02 May 2023

Do microglia thwart neurodegenerative disease, or help it along? Do they keep amyloid in check with one hand, while goading tau entanglement with the other? Do they protect neurons early on in disease, but sour into synaptic slayers later? As the field gears up to target these reactive immune cells in clinical trials (see Part 8 of this series), these fundamental paradoxes remain unsettled. At the International Conference on Alzheimer’s and Parkinson’s diseases, held March 28 to April 1 in Gothenburg, Sweden, scientists leveraged longitudinal data from human cohorts to get at these questions.

A load of findings came from the Montreal-based Translational Biomarkers of Aging and Dementia cohort. TRIAD deploys a trio of PET scans to map amyloid plaques, tau tangles, and microglial activation within a person's brain. Its data indicate that ApoE4 goads microglial activation, which, in turn, appears to worsen tau pathology and neurodegeneration. In TRIAD, fluid markers of phosphorylated tau tracked with heightened microglial activation in brain regions where tangles accumulate early in AD. Curiously, people with revved-up microglia tended to be more irritable than those with calmer microglia.

While these TRIAD results cast microglia as bad actors, data shown at AD/PD from a Swedish cohort implied the opposite conclusion. Based in Lund, BioFinder used CSF markers to gauge microglial activity, and those results associated microglial responses to reduced tau accumulation and slower cognitive decline.

“These clinical findings are really important. We have a clear divide in the microglial field between whether the microglia responses seen in the AD brain protect against aspects of the pathology, or contribute toward them,” commented Kim Green of the University of California, Irvine. “In reality, it is likely disease stage- and brain region-specific.” To study this, Green said the field sorely needs animal models that develop the spectrum of AD pathologies.

Pedro Rosa-Neto of McGill University in Montreal heads TRIAD. This cohort is unique in that participants periodically undergo three scans with second-generation PET tracers: [¹⁸F]AZD4694 for amyloid, [¹⁸F]MK6240 for tau, and [¹¹C]PBR28 for TSPO. TSPO is an outer mitochondrial membrane protein that cranks up as microglia become activated.

Including this PET scan comes at a cost. A polymorphism in the gene encoding TSPO influences how tightly the protein binds the PET tracer. Therefore, TRIAD participants are screened for these polymorphisms, and only carriers of the high TSPO-binding version—about 60 percent of participants in this predominantly non-Hispanic white population—undergo TSPO scans, Rosa-Neto told Alzforum. Rosa-Neto believes that the extra screening is worth the trouble, as zeroing in on how microglia, Aβ, and tau interact at the regional level is critical for understanding how they influence AD pathogenesis. In an example of this, Rosa-Neto and Tharick Pascoal, now at the University of Pittsburgh, previously reported that microglial activation arises in step with Braak stage regions, just before tau tangles inundate a given region (Sep 2021 news).

At AD/PD, the scientists added ApoE4 to this heady mix. Setting microglia aside for a moment, João Pedro Ferrari-Souza’s talk focused on the role of ApoE4 in potentiating the effect of Aβ plaques on the subsequent spread of tau tangles. A graduate student in Pascoal’s lab, Ferrari-Souza studied amyloid- and tau-PET scans from 104 TRIAD participants, including 72 cognitively unimpaired people, 25 with MCI, and seven with AD dementia. Ferrari-Souza found that relative to noncarriers with or without amyloid at baseline, ApoE4 carriers with amyloid had dramatically higher levels of tau deposition in medial temporal lobe regions over the following two years. A mediation analysis then suggested that ApoE4 exacerbated the connection between Aβ pathology and tau entanglement. Notably, serial measurements of plasma p-tau217 in the same participants indicated that the synergistic effect of Aβ and ApoE4 on tau tangles happened via phosphorylated tau.

“Indeed, the ApoE4 potentiated effects of Aβ on tau tangle accumulation occurs through tau phosphorylation,” Ferrari-Souza told the audience.

E4 Sparks Tau, but How? In ApoE4 noncarriers (blue), baseline Aβ burden did not correlate with tau tangle deposition two years later. In carriers (pink), baseline Aβ burden predicted a rise in plasma p-tau217, which, in turn, predicted tau tangle accumulation. [Courtesy of Ferrari-Souza, University of Pittsburgh.]

How does ApoE4 influence tau tangle accumulation? Ferrari-Souza and colleagues wove microglial activation into the equation. The study included 118 participants across the AD spectrum, including 79 who were cognitively normal, 23 with MCI, and 16 with AD dementia. At baseline, ApoE4 carriers had significantly higher microglial activation, according to TSPO-PET, than noncarriers. This E4 effect cropped up predominantly in areas corresponding to Braak stage regions, and it was strongest in early Braak regions. People with the “hottest” TSPO-PET signals in Braak regions at baseline tended to decline more steeply on cognitive tests, and their hippocampi shrank more over the following year. This suggested that ApoE4-triggered microglial responses speed up neurodegeneration.

E4 Riles Microglia. TSPO-PET scans (left) reveal that ApoE4 carriers have more activated microglia than noncarriers in medial temporal regions. The difference is greatest in early Braak stage regions (right, percent area showing E4-related TSPO-PET boost). [Courtesy of Ferrari-Souza et al., Science Advances, 2023.]

Why would ApoE4 prod microglia more in some regions than others? The researchers got a hint from the Allen Human Brain Atlas. Its spatial RNA sequencing data revealed that ApoE mRNA expression tracked with Braak stage regions, with expression being highest in the earliest-stage regions, including the transentorhinal cortex, entorhinal cortex, and hippocampus. Importantly, these ApoE expression patterns predicted the strength of the TSPO-PET signal across brain regions among TRIAD participants, Ferrari-Souza reported.

It remains unclear what factors dictate the distribution of ApoE expression, or even which cells are responsible. Production of the apolipoprotein by astrocytes, activated microglia, and even stressed neurons can reportedly jolt microglia out of their homeostatic state (Oct 2019 news; Apr 2021 news; Feb 2023 news). Still, the findings suggest that microglial activation depends on levels of ApoE expression.

Notably, ApoE4's microglia-stoking effect in the medial temporal lobe was observed regardless of the person's global Aβ burden, local tau tangle burden, or clinical diagnosis. The tie between ApoE4 and microglial activation in these early Braak regions was strongest among people with a higher burden of tangles in those same regions, suggesting that E4-triggered microglia promote tau pathology.

Ferrari-Souza used a statistical method called structural equation modeling to tease out the causal relationships among all these contributors to AD. He reported that heightened TSPO-PET signals in the medial temporal lobe were partially responsible for ApoE4’s promotion of tau pathology. In a separate, parallel pathway, amyloid burden also contributed to a boost in tau tangles. Both the Aβ-dependent and independent contributors to tau pathology were tied to hippocampal shrinkage and steeper cognitive decline. The findings were published in Science Advances on April 6 (Ferrari-Souza et al., 2023).

Parallel Paths to AD? Statistical modeling calculates relative causal relationships among different pathological contributors. Larger numbers (β-estimates) signify stronger causal ties. Black arrows represent significant effects; grey arrows, nonsignificant. [Courtesy of Ferrari-Souza et al., Science Advances, 2023.]

At AD/PD, Nesrine Rahmouni, a graduate student in Rosa-Neto’s lab and coordinator of the TRIAD cohort, presented fluid biomarker findings from the TRIAD cohort. They dovetailed with Ferrari-Souza’s work. Essentially, Rahmouni found that CSF markers of phosphorylated tau, including p-tau181, p-tau217, and p-tau231, all correlated with TSPO-PET signals within the medial temporal lobe.

In cognitively normal people, this association was independent of Aβ or tau burden, as gauged by PET scans. However, among those with MCI or AD dementia, the relationship between CSF p-tau and TSPO-PET was largely dependent on tangles, Rahmouni reported. In statistical models, a combination of CSF p-tau and tau-PET best predicted microglial activation as measured by TSPO-PET. Her interpretation? CSF p-tau biomarkers—which reflect the infancy of tau pathology—have an independent relationship with brain inflammation early in disease, but at later stages, tau tangles take precedence in mediating this effect.

Marc Diamond of UT Southwestern Medical Center in Dallas broached the chicken-and-egg conundrum: “Is neuroinflammation causing tau pathology, or the other way around?”

“That’s the million-dollar question,” Rahmouni responded. “We think it’s both.” She said that inflammation may help at first, only to become chronic, and therefore deleterious, later on. A deeper knowledge about inflammatory pathways at work in the brain will be needed to understand how and when that switch occurs, she said.

Rosa-Neto believes the findings are consistent with several parallel pathological pathways contributing to AD, as opposed to a sequential cascade of single events. “Perhaps AD is a tale of two proteinopathies that start to cause disease when they converge,” Rosa-Neto said. Microglia represent one point of this convergence, he said, as "well-meaning" cells eventually transform into harbingers of degeneration. In ApoE4 carriers, the path to this damage-prone state might be shortened both by microglial reactivity as well as by higher vulnerability among neurons.

Pascoal agrees that multiple players are likely involved in the overall progression of AD. He thinks future studies will become more granular at every level, and in that way zero in on cell type- and disease-stage specific contributors that converge on neurodegeneration.

The TRIAD results jibe with mouse studies from David Holtzman’s lab, in which ApoE4 dramatically worsened tau pathology in a way that depended upon microglia (Sep 2017 news; Oct 2019 news). “This new human data would argue that microglial reactivity may be useful in the future in predicting outcome, and be useful to measure in clinical trials,” Holtzman commented to Alzforum. “It would be very helpful going forward to have even more specific markers that we can use in humans to determine the particular microglial state that is present, whether via imaging or fluids, to better get at the details of how the microglial reactive state is changing.”

As microglia take blame for promoting tau propagation (see Part 13 of this series), how can scientists square this heinous act with the fact that loss-of-function variants in TREM2—the receptor that controls myriad microglial responses—boost a person's risk of AD? This contradiction has sparked confusion in the field, Joana Pereira of the Karolinska Institute in Stockholm said at AD/PD.

To investigate how TREM2-mediated signaling in microglia influences tau pathology and other aspects of AD, Pereira looked in CSF for indicators that microglia had transitioned to the disease associated microglia-2 (DAM2) state. This transcriptional state relies on TREM2 signaling, and is characterized by enhanced phagocytosis, mediated at least in part through TAM receptors Axl and MerTK (Jun 2017 news; Apr 2021 news). Pereira hunted for proteins churned out by DAM2 cells, including soluble TREM2 (sTREM2), Axl, and MerTK along with their ligand, Gas6, as well as LPL, CST7, SPP1, and CSF1.

Her analysis included 344 cognitively unimpaired participants from the BioFinder cohort, split into three groups based on results of their baseline amyloid and tau-PET scans: 121 had evidence of amyloid plaques but not tau tangles, 64 had both plaques and tangles, and 159 had neither. Participants underwent repeated scanning, CSF sampling, and cognitive testing two and four years later.

What did they find? Starting with soluble TREM2, Pereira reported that among all participants with amyloid at baseline, higher levels of sTREM2 predicted a slower rise in amyloid over the following four years. Among those who started the study with some evidence of tau pathology, Pereira found the same beneficial effect of higher sTREM2 on tau accumulation.

For the other DAM2 markers, Pereira found no association with future amyloid accumulation. Alas, she did find that higher CSF concentrations of three—Gas6, Cst7, and CSF1—predicted a slower progression of tau pathology. This was true in Braak III/IV and Braak V/VI regions, which correspond to neocortical tangles. These associations were independent of amyloid. Meshing with her tau findings, Pereira also reported that higher CSF DAM2 markers predicted slower cognitive decline as measured by MMSE scores. Curiously, this salubrious effect on cognition was stronger in women than in men.

SPP1 was a notable exception. High levels of this cytokine foreshadowed a faster spread of tau, and a steeper drop in cognition. To Pereira's mind, SPP1’s outlier status makes sense in light of the finding that SPP1 incites perivascular macrophages to nosh on synapses (Feb 2023 news). “Not all myeloid responses in the brain are beneficial,” Pereira said. The findings were published in Nature Aging (Pereira et al., 2022).

TREM2 Transition. When microglia stay in a homeostatic state (top), Aβ plaques and tau tangles can more quickly spread across the brain. When the cells transition into the DAM2 state, they keep AD pathology in check. [Courtesy of Pereira et al., Nature Aging, 2022.]

How to reconcile the helpful responses Pereira saw with the TRIAD results, which cast microglia as accomplices to tau propagation? Potential explanations abound. They include differences in the number and disease stage of participants in the cohorts, and the modalities used to gauge the microglial mood. Pereira hypothesized that, similar to the nefarious SPP1 cytokine in her study, perhaps TSPO-PET illuminates destructive microglia or macrophages. “I think TSPO-PET is probably detecting a harmful microglial phenotype that is associated with synaptic phagocytosis and degeneration, whereas DAM2 are more associated with protective effects,” she wrote to Alzforum.

A way to answer this question is to track both TSPO-PET and DAM2 CSF biomarkers in the same cohort, said Henrik Zetterberg of the University of Gothenburg in Sweden, who co-authored both papers.

To Pascoal’s mind, the TRIAD and BioFinder results don’t necessarily conflict. Instead, they offer a glimpse at the dramatic heterogeneity of microglial states within the human brain. Alluding to single-cell RNA sequencing studies, Pascoal said that not only are the cells in different states at any given time, but these states themselves change with time, age, and disease stage. Cracking open this heterogeneity is the focus of intense research (see Part 14 of this series). Just like the false dichotomy of M1 versus M2 phenotypes of the past, labeling microglia as “good” or “bad” is a gross oversimplification.—Jessica Shugart

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References

News Citations

1. All Roads Lead to TREM2: Gearing Up to Target This Receptor 14 Apr 2023
2. PET Firms Up Amyloid Cascade: Plaques, Inflammation, Tangles 3 Sep 2021
3. In Tauopathy, ApoE Destroys Neurons Via Microglia 17 Oct 2019
4. Squelching ApoE in Astrocytes of Tau-Ravaged Mice Dampens Degeneration 10 Apr 2021
5. Secreted by Neurons, ApoE4 Makes Tangles and Degeneration Worse 24 Feb 2023
6. ApoE4 Makes All Things Tau Worse, From Beginning to End 20 Sep 2017
7. From Phagocytosis to Exophagy: Microglia's Digestive Tract Dissected 25 Apr 2023
8. Hot DAM: Specific Microglia Engulf Plaques 9 Jun 2017
9. Microglia Build Plaques to Protect the Brain 21 Apr 2021
10. When Perivascular Macrophages Spew SPP1, Microglia Eat Synapses 10 Feb 2023
11. With Microglia, It Takes a Village to Connect the Dots 25 Apr 2023

Mutations Citations

1. APOE C130R (ApoE4)

Paper Citations

1. Ferrari-Souza JP, Lussier FZ, Leffa DT, Therriault J, Tissot C, Bellaver B, Ferreira PC, Malpetti M, Wang YT, Povala G, Benedet AL, Ashton NJ, Chamoun M, Servaes S, Bezgin G, Kang MS, Stevenson J, Rahmouni N, Pallen V, Poltronetti NM, O'Brien JT, Rowe JB, Cohen AD, Lopez OL, Tudorascu DL, Karikari TK, Klunk WE, Villemagne VL, Soucy JP, Gauthier S, Souza DO, Zetterberg H, Blennow K, Zimmer ER, Rosa-Neto P, Pascoal TA. APOEε4 associates with microglial activation independently of Aβ plaques and tau tangles. Sci Adv. 2023 Apr 5;9(14):eade1474. PubMed.
2. Pereira JB, Janelidze S, Strandberg O, Whelan CD, Zetterberg H, Blennow K, Palmqvist S, Stomrud E, Mattsson-Carlgren N, Hansson O. Microglial activation protects against accumulation of tau aggregates in nondemented individuals with underlying Alzheimer’s disease patholog. Nat Aging 2, 1138–1144 (2022)

Further Reading

News

1. ApoE4 and Tau in Alzheimer’s: Worse Than We Thought? Especially in Women 1 Nov 2019