Much of the genetic risk of Alzheimer’s disease plays out in microglia. But exactly how do risk variants change these cells? At the 14th International Conference on Alzheimer’s and Parkinson’s Diseases, held March 27–31 in Lisbon, Portugal, speakers filled in some gaps, and discussion of this question buzzed in the hallways all week. Christian Haass of the German Center for Neurodegenerative Diseases in Munich characterized progranulin as TREM2’s opposite. While mutations in TREM2 suppress microglial activation, mutations in progranulin permanently rev them up into voracious, toxin-spewing machines. Other speakers associated GWAS hits in general, and the MS4A gene cluster in particular, with hyperactive, damaging microglia. Researchers also highlighted potential therapeutic targets among microglial genes. Two talks presented evidence that suppressing the CD33 receptor can contain amyloidosis (see Part 5 in this series), while another fingered BIN1 as a key culprit in propagating phosphorylated tau.

  • Progranulin mutations trap microglia in a hyperactivated state.
  • The MS4A locus controls sTREM2 levels, which appear to protect against AD.
  • Microglial BIN1 may promote the spread of phosphorylated tau.

Modulating microglia for therapeutic purposes will be complicated, because these cells are exquisitely sensitive to their environment and can assume numerous different activation states depending on the stimuli around them. For example, Beth Stevens of Boston Children’s Hospital recently reported that different types of injury, such as demyelinating lesions and amyloid plaques, induce distinct microglial phenotypes (Dec 2018 news). In a plenary talk in Lisbon, Stevens noted, “There are disease-specific signatures. We need to be able to target these microglial subpopulations to switch them from a detrimental to a healthy state.”

GWAS Hits in Microglial Activation: An Up-Down Affair
Haass’ data suggests that interventions will need to be finely tuned. His lab previously reported that TREM2 normally activates microglia to respond to injury, and disease-linked variants disrupt this function, locking microglia into a homeostatic, unresponsive state (May 2017 news). In Lisbon, Haass showed that the opposite extreme is equally harmful. Julia Götzl, Matthias Brendel, and Georg Werner from his group, along with Anja Capell at Ludwig-Maximilians University in Munich, analyzed gene expression in microglia from progranulin knockout mice. The cells’ profile was diametrically opposed to that of TREM2 knockouts, with homeostatic genes suppressed and inflammatory genes up.

Polar Opposites. In aged TREM2 loss-of-function mice (left), microglia remain abnormally quiescent by TSPO scan, while in progranulin loss-of-function mice (right), they fire up. [Courtesy of Christian Haass.]

In keeping with this, microglia were more active sans progranulin. Progranulin-negative microglia gobbled up more bacteria than did wild-types in a dish, and migrated further in brain-slice cultures. This happened in mouse brain, as well. When APPPS1 mice were crossed with progranulin knockouts, many more microglia clustered around amyloid plaques in the progranulin-negative offspring. Both humans and mice with progranulin mutations had more hyperactivated microglia in their cortices, as seen by TSPO PET.

Importantly, however, these overly aggressive microglia were just as damaging to diseased brains as were the abnormally quiescent TREM2 knockouts. In essence, they mark the tips of a spectrum going from too little microglial response to amyloidosis to too much. Both types of knockout had weak brain glucose metabolism (Götzl et al., 2019, in press). The data suggest a narrow therapeutic window for tweaking microglia, where pushing too far in either direction could cause harm, Haass noted. Progranulin mutations cause frontotemporal dementia, not AD, although Haass’ group found that the protein rises in the cerebrospinal fluid of AD patients as disease progresses (Nov 2018 news). 

Progranulin and TREM2 are far from the only genes associated with damaging microglial states. Nicola Fattorelli, working with Carlo Sala Frigerio, Leen Wolfs, and Bart De Strooper at the University of Leuven, Belgium, performed RNA-Seq on more than 10,000 single microglia isolated from the cortices and hippocampi of young and old APP NL-G-F knock-in and wild-type control mice. In Lisbon, Fattorelli showed that the gene-expression profiles defined three distinct types of microglia: homeostatic cells, which were most abundant in young mice; interferon-response microglia, which increased with age equally in wild-type and AD mice; and amyloid-response microglia (ARM).

Separate Fates. Gene-expression analysis of mouse microglia showed that homeostatic cells can transform into two distinct forms, one associated more with age (IRM), the other more with amyloidosis (ARM). [Courtesy of Cell Reports, Sala Frigerio et al.]

ARM were mostly absent in young wild-type mice, and increased to 10 percent of the microglial population with age. In young APP NL-G-F mice, already one-third of microglia were in the ARM state, and their numbers mushroomed to half by one year of age. This progression toward ARM happened faster in females than males.

Importantly, the expression profile of ARM cells was enriched for Alzheimer’s GWAS hits. ApoE and TREM2 were up, whereas BIN1, MS4A6B, CD33, PICALM, and INPP5D were down in these cells. The researchers found a similar expression profile in microglia from APP/PS1 mice, and also in the Accelerating Medicines Partnership-AD transcriptomic database of human brain samples. In the latter, ARM transcriptional changes correlated with plaque burden.

In mouse brain, immunostaining revealed that microglial ApoE expression mainly occurred around amyloid plaques, dropping off gradually in microglia that were farther away. To determine ApoE’s function here, the scientists examined aged APP/PS1 mice lacking ApoE. They found almost no ARM in them, suggesting that ApoE is required to induce this microglial state. The paper appears today in Cell Reports (Sala Frigerio et al., 2019). 

Fattorelli told Alzforum that ARM resemble the disease-associated (DAM) or neurodegenerative phenotype (MGnD) microglia identified in other studies (Jun 2017 newsSep 2017 news). 

Haass noted that these findings fit with recent research from his group showing that microglia are the main source of ApoE in plaques (Jan 2019 news). 

Meanwhile, Amanda McQuade, working with Mathew Blurton-Jones at the University of California, Irvine, implicated the microglial receptor MS4A6A in damaging activation states. How variants in the MS4A gene cluster act in neurodegeneration is unknown. Rather than mouse cells, McQuade used microglia generated from human iPSCs (Jul 2016 news; McQuade et al., 2018). When she knocked out MS4A6A in these cells, 28 out of 53 AD-linked genes significantly changed expression. These genes affect phagocytosis, chemotaxis, and immune response. In line with this, phagocytosis became sluggish.

Overall, the cells assumed a DAM-like gene expression profile, McQuade said. In ongoing work, she is transplanting these human cell lines into mouse brain to examine the consequences of MS4A6A knockout in vivo. In a new review, the scientists call for better microglial models that mimic the natural in vivo environment, in order to determine which microglial functions actually affect disease (McQuade and Blurton-Jones, 2019). 

Where are the Drug Targets in All This?
MS4A may also affect disease through TREM2. Carlos Cruchaga previously reported that a noncoding variant, or eQTL (see Part 2 of this series), that boosts expression of MS4A4A and MS4A6A in blood and brain also raises levels of soluble TREM2 (sTREM2) in cerebrospinal fluid, while lowering AD risk and delaying age of onset (Jul 2018 news). In Lisbon, Cruchaga, of Washington University in St. Louis, added data supporting this relationship. In cell culture, he showed, overexpressing MS4A4A increased release of sTREM2, while knocking down MS4A4A suppressed it. This suggests that MS4A affects AD risk by modifying sTREM2 levels, Cruchaga believes. He noted that the strength of the association was comparable to ApoE’s effect on CSF Aβ42, and concluded that MS4A4A could offer a therapeutic handle.

Curiously, other data on the relationship between MS4A protein expression and sTREM2 conflict with Cruchaga’s. In Lisbon, Alison Goate of the Icahn School of Medicine at Mount Sinai, New York, described a single-nucleotide polymorphism in the MS4A locus that removes a repressive element, unleashing MS4A4A and MS4A6A expression. This SNP is a risk allele for AD, hinting that too much of these proteins could be harmful, not protective. What gives? Cruchaga noted that an eQTL for MS4A4A seems to nudge expression in opposite directions in blood and brain samples, which may explain some of the contradictory data. He is currently looking at what this eQTL does in brain-specific cell types to pin down a more definitive answer. Despite the conflicting data, both studies agree that MS4A variants that protect against AD raise sTREM2 levels.

Other research takes a closer look at the effect of sTREM2 on disease. The shedded protein rises in CSF during aging as well as in the prodromal stages of AD, and its levels correlate with CSF p-tau and t-tau (Mar 2016 news). Is sTREM2 beneficial or harmful? Haass believes it’s the former. Michael Ewers, Nicolai Franzmeier, and Marc Suárez-Calvet in his group compared ADNI data from 100 healthy controls with that of 285 people with amyloid and tau accumulation in their brains. Among the latter, 35 people were cognitively normal, 184 were mildly impaired, and 66 had dementia. Regardless of clinical diagnosis, people with AD pathology whose baseline CSF sTREM2 was high declined more slowly on memory and global cognition tests over four years. Also, their hippocampi shrank more slowly, compared with people with low CSF sTREM2 at baseline. Moreover, a high ratio of sTREM2 over p-tau181 correlated with slower progression to cognitive impairment or dementia (Ewers et al., 2019, in press). “I have a feeling that sTREM2 reflects protective microglial activation around the time tangles appear,” Haass said in Lisbon. He told Alzforum that sTREM2 itself may not be the key factor, though. Instead, sTREM2 may be a marker for successful signaling through full-length, membrane-bound TREM2.

While most talks in Lisbon investigated microglia for how they responded to amyloid, Andrea Crotti of Biogen in Cambridge, Massachusetts, is looking at how they handle tau pathology. After ApoE, BIN1 has the largest association with late-onset AD, and it is also expressed in microglia. This protein regulates membrane curvature and endocytosis. Because a previous study found that the p-tau181 in cerebrospinal fluid was hitched to exosomes, Crotti wondered if BIN1 might play a role in the spreading of pathologic tau from cell to cell (Saman et al., 2011). To investigate, he purified exosomes from human CSF, and found that three of 10 patient samples contained exosomes with phosphorylated or oligomeric tau that seeded aggregates in cell culture. Electron microscopy spotted BIN1 on the surface of these vesicles and, lo and behold, revealed that it was an isoform specifically expressed in microglia.

To look for in vivo effects, Crotti turned to mice. The researchers crossed a conditional knockout lacking microglial BIN1 with tau P301S mice, then injected synthetic tau fibrils into the brains of the offspring to accelerate tangle formation. Ninety days later, the BIN1 knockouts had but half as much phosphorylated tau in exosomes, and half as much staining for phosphorylated tau in hippocampal sections as the P301S mice did, leading him to conclude that microglial BIN1 promotes the spreading of pathologic tau via vesicles.

Previous studies have linked BIN1 expression with tangles and tau toxicity (Aug 2012 conference news; Holler et al., 2014; Apr 2015 conference news). These studies did not distinguish between neuronal and microglial BIN1, and at times generated conflicting findings regarding whether BIN1 alters amyloid or tau, once again highlighting the importance of dissecting out the microglial contribution to AD (Apr 2015 conference news).—Madolyn Bowman Rogers


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News Citations

  1. Could CD33 Be the Microglial Target for Stimulating Phagocytosis?
  2. Microglia Reveal Formidable Complexity, Deep Culpability in AD
  3. Paper Alert: TREM2 Crucial for Microglial Activation
  4. Progranulin: A Mark of Worsening Alzheimer’s?
  5. Hot DAM: Specific Microglia Engulf Plaques
  6. ApoE and Trem2 Flip a Microglial Switch in Neurodegenerative Disease
  7. Without TREM2, Plaques Grow Fast in Mice, Have Less ApoE
  8. Induced Microglia Make Debut at Keystone Symposium
  9. Expression, Expression, Expression—Time to Get on Board with eQTLs
  10. MS4A Alzheimer’s Risk Gene Linked to TREM2 Signaling
  11. Microglial Marker TREM2 Rises in Early Alzheimer’s and on Western Diet
  12. GWAS Mega-Meta Yields More Risk Genes, BIN1 Binds Tau?
  13. The Feud, Act II: Do Alzheimer’s Genes Affect Amyloid or Tau?

Research Models Citations

  1. APPPS1
  2. APP NL-G-F Knock-in
  3. Tau P301S (Line PS19)

Paper Citations

  1. . The Major Risk Factors for Alzheimer's Disease: Age, Sex, and Genes Modulate the Microglia Response to Aβ Plaques. Cell Rep. 2019 Apr 23;27(4):1293-1306.e6. PubMed.
  2. . Development and validation of a simplified method to generate human microglia from pluripotent stem cells. Mol Neurodegener. 2018 Dec 22;13(1):67. PubMed.
  3. . Microglia in Alzheimer's Disease: Exploring How Genetics and Phenotype Influence Risk. J Mol Biol. 2019 Apr 19;431(9):1805-1817. Epub 2019 Feb 7 PubMed.
  4. . Exosome-associated tau is secreted in tauopathy models and is selectively phosphorylated in cerebrospinal fluid (CSF) in early Alzheimer's Disease. J Biol Chem. 2011 Nov 4; PubMed.
  5. . Bridging integrator 1 (BIN1) protein expression increases in the Alzheimer's disease brain and correlates with neurofibrillary tangle pathology. J Alzheimers Dis. 2014;42(4):1221-7. PubMed.

Further Reading