19 Jul 2024

Ever since loss-of-function variants in the gene for PILRA, short for paired immunoglobulin-like type 2 receptor alpha, turned up as protecting against Alzheimer’s disease, scientists have wondered why. Now, a cell biology “omics” study proffers some answers.

In a manuscript uploaded to the Research Square preprint server, researchers led by Kathryn Monroe at Denali Therapeutics, South San Francisco, and Mathew Blurton-Jones at the University of California, Irvine, report that axing the gene in microglia gives these immune cells a boost. Despite oozing with lipid droplets, which have been previously associated with disease, these PILRA-free calls flip fat metabolism in favor of antioxidant lipids, and they refrain from spewing inflammatory cytokines. Their mitochondria churn out ATP, all the while producing few reactive oxygen species, and their lysosomes readily chew up protein. All told, the microglia seem to be fitter, if fatter, without this immunoglobulin receptor. An antibody to PILRA mimics these effects in wild-type microglia, the scientists report.

Others consider the work important. “I think this paper nicely illustrates that proper lipid turnover may have a key role in microglia and in AD,” Tony Wyss-Coray, Stanford University, California, wrote to Alzforum. His lab was the first to recognize lipid-associated microglia as dysfunctional and inflammatory in disease.

“It also illustrates that the field needs to start talking about what is in lipid droplets,” Wyss-Coray added. “Broadly speaking it seems cholesterol is good and neutral lipids, as we described, are bad. But maybe this is too simplistic.”

“This really exciting study is moving the needle in how we understand the impact of AD risk loci on cellular pathways in the brain that contribute to disease,” wrote Celeste Karch, Washington University, St. Louis (comment below).

Cells of the peripheral immune system, including neutrophils and myeloid cells, express PILRA, as do microglia in the CNS. The receptor binds a wide range of sialylated glycoproteins; however, the common G78R PILRA variant does so only weakly. This most likely explains a protective variant at the ZCWPW1/NYAP1 locus that emerged in genome-wide association studies (Bellenguez et al., 2019; Rathore et al., 2018). The two are always co-inherited.

To figure out how this loss-of-function variant might affect microglia, co-first authors Tanya Weerakkody and Hanna Sabelström at Denali knocked out the gene in human iPSC-derived microglia (iMG). Then they characterized changes to the transcriptome, metabolome, and lipidome in iMG cultures and in mice that had their own microglia replaced with the human PILRA knockout microglia. Scientists in Blurton-Jones' lab engineered these chimeric mice (Aug 2019 news).

Unlike wild-type microglia, PILRA-less iMG barely changed gene expression in response to interferon-γ or lipopolysaccharide (LPS), suggesting they tone down their response to inflammatory stimuli. In keeping with this, they released scant TNF-α, IL-6, IL-1β, and IP-10—all proinflammatory cytokines.

Their metabolome told a similar story. They produced less sphingosine, dimethylarginine, and imidazoleacetic acid than did wild-type cells, and LPS barely shifted this pattern. These metabolites modulate immune responses. On the lipidomic front, PILRA-less iMG favored antioxidant glycerophospholipids, specifically ethanolamine plasmalogens, which are reportedly depleted in AD brain, and shunned lysophosphatidylcholines, products of phospholipases activated by inflammation (Ginsberg et al., 1995; Han et al., 2001). All told, omics analyses painted a picture of less reactive microglia.

Knocking out PILRA induced enzymes involved in making cholesterol and its esters, which cells typically make to store this lipid. Looking into this further, the scientists found the microglia accumulated lipid droplets. Because lipid-accumulating microglia (LAMs) emerge in aging brains and around amyloid plaques, the scientists wanted to know more (Aug 2019 news). They found that, sans PILRA, microglia dialed down expression of transporters that shuttle lipids out of the cell, such as ABCA1 and ABCG1, and that they imbibed more ApoE. Increasing this further by dousing the cells with an ApoE4/cholesterol ester/high density lipoprotein mix, these cells held onto their noninflammatory state, however, suggesting that these lipid-loving microglia are better able to cope with an inflammatory pressure than cells expressing PILRA.

The authors think this helps explain why the G78R variant appears to protect against AD in APOE4 carriers only (Lopatko Lindman et al., 2022). They confirmed this by analyzing data from the Religious Orders Study/Memory and Aging Project in Chicago. Among 277 APOE4 carriers, cognition was normal in 13 percent of those with the wild-type PILRA allele. This jumped to 23 percent in people with one G78R allele, and to 37 percent in those with two copies of the loss-of-function variant. “Therefore, in an independent human cohort, we validated that increasing copies of the PILRA G78R allele exerts an AD protective effect selectively in APOE4 carriers,” the authors concluded.

In people of African descent about 10 percent carry this minor allele, while about 38 percent of Europeans do. In East Asia, it is the major allele, carried by 65 percent of that population.

There’s more. Complementing the PILRA-less iMG’s anti-inflammatory profiles, their organelle function also appeared to be reinforced. Mitochondrial respiration improved, as expression of respiratory chain components and mitochondria fusion machinery ticked up—mitochondrial fission typically indicates cell stress. These cellular powerplants made more ATP and fewer reactive oxygen species. Lysosomes more readily digested the fluorescent marker DQ-BSA than did wild-type microglia, even when the cells were treated with the ApoE/cholesterol/HDL concoction.

The cells also migrated faster toward a chemotactic agent, suggesting better motility (image below). This depended on the transcription factor STAT3. Indeed, in an unbiased look at the phosphoproteomes in wild-type and PILRA KOs, Weerakkody and colleagues found that the latter made more STAT3 and STAT1. Both help regulate inflammation and oxidative damage, while STAT3 also regulates cell migration (Debidda et al., 2005). 

Taking their findings in vivo, the authors used 5xFAD amyloidosis mice but replaced its microglia with human wild-type or human PILRA knockout microglia. At 6.5 months, when these chimeric mice have widespread amyloid pathology, transcriptomics analyses suggested no major difference in cell state between the two types of microglia. That said, the PILRA KOs had begun to ramp up proteins involved in mitochondrial respiration, ROS metabolism, and cell migration. The authors reported no effect on plaque burden. The data hint that a similar response occurs in vivo as in iMG in culture, and indeed in human microglia. They did not examine what happens as the mice age.

To learn if these findings support a therapeutic approach, the Denali scientists have developed an anti-PILRA antibody that binds both the G78 and R78 variants, but not the closely related PILRB receptor. This anti-PILRA antibody blocked a range of protein ligands from binding the PILRA immunoglobulin receptor. Treating iMG cultures with it boosted their mitochondrial respiration, tempered cytokine production, and sped migration.

The scientists plan to couple the antibody to the company’s transferrin receptor-targeting “antibody transport vehicle,” to ferry it across the blood-brain barrier. They will then test it alone, or in combination with anti-amyloid therapies for AD.—Tom Fagan

Comments

1. • Celeste Karch
     Washington University in St Louis
   • Posted: 19 Jul 2024

   This really exciting study is moving the needle in how we understand the impact of AD risk loci on cellular pathways in the brain that contribute to disease.

   An interesting finding from this work is the way in which PILRA may interact with APOE to impact microglia function. That PILRA impacts lipid droplets, mitochondrial function, lysosomal, and microglia function places it among an increasingly long list of AD risk genes that regulate these critical and inter-related pathways.

   View all comments by Celeste Karch

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References

News Citations

1. Human Microglia Make Themselves at Home in Mouse Brain 1 Aug 2019
2. Newly Identified Microglia Contain Lipid Droplets, Harm Brain 23 Aug 2019

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Further Reading

News

1. Microglial Master Regulator Tunes AD Risk Gene Expression, Age of Onset 18 Jun 2017
2. Largest AD Whole-Exome Study to Date Finds Two New Risk Genes 17 Aug 2018
3. AD Genetic Risk Tied to Changes in Microglial Gene Expression 9 Aug 2019