Arriving at the same conclusion, essentially at the same time, three research groups have independently mapped the site where proteases snip off the extracellular portion of TREM2. Two papers in the August 30 issue of EMBO Molecular Medicine and one under review and posted on the BioRχiv preprint server report that the site coincides exactly with a mutation in the microglial receptor, H157Y, that boosts AD risk by as much as 11-fold. “It’s incredible that all three results are essentially identical,” said Christian Haass of Ludwig-Maximilians-Universität, Munich, the senior author on one of the EMBO Molecular Medicine papers. Peter St. George-Hyslop of the University of Toronto, who co-led the second published study with Damian Crowther and Iain Chessell of AstraZeneca in Cambridge, U.K., presented their findings earlier this year at the 13th International Conference on Alzheimer’s and Parkinson’s Diseases in Vienna (Apr 2017 news). Ulf Neumann at Novartis in Basel, Switzerland, led the third group. 

TREM2 binds anionic lipids released during neuronal and glial damage. It supports microglial metabolism and it promotes the migration, cytokine release, phagocytosis, proliferation, and survival of the cells (Aug 2017 newsFeb 2015 news). Evidence suggests that TREM2 spurs microglia to form a neuroprotective barrier around amyloid plaques and to clear Aβ (May 2016 news; Jul 2016 news). Motivated by the discovery of roughly a dozen TREM2 genetic variants that increase the risk of frontotemporal dementia, AD, and possibly other neurodegenerative diseases, including amyotrophic lateral sclerosis and Parkinson’s disease, researchers are searching for ways to bolster TREM2’s protective function. Thinking that limiting ectodomain shedding might improve TREM2 signaling (see image below), the three research groups set out to find the cleavage site.

Snip Site. ADAM10, or other proteases, may shed TREM2’s extracellular ligand binding domain, abolishing TREM2-mediated signaling. (Courtesy of Yeh et al., 2017, Trends Mol Med.)

In Haass’s lab, first author Kai Schlepckow focused on the TREM2 C-terminal fragment (CTF) left over after shedding. Because γ-secretase quickly chews up the CTF in microglia, much as it does the C-terminal fragment of amyloid precursor protein in neurons, Schlepckow generated human embryonic kidney 293 (HEK293) cells expressing TREM2 and treated them with DAPT, a γ-secretase inhibitor. After immunoprecipitating the TREM2 CTF, he analyzed it by mass spectrometry. The result was crystal clear: a single major peak corresponding to a fragment with an N-terminus at serine 158. That amino acid lies in the extracellular domain of TREM2, 17 amino acids from the predicted transmembrane domain. “I’ve had experience mapping other cleavage sites and this one is super-precise. I’ve never seen something like it,” said Haass. 

Indeed, the result was so clean, Haass wondered if it was real. He had the researchers analyze the sequence on the other side of the break. Because sTREM2, the soluble extracellular domain, is too big to analyze by mass spectrometry, Schlepckow created a TREM2 construct with a tobacco etch virus protease cleavage site shortly before the proposed sheddase site. That way, the researchers could immunoprecipitate sTREM2 from the cell medium, shorten it with the TEV protease, and then determine its mass. Consistent with the CTF results, they obtained a single sharp peak corresponding to a peptide terminating at histidine 157. The researchers got the same results using human THP-1 cells, which are similar to monocytes, the cells that give rise to microglia.

Crowther’s group also relied on mass spectrometry. “First we used a library of peptide protease inhibitors to get a quick-and-dirty answer to where the site might be,” said Crowther. First author Peter Thornton at AstraZeneca synthesized a set of D-amino acid polypeptides that overlapped TREM2 amino acids 140-176, where they thought metalloprotease likely cleaved. Then they used these peptides to compete with TREM2 for the protease in primary human macrophages.

All peptides that spanned TREM2 amino acids 158-160 reduced TREM2 ectodomain shedding, whereas peptides mapping to nearby regions did not. Interestingly, the most effective peptide worked equally well when its sequence was reversed, suggesting that the responsible metalloproteases recognize biophysical properties, such as charge, to target a specific sequence. To map that sequence more precisely, Thornton immunoprecipitated sTREM2 from the conditioned media of several cell types, isolated it by gel chromatography, digested it with trypsin and analyzed the fragments by mass spectrometry. From human macrophages, primary murine microglia, and HEK293 cells expressing human TREM2, H157 surfaced as the most likely sheddase site.

Neumann’s group tackled the question by generating a series of TREM2 constructs with deletions or amino acid replacements in the stalk region, located between the transmembrane and Ig-like domains (see image above). They identified two regions, amino acids 169-172 and 156-164, in which mutations strongly reduced TREM2 cleavage induced by the protein kinase C activator PMA. To pinpoint the cleavage site, they designed a series of labeled peptides spanning the stalk region, incubated them with the metalloprotease ADAM17 in a test tube, and analyzed the resulting fragments using high-performance liquid chromatography and mass spectrometry. The researchers used ADAM17 because their prior data indicated it was a major TREM2 sheddase.

The  data also revealed the H157-S158 bond as the cleavage site, said Dominik Feuerbach, first author of the study. The researchers repeated the experiments using liquid chromatography and mass spec to analyze sTREM2 generated by HEK293 cells expressing human TREM2 and TYROBP, which forms a complex with the cytoplasmic portion of TREM2 (see image above). Feuerbach said they included TYROBP to mimic TREM2’s physiological state as closely as possible. Again, they found cleavage occurred at the H157-S158 site.

Researchers have reported that a histidine to tyrosine mutation at position 157 increases the risk for late-onset AD in Han Chinese (Jiang et al., 2016). How might this change affect TREM2 processing? Haass and Crowther’s groups compared shedding in cells expressing wild-type or the H157Y mutant. Crowther found that the extracellular domain of the wild-type protein has a half-life of less than one hour on the cell’s surface. Although the researchers didn’t measure the mutant extracellular domain half-life directly, they found cells more rapidly pumped mutant sTREM2 into the conditioned medium. “Shedding is fast in healthy cells, but it gets even faster with the variant,” he said. Haass’ group obtained similar results. “This was a surprise,” said Haass, who was expecting the H157Y mutation to reduce sTREM2 production, just like other mutations that increase risk for neurodegeneration. The T66M and Y38C mutations associated with frontotemporal dementia, for example, preclude TREM2 from reaching the cell surface where shedding predominantly occurs, shutting down production of sTREM2. “We got exactly the opposite of what we expected,” said Haass.

On further reflection, Haass and colleagues realized that increased shedding could have the same biological effect as reducing cell surface TREM2 because it reduced the amount of signaling-competent TREM2 on the cell surface. Indeed, Schlepckow found that monocytes expressing H157Y TREM2 phagocytosed a third less Escherichia coli than monocytes expressing the wild-type receptor. These findings support the idea that loss of function is key to the risk associated with H157Y TREM2, in line with most other TREM2 variants whose mechanisms have been dissected. Marco Colonna of Washington University in St. Louis noted that except for two TREM2 variants that increased ligand binding but were only weakly tied to AD risk, all other variants dampened TREM2 function, either by decreasing ligand binding, preventing TREM2 from reaching the cell surface, or, in this case, increasing TREM2 shedding. Feuerbach said the case for trying to therapeutically boost membrane-bound TREM2 is more compelling than ever. “From a genetic point of view, this is one of the most attractive targets,” he said.

To identify the proteases responsible for wild-type and mutant TREM2 shedding, Thornton and colleagues used various protease inhibitors, as well as siRNA, to block or knock down the expression of ADAM17 or ADAM10, a.k.a. a-secretase, which process the Aβ precursor protein (APP). Previous work implicated ADAM10 in TREM2 cleavage (Kleinberger et al., 2014). Lowering or blocking ADAM10, but not ADAM17, reduced TREM2 shedding. However, neither an inhibitor, nor ADAM10 siRNA, blocked shedding of the H157Y mutant as effectively as they blocked that of the wild-type. Crowther hypothesized the existence of a novel sheddase to account for the difference, but also acknowledged the mutant site might simply be more prone to ADAM10 cleavage and, thus, harder to fully block. “It could just be that the H157Y conformation is so tasty, that ADAM10 just continues to nibble away,” he said. Haass favors this latter possibility.

Feuerbach, however, said his data points to ADAM17, rather than ADAM10, as the major TREM2 sheddase. In contrast to Crowther’s group, which used HEK273 cells expressing human TREM2, but not TYROBP, Feuerbach used Chinese hamster ovary cells expressing both proteins, as well as human M2A macrophages, which are related to microglia and endogenously express TREM2 and TYROBP. To probe TREM2 cleavage, he used protease inhibitors, and ADAM10 or ADAM17 knockouts. His results indicate that TREM2 shedding depends much more on the activity of ADAM17. “We would not exclude ADAM10 or other proteases from playing a role, but I’d say ADAM17 probably accounts for 90 percent of the shedding,” he said. Colonna and Thomas Brett, also at Washington University, thought that differences in cell types might account for the discrepancy. “Different proteases might be more prevalent in different cells—probably multiple proteases can do the job,” said Colonna. Also, Feuerbach pointed out that TYROBP might affect the conformation of the cleavage site.

From a therapeutic standpoint, researchers agreed the proteases matter less than the location of the TREM2 cleavage site. “No one is interested in developing an ADAM10 inhibitor for AD,” said Crowther, noting it would have too many undesirable secondary effects. Indeed, ADAM10’s cutting of APP prevents the generation of Aβ, noted Terrence Town, University of Southern California, Los Angeles. Hoping to more specifically target TREM2 clipping, Haass has begun generating antibodies against the TREM2 cleavage site. Not only could such antibodies help people with the rare H157Y mutation, but they might also help nearly anyone with AD, he believes. “Antibodies against the TREM2 cleavage site could have a very potent effect,” said Crowther. A boost in membrane-bound TREM2 that would rev up Aβ phagocytosis would be beneficial. Indeed, evidence suggests people with sporadic and familial AD have increased levels of freewheeling sTREM2 in their spinal fluid, which may reflect an uptick in TREM2 shedding (Jan 2016 newsSuarez-Calvet et al., 2016). Nonetheless, Haass cautioned that it will be complicated to develop such a therapy because excessive TREM2 activity could be harmful.

Furthermore, any therapy that keeps TREM2 from casting off its extracellular domain will also cause a drop in sTREM2 levels. Brett wondered about the consequences, noting that, at least according to one study, sTREM2 causes inflammation, which is harmful, but also promotes microglial survival (Feb 2017 news). Colonna agreed. “At the end of the day, we don’t know if blocking sTREM2 production will have a positive, negative, or neutral effect,” he said.

Regardless of whether regulating TREM2 processing has a future in the clinic, the new studies offer important basic insights, said Colonna. “By looking at every single mutation, we’ll understand TREM2 better.”—Marina Chicurel


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News Citations

  1. New Evidence Confirms TREM2 Binds Aβ, Drives Protective Response
  2. Without TREM2, Microglia Run Out of Gas
  3. TREM2 Buoys Microglial Disaster Relief Efforts in AD and Stroke
  4. Barrier Function: TREM2 Helps Microglia to Compact Amyloid Plaques
  5. TREM2 Helps Phagocytes Gobble Up Aβ Coated in Antibodies
  6. TREM2 Goes Up in Spinal Fluid in Early Alzheimer’s
  7. Does Soluble TREM2 Rile Up Microglia?

Alzpedia Citations

  1. ADAM10

Paper Citations

  1. . A rare coding variant in TREM2 increases risk for Alzheimer's disease in Han Chinese. Neurobiol Aging. 2016 Jun;42:217.e1-3. Epub 2016 Mar 3 PubMed.
  2. . TREM2 mutations implicated in neurodegeneration impair cell surface transport and phagocytosis. Sci Transl Med. 2014 Jul 2;6(243):243ra86. PubMed.
  3. . Early changes in CSF sTREM2 in dominantly inherited Alzheimer's disease occur after amyloid deposition and neuronal injury. Sci Transl Med. 2016 Dec 14;8(369):369ra178. PubMed.

Further Reading


  1. . TREM2, Microglia, and Neurodegenerative Diseases. Trends Mol Med. 2017 Jun;23(6):512-533. Epub 2017 Apr 22 PubMed.
  2. . TREM2 in Neurodegenerative Diseases. Mol Neurodegener. 2017 Aug 2;12(1):56. PubMed.

Primary Papers

  1. . TREM2 shedding by cleavage at the H157-S158 bond is accelerated for the Alzheimer's disease-associated H157Y variant. EMBO Mol Med. 2017 Oct;9(10):1366-1378. PubMed.
  2. . An Alzheimer-associated TREM2 variant occurs at the ADAM cleavage site and affects shedding and phagocytic function. EMBO Mol Med. 2017 Oct;9(10):1356-1365. PubMed.