Microglia in CNS Development

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Image Source: London, Cohen, Schwartz (2013)

The development of the central nervous system (CNS) is highly influenced by neuroimmune effects. The primary cellular effectors are microglia, the resident mononuclear macrophages of the brain. Long thought to only be involved in pathogenic and damaged states, microglia are rapidly being revealed to be far more involved in all aspects of the CNS[1]. These cells have critical roles in neuronal differentiation and proliferation, synaptic networking modification/pruning, synaptogenesis, and even CNS angiogenesis[1],[2].Microglia execute their tasks by virtue of expressing a wide variety of immune molecules, including the major histocompatibility complex (MHC), toll-like receptors (TLRs), and a multitude of cytokines and receptors[9]. In addition to immune response specific molecules, microglia also secrete neurotrophic factors that both support and assist in CNS development.

1 Microglial Developmental Origins

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A. YS origin of microglia B. Pu.1 KO eliminates microglia and resulted in diminished CNS
vasculature in mouse embryos. Image Source: Arnold & Betscholtz (2013)

The origin of microglial cells has a long history of controversy and only recently has there been some consensus reached on the issue. First, microglial cells demonstrate homology with monocytes and macrophages, expressing a range of markers—F4/80, Fc receptors, and CD11b in mice, along with FcGRI and CD11b homologs in humans[1]. Monocytes and macrophages have an established myeloid origin, which was also confirmed for microglia using a knockout mice for a vital myeloid transctiption factor, PU.1[1],[3]. Current views hold that microglia themselves arise from embryonic hematopoietic precursors that line the CNS before bone marrow hematopoesis. The main controversy that existed was in relation to the origin of these pre-cursors, as there are two embryonic tissue regions that undertake hematopoiesis in early embryogenesis —the yolk sac (YS) and the fetal liver. Initially the YS produces primitive macrophages that bypass the mononucleation phase and colonize (as soon as the circulatory system is established, unique to mice) their target tissue, whereas a second mesodermal derived population is generated in mice in the aorta, gonads, and mesonephros (AGM). Both lines then colonize the fetal liver, where all hematocytes are generated. The YS line is distinct in that it undergoes primitive (independent of the transcription factor Myb) hematopoiesis as compared to true for the AGM line. Looking toward adult-hood and maintaining microglial populations, it is clear that local proliferation is the overwhelming contributor. It should be noted that under intensive states of pathology bone marrow derived microglia can arise, but this is very uncommon. Humans demonstrate similar developmental processes to their murine counterparts[1].

2 CNS Developmental Processes

2a Neurogenesis, Neuronal Maturation and Survival

Neurons undergo a number of stages before they reach their final destinations within the CNS. This maturation process is highly influenced by microglia. Microglia release a variety of factors that are associated with non-committed neural precursor cells. In particular, epidermal growth factor (EGF) treated cells demonstrate directed guidance both in culture and Boyden chambers (chemotaxis assay). Microglia are thus indicated in assisting nascent neurons in path finding to their targets[4].

Similarly, IGF1, a neurotrophic factor expressed by microglia has been indicated in the survival of cortical neurons in vitro and layer V cortical neuron survival in vivo in mouse models, allowing for the establishment of neuronal circuits. Mutant models simulating microglial loss demonstrate diminished IGF1 levels, further supporting the neurotrophic importance of microglia. In fact, neurons actively recruit microglia, via secreting the chemokine fractalkine (FKN)-CX3CL1 for which only microglia have the receptor CX3CR1 in the CNS[5].

Interestingly microglia also play a fundamental role in neurogenesis. The subgranular zone (SGZ) is a known site of neuronal birth in the adult brain, and a hallmark study site for plasticity within the brain. However, the majority of new neurons generated in the SGZ are in fact eliminated and not incorporated into the existing neuronal framework of the hippocampus. Elevated levels of CD11b and CD68, normally considered markers of inflammation, are observed in the SGZ. These antigens expressed by microglia are linked to phagocytic activity to clean up apoptotic residues of neurons which do not reach maturity and integrate into existing circuitry[1].

2b Synaptic Pruning and Modification

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Various proposed avenues for synaptic pruning and modification
by microglia. Image Source: Miyamoto et al. (2013).

Synapses within the CNS have various strengths dependent upon their input, and these variations correspond to which which are maintained. Weak synapses are pruned, allowing, the stronger ones to dominate and flourish[7]. The most well-studied route by which microglia may mediate synaptic pruning is their FKN receptor. The FKN receptor is only found on microglia in the CNS and binds FKN which is usually membrane bound but can also be secreted by neurons in a soluble form. Microglia have been shown phagocytose synapses and degrade them during development. In knockouts for Cx2cr1, significant aberrant electrophysological recordings associated with juvenile synapses and disrupted synaptic pruning in early development are observed[8]. The exact mechanism by which microglia can differentiate damaged neurons and synapses from developing ones using this paradigm remains in contention.

Alternatively, microglia may identify pruning targets utilizing the multiple histocompatibility complex (MHC) I as well as other aspects of the innate immune cascade. One suggested and observed avenue involves astrocyte's activating C1q, a classical immune response starter, activating C3b which binds to neurons marking them for degradation. Microglia would then interact with the C3b via a CR3 receptor to ultimately be degraded[9]. As in the case of the FKN mediated pruning, when the immune complement system is removed via a CR3 KO or other modifications also altered synaptic network modification in line with disrupted synaptic pruning is observed[10]. Interestingly, the FKN mediated synaptic pruning is only present in the early stages of life, as other processes undertake synaptic pruning later; however, the complement mediated avenue has sustained effects through till adulthood[8],[9],[10]. Additionally both cases resulted in significantly greater levels of spines on axons, again indicative of a failure to prune[8],[10].

Additionally, pathways have been proposed that involve activating microglia modifying synaptic networks. In particular, one pathway suggests ATP released by microglia stimulates astrocytes to release glutamate which retroactively increases pre-synaptic release via binding to mGluR5s. This increases the strength of the synapse, demonstrating a role for microglia in maintaining and altering synapses[16]

2c Vasculature

The developing CNS unlike other systems does not have its own innate vasculature and instead relies on angiogenesis, the formation of new blood vessels from pre-existing ones, stimulated by proangiongenic signals. These signals essentially increase the levels of sprouting from perineuronal vessels allowing an extensive networks to be established. The primary factors involved in this process are vascular endothelial growth factor (VEGF) A and notch, which have an intricate interplay that is highly modulated. Microglia themselves are not essential sources of these factors but, have a fundamental role in increasing sprouting and thus the vascular density. Studies in mice looking at diminished levels of microglia demonstrate a clear decrease in vasculature during development as did mice lacking them from the beginning[11],[12]. The specific importance of microglia during development is reinforced by the fact that mice lacking them initially, regained vasculature levels later. Thus, microglia are likely particularly important in remodelling and fine tuning of the vasculature[12]. An interesting contrast arises when looking at deep retinal vasculature in the CNS. Some studies indicate a null affect when microglia are depleted, whereas others display an increase in sprouting[13],[14]. Increased sprouting was linked to microglia being positioned as inhibitors of angiogenesis via Wnt-Flt1signalling[14]. These conflicting results display the wide diversity of microglial effects in CNS vasculature[15] as well as the need for greater study.

Bibliography
1. Ginhoux, F., Lim, S., Hoeffel, G., Low, D., Huber, T. Origin and differentiation of microglia. Front Cell Neurosci 7(45), 1-14 (2013).
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3. McKercher SR, Torbett BE, Anderson KL, Henkel GW, Vestal DJ, Baribault H, Klemsz M, Feeney AJ, Wu GE, Paige CJ, Maki RA. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J 15(20), 5647-58 (1996).
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6. Sierra A, Encinas JM, Deudero JJ, Chancey JH, Enikolopov G, Overstreet-Wadiche LS, Tsirka SE, Maletic-Savatic M. Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis. Cell Stem Cell 7(4), 483-95 (2010).
7. Kerschensteiner D, Morgan JL, Parker ED, Lewis RM, Wong RO. Neurotransmission selectively regulates synapse formation in parallel circuits in vivo. Nature 460(7258), 1016-20 (2009).
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9. Zabel, MK. Kirsch, WM. From development to dysfunction: Microglia and the complement cascade in CNS homeostasis. Ageing Res Rev 12(3), 749–756 (2013).
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11. Greenberg DA, Jin K. From angiogenesis to neuropathology. Nature438(7070), 954-959 (2005).
12. Santos AM, Calvente R, Tassi M, Carrasco M-C, Martín-Oliva D, Marín-Teva JL. Embryonic and postnatal development of microglial cells in the mouse retina. J Comp Neurol 506(2), 224-239 (2008).
13. Checchin D, Sennlaub F, Levavasseur E, Leduc M, Chemtob S. Potential role of microglia in retinal blood vessel formation. Invest Ophthalmol Vis Sci 47(8), 3595-3602 (2006).
14. Kurz H, Christ B. Embryonic CNS macrophages and microglia do not stem from circulating, but from extravascular precursors. GLIA 22(1), 98-102 (1998).
15. Arnold T and Betsholtz C. The importance of microglia in the development of the vasculature in the central nervous system. Vasc Cell 5(1), 4 (2013).
16. Miyamoto A, Wake H, Moorhouse AJ, Nabekura J. Microglia and synapse interactions: fine tuning neural circuits and candidate molecules. Front Cell Neurosci 7, 70 (2013).

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