As the brain’s resident immune cells, microglia gobble up dangerous gunk. However, according to a March 1 paper in Molecular Neurodegeneration, the cells may sometimes do more harm than good, at least in the earliest stages of Alzheimer’s disease. Researchers led by Charles Glabe at the University of California, Irvine, reported that using the drug PLX3397 to selectively kill off microglia in an AD mouse model reduced accumulation of Aβ inside neurons, and prevented the formation of neuritic plaques. The treated animals also dodged some of the behavioral deficits documented for this mouse line. The researchers propose a cascade in which microglia somehow promote the toxic accumulation of intraneuronal Aβ, which ultimately kills neurons and triggers the formation of neuritic plaques.
- Treating 5xFAD mice with PLX3397, a CSF1R inhibitor that ablates microglia, prevented neuritic plaque formation.
- This lessened accumulation of Aβ inside cells.
- Treated mice performed better on a memory test.
Glabe told Alzforum that he sought to understand the role microglia play in the development of neuritic plaques. Intermeshed with remnants of dead neurons and surrounded by swollen, dystrophic neurites, this form of plaque likely originates from what were once intracellular aggregates of Aβ, Glabe believes. A distinctive and, to Glabe’s mind, suspicious, halo of microglia surrounds neuritic plaques, prompting him to probe whether the cells promoted this pathology or were merely looky-loos.
To address this question, first author Justyna Sosna and colleagues ablated microglia in 5xFAD mice using PLX3397. This small molecule inhibits colony-stimulating factor 1 receptor (CSF1R), which microglia need to survive. The drug drastically reduces numbers of microglia, circulating monocytes, and resident macrophages in other organs; it has been approved for human use to treat some cancers.
Previous work from Kim Green’s lab, also at UC Irvine, found that microglial populations rapidly replenished following ablation. In Green’s lab, treating 10-month-old, plaque-ridden 5xFAD mice with PLX3397 did not reduce plaque burden, yet protected synapses (Apr 2014 news; Spangenberg et al., 2016).
Sosna asked what would happen if the mice took the drug earlier, at a time when neurons harbor Aβ aggregates but few neuritic plaques have formed yet. The researchers therefore started treating two-month-old mice and continued for three months. At five months, immunofluorescence of brain sections from untreated animals showed widespread Aβ deposition, the vast majority in the form of neuritic plaques. In contrast, animals fed with PLX3397-laced chow had up to 80 percent fewer microglia and 90 percent fewer neuritic plaques throughout the brain, including hippocampus, cortex, and amygdala. The few neuritic plaques that did exist were smaller and more compact than those in control mice, and remaining microglia surrounded them.
The microglial ablation also drastically reduced accumulation of intraneuronal Aβ, to a similar degree as it did neuritic plaques.
Using conformation-dependent antibodies that distinguish different types of Aβ oligomer, the researchers investigated how PLX3397 influenced these soluble Aβ species. They found that compared with wild-type mice, five-month-old 5xFADs had more so-called “fibrillar oligomers” in the hippocampus and cortex; these tend to be stable and contain parallel β-sheet structures. In the plasma of 5xFAD animals, the scientists found an abundance of “prefibrillar oligomers,” which are less stable and have anti-parallel β-sheet structure. PLX3397 substantially reduced levels of both of these species of Aβ.
Would this ward off memory problems? The researchers conducted two tests of hippocampal memory: the Y-maze test of spatial memory, and the fear-conditioning test of contextual memory. Five-month-old 5xFAD mice had slight deficits in both relative to wild-type. PLX3397-treated 5xFAD mice performed at wild-type levels on the contextual-fear-memory test, but showed no benefit on the spatial-memory test. Glabe told Alzforum that perhaps the treatment had no extensive benefits because at just five months of age, memory problems in 5xFAD mice are subtle.
The findings point to a role for microglia in the formation of neuritic plaques, Glabe said. Given Green’s previous finding that removing microglia in older, plaque-ridden mice had no effect on Aβ pathology, Glabe’s study suggests that microglia play a formative role early on but not later.
Glabe was surprised that microglia seemed to promote the accumulation of intraneuronal Aβ, and he wasn’t alone. “These results are interesting because they provide an additional line of evidence for the beneficial impact of targeting microglial numbers in AD, but also a bit puzzling because, after all, it is difficult to understand how Aβ production could have been ‘switched-off’ in a transgenic model where the promoters are not microglia or CSF1R-dependent,” commented Diego Gomez-Nicola of the University of Southampton, England. “Data analyzing the levels of APP expression and/or protein levels of Aβ could have clarified this issue, alongside analysis of the proteins involved in APP processing,” he added.
Glabe proposed that microglia might send signals to neurons that coax them into storing intraneuronal Aβ, which ultimately reaches a toxic threshold that kills the neurons; this would create fodder for future neuritic plaques. Future studies should identify those signals, he said.
Alternatively, CSF1R signaling on neurons might promote intraneuronal Aβ accumulation, Glabe offered. A previous study reported that some neurons express the receptor, and that it provides neuroprotective signals (Luo et al., 2013).
Gary Landreth of Indiana University School of Medicine in Indianapolis also raised this issue. “One of the difficulties with the manuscript arises from the use of PLX3397 to ablate microglia, since it inhibits CSF1R/kit found both on microglia and in a subset of neurons,” he wrote. “Thus, it is not possible to determine if the observed effects on pathology arise from salutary effects of the drug on neurons or from the loss of microglia in the brain.” He added that a careful counting of neuronal numbers would have helped clarify this conundrum.
Landreth also wondered about the nature of the plaque-associated microglia that somehow resisted ablation, speculating that these CSF1R-independent survivors could be endowed with beneficial properties.
“Overall this paper highlights some exciting ideas; however, it falls short on the delivery of clear results implicating microglia in the observed phenotypes and behavioral improvement,” he wrote.
For PLX3397 studies in the brain, mixed results are par for the course. A recent study found that PLX3397 treatment exacerbated neuronal loss in a Parkinson’s mouse model, while a paper submitted to BioRxiv reported the drug was beneficial in a model of epilepsy (Yang et al., 2018; Srivastava et al., 2017).
The explanation for these contradictory findings may lie in the growing realization that the role of microglia may change depending on disease stage. Researchers increasingly acknowledge that they need to learn to characterize context-dependent microglial phenotypes, and as they do so, expect to discover a variety of distinct microglial populations. Consider yet another new study, in a model of amyotrophic lateral sclerosis. In inducible TDP-43 mice, microglia stood idly by while pathology ramped up, but when TDP-43 expression was shut off, the cells rose up to expand, change gene expression, and prevent neurodegeneration. Giving PLX3397 during this “recovery phase” blocked the mice from regaining motor function (Mar 2018 news).—Jessica Shugart
Research Models Citations
- Microglial Magic: Drug Wipes Them Out, New Set Appears
- In TDP-43 Mouse, Hesitant Microglia Eventually Swoop to the Rescue
- Spangenberg EE, Lee RJ, Najafi AR, Rice RA, Elmore MR, Blurton-Jones M, West BL, Green KN. Eliminating microglia in Alzheimer's mice prevents neuronal loss without modulating amyloid-β pathology. Brain. 2016 Apr;139(Pt 4):1265-81. Epub 2016 Feb 26 PubMed.
- Luo J, Elwood F, Britschgi M, Villeda S, Zhang H, Ding Z, Zhu L, Alabsi H, Getachew R, Narasimhan R, Wabl R, Fainberg N, James ML, Wong G, Relton J, Gambhir SS, Pollard JW, Wyss-Coray T. Colony-stimulating factor 1 receptor (CSF1R) signaling in injured neurons facilitates protection and survival. J Exp Med. 2013 Jan 14;210(1):157-72. PubMed.
- Yang X, Ren H, Wood K, Li M, Qiu S, Shi FD, Ma C, Liu Q. Depletion of microglia augments the dopaminergic neurotoxicity of MPTP. FASEB J. 2018 Jun;32(6):3336-3345. Epub 2018 Jan 22 PubMed.
- Srivastava PK, van Eyll J, Godard P, Mazzuferi M, Danis B, Vandenplas C, Foerch P, Leclercq K, Mairet-Coello G, Vanclef F, Shkura K, Laaniste L, Delahaye-Duriez A, Kaminski RM, Petretto E, Johnson MR. A Systems-Level Framework For Drug Discovery Identifies Csf1R As A Novel Anti-Epileptic Drug Target. bioXriv, May 22, 2017
No Available Further Reading
- Sosna J, Philipp S, Albay R 3rd, Reyes-Ruiz JM, Baglietto-Vargas D, LaFerla FM, Glabe CG. Early long-term administration of the CSF1R inhibitor PLX3397 ablates microglia and reduces accumulation of intraneuronal amyloid, neuritic plaque deposition and pre-fibrillar oligomers in 5XFAD mouse model of Alzheimer's disease. Mol Neurodegener. 2018 Mar 1;13(1):11. PubMed.