Protective AD Variant Pinpoints Sweet Spot for Microglial Activation

Microglia can help or hinder in Alzheimer’s disease. A protective variant of the microglial gene phospholipase C-γ2 (PLCG2) may shed light on what makes the difference. In the September 11 Molecular Neurodegeneration, researchers led by Mikko Hiltunen at the University of Eastern Finland, Kuopio, and Christian Haass at Ludwig-Maximilians University in Munich report that mice carrying the protective P522R PLCG2 variant accumulate more activated microglia as they age. These microglia boast a gene expression profile that overlaps with the previously described DAM, or disease-activated microglia. The mobilized cells appear sensitized to defend the brain, because they respond better to inflammatory stimuli than do microglia from wild-type mice. P522R PLCG2 cells also survive longer and gobble up particles more readily.

  • P522R PLCG2 variant reduces a person’s risk of AD.  
  • In P522R PLCG2 knock-in mice, more microglia activate with age than in wild-types.
  • The variant acts downstream of TREM2, boosting phagocytosis.

Notably, PLCG2 acts downstream of TREM2, a major AD risk gene. “That AD mutations from different genes fall into the same pathway tells us this pathway is causally involved, and that activating it could be therapeutic,” Haass told Alzforum. In future work, he will test this idea by crossing the knock-in mice with models of amyloidosis.

The P522R variant was found in especially long-lived people who had a low risk of AD, dementia with Lewy bodies, and frontotemporal dementia (Aug 2017 conference news; Sims et al., 2017; May 2019 news).  

Pathway to Watch. Plcg2 acts downstream of the TREM2 receptor to snip lipids, activating numerous signaling pathways and modulating gene transcription. [Courtesy of Takalo et al., Molecular Neurodegeneration.]

Recently, researchers led by Joseph Lewcock at Denali Therapeutics in South San Francisco used microglial cultures generated from iPS cells to show that TREM2 activation switched on PLCG2’s phospholipase activity, which in turn regulated lipid processing. The P522R variant greased phospholipase activity and lipid processing, while knocking out PLCG2 caused lipid debris to build up inside microglia (Jun 2020 news). 

To examine what the variant does in vivo, Haass and colleagues generated a P522R PLCG2 knock-in mouse. At one year of age, these mice had the same number of microglia, with the same morphology, as did wild-types. However, TSPO PET imaging showed that the knock-ins had more activated microglia in all brain regions. Microglia in the knock-ins also expressed more of the purinergic receptor, P2RY12, which mediates phagocytosis.

Gene expression analysis showed that, like DAMs, P522R PLCG2 knock-in microglia expressed more APOE, TYROBP, CCL3, CST7, and CLEC7A. However, unlike DAMs, P522R PLCG2 cells did not upregulate TREM2, ITGAX, or CD68 (Jun 2017 news). This indicates a partial overlap between the phenotypes. Because the P522R variant is protective, Haass believes this expression profile represents a signature of therapeutic microglia. Studying this signature could help guide anti-inflammatory treatment strategies, he said.

First author Mari Takalo, working in Kuopio, isolated peripheral macrophages from the knock-in mice. Compared to wild-type macrophages, the cells were less prone to apoptosis when Takalo withdrew growth factors. After an inflammatory stimulus, the knock-in cells released more of the proinflammatory cytokines TNFα, IL-6, and IL-1β, but not NO, which has apoptotic effects. The knock-ins were better phagocytes, swallowing about 50 percent more test particles than did wild-types. In a mouse microglial cell line, P522R PLCG2 had an even greater effect, doubling phagocytosis. The data are exactly the opposite of what Denali reported in PLCG2 knockout microglia.

“These findings are entirely consistent with the results from our recent manuscript,” Lewcock wrote to Alzforum (full comment below). “This study is the first to examine how the P522R variant impacts microglial activity in vivo, and provides additional data to substantiate the idea that this variant acts as a mild hypermorph.”

Marco Colonna and Yingyue Zhou at Washington University in St. Louis agreed that the results confirm that P522R acts as a gain-of-function mutation, but noted that the disease relevance is still unclear. “How the mutation affects microglial functions in a neurodegenerative condition, such as AD, remains an important question to be answered,” they wrote (full comment below).

Hiltunen said this is the next goal. In their knock-in/amyloidosis crosses, the researchers will examine whether the variant mitigates pathology as animals age. So far, the data suggest that P522R hits the sweet spot of boosting microglia activity just enough to be helpful. That is no foregone conclusion, however, because too much activation of the TREM2 pathway, as happens with progranulin mutations, can turn microglia into dangerous, proinflammatory monsters that trigger neurodegeneration (Apr 2019 news). 

Given this tricky balance, could boosting PLCG2 function ever be a therapeutic approach? Lewcock said finding direct activators for this enzyme would be challenging. In addition, activating PLCG2 would have broader effects than activating TREM2 alone, because PLCG2 also mediates pro-inflammatory signaling through Toll-like receptors.

Haass agrees that the best approach is to target TREM2, rather than PLCG2. “TREM2 is very selective, and that’s a big advantage,” he said.

Haass collaborates with Denali to develop a TREM2-activating antibody (May 2019 conference news). Alector has a TREM2 antibody in a Phase 1 trial.—Madolyn Bowman Rogers

Comments

  1. A P522R coding variant in the PLCγ2 gene was recently demonstrated to reduce the risk of Alzheimer’s disease (AD). This study by Takalo et al. is the first to examine how the P522R variant impacts microglial activity in vivo and it provides additional data to substantiate the idea that this variant acts as a mild hypermorph. Overall, it is a very interesting study that provides new insight into PLCγ2 function and how it is impacted by AD-relevant genetic variants.

    The authors begin by showing that macrophages or BV2 cells expressing the P522R variant demonstrate improved survival and increased phagocytosis. They go on to examine gene expression, microglial number/morphology, and other phenotypes in homozygous P522R mice. While no changes in microglial number were observed, there was an increase in signal for F18-FEPPA, a TSPO PET tracer, in 1-year-old P522R animals suggesting an increase in activated microglia. The exact nature of this activation will require further study, though the protective effect of this variant on disease risks suggests it is likely to be beneficial rather than deleterious.

    These findings are entirely consistent with the results from our recent manuscript (Andreone et al., 2020) examining the impact of PLCG2 and TREM2 activity in human iPS-derived microglia (iMG). In our work, PLCG2 or TREM2 knockout iMG demonstrate the opposite phenotypes as well as defects in lipid metabolism, while the P522R mutants displayed improved clearance of phagocytosed lipids. Taken together, these findings make a strong argument that PLCG2 signals downstream of TREM2 in microglia to regulate a range of microglial functions.

    Takalo et al. also observed elevated cytokine production in cells expressing the P522R variant of PLCG2 following LPS treatment. This is perhaps not surprising as PLCG2 is known to signal downstream of a range of cell surface receptors, including TLRs, in other immune cell types. This has been a consistent finding as well, and we found that PLCG2-null iMG display reduced cytokine production. The findings from this study of P522R mutants are particularly important, as they imply  that there is not something qualitatively different about the P522R mutation in PLCG2 as compared to other mutations that are known to cause inflammatory disorders. Therefore, the lack of detrimental phenotypes observed in P522R is likely a result of the mutation having less of an impact on enzymatic activity rather than impacting the TREM2 signaling axis selectively.

    From a therapeutic perspective, this suggests that targeting PLCG2 directly through increasing its activity may more broadly impact microglial function than would the recently reported TREM2 agonist antibodies. Based on the results shown in this study, the effects may include increased pro-inflammatory cytokine production, but confirmation of this, as well as the impact on disease, will require further study.

    References:

    Andreone BJ, Przybyla L, Llapashtica C, Rana A, Davis SS, van Lengerich B, Lin K, Shi J, Mei Y, Astarita G, Di Paolo G, Sandmann T, Monroe KM, Lewcock JW. Alzheimer's-associated PLCγ2 is a signaling node required for both TREM2 function and the inflammatory response in human microglia. Nat Neurosci. 2020 Aug;23(8):927-938. Epub 2020 Jun 8 PubMed.

    View all comments by Joseph Lewcock

  2. Takalo et al. present an interesting paper on an AD-associated protective variant in PLCG2 by making a Plcγ2-P522R knock-in (KI) mouse model. The authors showed that the Plcγ2-P522R mutation increased PLCγ signaling and promoted phagocytosis, survival, and pro-inflammatory responses to lipopolysaccharide and interferon-γ in bone-marrow-derived macrophages. No obvious morphological changes were observed in KI microglia in vivo, but increased TSPO PET signal in KI microglia indicated their mild activation.

    In addition, some disease-associated microglia transcripts were slightly more expressed in KI microglia versus controls, suggesting a potential beneficial effect of the P522R mutation in AD models.

    This is a nice short report proving P522R as a gain-of-function mutation of PLCg and serves as a great complement to the recent paper by Andreone et al. (Andreone et al., 2020). How the mutation affects microglial functions in a neurodegenerative condition, such as AD, is yet to be determined and remains an important question to be answered.

    References:

    Andreone BJ, Przybyla L, Llapashtica C, Rana A, Davis SS, van Lengerich B, Lin K, Shi J, Mei Y, Astarita G, Di Paolo G, Sandmann T, Monroe KM, Lewcock JW. Alzheimer's-associated PLCγ2 is a signaling node required for both TREM2 function and the inflammatory response in human microglia. Nat Neurosci. 2020 Aug;23(8):927-938. Epub 2020 Jun 8 PubMed.

    View all comments by Yingyue Zhou View all comments by Marco Colonna

  3. This is a very nice example of functional follow-up of a genetic association study finding.

    I am very happy that this is taking place (it’s why we are performing the studies) and of course I am pleased it looks like the genetic association studies were right in suggesting that this is a pathway that potentially would modulate and protect for more than AD.

    I do miss the investigation of the previously suggested link to the NLRP3 inflammasome.

    View all comments by Sven van der Lee

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References

News Citations

  1. Searching for New AD Risk Variants? Move Beyond GWAS
  2. The Mutation You Want: It Protects the Brain, Extends Life
  3. Janus-Faced PLCγ2? Alzheimer’s Risk Protein Toggles TREM2 and TLR Pathways
  4. Hot DAM: Specific Microglia Engulf Plaques
  5. Parsing How Alzheimer’s Genetic Risk Works Through Microglia
  6. Antibodies Against Microglial Receptors TREM2 and CD33 Head to Trials

Paper Citations

  1. Sims R, van der Lee SJ, Naj AC, Bellenguez C, Badarinarayan N, Jakobsdottir J, Kunkle BW, Boland A, Raybould R, Bis JC, Martin ER, Grenier-Boley B, Heilmann-Heimbach S, Chouraki V, Kuzma AB, Sleegers K, Vronskaya M, Ruiz A, Graham RR, Olaso R, Hoffmann P, Grove ML, Vardarajan BN, Hiltunen M, Nöthen MM, White CC, Hamilton-Nelson KL, Epelbaum J, Maier W, Choi SH, Beecham GW, Dulary C, Herms S, Smith AV, Funk CC, Derbois C, Forstner AJ, Ahmad S, Li H, Bacq D, Harold D, Satizabal CL, Valladares O, Squassina A, Thomas R, Brody JA, Qu L, Sánchez-Juan P, Morgan T, Wolters FJ, Zhao Y, Garcia FS, Denning N, Fornage M, Malamon J, Naranjo MC, Majounie E, Mosley TH, Dombroski B, Wallon D, Lupton MK, Dupuis J, Whitehead P, Fratiglioni L, Medway C, Jian X, Mukherjee S, Keller L, Brown K, Lin H, Cantwell LB, Panza F, McGuinness B, Moreno-Grau S, Burgess JD, Solfrizzi V, Proitsi P, Adams HH, Allen M, Seripa D, Pastor P, Cupples LA, Price ND, Hannequin D, Frank-García A, Levy D, Chakrabarty P, Caffarra P, Giegling I, Beiser AS, Giedraitis V, Hampel H, Garcia ME, Wang X, Lannfelt L, Mecocci P, Eiriksdottir G, Crane PK, Pasquier F, Boccardi V, Henández I, Barber RC, Scherer M, Tarraga L, Adams PM, Leber M, Chen Y, Albert MS, Riedel-Heller S, Emilsson V, Beekly D, Braae A, Schmidt R, Blacker D, Masullo C, Schmidt H, Doody RS, Spalletta G, Jr WT, Fairchild TJ, Bossù P, Lopez OL, Frosch MP, Sacchinelli E, Ghetti B, Yang Q, Huebinger RM, Jessen F, Li S, Kamboh MI, Morris J, Sotolongo-Grau O, Katz MJ, Corcoran C, Dunstan M, Braddel A, Thomas C, Meggy A, Marshall R, Gerrish A, Chapman J, Aguilar M, Taylor S, Hill M, Fairén MD, Hodges A, Vellas B, Soininen H, Kloszewska I, Daniilidou M, Uphill J, Patel Y, Hughes JT, Lord J, Turton J, Hartmann AM, Cecchetti R, Fenoglio C, Serpente M, Arcaro M, Caltagirone C, Orfei MD, Ciaramella A, Pichler S, Mayhaus M, Gu W, Lleó A, Fortea J, Blesa R, Barber IS, Brookes K, Cupidi C, Maletta RG, Carrell D, Sorbi S, Moebus S, Urbano M, Pilotto A, Kornhuber J, Bosco P, Todd S, Craig D, Johnston J, Gill M, Lawlor B, Lynch A, Fox NC, Hardy J, ARUK Consortium, Albin RL, Apostolova LG, Arnold SE, Asthana S, Atwood CS, Baldwin CT, Barnes LL, Barral S, Beach TG, Becker JT, Bigio EH, Bird TD, Boeve BF, Bowen JD, Boxer A, Burke JR, Burns JM, Buxbaum JD, Cairns NJ, Cao C, Carlson CS, Carlsson CM, Carney RM, Carrasquillo MM, Carroll SL, Diaz CC, Chui HC, Clark DG, Cribbs DH, Crocco EA, DeCarli C, Dick M, Duara R, Evans DA, Faber KM, Fallon KB, Fardo DW, Farlow MR, Ferris S, Foroud TM, Galasko DR, Gearing M, Geschwind DH, Gilbert JR, Graff-Radford NR, Green RC, Growdon JH, Hamilton RL, Harrell LE, Honig LS, Huentelman MJ, Hulette CM, Hyman BT, Jarvik GP, Abner E, Jin LW, Jun G, Karydas A, Kaye JA, Kim R, Kowall NW, Kramer JH, LaFerla FM, Lah JJ, Leverenz JB, Levey AI, Li G, Lieberman AP, Lunetta KL, Lyketsos CG, Marson DC, Martiniuk F, Mash DC, Masliah E, McCormick WC, McCurry SM, McDavid AN, McKee AC, Mesulam M, Miller BL, Miller CA, Miller JW, Morris JC, Murrell JR, Myers AJ, O'Bryant S, Olichney JM, Pankratz VS, Parisi JE, Paulson HL, Perry W, Peskind E, Pierce A, Poon WW, Potter H, Quinn JF, Raj A, Raskind M, Reisberg B, Reitz C, Ringman JM, Roberson ED, Rogaeva E, Rosen HJ, Rosenberg RN, Sager MA, Saykin AJ, Schneider JA, Schneider LS, Seeley WW, Smith AG, Sonnen JA, Spina S, Stern RA, Swerdlow RH, Tanzi RE, Thornton-Wells TA, Trojanowski JQ, Troncoso JC, Van Deerlin VM, Van Eldik LJ, Vinters HV, Vonsattel JP, Weintraub S, Welsh-Bohmer KA, Wilhelmsen KC, Williamson J, Wingo TS, Woltjer RL, Wright CB, Yu CE, Yu L, Garzia F, Golamaully F, Septier G, Engelborghs S, Vandenberghe R, De Deyn PP, Fernadez CM, Benito YA, Thonberg H, Forsell C, Lilius L, Kinhult-Stählbom A, Kilander L, Brundin R, Concari L, Helisalmi S, Koivisto AM, Haapasalo A, Dermecourt V, Fievet N, Hanon O, Dufouil C, Brice A, Ritchie K, Dubois B, Himali JJ, Keene CD, Tschanz J, Fitzpatrick AL, Kukull WA, Norton M, Aspelund T, Larson EB, Munger R, Rotter JI, Lipton RB, Bullido MJ, Hofman A, Montine TJ, Coto E, Boerwinkle E, Petersen RC, Alvarez V, Rivadeneira F, Reiman EM, Gallo M, O'Donnell CJ, Reisch JS, Bruni AC, Royall DR, Dichgans M, Sano M, Galimberti D, St George-Hyslop P, Scarpini E, Tsuang DW, Mancuso M, Bonuccelli U, Winslow AR, Daniele A, Wu CK, GERAD/PERADES, CHARGE, ADGC, EADI, Peters O, Nacmias B, Riemenschneider M, Heun R, Brayne C, Rubinsztein DC, Bras J, Guerreiro R, Al-Chalabi A, Shaw CE, Collinge J, Mann D, Tsolaki M, Clarimón J, Sussams R, Lovestone S, O'Donovan MC, Owen MJ, Behrens TW, Mead S, Goate AM, Uitterlinden AG, Holmes C, Cruchaga C, Ingelsson M, Bennett DA, Powell J, Golde TE, Graff C, De Jager PL, Morgan K, Ertekin-Taner N, Combarros O, Psaty BM, Passmore P, Younkin SG, Berr C, Gudnason V, Rujescu D, Dickson DW, Dartigues JF, DeStefano AL, Ortega-Cubero S, Hakonarson H, Campion D, Boada M, Kauwe JK, Farrer LA, Van Broeckhoven C, Ikram MA, Jones L, Haines JL, Tzourio C, Launer LJ, Escott-Price V, Mayeux R, Deleuze JF, Amin N, Holmans PA, Pericak-Vance MA, Amouyel P, van Duijn CM, Ramirez A, Wang LS, Lambert JC, Seshadri S, Williams J, Schellenberg GD. Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease. Nat Genet. 2017 Sep;49(9):1373-1384. Epub 2017 Jul 17 PubMed.

External Citations

  1. Phase 1 

Further Reading

Primary Papers

  1. Takalo M, Wittrahm R, Wefers B, Parhizkar S, Jokivarsi K, Kuulasmaa T, Mäkinen P, Martiskainen H, Wurst W, Xiang X, Marttinen M, Poutiainen P, Haapasalo A, Hiltunen M, Haass C. The Alzheimer's disease-associated protective Plcγ2-P522R variant promotes immune functions. Mol Neurodegener. 2020 Sep 11;15(1):52. PubMed.

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