23 Feb 2015

Ever since genetic studies identified TREM2 as a risk factor for Alzheimer’s and other neurodegenerative diseases in 2012, researchers have raced to understand why. The trouble thus far has been that findings conflict as often as not, and the results presented at “Neuroinflammation in Diseases the Central Nervous System,” a Keystone Symposium held January 25-30 in Taos, New Mexico, were no exception. At first glance, data from different labs appeared to disagree on which cells expressed TREM2 and how they affected amyloid plaques. However, as the meeting progressed, researchers found common ground and charted plans to clarify lingering discrepancies. They seemed to agree that TREM2 channels supportive signals to myeloid cells surrounding plaques in AD model mice, but what those cells are and what they do remained controversial.

Meeting co-organizer Chris Glass of the University of California, San Diego saw the TREM2 controversy as a teachable moment for the field. As researchers learn more about this receptor, they will likely reveal fundamental realities about which aspects of neuroinflammation are protective, and which are harmful. “TREM2 is a perfect example of how little we know about neuroinflammation, and sorting out its function will be extremely instructive,” he said.

As its name suggests, TREM2 (which stands for triggering receptor expressed on myeloid cells) primarily adorns myeloid cells, including microglia and macrophages elsewhere in the body. No one knows what triggers it, but ligands ranging from lipopolysaccharide to mitochondrial chaperones to apoptotic neurons have been proposed (see Yaghmoor et al., 2014). When activated, TREM2 seems to soothe inflammation and promote phagocytic activity in macrophages and microglia (see Hickman et al., 2014). Multiple variants of the gene have popped up in genetic studies of neurodegenerative disease, and the R47H variant triples a carrier’s risk of AD (see Nov 2012 news). To understand how TREM2 and neuroinflammation influence AD, researchers have generated TREM2-deficient mouse strains and crossed them to different AD mouse models. The results—some presented for the first time at the meeting—sparked lively debate at Keystone.

Taylor Jay was the first to put forward TREM2 data at the meeting. A graduate student in the labs of Bruce Lamb and Gary Landreth at Case Western Reserve University in Cleveland, Jay and colleagues had presented their findings at the Society for Neuroscience annual meeting in D.C. last November (see Dec 2014 conference coverage). Jay reported that TREM2-expressing cells surrounded amyloid plaques in 4-month-old APP/PS1 mice. These cells highly expressed CD45, a marker of peripheral macrophages, and little P2RY12, a marker associated with microglia. These macrophages were absent from the brain in TREM2-deficient APP/PS1 mice. On the other hand, resident microglia (singled out by their low expression of CD45 and high expression of P2RY12) expressed no TREM2. They remained just as evenly spaced throughout the brain in APP/PS1 mice as they were in controls, indicating they were not the cells gathering around plaques. Jay dropped a counterintuitive bomb when she reported lower plaque burden in the hippocampus of TREM2-deficient mice.

Researchers galore lined up behind the microphones to pepper Jay with questions after her talk. V. Hugh Perry of the University of Southampton in England seemed perplexed that resident microglia weren’t the ones expressing TREM2, as previous studies had suggested they did. Jay responded that in their hands, microglia indeed expressed TREM2 RNA transcripts, but not a detectable level of protein. Oleg Butovsky of Harvard University, who originally generated the antibody to P2RY12, commented that the marker was an unreliable way to label resident microglia in APP/PS1 mice because its expression plummets in the context of disease. Jay said the combination of other markers she used to identify the cells made her confident she was looking at microglia. Finally, Arnon Rosenthal of the biotech company Alector in San Francisco brought up the elephant in the room: How could the plaque burden Jay had presented explain data suggesting that loss of TREM2 function raises AD risk? The findings were indeed perplexing, Jay acknowledged. Perhaps generating TREM2 knock-in mice that harbor disease-associated mutations could help, she suggested.

Marco Colonna of Washington University, St. Louis, presented data painting TREM2 as a microglial survival factor in AD. Colonna crossed TREM2-deficient mice with 5xFAD mice. When the crosses reached 8 months of age, their hippocampal plaque burden outpaced those in 5xFAD animals. Colonna saw no difference in the cortex. These findings conflicted with Jay’s; however, both researchers were quick to point out that they used different mouse models and measured plaques at different time points. Jay speculated that TREM2 could promote plaques early on, and then help remove them later. While that idea will need to be tested, some researchers believe that there may be stark differences between inflammatory responses that occur early and late in the disease process (see Eikelenboom and Hoozemans comment on Matarin et al., 2015). A recent study using postmortem brain samples reported that TREM2 expression levels are highest in brain regions most vulnerable to AD pathology, such as the hippocampus, and that expression rises as the disease progresses (see Strobel et al., 2015).

Colonna and colleagues next took a close accounting of the cells surrounding plaques. He told the audience that Iba1-positive cells, which he interpreted as resident microglia, surrounded plaques in 5xFAD mice. In TREM2-deficient 5xFAD mice, the total number of microglia in the brain dropped, and Colonna observed few cells surrounding plaques. Those that did expressed apoptotic markers. Except for the proposed origin of the cells, these findings agreed with Jay’s, who had also reported fewer cells around plaques in TREM2-deficient mice.

How might TREM2 boost the number of cells surrounding plaques? Colonna reported that TREM2 cooperated with colony-stimulating factor 1 receptor (CSFR1) to promote microglial survival. He hypothesized that when microglia proliferate in response to an insult such as amyloid plaques, they deplete colony-stimulating factor and thus may require co-signaling by TREM2 to amplify the signal. In support of this idea, Colonna reported that microglia isolated from TREM2-deficient mice or TREM2-deficient 5xFAD mice died off in culture when colony-stimulating factor levels were low, whereas cells expressing TREM2 managed to survive. All in all, Colonna suggested that TREM2 supports the survival of microglia during disease.

This left an obvious question: What activates TREM2? Colonna tantalized the audience by suggesting that phospholipids do. He used a reporter cell line that expresses green fluorescent protein (GFP) under control of the NFAT transcription factor. NFAT translocates to the nucleus in response to calcium mobilization when TREM2 is activated. Some anionic and zwitterionic lipids, including phospholipids such as phosphatidylserine, turned on GFP. TREM2 antibodies blocked this. Perhaps most intriguingly, many lipids failed to switch on GFP in cells that expressed TREM2 with the R47H high-risk variant. Unlike other TREM2 mutations that associate with neurodegenerative disease, R47H does not impair the receptor’s transport to the cell surface or shedding of its extracellular domain, although it does alter the protein’s glycosylation status (see Alzforum webinar and Park et al., 2015). The loss of an arginine residue in the R47H mutation would theoretically prevent TREM2 from interacting with anionic lipids, thus reducing microglial survival during disease.

In response to audience questions, Colonna speculated that lipids such as phosphatidylserine, which are exposed on the surface of dying neurons, could trigger TREM2 when disease strikes (see Takahashi et al., 2005). Sphingolipids released during demyelination could also trigger the receptor, and a recent study reported that TREM2 is required for efficient clearance of myelin debris (see Cantoni et al., 2015). Furthermore, lipids are known to associate with Aβ and facilitate aggregation. While Colonna found no evidence that Aβ directly triggers TREM2, he speculated that lipids associated with Aβ or exposed or released by Aβ-induced damage could. The loss of TREM2 expression did not impair microglial phagocytosis in vitro, but Colonna noted that increased cell survival would have the effect of enhancing any microglial function.

David Holtzman of WashU, who collaborated with Colonna on the work, said it will be interesting to test whether another major AD risk factor, that is, apolipoprotein E, plays a role in lipid binding by TREM2. As its name suggests, ApoE binds lipids, and the E4 isoform promotes accumulation of Aβ. Whether TREM2 and ApoE4 increase the chances of getting AD by affecting similar lipid-related pathways will be the subject of future experiments, Holtzman said. Interestingly, a recent study reported that the ApoE4 isoform exacerbates the microglial inflammatory response and turns down expression of TREM2 (see Li et al., 2015). 

Holtzman’s group had previously reported no change in amyloid plaque burden in an APP/PS1 mouse heterozygous for TREM2 (see Jun 2014 news).

Given that three different groups have reported varying phenotypes of TREM2 deficiency related to plaques, Erik Musiek, also at WashU, commented that perhaps plaques are the wrong place to look. “We may have to look beyond our usual phenotype when assessing the function of TREM2,” he said. “If there were striking effects on plaques, I think it would be obvious.” Musiek noted that the meeting helped identify other potential neuroinflammatory phenotypes, such as microglial gene expression signatures (see Part 2 and Part 3 of this series). These may be useful in figuring out how TREM2 raises disease risk. “We need to start defining these signatures in the AD brain and find out which ones are important,” he said.

Regardless of whether cells gathered around plaques are infiltrating macrophages or resident microglia, scientists still need to figure out what they do and how TREM2 plays a role. Landreth hypothesized that Aβ tempers microglial phagocytosis and presented a way to change that. Landreth previously reported that bexarotene, a retinoid X receptor agonist, boosted phagocytosis and cleared plaques in mouse models of AD and even improved cognition, though other groups reported difficulty reproducing the results (see May 2013 news).

At Keystone, Landreth reported that bexarotene increased the expression of Axl and Mer, two surface receptors known to enhance phagocytosis, on cells surrounding plaques in mice. These Axl/Mer-expressing cells also expressed TREM2 and high levels of CD45, suggesting they were infiltrating macrophages. Bexarotene treatment reduced plaque burden in the mice, and Landreth found that in vitro, bexarotene stimulated phagocytosis. Landreth interpreted the findings as further evidence that the cells surrounding plaques in AD are not resident microglia, but infiltrating macrophages that express TREM2.

Richard Ransohoff of Biogen Idec in Cambridge, Massachusetts, who collaborated with Landreth, Lamb, and Jay, sought to find common ground among the variety of TREM2 data at the conference. While Ransohoff remains convinced by Jay’s staining data that the cells surrounding plaques are macrophages that infiltrate from the periphery and Colonna prefers the idea that they are resident microglia, a commonality is that those cells disappear in AD models. Regardless of their origin, researchers agreed that what matters most is what those cells are doing around the plaques.—Jessica Shugart

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References

Alzpedia Citations

1. TREM2

News Citations

1. Enter the New Alzheimer’s Gene: TREM2 Variant Triples Risk 14 Nov 2012
2. TREM2 Data Surprise at SfN Annual Meeting 5 Dec 2014
3. TREM2 Mystery: Altered Microglia, No Effect on Plaques 6 Jun 2014
4. Nature Versus Nurture: What Gives Microglia Their Identity? 19 Feb 2015
5. Microglia in Disease: Innocent Bystanders, or Agents of Destruction? 20 Feb 2015
6. Bexarotene Revisited: Improves Mouse Memory But No Effect on Plaques 23 May 2013

Research Models Citations

1. APPPS1
2. 5xFAD (B6SJL)

Webinar Citations

1. Mutations Impair TREM2 Maturation, Processing, and Microglial Phagocytosis 1 Jul 2014

Paper Citations

1. Yaghmoor F, Noorsaeed A, Alsaggaf S, Aljohani W, Scholtzova H, Boutajangout A, Wisniewski T. The Role of TREM2 in Alzheimer's Disease and Other Neurological Disorders. J Alzheimers Dis Parkinsonism. 2014 Nov;4(5) PubMed.
2. Hickman SE, El Khoury J. TREM2 and the neuroimmunology of Alzheimer's disease. Biochem Pharmacol. 2014 Apr 15;88(4):495-8. Epub 2013 Dec 16 PubMed.
3. Matarin M, Salih DA, Yasvoina M, Cummings DM, Guelfi S, Liu W, Nahaboo Solim MA, Moens TG, Paublete RM, Ali SS, Perona M, Desai R, Smith KJ, Latcham J, Fulleylove M, Richardson JC, Hardy J, Edwards FA. A genome-wide gene-expression analysis and database in transgenic mice during development of amyloid or tau pathology. Cell Rep. 2015 Feb 3;10(4):633-44. Epub 2015 Jan 22 PubMed.
4. Strobel S, Grünblatt E, Riederer P, Heinsen H, Arzberger T, Al-Sarraj S, Troakes C, Ferrer I, Monoranu CM. Changes in the expression of genes related to neuroinflammation over the course of sporadic Alzheimer's disease progression: CX3CL1, TREM2, and PPARγ. J Neural Transm. 2015 Jul;122(7):1069-76. Epub 2015 Jan 18 PubMed.
5. Park JS, Ji IJ, An HJ, Kang MJ, Kang SW, Kim DH, Yoon SY. Disease-Associated Mutations of TREM2 Alter the Processing of N-Linked Oligosaccharides in the Golgi Apparatus. Traffic. 2015 May;16(5):510-8. Epub 2015 Feb 24 PubMed.
6. Takahashi K, Rochford CD, Neumann H. Clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells-2. J Exp Med. 2005 Feb 21;201(4):647-57. PubMed.
7. Cantoni C, Bollman B, Licastro D, Xie M, Mikesell R, Schmidt R, Yuede CM, Galimberti D, Olivecrona G, Klein RS, Cross AH, Otero K, Piccio L. TREM2 regulates microglial cell activation in response to demyelination in vivo. Acta Neuropathol. 2015 Mar;129(3):429-47. Epub 2015 Jan 29 PubMed.
8. Li X, Montine KS, Keene CD, Montine TJ. Different mechanisms of apolipoprotein E isoform-dependent modulation of prostaglandin E2 production and triggering receptor expressed on myeloid cells 2 (TREM2) expression after innate immune activation of microglia. FASEB J. 2015 May;29(5):1754-62. Epub 2015 Jan 15 PubMed.

Further Reading

Papers

1. Jay TR, Miller CM, Cheng PJ, Graham LC, Bemiller S, Broihier ML, Xu G, Margevicius D, Karlo JC, Sousa GL, Cotleur AC, Butovsky O, Bekris L, Staugaitis SM, Leverenz JB, Pimplikar SW, Landreth GE, Howell GR, Ransohoff RM, Lamb BT. TREM2 deficiency eliminates TREM2+ inflammatory macrophages and ameliorates pathology in Alzheimer's disease mouse models. J Exp Med. 2015 Mar 9;212(3):287-95. Epub 2015 Mar 2 PubMed.