24 Apr 2019

ApoE4 impairs how the brain handles lipids. At the 14th International Conference on Alzheimer’s and Parkinson’s Diseases, held March 27–31 in Lisbon, Portugal, researchers argued that this effect of AD’s biggest risk gene is important in the pathogenesis of late-onset Alzheimer’s. Speakers linked harmful variants of both ApoE and TREM2 to impaired lipid processing, particularly in glia. Conversely, making ApoE “fattier” lowered amyloidosis in disease models, hinting that targeting lipids could have therapeutic potential.

The ApoE4 allele causes lipid buildup in microglia and astrocytes.

Lack of TREM2 does the same in microglia during a chronic phagocytic challenge.

A peptide mimetic of HDL restores ApoE4 function and Aβ42 clearance.

Scientists have long known that ApoE binds lipids and that, in the healthy brain, it is produced primarily by astrocytes. During neurodegeneration, however, microglia pump out ApoE too, Julia TCW of Icahn School of Medicine at Mount Sinai, New York, told the audience. Previously, TCW had reported that lipid processing goes haywire in glial cells carrying the E4 allele. ApoE4/4 microglia and astrocytes generated from human iPS cells dialed up genes for lipid synthesis, while suppressing genes for lipid transport and degradation. This combination caused cholesterol to accumulate in glial cells (Jul 2018 conference news).

In Lisbon, TCW fleshed out her finding with new in vitro data. Comparing E4/4 and E3/3 isogenic astrocyte lines, she found that cholesterol built up only in the former. Much of the astrocytes’ excess cholesterol got stored as cholesterol esters in lipid droplets, though total cholesterol and free cholesterol were also up in E4/4 cells. In healthy glia, ApoE ferried cholesterol from the cell, but the E4/4s made less ApoE protein and allowed less of it out than did E3/3s.

Fat Buildup. Homozygous human ApoE4 astrocytes (right) accumulate more free cholesterol (white) than do homozygous E3s (left). [Courtesy of Julia TCW, Nina Pipalia, and Frederick Maxfield.]

When TCW performed a pulse-chase experiment with low-density lipoproteins, she found that E4/4 astrocytes also took up less lipid from outside than did E3/3s. She traced this to a lack of LDL receptors on the E4/4 glia. “ApoE4 decouples lipid metabolism in microglia and astrocytes,” TCW concluded. In other words, production and clearance become unbalanced, resulting in accumulation of intracellular fats.

ApoE is far from the only gene required for correct lipid processing. In Lisbon, Alicia Nugent of Denali Therapeutics in South San Francisco made a case that the microglial receptor TREM2 plays a similar role. Extracellular lipids are known to bind TREM2 and activate signaling that helps these cells survive, respond to injury, and mop up debris. Notably, this function is disrupted by pathogenic mutations in TREM2 that raise the risk for late-onset AD. R47H and R62H weaken lipid binding, while H157Y speeds shedding of the extracellular portion of TREM2, abolishing intracellular signaling (Feb 2015 news; Kober et al, 2016; Aug 2017 news).

Nugent said that complete absence of TREM2 is even worse, preventing microglia from responding to lipid damage. In wild-type mice poisoned with cuprizone, an agent scientists use to model aspects of multiple sclerosis, oligodendrocytes die and axon tracts in the brain become demyelinated. Microglia clean up myelin debris and, once the toxin is gone, new myelin sheaths form. In TREM2 knockouts, however, remyelination is perturbed and axons degenerate (Poliani et al., 2015).

Why might this be? Nugent and colleagues fed cuprizone to wild-type or TREM2 knockout mice for 12 weeks, then isolated microglia from their brains by fluorescence-activated cell sorting (FACS), and analyzed gene expression. In wild-type microglia, genes for lipid metabolism and lysosomal degradation revved up. This included ApoE, which regulates cholesterol intake and efflux.

The analysis suggested that wild-type microglia challenged by cuprizone ramp up cholesterol metabolism pathways. To do that, the cells enter an activation state reminiscent of disease-associated microglia (DAM) in AD mice, Nugent said (Jun 2017 news). In TREM2 knockouts, this did not happen, indicating these cells cannot initiate the transcriptional programs that are needed for a healthy microglial response to injury.

The researchers confirmed these findings by analyzing the lipid profiles of brain slices from wild-type and TREM2 knockout mice. In the knockouts, cholesteryl esters, the storage form of cholesterol, rose during cuprizone treatment. Other tissue lipids were up as well, including bis(monoacylglycero)phosphate and gangliosides, suggesting a defect in lysosomal degradation of lipids. Lipids accumulated specifically in microglia, not in astrocytes. Nor did the researchers find accumulation in cerebrospinal fluid, which serves as a proxy for the brain’s extracellular space. Intriguingly, TREM2 knockout microglia still took up myelin debris, but they failed to clear it from their cell bodies, resulting in the buildup of cholesteryl esters stored in lipid droplets.

Why did the Denali researchers find lipid accumulation only in microglia, while TCW saw it in both microglia and astrocytes? TCW noted that TREM2 is expressed only in microglia, and thus manipulating it should change only these cells. ApoE, on the other hand, is highly expressed in astrocytes.

Nugent noted that Alois Alzheimer first reported seeing lipid inclusions in glial cells, and others since then have found high levels of cholesteryl esters in AD brain and mouse models (Chan et al., 2012; Morel et al., 2013). TREM2 regulates microglial cholesterol metabolism, and without it, microglia cannot process lipids properly, Nugent concluded. In answer to an audience question, she said it’s unclear if disrupted lipid metabolism is a cause of AD or a consequence.

Could improving lipid metabolism alleviate pathology? The enzyme acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) converts free cholesterol into cholesteryl esters. When the researchers inhibited ACAT1 in macrophages from TREM2 knockouts, cholesteryl esters did not accumulate. Other studies have found that ACAT1 inhibition lowered both amyloid and tau pathology in AD mice (Puglielli et al., 2001; Hutter-Paier et al., 2004; Bryleva et al., 2010; Shibuya et al., 2015). This line of work did not advance to human trials.

Lipidation, Please. ApoE needs to be properly lipidated to clear Aβ. The E4 allele and aggregated Aβ interfere with this, while “good” cholesterol facilitates it. [Courtesy of Ling Li.]

Some researchers are tackling the problem of blocked lipid metabolism from the other end, looking to speed clearance in the periphery rather than the central nervous system. Peripheral apolipoprotein A-1 (ApoA1) is the main protein component of high-density lipoprotein, the so-called “good” cholesterol. HDL pulls cholesterol from tissues and ferries it to the liver for disposal. Cheryl Wellington of the University of British Columbia in Vancouver, Canada, is using tissue engineering and a reactor system with circulating fluid to build a model of cerebral blood vessels. This allows her to measure trafficking of molecules across the blood-brain barrier. With this system, she previously showed that HDL speeds the flushing of Aβ42 from brain, and can even compensate for ApoE4’s harmful effects on clearance (Oct 2017 news).

In Lisbon, Wellington showed that HDL helps prevent Aβ42 from sticking to collagen and becoming trapped in vessel walls as cerebral amyloid angiopathy (CAA). HDL also suppresses the inflammation of vascular endothelial cells caused by Aβ. HDL levels fall during aging, perhaps contributing to the development of AD, Wellington said.

Peptide Mimetic. 4F mimics the structure of HDL cholesterol, but enters the brain more readily. [Courtesy of Ling Li.]

Could increasing HDL help prevent AD? Ling Li of the University of Minnesota, Minneapolis, studies this question. Li previously found that overexpressing ApoA1 in APP/PS1 mice halved CAA and astrogliosis and restored learning and memory (Oct 2010 news). However, ApoA1 is impractical as a therapeutic, in part because it is large and expensive to make, Li noted.

As an alternative, cardiovascular researchers synthesized an 18-amino acid peptide that mimics HDL. Nicknamed 4F after its four phenylalanines, this mimetic forms amphipathic helices similar to those of ApoA-1. Made from D-amino acids to resist digestion, 4F has undergone a small amount of testing for cardiovascular disease (Navab et al., 2006; Van Lenten et al., 2009; Sherman et al., 2010; Dunbar et al., 2017).

Could 4F be a preventive treatment for AD? Li found that 4F passes through the blood-brain barrier more easily than ApoA1 does, achieving 500 times the concentration of ApoA1 in several mouse-brain regions. In primary astrocyte cultures from mice and human iPSCs, 4F enhanced ApoE secretion and lipidation. The molecule also countered the inhibitory effect of aggregated Aβ42 on ApoE secretion (Chernick et al., 2018; Wang and Zhu, 2018).

In more recent work, Li and colleagues found that 4F treatment changed the lipidation of ApoE4 to that of ApoE2 in primary astrocytes from transgenic mice. To explore 4F in a more complex system, the researchers made brain organoids from human iPSCs (Aug 2013 news; Lindborg et al., 2016). As it did in cell culture, 4F treatment promoted ApoE secretion. In addition, it dose-dependently inhibited Aβ42 aggregation. In a cellular model of the blood-brain barrier, 4F doubled Aβ42 efflux, Li reported in Lisbon.

These data led Li to conclude that HDL-based therapeutic strategies might protect against AD. She noted that the findings once again support the maxim, “What’s good for the heart is good for the brain.”—Madolyn Bowman Rogers