Different variants in the gene encoding phospholipase C-γ2—an enzyme expressed in microglia—can either raise the risk of AD, or protect against it. According to a study published September 12 in Immunity, the respective variants shape how microglia respond to Aβ plaques. Scientists led by Gary Landreth at Indiana University School of Medicine in Indianapolis report that in a mouse model of amyloidosis, microglia expressing a protective PLCγ2 variant rallied around plaques and compacted them, sparing synapses and memory loss. In mice expressing an AD risk-raising variant, microglia stalled in their transition to a neuroprotective state, mounting but a sluggish response to plaques. Overall, the findings suggest that PLCγ2 bolsters the way microglia manage amyloid.
- The M28L variant in PLCγ2 worsens plaque load, synaptic dysfunction, and memory loss.
- The protective P522R variant does the opposite.
- PLCγ2 signaling promotes microglial transition to a neuroprotective state.
“While mutations in PLCG2 have been extensively linked to late-onset AD risk, little is known about the in vivo functions of PLCγ2 in neurobiology or AD pathoetiology,” wrote John Lukens of the University of Virginia School of Medicine (comment below). “This study helps to bridge this gap.”
Expressed by microglia in the brain, and by various immune cells in the blood, PLCγ2 serves as a signaling hub for immune receptors, including the microglial cell-surface receptor, TREM2. Upon activation, the lipase cleaves its substrate, phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2), forming diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). This triggers intracellular calcium release and its numerous signaling cascades.
In 2017, scientists discovered a rare variant in the gene—P522R—that protected against AD, and later, that it protects against related diseases and even extends lifespan (Aug 2017 conference news; May 2019 news). The P522R version of PLCG2 reportedly cuts lipid substrate with more gusto than does the wild-type lipase, readying microglia to respond with great vigor when needed (Sep 2020 news).
How does PLCγ2 activity influence AD pathogenesis? To get a better idea, first author Andy Tsai and colleagues reanalyzed a genome-wide association study and sequencing data from several European-American cohorts to hunt for more PLCG2 variants (Mar 2019 news; Olive et al., 2020). In both studies, they identified a new one—M28L—that slightly elevated AD risk, although the association only reached statistical significance in the GWAS. Still, taking advantage of these opposing variants, the researchers used CRISPR to edit the protective PLCγ2-P522R or the risk-raising PLCγ2-M28L into mice. They crossed these mice onto a 5xFAD background to study how the variants influenced amyloidosis.
Relative to levels in 5xFAD mice, neither mutation changed how much PLCγ2 mRNA there was in the cortex. However, PLCγ2-M28L protein level was about half that of the wild-type, suggesting that, somehow, the M28L mutation dampened translation or stability of PLCγ2. The underlying mechanism is unknown.
The PLCγ2 variants had opposite effects on plaque load and density. While M28L boosted plaque load by 45 percent, P522R roughly halved load relative to 5xFAD controls. PLCγ2 M28L increased the proportion of diffuse plaques, while PLCγ2 P522R shifted the balance toward compact aggregates. The findings mesh with studies linking TREM2 signaling to plaque compaction, and support the notion that diffuse plaques are more toxic than their compacted counterparts (May 2016 news; Apr 2021 news).
The variants influenced microglia differently. While the risk variant hobbled their response to plaques, the protective variant rallied them to the scene. Once there, the PLCγ2-P522R-expressing microglia appeared to glom onto amyloid with more appetite than their counterparts in wild-type mice (image below).
Slack versus Mobilized. Microglia (green) surround plaques (blue) in 5xFAD mice expressing different variants of PLCG2. The M28L risk variant (middle) reduces this recruitment, while the protective P522R variant (right) ramps it up. [Courtesy of Andy Tsai.]
The findings suggested that PLCγ2 activation supports microglial containment of Aβ plaques. How so? To investigate, the scientists conducted a slew of gene-expression studies. In one, single-nuclei RNA sequencing unearthed five distinct transcriptional clusters of microglia across the control and PLC-variant 5xFAD mice. They included microglia in homeostatic, transitioning, activated A, activated B, and IFN-responsive states. Both activated clusters expressed a DAM-like profile, with the A group upregulating genes involved in endocytosis and inflammation, such as Itgax, Cd9, and Axl, and the B group ramping up expression of genes involved in apoptosis, lipid metabolism, and plaque compaction, including Lpl, ApoE, Lgals3, and Lilr4b. Relative to controls, PLCγ2-M28L mice had more microglia in the homeostatic and transitioning states, and fewer microglia in the activated states. The opposite was true for PLCγ2-P522R mice, in which more microglia had fully transitioned into the active states. The findings suggested PLCγ2 activity greases the wheels for microglial mobilization.
Ultimately, neurons reaped the benefits of stepped-up PLCγ2 activation. PLCγ2 P522R prevented memory problems, and it restored multiple measures of synaptic function to wild-type levels. Mice expressing the M28L protein fared no better on these measures, but also no worse, than controls.
Tsai considers making PLCg2 more active an attractive therapeutic strategy for AD and other neurodegenerative diseases. So does Mikko Hiltunen of the University of Eastern Finland in Kuopio. Hiltunen was impressed by the rigor of the study. Previously he reported that in P522R knock-in mice, microglia had transitioned into a state of apparent readiness—expressing a mix of homeostatic and DAM signature genes—even without amyloid around. The new findings in plaque-ravaged mice confirm that the protective variant had indeed rendered the microglia more responsive, Hiltunen said.
Still, he cautioned that too much activation of PLCγ2 can be deleterious. Rare mutations that constitutively activate the lipase cause immunodeficiency and autoimmune disorders (Ombrello et al., 2012). Because TREM2 signaling activates PLCγ2, activating the receptor signaling with antibodies is another way to promote PLCγ2 signaling. Led by UEFs Mari Takalo, Hiltunen's team is investigating how TREM2 and other immune receptors influence the protective effect of the P522R variant (Jul 2023 news).
Kathryn Monroe of Denali Therapeutics, San Francisco, discovered that PLCG2 signaling is required for the protective effects of TREM2. She said the new findings support that idea (Jun 2020 news). “However, because PLCG2 signals downstream of several immune receptors, it is possible that other signaling pathways contribute,” she added. “Either way, this data supports an exciting and novel therapeutic hypothesis to mildly agonize PLCG2 directly, based on the functional implications of the human genetic AD variants,” Monroe and Joe Lewcock wrote to Alzforum. Denali’s DNL919 has recently completed a phase 1 clinical study, but has been discontinued (8 Aug press release). Other TREM2 antibodies by Alector and Vigil Neuroscience are in clinical trials. —Jessica Shugart
- Searching for New AD Risk Variants? Move Beyond GWAS
- The Mutation You Want: It Protects the Brain, Extends Life
- Protective AD Variant Pinpoints Sweet Spot for Microglial Activation
- Paper Alerts: Massive GWAS Studies Published
- Barrier Function: TREM2 Helps Microglia to Compact Amyloid Plaques
- Microglia Build Plaques to Protect the Brain
- Brain Biopsies and FinnGen Form Wellspring for Functional Genomics
- Janus-Faced PLCγ2? Alzheimer’s Risk Protein Toggles TREM2 and TLR Pathways
Research Models Citations
- Olive C, Ibanez L, Farias FH, Wang F, Budde JP, Norton JB, Gentsch J, Morris JC, Li Z, Dube U, Del-Aguila J, Bergmann K, Bradley J, Benitez BA, Harari O, Fagan A, Ances B, Cruchaga C, Fernandez MV. Examination of the Effect of Rare Variants in TREM2, ABI3, and PLCG2 in LOAD Through Multiple Phenotypes. J Alzheimers Dis. 2020;77(4):1469-1482. PubMed.
- Ombrello MJ, Remmers EF, Sun G, Freeman AF, Datta S, Torabi-Parizi P, Subramanian N, Bunney TD, Baxendale RW, Martins MS, Romberg N, Komarow H, Aksentijevich I, Kim HS, Ho J, Cruse G, Jung MY, Gilfillan AM, Metcalfe DD, Nelson C, O'Brien M, Wisch L, Stone K, Douek DC, Gandhi C, Wanderer AA, Lee H, Nelson SF, Shianna KV, Cirulli ET, Goldstein DB, Long EO, Moir S, Meffre E, Holland SM, Kastner DL, Katan M, Hoffman HM, Milner JD. Cold urticaria, immunodeficiency, and autoimmunity related to PLCG2 deletions. N Engl J Med. 2012 Jan 26;366(4):330-8. Epub 2012 Jan 11 PubMed.
No Available Further Reading
- Tsai AP, Dong C, Lin PB, Oblak AL, Viana Di Prisco G, Wang N, Hajicek N, Carr AJ, Lendy EK, Hahn O, Atkins M, Foltz AG, Patel J, Xu G, Moutinho M, Sondek J, Zhang Q, Mesecar AD, Liu Y, Atwood BK, Wyss-Coray T, Nho K, Bissel SJ, Lamb BT, Landreth GE. Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer's disease. Immunity. 2023 Sep 12;56(9):2121-2136.e6. Epub 2023 Sep 1 PubMed.