 [
42]. In contrast, such exposure also increases inflammatory cytokines such as TNF-α and IL-1β in the CNS [
43] and IL-6 in plasma [
44]. Additionally, increased systemic inflammation causes neuronal cell death and an imbalance between clearance and production of ROS, severely damaging synaptic plasticity as well (
Figure 2 and
Figure 3). Many of these alterations in ageing brains include impairment in basal autophagy that begins with cellular stress. Ageing human brain analysis has shown a reduction in autophagy genes, including
Atg5,
Atg7 and
Becn1 [
45], and similar downregulation in Atg-proteins has been evident in ageing mouse brains. In contrast, ageing has been found to upregulate mTORC1 [
45,
46] and accelerated mTOR reduces macroautophagy and promotes aggregated protein and metabolic disturbances during ageing. These data have been supported by the rapid development of neurodegeneration in
Atg5- [
47] and
Atg7- depleted [
48] mice. Microglia, as the first line of host defence, selectively activate autophagy to entrap threatening molecules into autophagosomes and clear them via autophagic degradation. For example, microglial TLR4-induced activation of nuclear factor κB (NF-κB) upregulates p62/SQSTM1 signalling, which degrades misfolded α-syn proteins via autophagy and protects against midbrain dopaminergic neuronal loss [
49]. Furthermore, ageing-mediated alteration in microglial functions disrupts microglial regulation of autophagy and promotes neuronal loss.
Ageing has been recognised as a major risk factor for many neurodegenerative disorders. Advanced ageing includes several hallmarks indicating risks of developing neurodegenerative diseases such as Alzheimer’s (AD), Parkinson’s (PD), Huntington (HD) and frontotemporal lobar (FTD) disease. Furthermore, microglial cells change with ageing, which is one of the major risk factors for age-related development of neurodegeneration. Although neurodegenerative diseases are multifactorial conditions, and their complexity is not yet well understood, there has been scientific agreement on the degenerative diseases and age-related changes they can cause in the neural microenvironment.
Ageing produces the common feature of high heterogeneity in microglia, which is also a common phenotype of different neurodegenerative diseases [
39]. Moreover, the pattern of microglial gene expression changes with ageing and neurodegenerative conditions [
50]. The major phenotypic changes in ageing microglia are increased soma volume, a retraction in processes and a loss in uniform tissue distribution [
51]. Furthermore, microglial activation slows with age, reducing sensing activity and impairing synaptic contact [
6]. This process of ageing microglial activation is distinct from classical activation and is referred to as microglial dystrophy; the anomalous activation is more likely to be senescent rather than a classical phenotype [
52,
53]. Moreover, a new phenotype of microglia has been defined—dark microglia—characterised by condensed electron-dense cytoplasm and nucleoplasm, nuclear chromatin remodelling and high levels of synaptic stripping activity and oxidative stress [
54,
55]. Interestingly, these phenomena have not only been observed in microglial populations associated with chronic stress or diseases such as AD but also in the microglia in normally ageing brains [
54]. Although knowledge of these molecular structural changes is still in its infancy, it has already been established that ageing microglia are highly granular and present an uncharacteristic dark appearance in immunohistological preparations. Another phenotypic change in aged microglia is defective lysosomal digestion. This defect largely privileges accumulation of indigestible material composed of lipofuscin and other autofluorescent pigments [
56,
57]. Therefore, the use of immunofluorescence or flow cytometry has become familiar among researchers to distinguish between normal and aged microglia. Accumulation of such autofluorescent pigments and lipofuscin is believed to be a by-product of impaired disposal mechanisms and purported to have a direct relation to several neurodegenerative diseases, including AD [
58,
59].
Microglial changes with age do not follow one specific process but, rather, change throughout one’s life; after reaching a certain age, threshold impacts will appear. A transcriptome analysis of the frontal cortex region of post-mortem healthy brains across a wide age range (from young teenagers to people over 80 years old) showed that microglial gene markers assemble into a transcriptional module in a gene co-expression network [
60], and this expression pattern negatively correlates with age. Another study revealed that genes that encode microglia surface receptors for neuronal and/or microglial crosstalk are particularly affected. Several brain-expressed transcription factors, including RUNX1, IRF8, PU.1 and TAL1, are the master regulators of age-dependent microglial modulation [
39]. This raises the question of how important it is to identify age-dependent genetic modulation in adulthood to understand neurodegenerative disease pathology. Identification at the beginning of genetic changes in middle or late-middle age might correlate several chronic neurodegeneration initiations, and thus may help stall disease progression.
5. Microglia as a Therapeutic Possibility in Neurodegenerative Diseases
Microglia play vital roles at different brain development phases. With age or due to aberrant endogenous or exogenous stimuli, they begin losing their normal physiological functions. Thus, homeostasis in CNS microglia is necessary to disrupt neurodegenerative disease pathology and progression. In addition, identification of critical microglial markers is important to find new therapeutic strategies. Initial studies have suggested an M1 and M2 activation paradigm, in which M1 activation promotes inflammatory cytokines, and M2 activation promotes neurotrophic factor release. However, advanced studies have found that this paradigm does not fit during neurodegeneration. That is, M1/M2 activation does not always function as expected. For instance, in regular cases, M1 activation produces neurotoxicity via proinflammatory release, whereas in some cases, this activation promotes axonal regeneration [
118]. An AD model study showed that M1 activation promotes Aβ plaque clearance, and, in contrast, M2 activation may ease amyloid spread [
119].
Microglia are emerging as a cell type used to understand neurodegenerative diseases, but the major challenge is studying human microglia in vitro. For an in vitro neurological disease model study, microglial cells could possibly be developed by differentiating iPSCs or monocytes [
120,
121], which has recently been demonstrated. In addition, authors have shown that human microglial-like cells (iMGLs) have phenotypic similarity to in vivo microglia, such as through inflammatory cytokine release or CNS substrate phagocytosis. On the other hand, human monocyte-derived, microglia-like (MDMi) cells have not only presented with microglia phenotype and functions but have also presented with altered expression of gene loci related to neurodegenerative diseases such as AD, PD and MS. These two in vitro microglial cell models could beneficiate therapeutics screening in vitro for neurodegenerative diseases. In addition, genetic defects in microglia could be edited by replacing allogenic or autologous stem cells or monocytes through bone marrow transplantation. Although the latter has not been successfully established for all neurological diseases—though it has for X-linked adrenoleukodystrophy—a recent study showed that brain-engrafted bone marrow derived microglia after a long time in AD mice [
122,
123]. It has been suggested that peripheral myeloid cells constitute a heterogeneous cell population that is more effective at clearing Aβ plaque than CNS resident microglia. Extrapolating this therapy with additional triggering could bring success or could be useful for studying other neurological diseases.
Microglial phagocytosis could be another option, but therapeutic agents that target microglial phagocytosis can have both beneficial and detrimental effects—another double-edged sword in neurodegeneration. Microglial phagocytosis opsonises misfolded protein plaques, including Aβ, via the Fc receptor to help antibodies that target misfolded proteins [
7,
124,
125]. In the same way, defective microglia activation in
Trem2-deficient mice showed a lack of effectiveness toward the anti-Aβ antibodies [
126]. Several antibodies currently used in autoimmune diseases may be beneficial in neurodegenerative diseases as well because they target specific proinflammatory cytokines, such as IL-6 and IL-1, or their receptors. Compounds targeting CSF1R can affect proinflammatory microglia activation in AD [
127,
128] and reduce microglia-induced inflammation and/or neuronal death [
129] in neurodegenerative diseases. In the same vein, IL-34 and CSF1, ligands of CSF1R, may provide neuroprotection and promote neuronal cell survival shown in neurodegenerative models by activating CSF1R in neuron populations but not in microglia [
130].
In addition, bexarotene-induced
Trem2 expression in microglia is, at least in part, mediated by
ApoE/
Trem2 signalling activation [
131]. Thus, developing anti-
ApoE antibodies in carriers of the ApoE4 allele may help to prevent amyloid deposition and its consequences [
132]. Moreover, neuronal autophagy has been shown to be useful in neurodegenerative diseases for clearing misfolded proteins and reducing inflammatory cytokines [
133]. However, very little is known about the role of microglia in autophagy. In one study, loss-of-function mutations of TBK1 affected autophagy in myeloid cells and increased susceptibility to ALS [
134]. Beyond this, microglial autophagy facilitates Aβ clearance and reduces NLRP3 inflammasome activation [
135]. Therefore, further study of autophagy in microglia may enhance the understanding of whether drugs activating autophagy have beneficial or detrimental impacts on neurodegenerative diseases.