Regulation of microglial homeostasis

Several of the factors that promote microglial identity are also required for the survival, self-renewal and homeostatic state of microglia. Steady-state microglia are typically considered to be the mature, ramified and dynamic cells that actively survey their local microenvironments without overt increase in inflammatory response (Tay et al., 2017a). Reactive microglia (which appear ameboid and upregulate proinflammatory proteins and antigen-presenting markers), primed microglia (which produce an exaggerated inflammatory response to a second stimulus) and senescent microglia (which have enlarged soma and thick processes, and accumulate cellular debris) are generally considered dysfunctional and associated with pathology and aging. Notably, the proliferative, phagocytic or ameboid microglia that appear with spatial and temporal specificity during normal CNS development are integral to the diverse functions performed by microglia and should not be confused with undesirable microglial states that lead to neurotoxicity (Tay et al., 2017a). Stable Tgfβ signaling maintains the unique homeostatic microglial transcriptomic signature comprising Sall1, Hexb, transmembrane protein 119 (Tmem119), purinergic receptor P2RY12 (P2ry12), Tgfbr1, Fcrls and olfactomedin-like 3 (Olfml3) (Fig. 2), as well as immune reactivity (Butovsky et al., 2014; Zöller et al., 2018). In mice that lack expression of Tgfβ1 in the CNS, no microglia that express the microglial markers ionized calcium-binding adapter molecule 1 (Iba1) and P2ry12 are observed in the brains of P20 and older animals (Butovsky et al., 2014). Epigenetic factors, such as the class I histone deacetylases Hdac1 and Hdac2, are required for microglial survival during development, but are functionally redundant in homeostatic adult microglia (Datta et al., 2018). Microglial homeostasis and function are also post-transcriptionally regulated by microRNAs (miRNAs) in an age-dependent manner (Varol et al., 2017). For instance, knocking out Dicer (Dicer1), a protein that processes miRNAs, in Cx3cr1⁺ yolk sac EMPs leads to a global increase in DNA damage in P0 newborn microglia, but microglial depletion of Dicer in adults does not have the same outcome. Instead, loss of Dicer in adult microglia drives hyper-reactivity and results in hippocampal neuronal impairment (Varol et al., 2017). Maintaining microglial self-renewal and normal tiling pattern in the CNS requires Csf1r signaling, largely through Il34 (Greter et al., 2012). Regeneration of microglial homeostasis and cell density after transient chemical ablation by a Csf1r inhibitor in the adult mouse brain requires a stepwise maturation process reminiscent of CNS development (Zhan et al., 2019). However, the requirement for the activation of NF-κB signaling, and the upregulation of the transcription factor Mafb, may be specific to the regulation of adult microglial homeostasis and is independent of the developmental and perinatal maturation programs (Matcovitch-Natan et al., 2016; Zhan et al., 2019).

Microglia-specific inactivation of Sall1 causes the adoption of an inflammatory phenotype in steady-state microglia that leads to altered brain development, disrupted tissue homeostasis and impaired neurogenesis (Buttgereit et al., 2016). miR-124 reportedly modulates mouse and zebrafish microglial phagocytosis and motility as the cells switch from the ameboid to the ramified phenotype (Ponomarev et al., 2011; Svahn et al., 2016). Furthermore, a study of juvenile rodent microglia has reported that the ramified surveillant phenotype of motile microglia is regulated by the two-pore domain potassium channel Thik-1 (Kcnk13) (Madry et al., 2018). Aside from cell-intrinsic aspects that mediate microglial homeostasis, we next examine the non-cell-autonomous signals that modulate the steady state phenotype. Signaling between neurons and microglia via Cx3cr1 is important for maintaining microglia in their non-reactive state. Notably, Cx3cr1 is also necessary for regulating microglial behavior, including their timely recruitment into the CNS, cell density in the early postnatal stage and phagocytic activity that impact their role in physiological synaptic pruning and synaptic plasticity (Hoshiko et al., 2012; Paolicelli et al., 2011; Rogers et al., 2011; Ueno et al., 2013; Zhan et al., 2014). Furthermore, the dysregulation of microglial activity does not necessarily involve a switch to an inflammatory state. In fact, elevated mammalian target of rapamycin (mTOR) signaling in microglia activates a reactive phenotype, independently of inflammatory responses, and is correlated with early-onset seizures or death in juvenile mice (Zhao et al., 2018).

In summary, several endogenous and external elements (Box 1) act independently or in concert to regulate microglial homeostasis and function during CNS development and maturation.

Neural precursor cells and neurogenesis

Neural precursor cells (NPCs) crucially give rise to both neurons and a variety of glial cell populations (Martínez-Cerdeño and Noctor, 2018). Early work documenting the presence of microglia during normal development, and subsequent cell death in both mice and birds, first hinted at the importance of microglia in shaping the newly formed CNS (Antony et al., 2011; Lang and Bishop, 1993; Marín-Teva et al., 1999). Recent research has further identified key interactions between microglia and NPCs, which have profound effects on neurogenesis and overall brain development in vertebrates. Microglia regulate developmental neurogenesis in multiple proliferative brain regions via phagocytosis (Fig. 3A). For instance, microglia in the subgranular zone of the murine dentate gyrus phagocytose apoptotic neuroblasts in the early postnatal period to regulate their incorporation into hippocampal circuits (Sierra et al., 2010). Dysregulation of Sall1 abrogates neuroblast differentiation in the murine dentate gyrus via disruption of microglial gene expression, morphology and reactivity (Buttgereit et al., 2016). During embryonic development, microglia also colonize the subventricular zone (SVZ) and inner SVZ of macaque and rat brains to phagocytose NPCs and regulate NPC population density (Cunningham et al., 2013). Notably, the phagocytic capacity of microglia is heavily context dependent; during zebrafish neurodevelopment, a phenotypically distinct microglial subpopulation, called neurogenic-associated microglia (Table 1), localizes to the neurogenic regions of the optic tectum and primarily phagocytoses dead neurons. In contrast, microglia located in the zebrafish midbrain and hindbrain, called synaptic-region associated microglia (Table 1), express genes associated with phagocytosis of synaptic material and complement pathway activation (Silva et al., 2021). Taken together, these findings emphasize the multi-faceted ways microglia can regulate neurogenesis via phagocytosis.

Although the mechanism(s) that drive microglial selection of NPC phagocytosis remain unclear, an increasing body of work has elucidated the role of microglial secreted factors in regulating NPCs and neurogenesis to support brain development. When cultured alone, murine NPCs lose the capacity to produce committed neuroblasts, but co-culture with either microglia or microglial-conditioned media rescues this ability. These findings suggest that microglial-secreted factors are sufficient to promote SVZ neurogenesis (Walton et al., 2006). Indeed, microglial reactivity and the subsequent secretion of inflammatory cytokines [e.g. Il1β, Il6, tumor necrosis factor α (Tnfα) and Ifnγ], in early postnatal development promotes both neurogenesis and oligodendrogenesis in the rat SVZ (Shigemoto-Mogami et al., 2014) (Fig. 4A). Recent work has suggested that the crosstalk between microglia and NPCs may also be bidirectional; basal progenitor cells in the murine ventricular zone and SVZ actively recruit microglia through the expression of Cxcl12 during cortical development. In turn, disruption of essential microglial Csf1r signaling reduces the population of basal progenitor cells, implicating microglia in proper progenitor cell maintenance (Arnò et al., 2014). Furthermore, embryonic murine microglia are recruited by Cxcl12 to both the SVZ and meninges from the cortical plate. The absence of microglia-secreted factors (e.g. Il6 and type 1 interferons) in the cortical plate is instrumental in the proper maturation of post-migratory cortical plate neurons (Hattori et al., 2020). These studies highlight both the phagocytic (Fig. 3A) and secretory (Fig. 4A) mechanisms by which microglia mediate NPC development and neurogenesis. Future studies linking bidirectional signaling mechanisms, secretory or otherwise, to phagocytosis will clarify the complex crosstalk between NPCs and microglia during neurodevelopment.

Neurons

Microglia are central regulators of the survival and activity of newborn neurons after embryonic development, both promoting and inhibiting cell death when appropriate. To highlight this, numerous studies have investigated the role of microglia during pre- and postnatal eye development (Anderson et al., 2019; Jobling et al., 2018; Lang and Bishop, 1993; Marín-Teva et al., 1999). During murine prenatal development, retinal microglia primarily associate with newborn retinal ganglion cells (RGCs) and regulate neuronal elimination via complement-dependent phagocytosis (Anderson et al., 2019) (Fig. 3B). During postnatal murine eye development, microglial fractalkine signaling modulates microglial-cone contacts, as well as proper cone photoreceptor cilium gene expression and maturation. Genetic ablation of Cx3cr1 signaling induces retinal dysfunction and the loss of cone photoreceptors (Jobling et al., 2018), demonstrating the crucial role of microglia in cell survival. Recent studies have also demonstrated the importance of microglial cytokines for neuronal survival. Previously, microglial-derived Tnfα (Sedel et al., 2004), nerve growth factor (Ngf) (Frade and Barde, 1998) and superoxide ions (Marín-Teva et al., 2004) have been identified as salient mediators of neuronal survival during mouse development (Fig. 4B). More recent work has found that microglia postnatally accumulate near subcerebral and callosal projection axons and that microglial-derived Igf1 is crucial in preventing the apoptosis of layer V cortical neurons (Ueno et al., 2013). Moreover, disruption of microglial Tgfβ signaling (achieved by αVβ8 integrin deletion in the CNS) induces the formation of dysmature microglia, which impair oligodendrocyte maturation and promote the loss of GABAergic interneurons, ultimately leading to severe motor dysfunction and death (Arnold et al., 2019) (Fig. 4C). In addition to secretory mechanisms, microglial processes can directly contact neuronal somata to modulate neuronal function through purinergic junctions in both adult mice and humans (Cserép et al., 2020) (Fig. 3C). Collectively, these findings indicate that microglia are vital to sustain numerous neuronal populations, yet, crucially, microglia are also imperative to the establishment and refinement of the neuron–neuron communication that forms the foundation of neural signaling.

Synaptogenesis, synaptic activity and pruning

Microglia are also involved in the formation of functional neural circuits by modulating synaptogenesis, synapse maturation and synaptic activity. A number of early studies have demonstrated that the loss of functional microglia can alter both excitatory and inhibitory synaptic activity, as well as long-term potentiation (Béchade et al., 2013; Bessis et al., 2007; Roumier et al., 2004). More recent work has elucidated novel mechanisms underlying microglial-mediated alterations to synaptic activity in different cellular, spatial and temporal contexts. For example, microglial loss in prenatal mice can disrupt the proper laminar positioning of a subset of neocortical Lhx6⁺ inhibitory interneurons, which is required for circuit function (Squarzoni et al., 2014). Alternatively, microglia can physically interact with the dendrites of pyramidal neurons within the somatosensory cortex to initiate actin accumulation and augment calcium ion transients, allowing for filopodia and spine formation in mice. Pharmacological blockade of microglia decreases spine density and excitatory synapse formation (Miyamoto et al., 2016). In contrast, microglial depletion in postnatal development increases excitatory and inhibitory synapse number in the somatosensory cortex by disrupting the preferential pruning of inhibitory parvalbumin interneuron synapses by GABA-receptive microglia (Favuzzi et al., 2021) (Table 1). Notably, microglial depletion in utero increases inhibitory parvalbumin interneuron synaptic connections with layer IV neurons in the juvenile (P20) barrel cortex, but decreases them in adults (P60) (Thion et al., 2019). Adding further to this complexity, microglial depletion in adult mice increases synapse number, as well as the activity, of excitatory neurons and inhibitory parvalbumin interneurons in the visual cortex (Liu et al., 2021) (Fig. 3D). These seemingly contradictory findings saliently highlight the necessity of studying the temporal and spatial heterogeneity of microglia during development.

As with neurogenesis, microglia can secrete cytokines as a mechanism to aid developmental synaptogenesis and synaptic maturation (Fig. 4D). In vitro, microglial-secreted Il10 increases the number of hippocampal dendritic spines, excitatory synapses and inhibitory synapses of rat hippocampal neurons (Lim et al., 2013). In vivo, microglial brain-derived neurotrophic factor (Bdnf) signaling in juvenile mice promotes motor learning-related synapse formation and structural plasticity, and ultimately supports glutamatergic synaptic activity and cognitive function (Parkhurst et al., 2013). Postnatal microglial recruitment to thalamocortical synapse clusters in the murine somatosensory cortex is mediated by fractalkine signaling and genetic ablation of this signaling delays the maturation of postsynaptic glutamate receptors at these synapses (Hoshiko et al., 2012). Intriguingly, during human brain development, integrated scRNA-seq has revealed a human cytokine-associated microglia subtype, and xenotransplantation of human prenatal microglia in cerebral organoids increases the synchronization and frequency of neural activity, suggesting a role for microglia in circuit formation and accelerated synaptic maturation (Popova et al., 2021).

Reciprocal crosstalk between microglia and neurons is another key component regulating synaptic activity. Activated hippocampal neurons in adolescent mice secrete Il33, which induces experience-dependent microglial spine remodeling and engulfment of the extracellular matrix, which in turn augments excitatory postsynaptic current frequency (Nguyen et al., 2020). In adult mice, striatal microglia migrate to the synapses of activated thalamocortical projection neurons that secrete ATP. Microglia then convert this ATP to adenosine, which limits neuronal synchrony and firing frequency to prevent seizure (Badimon et al., 2020). However, when hippocampal microglia release ATP, it can be amplified by P2y1r-expressing astrocytes, leading to increased excitatory postsynaptic currents (Pascual et al., 2012). Using several transgenic mouse models to disrupt microglial homeostasis during CNS development, another study has demonstrated that microglia are important for the negative regulation of neuronal activity in an ATP-dependent manner to prevent overactivation and seizures (Badimon et al., 2020).

In addition to building functional circuits, microglia are also essential for synaptic refinement, in which established synapses are either eliminated or reinforced and maintained (Schafer et al., 2013). Surveilling microglia phagocytose live synaptic material in the hippocampi of young mice and microglial loss leads to an increase in dendritic spines and the development of immature synapses (Paolicelli et al., 2011). Microglial loss and impaired synaptic pruning during neurodevelopment has also been associated with autism spectrum disorder; Cx3cr1 knockout mice exhibit a reduced number of excitatory synapses per axon, which is associated with deficits in long-range hippocampal connectivity, decreased social interactions and increased repetitive behavior (Zhan et al., 2014). These works suggest a neuroprotective role for synaptic pruning. Indeed, reactive microglia in adult mice physically disrupt inhibitory presynaptic terminals at cortical neuron somata (Fig. 3C) and the subsequent increase in neuronal activity prevents neuronal apoptosis post-injury (Chen et al., 2014).

Synaptic pruning in the postnatal retinogeniculate system depends on both intact microglial complement signaling and neuronal signaling. In mice, the C1q protein initiates classical complement signaling and is expressed by RGCs in the dorsal lateral geniculate nucleus (dLGN) in response to astrocyte exposure. Complement pathway activation ultimately leads to the deposition of the C3 complement proteins, which mark cells for phagocytosis, and thus promotes dLGN synapse refinement and proper segregation of RGC inputs from each eye (Stevens et al., 2007). Microglia in the dLGN preferentially prune less-active RGC presynaptic terminals and loss of microglial complement signaling induces increased synaptic density and impaired eye segregation (Schafer et al., 2012), thereby demonstrating that synaptic pruning is essential for proper eye development. However, in a murine multiple sclerosis model, microglia engulf synapses in the dLGN during early-stage disease. Inhibition of synaptic C3 prevents pruning and rescues vision loss independently of demyelination and C1q (Werneburg et al., 2020), suggesting that alterations to synaptic pruning may underlie the neurodegeneration associated with multiple sclerosis. In summary, microglia control synaptogenesis and synapse refinement in both health and disease through numerous mechanisms depending on their unique cellular, spatial and temporal contexts.

Beyond the synapse: axonogenesis, myelinogenesis and oligodendrocyte precursor cells

In addition to the synapse, microglia are known to influence the generation and pruning of neuronal axons and the myelin that ensheathes them. Studies in Drosophila initially provided evidence that glial cells can alter axons. These glia engulf the fragmented axons of mushroom body γ neurons in a microglial-like manner to facilitate proper larval development (Watts et al., 2004). In the prenatal murine forebrain, microglia regulate the outgrowth of dopaminergic axons (Squarzoni et al., 2014). In the context of optic nerve injury, RGC axonal regeneration requires Clec7a (also known as dectin-1), which is expressed only by myeloid cells (including microglia) in the murine retina (Baldwin et al., 2015), thus suggesting that microglia are instrumental for proper axonogenesis. However, in embryonic human brains, reactive microglia are present in the pons, olivary bodies, hippocampus, optical tract and anterior limb of the internal capsule, but are absent from most regions with growing axons, indicating that reactive microglia may limit axonogenesis (Cho et al., 2013). These works shed light on both pro- and anti-axonogenic roles for microglia during neurodevelopment and further work is necessary to fully elucidate the context-specific nuances in microglial regulation of axonogenesis.

Microglia are also crucial regulators of myelinogenesis, which is the process of myelin formation around bare axons. Myelin is a lipid-rich sheath produced by oligodendrocytes that enwraps neuronal axons to insulate and promote the saltatory conduction of action potentials. As such, the differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes and properly formed myelin is crucial for healthy brain development. Genetic or pharmacological ablation of microglia soon after birth in mice leads to decreased numbers of OPCs and oligodendrocytes and a transient decrease in white matter-specific myelinated axons (Hagemeyer et al., 2017) (Fig. 3E). Aberrant perinatal activation of inflammatory signaling in microglia is associated with white matter dysregulation of myelination; however, blocking the microglial intracellular protein complex (known as the inflammasome) in mouse brain explants can reverse the developmental hypomyelination (Holloway et al., 2021). Furthermore, murine microglial ablation in adults results in a rapid decrease in OPCs of the corpus callosum (Hagemeyer et al., 2017). The type 1 integral membrane protein neuropilin 1 (Nrp1), expressed specifically by white matter-associated microglia, facilitates OPC proliferation during development and remyelination after acute demyelination via PDGF-AA signaling (Sherafat et al., 2021), thus underscoring the potent role of microglia in oligodendroglial lineage cell maintenance throughout life. Microglia migrating from the ventricular zone can phagocytose viable OPCs in the murine corpus callosum, a process that is reduced with knockdown of fractalkine signaling; this reduced OPC phagocytosis resulting from fractalkine signaling knockdown is subsequently associated with an increase in adulthood of mature oligodendrocytes that produce thinner myelin, thereby suggesting that microglia contribute to myelin and myelin-forming cell homeostasis (Nemes-Baran et al., 2020). Indeed, in zebrafish, microglia phagocytose excess myelin in the optic tectum to ensure proper developmental myelination in a neuronal activity-dependent manner (Hughes and Appel, 2020). Furthermore, young human patients who carry homozygous mutations for the CSF1R completely lack the myelinated nerve bundles that connect both hemispheres of the brain (Oosterhof et al., 2019).

Another mechanism by which microglia regulate developmental myelination is through cytokine release (Fig. 4E). Suppression of microglial reactivity with minocycline in early life decreases secretion of inflammatory cytokines and oligodendrogenesis in the SVZ (Shigemoto-Mogami et al., 2014). Intriguingly, though a Cd11c⁺ subset of microglia has been shown to poorly produce proinflammatory cytokines during experimental autoimmune encephalitis in mice (Wlodarczyk et al., 2014), a Cd11c⁺ postnatal microglial population (Table 1) can express Igf1 and other neurogenic signals implicated in developmental myelination (Wlodarczyk et al., 2017) (Fig. 4E). Finally, as in neurogenesis, CNS-specific deletion of murine αVβ8 integrin causes dysmature microglia, leading to deficits in oligodendrocyte maturation (Arnold et al., 2019). Taken together, these works emphasize pivotal neuron-glia and glial-glial interactions that support healthy CNS development.

Astrocytes and astrogenesis

Prenatal and postnatal interactions between microglia and astrocytes also underscore the essential role of microglia for glia-glia interactions pertaining to development. Experiments culturing NPCs from Spi1^−/− mice, which lack microglia, have reduced staining for glial fibrillary acidic protein-positive (Gfap⁺) cells relative to mice with intact microglia, thus suggesting that microglia may be involved in astrogenesis (Antony et al., 2011). Further work has specified how secreted factors may regulate astrogenesis and astrocyte development (Fig. 4F). Recombinant, proinflammatory cytokine Il1 increases Gfap⁺ cell number in embryonic mixed-glial cerebral cortex cultures (Giulian et al., 1988). Likewise, culturing embryonic rat SVZ cells in microglial conditioned media, which specifically contains Il6 and leukemia inhibitory factor (Lif), also increases the percentage of Gfap⁺ cells (Nakanishi et al., 2007). Nitric oxide has also been implicated in the differentiation of hippocampal radial glia into mature astrocytes. Nitric oxide synthase 2 (Nos2) is highly upregulated by inflammatory microglia and a key producer of nitric oxide; genetic ablation of Nos2 shows no evidence of disrupting the generation of glutamine synthetase-positive astrocytes, but does reduce astrocyte differentiation in mice as measured by reduced Gfap expression (Béchade et al., 2011). Thus, microglial secreted factors are a primary mechanism by which microglia regulate putative astrocyte development.

Finally, recent work has drawn attention to the reciprocal nature of crosstalk between microglia and astrocytes that supports cellular survival. Microglial survival ex vivo requires a number of astrocyte-derived factors including Csf1, Il34, Tgfβ2 and cholesterol (Bohlen et al., 2017). Microglial-astrocyte communication has functional implications in a neurodegenerative context as well; neuroinflammatory microglia secrete Il1α, Tnf and C1q, which induce A1 astrocyte reactivity (Fig. 4F). In mice, A1 astrocytes fail to promote multiple regenerative mechanisms ranging from neuronal process outgrowth and synaptogenesis to phagocytosis, leading to neuronal and oligodendrocyte death (Liddelow et al., 2017). As such, microglial-astrocyte crosstalk represents yet another axis for microglial maintenance of brain homeostasis.

This Review is in memory of C. Mehl and M. Spelleken. All figures were created with BioRender.com.