Evidence suggests that microglia compact Aβ in brain parenchyma into dense-core plaques, but their effect on vascular deposits of Aβ, namely cerebral amyloid angiopathy (CAA), has been unclear. Now, researchers led by Mathew Blurton-Jones, University of California, Irvine, report that CAA runs amok in a mouse model of amyloidosis that lacks microglia from birth. In the June 14 Cell Reports, they reported that these animals had mostly diffuse, not dense-core, plaques and that these accumulated in and around the tiny blood vessels of the brain.
Without microglia, endothelial integrity faltered, calcium crystals accumulated in the thalamus, and the blood vessels burst. Most animals died by 6 months old. Injecting microglia into the brains of 2-month-old mice repopulated the whole brain within a few months, and this attenuated the pathology. “This fascinating study advances our understanding of the role of microglia in cerebral amyloidosis,” Mathias Jucker, University of Tübingen, Germany, wrote to Alzforum.
- Mice without microglia have diffuse plaques surrounding blood vessels.
- In the brain, endothelial genes are downregulated, and calcium forms deposits.
- Brain tissue from people with CAA also contained calcium crystals.
Researchers have studied microglial effects on amyloid pathology by asking what happens when the cells are ablated. They wiped out microglia by feeding adult mice inhibitors of colony-stimulating factor 1 receptor, which these cells need to survive and proliferate. In some cases this reduced plaques, slowed neurodegeneration, and improved cognition (Mar 2018 news; Apr 2014 news). Others saw plaques mass at blood vessels and neurite dystrophy worsen (Sept 2019 news). Oddly enough, blocking CSF1R had no effect on lifespan, perhaps because the inhibitors did not completely suppress microglia or because they were given once the mice were adults. CSF1R inhibitors may also deplete macrophages, making the findings tricky to interpret.
With or Without. In the brains of 5xFAD mice (left), microglia (green) surround blood vessels (red lines) and diffuse amyloid deposits to mop up Aβ and package it into dense-core plaques (gray). When the brain has no microglia (right), it accumulates diffuse amyloid plaques (gray squiggles), cerebral amyloid angiopathy (gray branches), cerebral bleeds (red blotches), and calcium deposits (white). [Courtesy of Kiani Shabestari et al., Cell Reports, 2022.]
To specifically and completely eliminate microglia, first author Sepideh Kiani Shabestari and colleagues turned to mice that lack the fms intronic regulatory element, a major driver of CSF1R expression. Without FIRE, mice make no microglia, though they still have meningeal and perivascular macrophages. The animals seem to develop normally, and live as long as do wild-type mice (Rojo et al., 2019). Kiani Shabestari found they carry a normal complement of peripheral immune cells in their cervical lymph nodes, spleen, and bone marrow. “This mouse model allows researchers to study microglial function more cleanly and in a much more sophisticated way than before,” Oleg Butovsky, Brigham and Women's Hospital, Boston, told Alzforum.
How did lack of FIRE affect plaque pathology? The scientists crossed FIRE mice with 5xFAD transgenic animals, then analyzed cortical, hippocampal, and thalamic tissue from 5-month-old offspring. They measured plaque load using confocal microscopy and insoluble Aβ levels with by ELISA after extraction.
The 5xFAD-FIRE mice had as many plaques in the parenchyma as did their 5xFAD counterparts, but the former were diffuse while the latter had dense cores (see image below). The crosses had one-quarter of the insoluble Aβ40 and Aβ42 and five times as many plaques surrounding endothelial cells as did 5xFAD mice. The authors interpreted this as amyloid accumulating around the vasculature rather than being packed into plaques by microglia.
FIRE and Mice. Microglia (green) tile hippocampal tissue in 5xFAD mice (left) but are absent from 5xFADs lacking FIRE (middle). While both had a similar number of amyloid deposits (white), 5xFAD mice accumulated dense-core plaques (top right), while in 5xFAD-FIRE mice they were diffuse (bottom right). [Courtesy of Kiani Shabestari et al., Cell Reports, 2022.]
Unlike 5xFAD mice, 5xFAD-FIRE animals and FIRE mice with no amyloid pathology had extensive blood vessel damage, most notably in the thalamus where small and large hemorrhages erupted. Single-nucleus RNA sequencing of vascular cells, glia, and neurons from 5xFAD-FIRE mice identified many transcriptional changes in genes associated with endothelial cells and blood-brain barrier integrity. The expression of two key growth factors, PDGF-β and TGF-β, dropped. Produced by microglia, these proteins support pericyte and endothelial cell growth and maintenance. The authors believe that, without microglia, deficiency of these growth factors leads to breakdown of the BBB.
Interestingly, mutations in PDGF-β cause primary familial brain calcification, a parkinsonian-like neurodegenerative disorder where calcium crystals accumulate in none other than the thalamus (Tadic et al., 2015). In a mouse model of this disease, CSF1R inhibitors exacerbated the calcification of blood vessels in the brain (Zarb et al., 2021). To see if something similar might be going on in 5xFAD-FIRE mice, Kiani Shabestari labelled brain slices with alizarin red, a dye that precipitates free calcium, and fluorescent risedronate, an osteoporosis drug that binds calcium crystals. Both molecules revealed calcium deposits mostly in the thalamus of 5xFAD-FIRE but not 5xFAD mice.
The most striking phenotype was early death. More than 70 percent of the 5xFAD-FIRE mice died by 6 months, while 5xFAD mice usually lived until they were at least 15 months old. “The combination of microbleed tendency of the FIRE mice along with CAA seems to be killing the crossed animals,” Charles Glabe of UCI told Alzforum. Kim Green, also at UCI, told Alzforum that he sees similar premature death after pharmacologically knocking down microglia in 5xFAD mice that had been crossed with animals expressing mutant human tau. This emphasizes the importance of having functional microglia when both Aβ and tau pathologies are in the picture.
Why wasn’t this early demise previously seen in mice given CSF1R inhibitors? Some think it is because this treatment does not deplete all the microglia. Blurton-Jones agreed. “The difference could be that FIRE mice never had microglia, whereas CSF1R-treated mice had them until early adulthood,” he said. Butovsky thought that, without any microglia to support other brain cells, their development and function may suffer in FIRE mice.
Others thought the mice’s young death may came down to the lack of CSF1R, which is also expressed by neurons and supports their survival (Nandi et al., 2012). “Genetic CSF1R and microglial ablation in the FIRE mice may ... predispose them to malfunctions … upon a pathological challenge, such as amyloid plaques, that cannot be properly resolved in the absence of microglia,” wrote Dave Holtzman and Yang Shi, Washington University, St. Louis (full comment below). However, Kiani Shabestari found no CSF1R mRNA in neurons from FIRE mice. Blurton-Jones added that FIRE is only expressed in microglia, hence the specific knockout of just those cells.
Could restoring microglia in 5xFAD-FIRE mice prevent pathology and increase survival? Kiani Shabestari injected the hippocampi and cortices of 2-month-old mice with microglia from wild-type animals. At 2 months, 5xFAD-FIRE mice show no signs of brain hemorrhaging or calcification. After 3 months, microglia had repopulated the brain to levels seen in 5xFAD animals, though the transplanted cells had fewer, stubbier branches than their 5xFAD counterparts.
At 5 months old, animals that received the transplants had less CAA, more dense-core plaques, and no cerebral bleeds or brain calcification. The repopulated microglia expressed TGF-β and PDGF-β and gathered near the vasculature, likely flocking to the CAA. “It is quite remarkable that CAA, brain calcification, and intracerebral hemorrhage were all resolved by microglia transplantation,” wrote Marco Colonna and Zhangying Cai, also at WashU (comment below). Glabe wondered about the therapeutic potential of infusing people with microglial precursors derived from their own stem cells in order to prevent downstream calcification and BBB instability.
Indeed, Kiani Shabestari found calcification in prefrontal cortex tissue from five AD cases with CAA, but none in five CAA-negative cases, suggesting that this phenomenon might occur in the AD brain also. Cortical tissue was more readily available than thalamic tissue from the UCI AD Research Center. People with CAA have more microhemorrhages, worse memory, and die sooner than people who have AD (Bell and Zlokovic, 2009; Arvanitakis et al., 2011).—Chelsea Weidman Burke
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