30 Oct 2024

Scientists broadly agree that both apolipoprotein E and microglia are necessary ingredients for amyloidosis. In mice devoid of ApoE, or of their microglia, scant plaques form. Now, a sweeping study published October 9 in Immunity unveils a mechanistic link between the two. Scientists led by Mikael Simons of the German Center for Neurodegenerative Diseases in Munich report that, within the bowels of microglial lysosomes, ApoE forms fibrillar aggregates alongside Aβ. There, the two festering fibrils team up, forming seeds with the potential to grow into Aβ plaques once microglia either spew them out, or die.

Gobs of these intra-lysosomal deposits formed when lysosomes were hobbled with inhibitors, or when interferon signaling was dialed up. The findings implicate a nexus of ApoE, microglia, and endolysosomal dysfunction as the driving force behind amyloidosis in AD.

“This is one of the better papers I’ve read in the past few years,” said David Holtzman of Washington University in St. Louis. He thinks it offers a convincing explanation for why both microglia and ApoE seem to be needed to seed plaques.

The study also underscores the primary role of lysosomal dysfunction in the etiology of AD, commented Ralph Nixon of New York University. Nixon’s research has primarily focused on how this pathology plays out in neurons, and this new work demonstrates the importance of parallel lysosomal crises unfolding in microglia. “This paper pulls together a lot of seemingly disparate observations in the field,” he said.

Jessica Young of the University of Washington Medical School, Seattle, shares this sentiment. “The endo-lysosomal, immune, and lipid trafficking pathways are highly interrelated as lipids and immune mediators utilize vesicular trafficking,” she wrote. “This study highlights how events that converge at the lysosome can initiate some of the earliest events in AD pathogenesis.” 

More papers than a writer could list here have shown that ApoE—the strongest genetic risk factor for AD—plays some part in amyloid plaque formation, and is itself integrated into Aβ plaques (e.g., Kim et al., 2012; Dec 2017 news). While microglia have long been credited with clean-up and containment of plaques, recent studies have pointed to a potential role as plaque builders, as well (Sep 2019 news; Apr 2021 news; Apr 2024 conference news).

Endolysosomal problems have also taken some blame as fueling AD pathogenesis. Some scientists favor the idea that endosomal traffic jams spark amyloidosis inside of neurons (Cataldo et al., 2000; Jul 2015 news; Jan 2010 news). Many AD risk genes are implicated in immune, lipid, and endolysosomal pathways.  

How do all these different puzzle pieces fit together? Co-first authors Seiji Kaji, Stefan Berghoff, and Lena Spieth and colleagues initially had their sights set on humbler questions relating to ApoE’s role of in remyelination. To answer them, they generated ApoE-Halo mice, which express a tagged version of ApoE that can be visualized with high resolution within cells.

Alas, when they crossed ApoE-Halo to 5xFAD amyloidosis mice, they were in for a surprise. As expected, they saw both Aβ and Halo-tagged ApoE co-mingling in most plaques in 3.5-month-old mice, as well as some plaques that contained mostly Aβ and little ApoE. However, the scientists also spotted an unexpected breed of plaque: one that contained mostly ApoE and hardly any Aβ. These “ApoE-intense” aggregates comprised 14 percent of all plaques. Like their more evenly mixed counterparts, they stained positive with methoxy-04, thiazine red, and thioflavin, suggesting their contents were fibrillar. In human AD brain samples, the scientists saw a similar repertoire of plaques, with varying compositions of Aβ and ApoE.

Intense Surprise. This confocal image of a 5xFAD/Halo-ApoE mouse brain shows two of three plaque types. An “Aβ intense” plaque in the top left corner contains mostly Aβ (turquoise) and no ApoE (yellow). The “ApoE-intense” plaque in the middle contains mostly ApoE and scant Aβ (MX04, white; Iba1, magenta). [Courtesy of Kaji et al., Immunity, 2024.]

Deploying biochemical and microscopic experiments to analyze these ApoE aggregates, the scientists found that, in sarkosyl-insoluble fractions isolated from ApoE-Halo/5xFAD mice, purified ApoE-Halo existed as multimers devoid of Aβ, suggesting they were able to form independently. Under the electron microscope, these ApoE multimers were spied twisting into wavy, snake-like fibrils. Similar structures appeared when the researchers isolated ApoE aggregates from postmortem brain samples of ApoE3/E3 and ApoE4/E4 carriers with AD. 

In seeding experiments, ApoE aggregates revved up amyloidosis in the mouse brain. For example, 2-month-young 5xFAD mice injected with ApoE aggregates extracted from other 5xFAD mice formed twice as many plaques over the following two months as controls. The ApoE aggregates even managed to seed plaques in ApoE knockout 5xFAD mice, which typically have few plaques. Human ApoE had the same plaque-seeding effect in human ApoE knock-in 5xFAD mice. When injected with ApoE aggregates extracted from human AD brain samples, plaque numbers doubled in ApoE3-KI and tripled in ApoE4-KI mice. This suggested that both isoforms of aggregated ApoE stoke amyloidosis, with ApoE4 doing so with more gusto.

How and where does ApoE aggregate in the first place? To investigate, Kaji and colleagues hunted them down in the ApoE-Halo/5xFAD mouse brain with confocal microscopy and three-dimensional imaging. In addition to the usual large extracellular plaques, ApoE aggregates also turned up in tiny punctate structures within Iba1+ microglia, where they overlapped with MX04+ aggregates (image below).

This microglial hide-out for ApoE aggregates appeared crucial for the formation of extracellular Aβ plaques. Consider what happened when the mice had no microglia thanks to the CSF-1R inhibitor, PLX5622. Without microglia around, injected ApoE aggregates had no plaque-seeding power in 5xFAD mice. Notably, when the researchers injected purified ApoE-Halo into ApoE knockout mice, and 5xFAD mice with or without microglia, they found that plaques only formed when both ApoE and microglia were present.

The scientists next explored how lysosomal function influenced the development of ApoE aggregates within microglia. Both in a cultured microglial cell line and in human iPSC-derived microglia, the scientists sparked intracellular aggregates by treating the cells with recombinant ApoE and fluorescently labeled Aβ42 peptides. Hobbling lysosomal function with a suite of inhibitors ramped up this aggregation. So did knockdown of the microglial receptor TREM2, which promotes lysosomal function. These same results were borne out in 5xFAD mice that received daily injections of the lysosomal acidification blocker chloroquine.

In contrast, shutting down the interferon response with the JAK-STAT inhibitor baricitinib had the opposite effect. It quelled production of ApoE aggregates within microglia. So far, the findings suggested that subpar lysosomal function, or overactive interferon signaling, could accelerate the process of ApoE/Aβ aggregation within microglia.

“The findings of this paper add to the accumulating evidence that dense-core, highly-aggregated Aβ plaques do not form spontaneously within the extracellular spaces of the AD brain, but rather are assembled intracellularly—within the acidic environment of microglial lysosomes,” wrote Greg Lemke of the Salk Institute in La Jolla, California.

As an apolipoprotein, ApoE’s main job is to hook up with lipids and deliver them to cells. To investigate how this process might relate to its aggregation within microglial lysosomes, the researchers added Aβ42 along with lipid-free or lipidated ApoE to microglial cultures. They found that microglia more readily internalized lipidated ApoE than its nonfat version, and therefore led to more aggregates within the lysosome. Further studies revealed that once inside the cells, lipidated ApoE proteins were rapidly stripped of their lipids.

Notably, in experiments with human ApoE, ApoE4 was more readily taken up by microglia, and it more efficiently lost its lipids, relative to ApoE3. Simons thinks that while associated lipids promote receptor-mediated uptake of ApoE, subsequent delipidation—which occurs within the acidic environment of the lysosome—renders ApoE more prone to aggregate. Aβ has also been shown to aggregate more readily in acidic compartments.

Holtzman told Alzforum that this idea is consistent with findings from his lab using an antibody specific for delipidated ApoE. It not only revealed that ApoE is largely delipidated within plaques, but that targeting this lipid-free ApoE with antibodies removed plaques (Apr 2018 news). Perhaps the acidic environment of the microglial lysosome is where this critical lipid stripping step occurs, Holtzman suggested. It also happens to be where the newly disrobed ApoE rendezvous with Aβ.

When the scientists disabled cholesterol biosynthesis in cell cultures, or in 5xFAD mice by conditionally knocking out a key sterol synthesis enzyme in microglia, the cells cranked up expression of lipoprotein receptors, and both intracellular lysosomal aggregates and extracellular plaques soared, according to methoxy-04 staining. This suggested that in a lipid-deprived state, microglia take up more ApoE, exacerbating intracellular aggregate formation and ultimately, Aβ plaques.

Jason Ulrich of Washington University in St. Louis wondered whether reduced receptor-mediated uptake of ApoE2 and of ApoE3-Christchurch isoforms by microglia might explain their protective effects. Simons has yet to test these variants.

Simons proposes a chain of events whereby microglia consume both Aβ and ApoE, which are typically degraded within the lysosome. When lysosomes become overwhelmed, as can happen due to any number of age-related and genetic risk factors, these two proteins might linger in the lysosomes long enough to aggregate.

“While the demonstration of ApoE and Aβ aggregates in microglial lysosomes is certainly exciting, it remains to be determined how the intracellular aggregates seed the extracellular plaques, and to what degree that contributes to the overall Aβ pathology,” noted Hui Zheng of Baylor College of Medicine in Houston.

The authors have yet to answer these questions. Simons proposed that the intracellular aggregates may be spewed out by microglia, or exposed once microglia die, possibly due to lysosomal damage inflicted by the aggregates. Recent studies suggest that microglia can form “lysosomal synapses” with the plasma membrane to release their contents (Apr 2023 conference news). Furthermore, when Simons recently presented this work at the Eibsee conference in Germany, John Hardy of University College London commented that these new findings harken back to “amyloid-related cells” spotted in AD brain samples more than three decades ago (Roher et al., 1988). These ARCs were likely microglia, and they contained amyloid filaments within intracellular vesicles. At the time, the authors proposed that these vesicles fused with the plasma membrane, releasing their acidic contents and fueling amyloidosis.

Nixon favors the idea that microglial cell death, caused by ruptured lysosomes, could give rise to extracellular plaques. He has reported such a phenomenon within neurons, which produce intracellular Aβ themselves (Jun 2022 news). Neurons also produce and take up ApoE when stressed, he noted (Feb 2023 news). Nixon suspects microglia may take up aggregated Aβ from dying neurons, and that this could contribute to its subsequent aggregation along with ApoE in microglial lysosomes. Simons told Alzforum that his lab only spotted ApoE/Aβ aggregates in microglia.

To Holtzman’s mind, the study provides compelling evidence that ApoE aggregation within microglial lysosomes is important in the early phase of amyloidosis. But would it contribute later on in disease, during the phase of amyloid-induced tauopathy? Holtzman and others have pegged ApoE, ApoE4 in particular, as exacerbating tauopathy-induced neurodegeneration (Sep 2017 news). In tauopathy models, microglia lysosomes are chock-full of lipids, and this accumulation requires ApoE. “This suggests that ApoE influences lysosomal function,” he said, implying that perhaps ApoE-induced lysosomal problems are a common feature of both Aβ and tau pathologies.—Jessica Shugart

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References

News Citations

1. ApoE4 Promotes Amyloidosis, But Only in Plaque-Free Mice 7 Dec 2017
2. Are Microglia Plaque Factories? 4 Sep 2019
3. Microglia Build Plaques to Protect the Brain 21 Apr 2021
4. Over the Span of AD, Roles of Astrocytes and Microglia Change 12 Apr 2024
5. Partners in Crime: APP Fragment and Endosomal Protein Impair Endocytosis 20 Jul 2015
6. Inside Out—Plaques May Have Intracellular Origin 22 Jan 2010
7. Human ApoE Antibody Nips Mouse Amyloid in the Bud 5 Apr 2018
8. From Phagocytosis to Exophagy: Microglia's Digestive Tract Dissected 25 Apr 2023
9. Behold PANTHOS, a Toxic Wreath of Perinuclear Aβ That Kills Neurons 5 Jun 2022
10. Secreted by Neurons, ApoE4 Makes Tangles and Degeneration Worse 24 Feb 2023
11. ApoE4 Makes All Things Tau Worse, From Beginning to End 20 Sep 2017

Mutations Citations

1. APOE C130R (ApoE4)

Paper Citations

1. Kim J, Eltorai AE, Jiang H, Liao F, Verghese PB, Stewart FR, Basak JM, Holtzman DM. Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of Aβ amyloidosis. J Exp Med. 2012 Nov 19;209(12):2149-56. PubMed.
2. Cataldo AM, Peterhoff CM, Troncoso JC, Gomez-Isla T, Hyman BT, Nixon RA. Endocytic pathway abnormalities precede amyloid beta deposition in sporadic Alzheimer's disease and Down syndrome: differential effects of APOE genotype and presenilin mutations. Am J Pathol. 2000 Jul;157(1):277-86. PubMed.
3. Roher A, Gray EG, Paula-Barbosa M. Alzheimer's disease: coated vesicles, coated pits and the amyloid-related cell. Proc R Soc Lond B Biol Sci. 1988 Jan 22;232(1269):367-73. PubMed.

Further Reading

News

1. Does Plaque ApoE Summon Microglia to Amyloid? 29 Sep 2023
2. Without TREM2, Plaques Grow Fast in Mice, Have Less ApoE 5 Jan 2019
3. Off-Balance Endocytosis Lays Groundwork for Disease 3 May 2019
4. Without SORL1, Endosomes Swell in Neurons but not Microglia 2 Jun 2020