Lipid-Laden, Sluggish Microglia? Blame Aβ.

06 Sep 2023

Alois Alzheimer reported lipid-filled glia surrounding amyloid plaques in the AD brain. But how and why they appear remains elusive. Now, two bioRxiv preprints blame Aβ. In one, posted June 6, scientists led by Gaurav Chopra of Purdue University, West Lafayette, Indiana, and Dimitrios Davalos at Ohio’s Cleveland Clinic reported that mouse microglia shift their lipid metabolism when exposed to Aβ fibrils in vitro. The cells form lipid droplets, courtesy of overzealous diglycerol acetyltransferase, aka DGAT2, an enzyme that creates triglycerides from free fatty acids. These droplet-filled microglia poorly phagocytosed Aβ. In the other manuscript, uploaded July 25, scientists led by Tony Wyss-Coray, Stanford University, described Aβ-induced triglyceride droplets in human microglia and attributed them to an uptick in ACSL1, an enzyme upstream of DGAT2.

APOE4 exacerbated lipid droplet formation in these cells, and lipid-filled microglia dotted hippocampal tissue from APOE4/4 carriers. The glia also exuded something neurotoxic, perhaps the lipids themselves, hiking up phospho-tau and programmed cell-death proteins produced by nearby neurons.

“This work brings into sharp focus the mechanisms underlying one of Alzheimer’s original observations, that lipids accumulate in microglia, which has received relatively scant attention to date,” wrote Anne Poljak, University of New South Wales in Sydney. Priyanka Narayan at the National Institute of Diabetes and Digestive and Kidney Diseases in Bethesda, Maryland, noted how far this research area has come over the past decade. “It is clear that lipids in neurodegenerative disease have entered a new era of importance; however, there is still a long way to go to understand the mechanisms that govern their contribution to different cell types in initiation and progression of the disease,” she wrote (comments below).

Lipids and Droplets. ACSL1 adds acetyl-CoA onto free fatty acids (FFA) to form Acyl-CoAs. Thioesterases (Them1 and Them2) can reverse the process, but Glycerol-3-phosphate acyl transferase (GPAT) and DGAT2 (not shown) can combine these acyl-CoAs to form triacylglycerols, aka triglycerides. TGs typically bind very low-density lipoproteins (VLDL), but they can also form lipid droplets (yellow circle). (Courtesy of Desai et al., 2018).

Microglia and macrophages make lipid droplets when stressed by inflammation. Scientists believe the lipids sustain the cells’ metabolic need, enabling them to respond to threats (van Dierendonck et al., 2022; reviewed by Olzmann and Carvalho, 2019). Wyss-Coray previously described lipid-droplet accumulating microglia (LDAMs) that spewed pro-inflammatory cytokines in the hippocampi of old wild-type mice (Aug 2019 news). Matthew Blurton-Jones, University of California, Irvine, found that human microglia placed into the brain of an amyloidosis mouse filled up with lipid droplets (Claes et al., 2021).

Could Aβ spur LDAM formation? Both research groups suggest as much. In vitro, an hour after adding synthetic Aβ42 fibrils to cultured wild-type mouse microglia, co-first authors Priya Prakash and Palak Manchanda in Chopra’s lab saw the lipid droplet content swell 4.5-fold. Co-first authors Michael Haney and Róbert Pálovics in Wyss-Coray’s lab saw a similar uptick in human induced pluripotent stem cell-derived microglia after 24 hours of Aβ42 fibril exposure.

In vivo, the teams spotted LDAMs near amyloid plaques in 6-month-old 5xFAD mice, and saw similar cells in hippocampal tissue from people who had had AD (see image below). In each case, the cells were filled with lipid droplets. Notably, Aβ fibrils provoked no lipid droplet formation in cultured 5xFAD microglia, perhaps, Davalos suggested, because they were already stuffed with lipids.

White Fat. High-resolution confocal microscopy of AD brain shows microglia (green) near amyloid plaques (blue) brimming with lipid droplets (red) that appear white in overlay images. Microglia far away were lipid-free (top). [Courtesy of Prakash et al., bioRxiv, 2023.]

Does Aβ change the microglial lipidome? Prakash reported that, after 24 hours with the fibrils, wild-type microglia contained few fatty acids but an abundance of triglycerides, mirroring the lipidome of 5xFAD LDAMs. Haney saw the same rise in triglycerides. These fats, along with cholesterol esters, constitute the core of lipid droplets, connecting the lipidomic and phenotypic changes (Listenberger et al., 2003; Fujimoto and Parton, 2011). Chopra thinks this ramped-up triglyceride synthesis could be the microglia’s way of controlling a flood of free fatty acids, which can easily be oxidized to become toxic.

What caused this shift? Both groups suspected enzymes in the triglyceride synthesis pathway. Wyss-Coray pegged changes to long-chain-fatty-acid—CoA ligase 1 (ACSL1), which tacks on CoA to free fatty acids. Chopra pegged diacylglycerol O-acyltransferase 2 (DGAT2), which turns the fatty-acyl-CoAs into triglycerides. ACSL1 overexpression induces lipid droplet formation in multiple cell types; DGAT2 flocks to lipid droplets, where it makes triglycerides (Zhao et al., 2020; Kuerschner et al., 2008). 

ACSL1 is implicated in ferroptosis. Instigated by oxidized lipids, this cell death pathway is linked to Alzheimer’s, Parkinson’s, and Huntington’s diseases (Bai et al., 2019; reviewed by Reichert et al., 2020).

In single-nucleus RNA-Sequencing analysis of AD cortical tissue, Haney et al. saw a subset of ASCL1-expressing LDAMs surrounding amyloid plaques. ASCL1 expression rose hand-in-hand with lipid droplet formation, indeed it was the most upregulated gene in microglia from AD cases compared to cells from controls. Likewise, Prakash et al. spotted DGAT2-positive LDAMs surrounding plaques in immunostained mouse and human brain tissue (image below). Both groups concluded that those enzymes likely drove lipid droplet formation.

Droplets and DGAT2. In AD hippocampal tissue (bottom), microglia (green) near amyloid plaques (blue) contained lipid droplets (pink) and DGAT2 (yellow). [Courtesy of Prakash et al., bioRxiv, 2023.]

The scientists next asked if APOE4 greases this process, since this lipoprotein strongly binds triglyceride-rich lipoprotein particles (Mahley, 2016). Indeed, they found that APOE4 homozygotes who had AD had more LDAMs in their brains than did APOE3 homozygotes with AD, and they expressed more ASCL1 in their microglia. APOE4 fit this picture in vitro, too. Human iPSC-derived APOE3/3 microglia had few lipid droplets with or without exposure to Aβ fibrils, whereas E4/4 cells had some at baseline, generated many more in the presence of Aβ, and ramped up ACSL1 expression.

Does the Fat Matter?
Lipid-laden APOE4/4 microglia seemed to poison neurons. Human iPSC-derived neurons bathed in media from E4/4 LDAMs expressed the apoptosis indicator caspase, made AT8-positive phospho-tau, and contained lipid droplets made of the same triglyceride species found in droplets from LDAMs. To Haney, this suggests that the LDAMs secrete these triglycerides, which nearby neurons take up.

Death by Fat? When microglia sense Aβ fibrils, among other inflammatory triggers, they upregulate ACSL1, creating an abundance of triglycerides that form lipid droplets with the help of ApoE. These LDAMs (red) secrete molecules, perhaps the lipids themselves, that are toxic to neurons. [Courtesy of Haney et al., bioRxiv, 2023.]

On the flip side, consider APOE knockout microglia. Aβ fibrils did not provoke lipid droplets or ACSL1 production in them, and the conditioned medium was harmless to neurons. To Julia TCW of Boston University, these results add weight to the notion that the APOE4 allele has a toxic gain of function (comment below).

Sarah Cohen at the University of North Carolina, Chapel Hill, agrees. In her lab’s preprint, uploaded in April 2023, Cohen saw ApoE4 astrocytes bloat with bubbles of triglycerides and found ApoE4 interacting with lipid droplets, attempting to control their size. The protein fumbled this lipid turnover, allowing large droplets to form (Windham et al., 2023). Cohen suspects the same happens in microglia exposed to Aβ. “Our data indicate that APOE plays a previously unrecognized role as a [lipid droplet] surface protein that regulates size and composition,” wrote Cohen and colleagues in their manuscript.

Is There a Drug Target in There?
Knocking down ACSL1 or DGAT2 might dissolve lipid droplets, though perhaps with unintended consequences. Previously, Li-Huei Tsai and Matheus Victor at MIT reported that blocking ACSL1 for two days killed microglia. “Lipid accumulation influences many processes and must be fine-tuned rather than abrogated,” wrote Tsai and Victor (comment below).

For their part, Prakash and colleagues saw, after applying a DGAT2 inhibitor for two hours to cultured 5xFAD microglia, a halving of their lipid droplet number and more Aβ phagocytosis. In 2-year-old 5xFAD mice, a week-long intraventricular infusion of a molecule that degrades DGAT2 cut LDAMs by one-third and amyloid plaques by 60 percent. Chopra was tight-lipped about this molecule, only saying that it is a bifunctional, small-molecule conjugate. He plans to upload a preprint on it to bioRxiv soon, he told Alzforum.

DGAT2 inhibitors are already being tested in people. Pfizer’s small-molecule drug ervogastat and Ionis’s ASO ION224 are both in Phase 2 trials for non-alcoholic steatohepatitis, a form of fatty liver disease that can cause the organ to fail (Amin et al., 2022; clinical trials.gov).

Others shared Tsai and Victor’s concern. Jörg Hanrieder, University of Gothenburg, said these lipid processing enzymes are crucial throughout the body. Ole Isacson of McLean Hospital, Belmont, Massachusetts, stressed that microglia need lipid droplets to initiate an immune response (Bosch et al., 2020). Edoardo Marcora, Icahn School of Medicine, Mount Sinai, New York, thinks lipid droplets may not be the “bad guy.” “Lipid droplets might just be a beneficial adaptive response to something that causes dyshomeostasis, and this must be understood before targeting them with drugs,” he said.—Chelsea Weidman Burke