An overview on microglial origin, distribution, and phenotype in Alzheimer’s Disease

Abstract

Alzheimer’s disease (AD) is the most common neurodegenerative disease that is responsible for about one-third of dementia cases worldwide. It is believed that AD is initiated with deposition of Ab plaques in brain. Genetic studies have shown that a high number of AD risk genes are expressed by microglia, the resident macrophages of brain. Common mode of action by microglia cells is neuroinflammation and phagocytosis. Moreover, it has been discovered that inflammatory marker levels are increased in AD patients. Recent studies advocate that neuroinflammation plays a major role in AD progression. Microglia have different activation profiles depending on the region of brain and stimuli. In different activation profile microglia can generate either pro-inflammatory or anti-inflammatory responses. Microglia defend brain cells from pathogens and respond to injuries; also, microglia can lead to neuronal death along the way. In this review we will bring the different roles played by microglia and microglia related genes in progression of Alzheimer’s disease.

Graphical Abstarct

Introduction

Alzheimer’s disease (AD) is the most common cause of dementia and the sixth leading reason of mortality in the United States with neuroinflammation identified as one of the key hallmarks of AD progression. The accumulation of Aβ plaques and Neurofibrillary Tangles (NFTs) is well known characteristic of AD pathogenesis (Ittner et al., 2010). Recently, several studies have focused on the activation of astrocytes and microglia contribution to AD development and progression (Cameron & Landreth, 2010; Katsumoto, Takeuchi, Takahashi, & Tanaka, 2018; Li, Li, Zheng, & Qin, 2019; Mandrekar-Colucci & Landreth, 2010; Spangenberg & Green, 2017; Yang & Zhang, 2020). Studies have shed light on the complex cyclical interplay between Aβ plaques and microglial release of pro-inflammatory cytokines to promote initiation of Amyloid Precursor Protein (APP) production (W. Y. Wang, Tan, Yu, & Tan, 2015). The role of Aβ plaques and NFTs in AD pathogenesis have recently come into question, given the failure of many pharmaceutical agents targeting these depositions, as well as the discovery of these depositions in normal human brains. Therefore, exploring and understanding novel mechanisms mediating AD pathogenesis are of great importance to future AD treatment.

Microglial activation in neuroinflammation may yield beneficial effects in AD. Studies implicate the development and progression of neurodegenerative diseases via chronic microglial neuroinflammation, through the release of neurotoxic factors and other inflammatory markers. In the initiation or progression of AD different inflammatory mediators may also be involved. Chronic neuroinflammation, persisting after an initial injury or insult, is self-perpetuating and microglial proinflammatory cytokines including TNF-α, IL-1 and IL-6 are strongly associated with the pathophysiology of AD (Kothur, Wienholt, Brilot, & Dale, 2016; W. Y. Wang et al., 2015). However, the pathology and underlying mechanism of dementia of AD caused by neuronal death is not fully understood. The extracellular deposition of amyloid-β (Aβ) plaques could be due to an increased production and/or lack of clearance of Aβ peptides derived from the cleavage of amyloid precursor protein (APP), as well as abnormal intraneuronal accumulation of hyperphosphorylated tau protein are associated with the initiation and progression of AD (Heneka et al., 2015; Mawuenyega et al., 2010; Sarlus & Heneka, 2017) (Johnson & Stoothoff, 2004).

It has also been proposed that the changes in microglial cell responses during AD progression are associated with both functional and morphological changes of the hippocampus (Henneman et al., 2009). Pattern recognition receptors (PRRs) in Aβ plaques bind to different Danger associated molecular patterns (DAMPs) or Pathogen associated molecular patterns (PAMPs) to induce microglial activation and an inflammatory response (Venegas & Heneka, 2017). Microglial cells are the first line of defense of the innate immune system in the brain where amyloid-like structures are expressed in microorganisms such as bacteria (Kreutzberg, 1996). This fact would give a potential explanation for the microglial response against Aβ. The role of PRRs-associated factors including, ApoE, TREM2, CD33, toll-like receptors (TLRs) and inflammasome has been reported in AD pathology (Kim, Basak, & Holtzman, 2009).

Recently, Disease associated microglia (DAM) have been recognized as a subset of resident brain macrophages involved in neurodegenerative diseases, such as Alzheimer’s disease, Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS) (Deczkowska et al., 2018; Dhib-Jalbut et al., 2006; Ferreira & Romero-Ramos, 2018; Keren-Shaul et al., 2017; Walker & Lue, 2015). The transition to DAM phenotype is a signature consequence of microglial-induced neuroinflammation, thus limiting microglial activation during AD is high priority for decreasing neuroinflammation. Studies elucidating the role of DAM in amyloid deposition are lacking, however the close proximity of DAMs to amyloid plaques and related DAM associated immune responses needs further investigation. Moreover, structural changes within the neurons associated with DAMs highlight the need for further study of microglia-mediated neuronal damage and potentially uncover new targets for future AD treatment (Fang et al., 2010; Hansen, Hanson, & Sheng, 2018; Hickman, Izzy, Sen, Morsett, & El Khoury, 2018).

Microglia Origin, Distribution and Phenotype

Microglial cells migrate from the yolk sac into the central nervous system (CNS) during embryogenesis (Ginhoux et al., 2010). They propagate and disperse in a non-heterogeneous manner around the CNS. In mice brain, a higher relative number of microglial cells is noticed in the dentate gyrus of the hippocampus, the substantia-nigra and parts of the basal ganglia, with the highest number of microglia appear in the olfactory telencephalon (Lawson, Perry, Dri, & Gordon, 1990). Microglia present different morphological features, (Gomez-Nicola & Perry, 2015), lysosome content (Majumdar et al., 2007), membrane composition (Button et al., 2014), electrophysiological activities (i.e., hyperpolarized resting potentials and differential membrane capacitance) (De Biase et al., 2017), and gene transcriptome profile (Chiu et al., 2013), depending on the specific anatomical structure or activation profile.

Microglial phenotype regulation is largely dependent on their interaction between membrane-bound pattern recognition receptors (PRRs) with molecules released by surrounding cells (neurons, microglial cells, astrocytes, etc.). These PRRs can be classified depending on their affinity for molecules associated to pathogens (Pathogen Associated Molecular Patterns, PAMPs) or cellular damage (Danger Associated Molecular Patterns, DAMPs) (Kettenmann, Hanisch, Noda, & Verkhratsky, 2011; Venegas & Heneka, 2017). Variety of receptors are also detected within microglial cells to bind other type of molecules such as hormones and neurotransmitters (Kettenmann et al., 2011). Traditionally, macrophage and microglial activation has been categorized in two different, opposite and interchangeable states: classic (M1) and alternative (M2). The pro-inflammatory state of M1 phenotype of microglial cells produce and release reactive oxygen species (ROS), nitrogen reactive species (NRS) and cytokines like tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) or interleukin 12 (IL-12). On the other hand, the anti-inflammatory state of M2 microglial phenotype, is involved in the production and release of trophic factors, such as tumor growth factor-β (TGF-β) and brain derived neurotrophic factor (BDNF) (Tang & Le, 2016). Regarding expression pattern of microglial cells (de Haas, Boddeke, & Biber, 2008), found throughout striatum, hippocampus, spinal cord, cerebellum, and cerebral cortex, a clear expression of CD11b, CD40,CD45, CD80, CD86, F4/80, TREM−2b, CXCR3 and CCR9, whereas no expression was found for either major histocompatibility complex II (MHCII) or CCR7 (Bachiller et al., 2018).

Microglia in other Neurological Diseases:

Microglia, macrophages-like cells in the central nervous system (CNS), are considered immune sentinels capable of orchestrating a potent inflammatory response. Microglia are also involved in phagocytosis of apoptotic cells in developing brain, myelin turnover, control of neuronal excitability, synaptic organization, trophic neuronal support during development, phagocytic debris removal as well as brain protection and the repair of the microglial response is pathology dependent and the first effects are on immune and metabolic systems (Bachiller et al., 2018). In this review, we will focus on microglial activation depending on the disease in context

A. Traumatic Brain Injury

Traumatic brain injury (TBI) is a serious cause of morbidity and mortality (Taylor, Bell, Breiding, & Xu, 2017). Loss of neurons, astrocytic gliosis, and microglial activation associate in complex neurological disorders, are the consequences of primary and secondary brain damage in TBI (Akamatsu & Hanafy, 2020; Loane & Faden, 2010). Even though irreversible primary brain damage is caused by mechanical damage during initial impact and reversible secondary damage results from delayed neurochemical process and intracellular signaling pathways. During the chronic phase of TBI, increased neuronal damage have been implicated in secondary brain damage and poor outcome (Bramlett, Dietrich, Green, & Busto, 1997; Colicos, Dixon, & Dash, 1996; Dietrich, Alonso, & Halley, 1994; Hicks, Soares, Smith, & McIntosh, 1996). Primary immune cell in the CNS, microglia, maintains CNS homeostasis by engulfing and clearing apoptotic neurons; they also participate in brain development and neuronal plasticity (Lawson, Perry, & Gordon, 1992; Marin-Teva et al., 2004; Tremblay et al., 2011). Microglia derived neuronal apoptosis via the release of superoxide anion (Salter & Stevens, 2017), nerve growth factor (Frade & Barde, 1998), and tumor necrosis factor (TNF) (Sedel, Bechade, Vyas, & Triller, 2004) was observed after TBI. Still, the role of microglia in TBI remains to be elucidated. It has been previously demonstrated that depletion of microglia significantly reduced neuronal apoptosis following moderate fluid percussion injury in rodents (Hanafy, 2013; C. F. Wang et al., 2020).

Bennett et al. (Bennett & Brody, 2014) demonstrated that inhibition of microglia did not involve acute axon degeneration after multiple concussive injury. Hanlon et al. (Hanlon, Raghupathi, & Huh, 2019) illustrated that microglia depletion with clodronate decreased the clearance of dying cells causing an increase in neurodegeneration in rat CC model. Therefore, depending on the type of injured cells and TBI models, the microglial impact may vary. Furthermore, inhibition of microglial activation may also contribute to the chronic spread of cell damage via apoptosis (Aungst, Kabadi, Thompson, Stoica, & Faden, 2014). TBI involves a highly complex pathology characterized by multiple interacting secondary injury cascades. Therefore, bidirectional translational research between preclinical and clinical investigations is justified to identify novel signaling pathways, target, and modify them, in the hope of identifying pharmaceuticals that may one day improve TBI outcome (Akamatsu & Hanafy, 2020).

B. Stroke

Subarachnoid hemorrhage (SAH) is responsible for about 50% mortality rate in stroke patients in the USA. Heme-induced cerebral inflammation contributes to many of the adverse sequelae seen in patients with subarachnoid hemorrhage (SAH). Although little is known about the mechanism; studies in mouse models have shown a critical role for macrophages/microglia (Schallner et al., 2015). The role of microglia in neuronal injury is a double-edged sword as these cells on one side can induce neuronal apoptosis via the triggering of inflammatory response (Hanafy, 2013) but can also show neuroprotection; which is not unlike macrophages in other tissues (Boscia et al., 2009; Montero, Gonzalez, & Zimmer, 2009; Vinet et al., 2012). The answer to which fate neurons will fall in response to activated microglia most likely depends on the etiology of the injury. Here, we define a new role for microglia in response to SAH. Microglia are the principal cell type responsible for phagocytosis and elimination of rogue erythrocytes after SAH (Schallner et al., 2015).

Early studies showed microglia to be critical in red blood cell-induced neuroinflammation (Hanafy, 2013), and most recently, we found microglial HO-1 to be neuro-protective after SAH in a mouse model (Schallner et al., 2015). We hypothesized that microglial/macrophage HO-1 is critical for DFX neuroprotection and that intracerebroventricular administration would provide superior neuroprotection in a mouse model of SAH. Data showed that intracerebroventricular DFX yields the greatest neuroprotection via a mechanism that is dependent on microglial HO-1 and possibly a protective microglial polarization. Our results indicate that the mechanisms by which DFX provides neuroprotection after SAH may involve microglial/macrophage HO-1 expression. Monitoring patient HO-1 expression during DFX treatment for hemorrhagic stroke may help clinicians identify patients that are more likely to respond to treatment (LeBlanc, Chen, Selim, & Hanafy, 2016).

C. Parkinson’s disease

Parkinson’s disease (PD) is characterized by motor impairment due to degeneration of dopaminergic neurons. Innate immune response in PD has been widely studied and compelling evidences demonstrate the role of neuroinflammatory response in the progression of PD (Perry, 2012). The principal pathological hallmark of PD is death of dopaminergic neurons in the Substantia nigra pars compacta (SNpc) which causes loss of these neuronal connections (Brettschneider, Del Tredici, Lee, & Trojanowski, 2015). The loss of dopaminergic neurons occurs in many different regions of brain such as: locus coeruleus (LC), dorsal motor of the vagus nerve, amygdala or hypothalamus (Dickson, 2012). The aggregation of misfolded α-synuclein in intraneuronal inclusions, called Lewy bodies, is another remarkable characteristic of the pathology. These inclusions are observed in neuronal somas [Lewy’s bodies (LB)] or in the neurites (Lewy’s neurites) (Spillantini et al., 1998). The α-synuclein inclusion can also be found in other pathologies such as Dementia and Lewy body (DLB) (McKeith, 2004). Another important aspect of the biology of PD are the different aggregation states α-synuclein can be in during pathogenesis of the disease. For example, oligomers of α-synuclein are toxic for neurons (Ingelsson, 2016).The neuroinflammatory response is mainly carried out by microglial cells and to a minor extent by astrocytes in PD (Perry, 2012). Mcgeer et al. (McGeer, Itagaki, Boyes, & McGeer, 1988) explained the reactivity of human leukocyte antigen (HLA) receptor in the SNpc of PD patients. Microglial MHCII expression has been largely correlated to neurons containing Lewy bodies, damage neurons and neurites in PD brains (Imamura et al., 2003). Moreover, the cells expressing MHCII were also positive for pro-inflammatory markers such us IL-6 or TNF-α. In addition, MHCII knockout mice showed less neuronal loss (Harms et al., 2013). GWAS studies in PD patients have also highlighted the MHCII-related SNP considered a risk factor (Pierce & Coetzee, 2017). Although microglial activation and consequent inflammatory response taken place in PD is a major component of the pathology but the exact mechanism of activation of microglial cells and how is that affecting to the neuronal survival in PD still remains unclear.

D. Multiple Sclerosis

Multiple sclerosis (MS) is a CNS disabling disease. In this disease, body’s immune system attacks myelin sheath and demyelinate nerve fibres. This disturbs brain’s communication to rest of the body. With time this can cause to permanent damage to nerves. In MS pathogenesis, measurement of Microglia exclusive marker TMEM 119 has shown that initial pool of phagocytic cells in early MS lesion progression is approximately 40% microglia (Satoh et al., 2016). Peripheral macrophages are incorporated with lesion progression (Zrzavy et al., 2017). Presence of P2RY12 ADP receptor infers that these microglia are not homeostatic (Bogie, Stinissen, & Hendriks, 2014; Moore et al., 2015; Zrzavy et al., 2017). In addition, nodules of activated microglia are also reported in normal appearing white matter of MS patients. Homeostatic or activated status of these microglia is however debated, as out of two, one study has shown a loss of P2RY12 gene while the other showed normal P2RY12 expression (van der Poel et al., 2019; Zrzavy et al., 2017). Progressive MS patients who demonstrate upregulation in white matter lipid processing genes and gray matter iron homeostasis genes are reported to have disease specific manifestation of this regional heterogeneity of microglia, that was originally reported in mice (Grabert et al., 2016; van der Poel et al., 2019). It is understood that in absence of demyelinating lesions, metabolic changes in microglia that mirror MS pathology are detected. This highlights the differential inflammatory processes seen in white and gray matter in MS (Guerrero & Sicotte, 2020). Evidence of involvement of microglia in balancing bone morphogenetic protein 4 and noggin are also reported. This involvement may impede remyelination (Harnisch et al., 2019). Microglia in animal models are reported to be in homeostatic state while in intermediate state in human brain (CD68 expression and reduced P2RY12). Therefore, due to higher systemic inflammation (at time of autopsy) human microglia may differ from microglia in other species (Lassmann, 2020; Zrzavy et al., 2017). In another study, microglia from normal appearing white matter in MS brain were unresponsive to LPS. In addition, other evidences of diminished inflammatory responsiveness were also reported despite their activated state (Melief et al., 2013). Recent studies have shown that in comparison to neurons or astrocytes, MS susceptibility genes are more frequently associated with microglia function (International multiple sclerosis genetics consortium). Microglia are found associated with pro-inflammatory functions throughout lesion formation. They contribute in synaptic loss. However, they are also known to play important roles in reducing inflammation and promote remyelination. Hence classifying microglia as good or bad cannot justify their enigmatic role in MS disease pathogenesis. New emerging microglia related biomarkers should help us to further unravel the role of these cells in CNS related disorders.

E. Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a neurodegenerative disease. It involves rapid degeneration of neurons in motor cortex (Al-Chalabi, van den Berg, & Veldink, 2017). This is evident from previous studies with CSF and blood of ALS patients that inflammatory responses are induced by degeneration of motor neurons and pathogenic cascades within the surviving neurons. These inflammatory responses involve activation of CNS resident microglia and astrocytes (Gupta, Prabhakar, Abburi, Sharma, & Anand, 2011; Lu et al., 2016; Tateishi et al., 2010). ALS related gene mutations are reported expressing in microglia. This can lead to speculations of microglia role in neurodegeneration; but further comprehensive studies are warranted to come to any conclusive finding (Cady et al., 2014). Although association between microglia and ALS has been shown in studies with post-mortem tissue; there is no single opinion on microglial activation to ameboid shape, increased proliferation and upregulation of regulatory pathways (Clarke & Patani, 2020). Researchers have reported microglia proliferation in a subset of ALS patients (Spiller et al., 2018; Tang & Le, 2016) as well as in both motor cortex and spinal cord (Dols-Icardo 2020, Brettschneider 2012, D’Erchia 2017). Much of what we know about mechanism involving microglia role in ALS have come from studies in animals, mainly SODIG93A mouse. These studies are summarized by Clarke and Patani in their review (Clarke & Patani, 2020). Studies have shown several factors linked to both microglial protection and toxicity associated with ALS. IL-4, IL-10 and G-CSF are reported altered in ALS patients and models (Jeyachandran, Mertens, McKissick, & Mitchell, 2015; Lu et al., 2016; Moreno-Martinez, Calvo, Munoz, & Osta, 2019). CD14, TLR2 and TLR4 expression is also shown to be increased in post-mortem ALS spinal cord tissues (Casula et al., 2011; Henkel et al., 2004). It is still not clear whether neuroinflammation in ALS is good or bad; this is determined by a number of factors across the course of the disease. Microglia has been shown to play neuroprotective as well as neurodegenerative roles in ALS progression (Chiu et al., 2013). Although microglia are not likely to be the main antagonist in neuronal degeneration in ALS, it may however play a modulatory role in the disease progression. Further detailed studies are required to simplify more of the complex functions of microglia in ALS progression.

Microglia in Aging and Alzheimer’s Disease

Two thirds of individuals currently living with Alzheimer’s disease (AD). Many evidence suggest a correlation of gender-specific risk for AD. Females may have higher AD pathology burden (Barnes et al., 2005; Hohman et al., 2018; Oveisgharan et al., 2018) and may be more susceptible than males as by higher pathophysiological downstream effects and worse clinical and cognitive outcomes, particularly among apolipoprotein E (APOE) ε4 allele carriers (Buckley et al., 2018; Damoiseaux et al., 2012; Koran, Wagener, Hohman, & Alzheimer’s Neuroimaging, 2017). On the contrary, females have performed better in verbal memory measures across the lifespan (Kramer, Delis, Kaplan, O’Donnell, & Prifitera, 1997),and there may be variations among genders in rates of decline in specific cognition in the U.S. Recent investigations manifest the continuation of verbal memory among female in early stages of AD, despite initial accumulation of pathology and neurodegeneration, including greater post-mortem tau pathology, hippocampal atrophy, and brain glucose hypometabolism (Digma et al., 2020; Sundermann et al., 2016; Sundermann et al., 2020). Another study explained the increased number of microglia with age in the neocortex of women (19–87 years), but not men (18–91 years), despite men having 28% more neocortical glial cells (Pelvig, Pakkenberg, Stark, & Pakkenberg, 2008).

Morphological changes in microglia due to age

Aging studies in microglia showed that disturbance of the brain’s homeostasis during aging can lead to glial activation, and through mitotic proliferation, reactive microglia increase in density to restore tissue equilibrium (Kettenmann et al., 2011). Higher amount of microglial activation have been found in the EC, CA1-CA4 hippocampal subfields, dentate gyrus (DG), and subiculum of elderly nondemented subjects (73 years) compared to adult controls (38 years) (DiPatre & Gelman, 1997). In cognitively normal older adults, HLA-DR microglia density was greater than young adults and super agers (Gefen et al., 2019).

Aged human brains exhibit higher numbers of intermediate and amoeboid morphologies in the neocortex and hippocampus and higher expression of CD68 and HLA-DR, an MHC II cell-surface antigen and marker for immune stimulation (Raj et al., 2017; Sheng, Mrak, & Griffin, 1998; Zotova et al., 2013). Though the functional relevance of rod-shaped microglia remains unknown, these are prevalent in aged human hippocampus and cortex, (Bachstetter et al., 2017; Styren, Civin, & Rogers, 1990). Elderly human brains also display non-activated dystrophic microglia with increased soma volume, abnormalities in the cytoplasmic structure, retracted, fragmented processes, and nonuniform tissue distribution (Streit, Braak, Xue, & Bechmann, 2009; Streit & Xue, 2009). Moreover, decrease in microglial length and branching suggests glial activation in the neocortex of aged humans (Davies, Ma, Jegathees, & Goldsbury, 2017).

Microglial activation and density in AD

Glial activation is linked with both Aβ and tau accumulation in AD pathology (Figure 1). Aβ peptides activate microglia, resulting in proliferation demonstrated by higher microglial density (Heneka et al., 2015). Normal aging increases the numbers of microglia in white matter, while the AD brain shows a selective increase in gray matter microglial density, suggesting a different mechanism of activation in normal aging versus AD (Rozemuller, Eikelenboom, & Stam, 1986; Sloane, Hollander, Moss, Rosene, & Abraham, 1999). Notably, increased microglial density and proliferation take place concomitant with Aβ plaques in the hippocampus of AD individuals (Ekonomou et al., 2015; Streit et al., 2009). However, many studies showed Aβ initiates microglial activation, whereas a study examining four humans with substantial plaque loads in absence of tau lesions found no evidence of microglial activation (Streit et al., 2009). Neuroinflammation also has been involved in aggregation of tau in humans (Bellucci, Bugiani, Ghetti, & Spillantini, 2011; Gebicke-Haerter, 2001). In post-mortem AD brains, microglia density increased linearly, even after amyloid burden stopped growing, and correlated with Neurofibrillary tangles (NFT) burden rather than plaque load (Serrano-Pozo et al., 2011). Moreover, increased microglial activation and proliferation was correlated with increased NFT numbers, specifically in the CA1 subfield of the AD hippocampus (Ekonomou et al., 2015). NFT burden also was certainly corresponded with HLA-DR-ir activated microglia density in a non-amnestic clinical AD variant called primary progressive aphasia (Ohm et al., 2021).

Figure 1.

Different mechanisms of association between microglial genes and AD. TLR4, TREM2 and LYN kinase play important roles in AD progression and Ab_1–42 initiated neuronal cell death. Please refer to text for detailed mechanism. Picture created using BioRender. (AD= Alzheimer’s disease, Ab= amyloid beta, TLR4= toll like receptor-4, LYN= Lck/Yes novel tyrosine kinase, TREM2= Triggering receptor expressed on myeloid cells 2).

Activated Microglia were found in brains of aged common marmosets that exhibited Aβ and tau depositions around dystrophic microglia, not activated microglia; contained hyperphosphorylated tau (Edler et al., 2018). In the neocortex of aged rhesus monkeys, fibrillar Aβ plaques and neuronal loss were associated with activated microglia, senile plaques, vascular Aβ, and NFT (Shah et al., 2010). Profound neuron loss, tau phosphorylation, and microglial activation and proliferation were observed when insoluble Aβ fibrils were microinjected into the cerebral cortex of old rhesus macaques, (Geula et al., 1998). Inhibition of activation of microglial with a macrophage/microglia inhibitory factor eliminated fibrillar Aβ toxicity in elderly rhesus macaques (Leung et al., 2011). Aβ oligomers injection in female cynomolgus monkeys developed microglial activation along with NFT formation, astrogliosis, and synapse loss (Forny-Germano et al., 2014). A recent study of 20 aged chimpanzees observed an elevated microglial activation in the hippocampus in association with Aβ42-positive plaques and vasculature but not NFT (Edler et al., 2018).

Rodents, such as mice and rats, do not naturally develop amyloid plaques or NFT; therefore, these pathologies are artificially induced using human transgenes. Microglial activation in the vicinity of Aβ plaques and vessels has been detailed in several transgenic mouse models of AD, which overexpress APP or Aβ but lack NFT formation (Frautschy et al., 1998; Rodriguez et al., 2013; Rodriguez, Witton, Olabarria, Noristani, & Verkhratsky, 2010).

In addition, intraperitoneal injection with LPS showed hyperreactive microglia surrounding dense-core plaques of 5XFAD (12 months) and APP23 (24 months) transgenic mice (Yin et al., 2017). Contrarily, in a more aggressive AD mouse model (TgCRND8), which develops diffuse and dense-core plaques as early as 9–10 weeks, microglia were associated with both types of plaques (Dudal et al., 2004).

Microglial homeostasis in AD

In AD, an activated state microglia are categorically correlated with dense-core Aβ plaques and NFT, and an increased expression of MHC II and HLA-DR antigens in the neocortex and hippocampus (Mattiace, Davies, Yen, & Dickson, 1990; McGeer et al., 1988; Perlmutter, Scott, Barron, & Chui, 1992). Moreover higher protein expression of HLA-DR-ir, microglia increased in number in the mid-temporal gyrus of AD patients compared to controls, and CD33-ir microglia density was definitely correlated with insoluble Aβ42 levels and plaque loads in AD brains (Carpenter, Carpenter, & Markesbery, 1993; Griciuc et al., 2013). In addition, during plaque accumulation in early-onset AD brains showed the rapid increase of CD11c-ir microglia (Yin et al., 2017). On the contrary, diffuse plaques are not correlated with microglia in AD, and humans with significant Aβ plaque deposition, but no tau lesions, displayed totally ramified microglia throughout the temporal lobe (Hendrickx, van Eden, Schuurman, Hamann, & Huitinga, 2017; Streit et al., 2009). Gene expression profiling of plaque-associated MHC II microglia from 5XFAD mice revealed a proinflammatory phenotype with upregulation of various markers for genes engaged in the immune response to external stimuli (e.g., CD63) and phagocytosis (e.g., CD11c) (Yin et al., 2017). In contradiction, CD11b, in double APP/PS1 transgenic mice was not upregulated in microglia (Howlett & Richardson, 2009).

In a study in canines with Cognitive dysfunction syndrome (CDS; AD equivalent in dogs), activated microglia with larger cell processes (i.e., intermediate) and dystrophic microglia with spheroidal or bulbous swellings and de-ramified or tortuous processes were appeared (Rofina et al., 2003). Conversely, it was reported the neuron loss was associated with substantial diffuse plaques with microglial clustering and Cerebral amyloid angiopathy in a 12-year-old dog (Edler et al., 2018).

AD associated microglial genes:

TREM2

TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) is a transmembrane receptor expressed in myeloid cells. In brain, this receptor is only found in microglia cells. It regulates transcription of anti-inflammatory genes like GAL1, GAL3 and progranulin. In healthy individuals, TREM2 act in support to hair follicles, bones, and microglia. Synaptic pruning is an important homeostatic mechanism supported by TREM2.TREM2 is crucial to the functioning of microglial cells, and variants of this protein are associated with a significantly increased risk of Alzheimer’s disease. High risk of developing Alzheimer’s disease (AD) is associated with variants of TREM2, a surface receptor required for microglial responses to neurodegeneration, including clustering, survival, proliferation, and phagocytosis (Gatz et al., 2006; Gratuze, Leyns, & Holtzman, 2018; Keren-Shaul et al., 2017).

Early studies have reported the accumulation brain microglia of around Ab plaques both AD patients (D’Andrea, Cole, & Ard, 2004; McGeer et al., 1988; Perlmutter, Barron, & Chui, 1990) and transgenic mouse models of AD (Dickson, 2012; Frautschy et al., 1998; Stalder et al., 2005). in the early phases of neurodegeneration, microglia contribute to Ab clearance (El Khoury et al., 2007); however, the ability of microglia to clear Ab may vary with age (El Khoury et al., 2007; Streit et al., 2009; Streit & Xue, 2009). At late stages of AD, microglia may paradoxically contribute to the disease by releasing pro-inflammatory cytokines in response to Ab deposition (Hansen et al., 2018; Hickman et al., 2018). Recent genome-wide association studies (GWASs) have displayed that a rare Arginine-47-Histidine (R47H) mutation (figure 1) of the triggering receptor expressed on myeloid cells 2 (TREM2) is associated with greater risk of developing AD (Guerreiro et al., 2013; Johnson & Stoothoff, 2004). Many evidences showed that TREM2 is essential for the microglial response to Ab plaques (Y. Wang et al., 2016).

TREM2 is a receptor of the innate immune system that is expressed specifically on microglia in the central nervous system, with variants of the gene found to be associated with a 2–4-fold increase in the risk of developing Late Onset (occurring after the age of 65) Alzheimer’s Disease (LOAD) [7]. The functions of TREM2 are a topic on which active work is still ongoing and studies have suggested that it is critical to the normal functioning of microglia viz a viz phagocytosis and clearance of bacterial and cellular debris including amyloid beta (Aβ).

TLR4:

Toll-like receptors play key roles in innate immunity. These are equipped to detect pathogen associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). TLR4 was the first TLR identified in humans. Recent studies have provided evidences of TLR4 association with AD (Gambuzza et al., 2014). Microglial TLR4 has been shown to be necessary for vasospasm in both in vitro and in vivo studies (Hanafy Khalid A, 2013). Another study has shown that a prototypic DAMP, HMGB1 cause neuronal degeneration via TLR4 mediated pathway. HMGB1 released by necrotic or hyper-excitatory neurons bind to TLR4 and activates MAP kinase. Activated MAP kinase induce phosphorylation in myristoylated alanine rich C-kinase substrate (MARCKS) at Ser46 inducing neuronal degeneration. This phosphorylation is present through the course of AD progression both human and mouse models (Fujita et al., 2016) (figure 1). Gene profile study revealed increased expression of TLR4, TNF and IL-6 genes in frontal cortex region of AD patients (Miron et al., 2018). In addition, studies in entorhinal cortex mouse showed overexpression of TLR4 and IL-1b during deafferentation phase. This overexpression was not seen in during reinnervation. This suggest the possibility of Aβ plaque or deafferentation processes regulating TLR4 dependent modulation of cytokines (Miron et al., 2018). Another mode for TLR4 to add to AD progression is by inhibition of anti-inflammatory pathways. APP/PS1 mice are models of cerebral amyloidosis. One study involving these models increased expression of TLR4 and TREM2 in cortex. LPS treatment resulted in increased cognitive impairment in these mice. Thus, indicating that addition of systemic inflammation to familial AD may result in increased AD progression. However, LPS treatment had no effect on TLR4 expression level but TREM2 was down-regulated. This suggested that TLR4 activation may downregulate inhibition of inflammation by TREM2. This would lead to inflammation and apoptosis in cortex region of these mice (Zhou et al., 2019). Recent reports have also linked TLR4 with Aβ oligomer mediated memory loss in AD progression (Balducci et al., 2017). Detection of LPS in hippocampus and neocortex of post-mortem AD brains also supports the notion that TLR4 plays significant role in AD (Zhou et al., 2019). Rodriguez et al showed that LPS induced increase in cytosolic Calcium ion concentration and apoptosis in rat hippocampal senescent neurons can be prevented by CAY10614, an antagonist of TLR4. These LPS induced effects are not seen in young neurons. Also, TLR4 expression levels in aged neurons are much higher than in young neurons. They reported that when treated with Aβo, TLR4 expression in aged rat hippocampal neurons is increased and so does the LPS induced Calcium ion effect and neuron degeneration (Calvo-Rodriguez et al., 2017).

LYN:

LYN kinases are member of SRC-family of tyrosine protein kinases (SFKs). LYN is expressed mainly in neuronal cells, adipose tissue, liver, and hematopoietic cells. SFKs have been known to play an important role in Alzheimer’s disease (AD) progression, but their mode of action is scarcely understood. Activated microglial LYN in the brain is involved in a crucial interplay with amyloid associated AD pathogenesis, suggesting microglial activation via LYN activity is cell lineage and disease microenvironment dependent (Dhawan & Combs, 2012). In addition, the role of LYN in neuronal cells in amyloid and tau pathologies is also not clearly understood. Elevated brain LYN activity is found in AD patients and was promoted in neuronal cells exposed to Ab 1–42 (Ab1–42). LYN mediates Ab1–42-initiated inflammatory signaling from CD36 in microglia and activates the NF-kB pathway to produce different cytokines promoting AD progression. (Figure 1) (12, 13, 46, 47). Also, Gwon et al., showed that LYN after interaction with Ab_1–42 phosphorylates Fcγ receptor IIb2 (FcγRIIb2) at Tyr273 and consequently leads to Aβ triggered neuron death and tau hyperphosphorylation (figure1) (https://pubmed.ncbi.nlm.nih.gov/30540497/).

Microglial activation is an important histologic characteristic of Alzheimer’s disease (AD) pathology. One hypothesis is that amyloid beta (Aβ) peptide serves as a specific stimulus for non-receptor tyrosine kinase-based microglial activation leading to pro-inflammatory changes that contribute to AD disease pathology. Aβ fibrils stimulated primary murine microglia through the activation of tyrosine kinase pathway involving Src kinase and this effect could be attenuated using dasatinib to decrease protein phosphotyrosine. Active Src-kinases, reactive microglia, and TNFα levels in the hippocampus and temporal lobe may be therapeutically useful targets and a novel anti-inflammatory treatment approach against AD (Dhawan & Combs, 2012; Manocha et al., 2015).

Gas6

Gas6 (Growth arrest specific 6) gene encodes for a gamma-carboxyglutamic acid (Gla) domain-containing protein. The encoded protein, Gas6 is a member of the vitamin K-dependent family of proteins expressed in many human tissues. This protein is known to be involved in regulation of various cellular processes like cellular proliferation, survival, and migration. To perform these functions, it binds to its receptors Tyro3, Axl and Mer (TAM) (https://pubmed.ncbi.nlm.nih.gov/29386018/) TAM are known for their role in phagocytosis and modulation of inflammation, and recent evidence suggests a complex relationship between TAM receptors kinases and microglial phagocytosis of amyloid plaques in AD. Treatment of cortical neurons with Gas6 causes a reduction in Aβ-induced apoptosis (Yagami et al., 2002). Administration of Gas6 has also shown benefits in reducing neuroinflammation and improving behavior in rodent models of stroke, acute cerebral hemorrhage, and multiple sclerosis (Goudarzi, Rivera, Butt, & Hafizi, 2016; Tong et al., 2017; Wu, McBride, & Zhang, 2018). Gas6 is produced and secreted from neurons in the CNS and activates downstream pathways that engage phagocytic machinery and suppress inflammation (Fourgeaud et al., 2016; Grommes et al., 2008). Interestingly, Gas6 binds amyloid plaques in murine models and its production is tightly regulated by Axl expression (Huang et al., 2021). Gas6, the primary CNS TAM ligand has been reported to reduce neuroinflammation and improve behavior outcomes in murine models of CNS disease.(Davra, Kimani, Calianese, & Birge, 2016; Lemke, 2013; Tondo, Perani, & Comi, 2019). However, Gas6 also showed a proinflammatory role in the context of Alzheimer’s disease pathology. Gas6 overexpression studies showed reduced plaque burden in male APP/PS1 mice with no effect on phagocytic mechanisms, but increased pro‐inflammatory microglial gene expression and worsened behavior outcomes in a sex dependent manner (Owlett et al., 2022).

Thus, Gas6 could be an important molecular target to control plaque pathology and AD associated inflammation and pathogenesis, but more understanding of TAM receptor signaling is warranted.

Exercise-induced microglial plasticity of AD model mice

Alzheimer’s disease (AD) considered the most common cause of dementia in older adults accompanies progressive cognitive decline and disability. Recent clinical trials involving new drugs targeting β-amyloid (Aβ) hypothesis and Tau hypothesis (165) have failed to delay memory and cognitive decline in patients with AD (Abbott, 2019). Although exact mechanisms are not yet understood, the benefits of physical exercise in prevention or delay of AD onset as well as improving cognitive function are well recognized, and widely advocated (Sobol et al., 2016). Long-term running exercise has improved learning and memory in AD mouse models (Cotman & Berchtold, 2007; Lin et al., 2015). Many clinical trials have showed benefits of running exercise in alleviating memory loss and other cognitive improvements in AD patients (Hernandez et al., 2015; Meng, Lin, & Tzeng, 2020) (Revilla et al., 2014; Tapia-Rojas, Aranguiz, Varela-Nallar, & Inestrosa, 2016).

Microglia may play an active role in exercise induced effects in AD patients via Triggering receptor expressed in myeloid cells 2 (TREM2) protein. TREM2 is expressed on microglia and is known to mediate microglial metabolic activity and brain glucose metabolism. The level of serum TREM2 decreased in the AD mouse model, APP/PS1 subjected to running exercise indicating inhibited TREM2 shedding. TREM2 protein levels, were directly correlated with promoting brain glucose metabolism, microglial glucose metabolism and morphological plasticity. However, the exact relationship between brain glucose metabolism and microglial metabolic activity during exercise still needs to be elucidated (Zhang et al., 2022).

Disease-Associated Microglia (DAM): Universal Immune Sensor of Neurodegeneration/ DAMs contribute to Aβ pathology

DAMs were initially identified in an AD mouse model that expresses 5XFAD; a molecular characteristic of immune cells expressing specific microglial markers like Iba1, Cst3, and Hexb. These microglial immune markers coincide with the downregulation of “homeostatic” microglial genes including P2RY12, P2RY13, CX3CR1, CD33, and Tmem119 (Keren-Shaul et al., 2017). DAM, or DAM-like phenotype has also been reported by several studies in other neurodegenerative diseases. The single-cell RNA-seq analysis of sorted immune cells (CD45+) from brains of 5XFAD mouse model show specific microglial transition from the homeostatic state to DAM phenotype, significantly different from age-sex matched wild type controls (Oakley et al., 2006).

DAM enrichment was observed as a biomarker reflecting senescence associated increase of neurodegeneration (Hu et al., 2021). An important challenge is to clarify the inhibitory or stimulatory role of DAM in neurodegenerative disorders including the identification of several as-yet-unknown cellular components affected by DAM, or in-turn activating DAM. There is a significant need for a broad molecular characterization of DAM and clearly identify other macrophage functions like phagocytosis, migration, and cytokine production of this specific type of microglia. Invitro functional analysis have shed some light on DAM phagocytosis of Aβ peptide (amyloid plaques) and tau protein (neurofibrillary tangles), DAM migration to the site of inflammation, DAM-neuron interactions, and DAM cytokine production during neuroinflammation in AD (Keren-Shaul et al., 2017). Altered microglial phagocytic ability in neurodegenerative diseases is well known, however any specific contribution of DAM to this pathology is still unclear.

Microglia migration towards the site of inflammation is important for the clearance of pathogens and abnormal proteins from the CNS (Miyamoto, Wake, Moorhouse, & Nabekura, 2013). Neuron-microglial signaling can be influenced by neuroinflammation, metabolic impairment, and microglial activation and errors in neuron-microglial interaction are known to lead to microglial phagocytosis of live neurons and excessive neuronal loss, potentially yielding poorer clinical outcomes. Neuron - microglial interactions and the factors affecting these interactions that can be affected in the brain related disorders such as AD, PD, MS, dementia, and chronic traumatic encephalopathy (CTE). Therefore, study on the DAMs will help to understand the AD pathogenesis and to develop novel strategies to treat this dementia.

Conclusion

Microglia are sentinels of Central Nervous System. These cells can concentrate in different densities in different parts of the brain, and function as part of the immune system. Microglia are also important in cleaning debris. They attack in numbers and phagocytose different debris around brain cells. This coming together of microglia cause inflammation. The inflammation arising from microglial response to first (primary) injury can sometimes lead to a secondary neuronal injury. In such conditions, classifying these cells as good or bad becomes cloudy. Activation profile of microglia vary according to disease states including stroke, PD, AD, MS etc. Evidence supports both pro-inflammatory and anti-inflammatory responses by microglia. In this review we focused on how microglia can help AD progression and some other neurogenerative diseases. Microglia and microglia associated gene contribute towards or against neuroinflammation through different mechanism. Our understanding of role of microglia in context of AD is lacking and further in depth studies are needed to simplify this complex relationship. Experimental AD models can help us elucidate role of microglia by using inhibitors. Also, newly discovered microglia markers can lead us deciphering role of these cells in CNS disorders. These different disease specific activation profiles and responses needs to be studied in detail to help find new targets for therapeutic interventions.