To Kill a Microglia: A Case for CSF1R Inhibitors

doi:10.1016/j.it.2020.07.001

Trends in Immunology

Volume 41, Issue 9, September 2020, Pages 771-784

https://doi.org/10.1016/j.it.2020.07.001 Get rights and content

Highlights

Microglial depletion animal models provide insight into microglial cell function in the healthy and diseased brain.
Microglia are dependent on colony-stimulating factor 1 receptor (CSF1R) signaling for survival in the vertebrate brain.
CSF1R inhibition allows rapid and titratable myeloid cell population manipulation in the rodent CNS.
We posit that optimal microglial depletion models (e.g., CSF1R inhibitors) should ideally enable the elimination of these cells for any duration of time in different cell subsets. These models might also stimulate microglial repopulation in any model or species, and act in a clinically relevant fashion.

Microglia, the brain’s immune sentinels, have garnered much attention in recent years. Researchers have begun to identify the manifold roles that these cells play in the central nervous system (CNS), and this work has been greatly facilitated by microglial depletion paradigms. The varying degrees of spatiotemporal manipulation afforded by such techniques allow microglial ablation before, during, and/or following insult, injury, or disease. We review the major methods of microglial depletion, including toxin-based, genetic, and pharmacological approaches, which differ in key factors including depletion onset, duration, and off-target effects. We conclude that pharmacological CSF1R inhibitors afford the most extensive versatility in manipulating microglia, making them ideal candidates for future studies investigating microglial function in health and disease.

Section snippets

Manipulating Microglia for Insight into Brain Function: Tools at Hand

Microglia are the primary immune cells of the brain and, together with perivascular, choroid plexus (see Glossary), and meningeal macrophages, comprise the macrophage compartment of the CNS. Under steady-state conditions, microglia are dynamic surveyors of the CNS, occupying distinct non-overlapping territories where they constantly extend and retract their processes to sample the local milieu and maintain tissue homeostasis [1., 2., 3.]. Recent studies have shown that these cells are

Toxin-Based Models of Microglial Depletion

Early approaches to deplete microglia involved the generation of Cd11b–HSVTK transgenic mice, which overexpress the herpes simplex virus-derived thymidine kinase (HSVTK) under the control of the Cd11b promoter (i.e., in cells of myeloid origin). In the presence of ganciclovir, HSVTK is activated and induces apoptosis in CD11b⁺ mitotic cells [14,15]. However, systemic and extended administration of ganciclovir leads to fatal anemia owing to loss of CD11b⁺ cells that are necessary for normal

Colony-Stimulating Factor 1 (CSF1) Receptor Signaling and the Evolution of Genetic Microglial Depletion Models

Extensive research over the past 50 years has demonstrated that signaling through CSF1 and its cognate receptor (CSF1R) is essential for microglial cell survival, and this pathway has played a fundamental role in the development of genetic models of microglial depletion. CSF1, also known as macrophage colony-stimulating factor (M-CSF), is a hematopoietic growth factor/cytokine that is involved in promoting macrophage proliferation, differentiation, and survival [34]. The sole receptor for CSF1

Pharmacology-Based Microglial Depletion: CSF1R Inhibitors Allow Investigations into Adult Microglial Homeostasis

Although the first generation of CSF1R-associated knockout mice (e.g., Csf1^−/−, Csf1r^−/−, Csf1^op/Csf1^op) revealed that CSF1R signaling is essential for the development of microglia, studies into the role of this axis in the adult mouse CNS were limited. Our research group initially identified two CSF1R inhibitors (CSF1Ri), PLX3397 and PLX647, that cross the BBB, and tested their ability to prevent microglial proliferation in mouse models of lipopolysaccharide (LPS)-induced neuroinflammation.

What Have We Learned from Microglial Depletion Models?

Although microglia have well-established roles in immunity and immunosurveillance, other homeostatic functions have come to light, including essential roles in synaptic refinement and sculpting, as well as in neurogenesis [3,100,101]. Studies in mice indicate that microglia are involved in regulating learning-induced synaptic modifications (in early adulthood) [27] and in phagocytosing apoptotic neuronal progenitors in CNS neurogenic niches [102,103]. Microglial depletion models have been

Concluding Remarks

Pharmacological CSF1R inhibitors provide unprecedented control over microglial population dynamics in animal models, attaining rapid and sustained depletion through oral administration in chow, without inducing notable compensatory mechanisms or debilitating health effects. Indeed, perhaps one of the most interesting findings from CSF1Ri research is that microglial loss does not induce any overt phenotypic or cognitive abnormalities in otherwise healthy adult mice. We and others [104]

Acknowledgments

This work was supported by the National Institutes of Health under awards R01NS083801 (NINDS), R01AG056768 (NIA), and P50AG016573 (NIA) to K.N.G., and F31NS108611 (NINDS) to J.D.C. L.A.H. was supported by an Alzheimer’s Association Research Fellowship (AARF-16-442762).

Glossary

5×FAD mouse model: a transgenic mouse model of Alzheimer’s disease (AD), in which mice express human amyloid precursor protein (APP) and presenilin 1 (PSEN1) transgenes harboring five familial AD (FAD)-causing mutations.
Agenesis of the corpus callosum: a rare brain disorder in which the corpus callosum, a white matter tract that connects the two brain hemispheres, is partially or completely absent.
Choroid plexus: a structure located in the ventricles that plays an important role in regulating immune

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Microglia are the residential immune cells in the central nervous system. Their roles as innate immune cells and regulators of synaptic remodeling are critical to the development and the maintenance of the brain. Numerous studies have depleted microglia to elucidate their involvement in healthy and pathological conditions. PLX3397, a blocker of colony stimulating factor 1 receptor (CSF1R), is widely used to deplete mouse microglia due to its non-invasiveness and convenience. Recently, other small rodents, including Syrian hamsters (Mesocricetus auratus) and Mongolian gerbils (Meriones unguiculatus), have been recognized as valuable animal models for studying brain functions and diseases. However, whether microglia depletion via PLX3397 is feasible in these species remains unclear. Here, we administered PLX3397 orally via food pellets to hamsters and gerbils. PLX3397 successfully depleted gerbil microglia but had no effect on microglial density in hamsters. Comparative analysis of the CSF1R amino acid sequence in different species hints that amino acid substitutions in the juxtamembrane domain may potentially contribute to the inefficacy of PLX3397 in hamsters.
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Age-related macular degeneration is the leading cause of irreversible visual impairment in the elderly. It manifests in two forms, wet and dry. However, the mechanisms underlying spontaneous dry age-related macular degeneration (SD-AMD) remain unclear. Herein, we constructed a single-cell retinal transcription atlas in aged non-human primates with SD-AMD. Retinal tissues affected by SD-AMD exhibited a more degenerative and dysfunctional transcriptomic landscape, with global activation of the oxidative stress response and apoptotic signaling pathway. We found two distinct Müller glia subtypes in normal aged and SD-AMD macaques, one exhibiting a photoreceptor-like transcriptome and the other exhibiting a typical Müller glia transcriptome. As SD-AMD progressed, the proportion of photoreceptor-like Müller glial cells decreased, and photoreceptor-function-associated genes were downregulated, indicating weaker Müller glia potential to transit into photoreceptor-like functional states. Microglial cells showed activated features, and the complement system was activated during disease pathogenesis. We also found that the disruption of iron homeostasis and ferroptosis could promote SD-AMD pathogenesis in neural cells. Further experimentation revealed that a ferroptosis inhibitor exerted a profound rescuing effect in SD-AMD mouse models. Based on these results, our study introduces a path toward understanding the pathogenesis of SD-AMD in a non-human primate model at single-cell resolution.
β-glucan, a specific immuno-stimulant, produces rapid antidepressant effects by stimulating ERK1/2-dependent synthesis of BDNF in the hippocampus
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A decline in microglia in the dentate gyrus of the hippocampus has recently been described as an important mechanism for the progression of depression. Reversal of this decline by innate immune system stimulants may represent a novel strategy to ameliorate the depressive phenotype in chronically stressed animals. β-glucan is a polysaccharide from Saccharomyces cerevisiae. It can efficiently stimulate microglia without inducing the production of pro-inflammatory cytokines. Therefore, β-glucan could be an ideal drug to ameliorate depressive phenotypes. In the present study, we found that a single injection of β-glucan reversed depression-like behaviors in mice induced by chronic unpredictable stress (CUS) in a dose-dependent manner, which was accompanied by a reversal of the CUS-induced decrease in brain-derived neurotrophic factor (BDNF) protein levels in the dentate gyrus. The crucial role of BDNF signaling in the antidepressant effect of β-glucan was demonstrated by experiments showing that infusion of an anti-BDNF antibody into dentate gyrus, construction of BDNF-Val68Met allele knock-in mice, or treatment with the BDNF receptor antagonist K252a abolished the antidepressant effect of β-glucan. The increased BDNF signaling induced by β-glucan was mediated by extracellular signal-regulated kinase1/2 (ERK1/2)-mediated BDNF synthesis, and inhibition of ERK1/2 by SL327 was able to abolish the antidepressant effect of β-glucan. Moreover, inhibition or depletion of microglia by minocycline or PLX3397 abolished the reversal effect of β-glucan on CUS-induced depression-like behaviors and CUS-induced impairment of ERK1/2-BDNF signaling. These results suggest that β-glucan exhibits antidepressant effects by stimulating microglia-mediated activation of ERK1/2 and synthesis of BDNF in the hippocampus.
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Trends in Immunology

OpinionSpecial Issue: Microglia and AstrocytesTo Kill a Microglia: A Case for CSF1R Inhibitors

Highlights

Section snippets

Manipulating Microglia for Insight into Brain Function: Tools at Hand

Toxin-Based Models of Microglial Depletion

Colony-Stimulating Factor 1 (CSF1) Receptor Signaling and the Evolution of Genetic Microglial Depletion Models

Pharmacology-Based Microglial Depletion: CSF1R Inhibitors Allow Investigations into Adult Microglial Homeostasis

What Have We Learned from Microglial Depletion Models?

Concluding Remarks

Acknowledgments

Glossary

Trends Immunol.

Cell Rep.

Neurobiol. Aging

Neuron

Immunity

Trends Immunol.

Brain Behav. Immun.

Cell

Neuron

Cell

Blood

Blood

Dev. Biol.

Am. J. Hum. Genet.

Neuron

Brain Behav. Immun.

Clin. Immunol.

Blood

Cell Rep.

Cell Rep.

Brain Behav. Immun.

Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo

Science

Microglia in steady state

J. Clin. Invest.

Proximal recolonization by self-renewing microglia re-establishes microglial homeostasis in the adult mouse brain

PLoS Biol.

Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution

Nature

Single-cell RNA sequencing of microglia throughout the mouse lifespan and in the injured brain reveals complex cell-state changes

Immunity

Single-cell transcriptomic analysis of Alzheimer’s disease

Nature

Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer's disease

Nat. Genet.

Brain cell type-specific enhancer–promoter interactome maps and disease-risk association

Science

TREM2 in neurodegeneration: evidence for association of the p.R47H variant with frontotemporal dementia and Parkinson's disease

Mol. Neurodegener.

TREM2 variant p.R47H as a risk factor for sporadic amyotrophic lateral sclerosis

JAMA Neurol.

Mouse model for ablation of proliferating microglia in acute CNS injuries

Glia

Experimental autoimmune encephalomyelitis repressed by microglial paralysis

Nat. Med.

Antiviral drug ganciclovir is a potent inhibitor of microglial proliferation and neuroinflammation

J. Exp. Med.

Acute reduction of microglia does not alter axonal injury in a mouse model of repetitive concussive traumatic brain injury

J. Neurotrauma

Microglial repopulation model reveals a robust homeostatic process for replacing CNS myeloid cells

Proc. Natl. Acad. Sci. U. S. A.

Impact of peripheral myeloid cells on amyloid-beta pathology in Alzheimer's disease-like mice

J. Exp. Med.

A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration

Nat. Methods

Tmem119–EGFP and Tmem119–CreERT2 transgenic mice for labeling and manipulating microglia

eNeuro

Competitive repopulation of an empty microglial niche yields functionally distinct subsets of microglia-like cells

Nat. Commun.

Cell-specific deletion of C1qa identifies microglia as the dominant source of C1q in mouse brain

J. Neuroinflammation

Comparative analysis of CreER transgenic mice for the study of brain macrophages: a case study

Eur. J. Immunol.

Inducible control of gene expression with destabilized Cre

Nat. Methods

Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications

J. Immunol. Methods

Microglial depletion with clodronate liposomes increases proinflammatory cytokine levels, induces astrocyte activation, and damages blood vessel integrity

Opinion
Special Issue: Microglia and Astrocytes
To Kill a Microglia: A Case for CSF1R Inhibitors