A Story of FIRE and Mice

Despite decades of research, scientists still grapple with the question of why neuronal health is compromised in neurodegenerative diseases. For a long time, scientists believed that Alzheimer’s disease resulted from toxic build-up of the proteins amyloid and tau, which normally aid neural growth, repair, and stability in the brain. Based on this idea, they developed therapies to break down these proteins, but those treatments did not completely improve patients’ cognitive abilities in clinical trials. This pushed scientists to explore other factors beyond protein toxicity that damage neuronal health.

In 2004, George Bartzokis, a neuroscientist at the University of California, Los Angeles, first hypothesized that myelin, the protective membrane that wraps around axons, might be the key to understanding and treating Alzheimer’s disease.¹ Myelin is essential for neuronal function. It provides axons with both the insulation and the nutrients they need to survive and thrive. Myelin is also central to efficient learning and memory encoding.² 

In the early 2000s, researchers studied the brains of rhesus monkeys that were older than 30 years of age (their average lifespan is ~35 years) to better understand aging. They found that myelin in the brains of aged nonhuman primates showed structural changes and breakdown. Strikingly, these changes correlated with the degree of cognitive decline in the monkeys.^3,4,5 When scientists conducted MRI studies in humans, they found that myelin shows damage up to 20 years prior to Alzheimer’s disease onset.⁶ Together, these studies hinted that myelin damage is intrinsically linked with cognitive decline and is an early event in Alzheimer’s disease progression.

Despite this evidence, there were no follow up studies to validate this hypothesis for almost two decades, partially because researchers assumed that the myelin changes resulted from, rather than caused, neuronal loss. But recently, researchers at the Third Military Medical University and the University of California, San Francisco observed myelin degeneration coupled with a high rate of myelin repair in a mouse model of Alzheimer’s disease. Although the high repair rate could not compensate for the ongoing damage, they found that enhancing myelination improved cognition and neuronal function, irrespective of the amount of amyloid buildup.^6,7 In fact, scientists at the Max Planck Institute for Multidisciplinary Sciences recently discovered that myelin damage itself can drive amyloid accumulation in mouse models of Alzheimer’s disease.⁸ These results finally provided evidence supporting Bartzokis’ hypothesis. ^9,10

The microglia and myelination connection

While these studies answered some questions, they raised several more. For example, if researchers could determine why myelin degenerates, they might be able to prevent dementia onset. The first step towards studying myelin degradation in diseases was to understand how myelin maintains its structure and function in a healthy brain. For this investigation, researchers turned to an internal partner: microglia.  

Microglia are a subset of glial cells that support neurons and keep the microenvironment in the central nervous system healthy and intact. Acting as soldiers on the brain frontlines, microglia function as immune cells, protecting the brain from pathogens, injury, or any harmful triggers. In the last decade, researchers have learned that microglia also contribute to healthy brain function. In fact, microglia consistently contribute to myelin health throughout the lifespan. 

Researchers recently found that young mice lacking microglia had less myelin in their developing brains than mice with intact microglia.^11,12 This suggested that microglia mediated myelination in the developing brain by driving the production of oligodendrocytes, which produce myelin. However, there was one key challenge with these studies. The tools available for eliminating microglia at the time also targeted another small population of central nervous system immune cells known as border-associated macrophages, which are found in the meninges, surrounding blood vessels, and in areas that contact cerebrospinal fluid. Scientists did not know the potential contributions of these cells to myelination.

In 2019, researchers at the University of Edinburgh created a game-changing new mouse model. By using CRISPR-Cas9, they deleted the FIRE sequence, which is a super enhancer in the gene encoding CSF1R, a receptor required for microglia development and survival.¹³ FIRE has redundant function in border-associated macrophages, so FIRE knockout mice (or FIRE mice) lack microglia but retain border-associated macrophages. We used this new model to unpack the specific roles of microglia in myelination for the first time. 

Microglia control myelin growth

In our lab at the University of Edinburgh, we eagerly investigated myelin in these FIRE mice. We hypothesized that the lack of microglia in the FIRE mice would cause deficient myelination during early development, an idea in line with results from previous studies that used global macrophage depletion models. But we were in for a surprise. 

In our analysis, we found no deficit in myelin formation in these mice. Perplexed, we spent months analyzing various myelin-associated proteins and characterizing oligodendrocyte numbers, but they all appeared unaffected. Finally, when we took a closer look at the ultrastructural level using electron microscopy, we had a breakthrough. We observed that myelin was present in FIRE mice, but in unexpected abundance. In the absence of microglia, young FIRE mouse brains formed excess myelin during development that remained in adult FIRE mice. 

These findings suggested that microglia actively engage in regulating myelin growth. To confirm our theory using another approach, we depleted microglia and macrophages in adult mice using the previous gold standard, a pharmacological drug that inhibits CSF1R. We saw the same result: FIRE mouse brains showed excess myelin formation after just one month of treatment. 

Next, we explored whether this phenomenon also occurred in humans. We obtained precious tissue from a rare human neurodegenerative disease known as adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) from Werner Stenzel, a neuropathologist at Charité-Universitätsmedizin Berlin. In patients suffering from ALSP, heterozygous mutations in the CSF1R gene result in approximately half the normal amount of microglia, specifically in myelin-enriched areas in the white matter. Patients with ALSP typically present with early-onset dementia and usually pass away in their 40s or 50s. In brain samples from patients with ALSP, we observed myelin overgrowth similar to what we saw in FIRE mice.

This revelation meant that microglia not only control myelin formation during development, but they also regulate myelin growth into adulthood. Moreover, myelin overgrowth when microglia were absent or reduced in number looked remarkably similar to the disrupted myelin observed in the aged nonhuman primates with cognitive decline. This suggested that in the absence of healthy microglia, myelin may age prematurely and affect cognition. 

We next assessed cognitive function in the FIRE mice by running the Barnes Maze test. In this test, mice learn to locate an escape hole in a maze over several trials. After a few days, we relocate the escape chamber; how the mice adapt to the new situation reflects their cognitive flexibility. The FIRE mice showed deficits in cognitive flexibility, which is one of the first cognitive functions that declines with age.^14,15 Previous studies have also suggested that the extent to which this ability is lost with age could predict development of Alzheimer’s disease.¹⁶

Microglia prevent myelin degeneration

Since aging perturbs myelin structure, and myelin degenerates in Alzheimer’s disease, we next wondered whether the myelin structural changes seen in the absence of microglia rendered myelin vulnerable to degeneration. In samples from patients with ALSP, we noticed some age-related differences in the myelin. A younger patient who died from an unrelated cause had abundant and extremely overgrown myelin. But in an older patient, much of the myelin had degenerated; any remaining myelin was overgrown. 

This led us to investigate the role of microglia in myelin maintenance. By six months of age in FIRE mice, we saw clear evidence of myelin degeneration. In the absence of microglia, myelin quickly went from overgrown to broken down. This was a strange phenomenon, and mechanistically, it didn’t make much sense. 

To find out whether myelin broke down due to the prolonged absence of microglia in FIRE mice, we pharmacologically depleted microglia and macrophages for one month in five-month-old wild type mice. We observed the same degenerated myelin in these mice, indicating that the function of microglia in maintaining myelin health is increasingly important as the brain ages. 

Microglia burnout

Burnout is characterized by utter exhaustion due to excessive workload and stress over a prolonged period of time. Microglia may also suffer from burnout, and understanding how this happens could be central to our fight against dementia. Numerous sophisticated single-cell transcriptomic sequencing studies have documented the appearance of a disease-associated microglia population in aging and in several disease contexts, including Alzheimer’s disease. This population may appear as a response to the increasing demands of the aging brain. With too many tasks to juggle, microglia may lose their ability to maintain myelin as well as they did when the brain was younger.

If we could rejuvenate microglia to their younger selves, perhaps we could prevent the degeneration of myelin and potentially the development of neurodegenerative disease in the aging brain. And if we can thwart this process, we might just be able to help our loved ones preserve their memories until well into their autumn years. 

Conflict of interest statement

Veronique E Miron has received consultancy or research funds from Novartis, Biogen, GSK, Astex Pharmaceuticals, Clene Nanomedicine, ReWind Therapeutics. 

Niamh McNamara recently completed her PhD on microglial regulation of myelin integrity at the University of Edinburgh. She is now pursuing postdoctoral research at the Netherlands Institute for Neuroscience in Amsterdam.

Veronique Miron is a neuroimmunology professor leading research laboratories at the University of Toronto and the University of Edinburgh that investigate what regulates myelin health and pathology across the lifespan. 

References

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