12 Jun 2020

Rare variants in TREM2 and PCLG2 influence a persons odds of developing Alzheimers disease, but that is far from all the two genes have in common. According to a study published June 8 in Nature Neuroscience, phospholipase C 2 acts downstream of TREM2 in a signaling pathway that supports critical microglial functions. Using human microglia derived from induced pluripotent stem cells, researchers led by Joseph Lewcock at Denali Therapeutics in South San Francisco reported that knocking out either gene product prevented the immune cells from efficiently processing lipids and neuronal debris. The researchers also found that, independently of TREM2, PLC2 is involved in a pro-inflammatory side hustle dictated by toll-like receptors, which, it so happens, is exacerbated by intracellular lipid build-up. Taken together, the findings strongly implicate faulty microglial lipid handling in the etiology of AD, and support therapeutic strategies that aim to rev up TREM2 signaling.

Using an impressive array of experimental conditions in gene-edited iPSC-microglia, [the authors] demonstrate that PLC2 is a downstream effector of TREM2 and a regulator of lipid metabolism. This exciting discovery directly connects PLC2 to well-established AD pathways involving APOE, TREM2, and microglial activation, commented Rik van der Kant, Vrije University, Amsterdam (full comment below). Florent Ginhoux of the Agency for Science, Technology and Research in Singapore, agreed. The study elegantly links TREM2 and PLC2 signaling pathways, and offers mechanistic insight into how variants in these genes affect the pathophysiology of AD, Ginhoux wrote (full comment below).

Double Dealing. When triggered by TREM2, PLC2 supports lipid metabolism and survival (left). When triggered by TLRs, PLC2 triggers inflammation. In TREM2 KO microglia (right), lipids accumulate and this exacerbates the pro-inflammatory, TLR-driven pathway. [Courtesy of Andreone et al., Nature Neuroscience, 2020.]

Since the discovery, in 2012, that rare variants in the coding region of TREM2 triple the risk of AD, researchers have pegged the receptor as supporting myriad microglial functions, including phagocytosis, walling off A plaques, and promoting an anti-inflammatory, neuroprotective environment (May 2016 news; Apr 2017 conference news;Jul 2018 conference news).

Separately, researchers discovered a rare variant in phospholipase C 2 (PLCG2) that protects against AD (Aug 2017 conference news on Sims et al., 2017). PLCs are a large family of intracellular enzymes that cleave the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3), a process that facilitates calcium signaling. In the brain, the 2 isoform is predominantly expressed by microglia, and initial studies suggest that the protective variant munches phospholipids with more gusto than the common one does (Zhang et al., 2014; May 2019 news).

Might the functions of TREM2 and PLC2 intersect in microglia? To study this question, co-first authors Benjamin Andreone and Laralynne Przybyla derived human microglia. They wove together elements from three recently developed protocols to coax so-called induced microglia (iMGs) from induced pluripotent stem cells (Muffat et al., 2016; Pandya et al., 2017; McQuade et al., 2018). They then used CRISPR to wipe out expression of TREM2 or PLCG2 in these cell-based models.

Under normal conditions, iMGs missing either TREM2 or PLCG2 appeared healthy and viable. When the going got toughi.e., when growth factors were depleted from the culture mediaboth types of knockout suffered a similar fate, dying sooner than their wild-type counterparts. The transcriptomes of each of the two iMG knockouts also differed from those of wild-type cells in similar ways. Specifically, half of the genes differentially expressed in TREM2 KO iMGs were similarly affected in PLCG KO iMGs. These common genes were part of signal transduction pathways downstream of DAP12, the adaptor protein that mediates TREM2 signaling. Using biochemical approaches, the researchers ultimately pieced together a signaling cascade by which lipids activate TREM2, leading to the phosphorylation of Syk2, which directly interacts with PLC2, unleashing its phospholipase activity and downstream signaling events.

Disabling the pathway, either by knocking out TREM2 or PLC2, had a dramatic impact on the processing of lipids, including cholesterol-laden myelin. All microglial lines in this study readily engulfed this type of fluorescently labeled debris; however, while wild-type cells had largely disposed of it after four days, TREM2 or PLCG2 knockouts were still chock-full of it by then. Tellingly, perhaps, the knockout cells failed to ramp up expression of several lipid processing genes in response to the myelin challenge.

Choking on Lipids? Wild-type microglia (left) readily digested lipids after treatment with myelin, while microglia lacking PLCG2 (middle) and TREM2 (right) accumulated the lipids. [Courtesy of Andreone et al., Nature Neuroscience, 2020.]

Lipidomics experiments revealed that the knockouts became burdened with a backlog of several subtypes of unprocessed lipid, including free cholesterol, cholesteryl esters, and myelin-derived ceramides. Similarly, in co-culture experiments with iPSC-derived neurons, both types of microglial knockout were unable to properly digest detritus from injured axons.

How might AD risk variants shift these phenotypes? The researchers generated iMGs that expressed the R47H variant of TREM2, or the protective P522R variant of PLCG2. As might be expected from prior findings on these variants, the R47H-TREM2 iMGs processed lipids more sluggishly than wild-type, whereas the P522R-PLCG2 microglia more deftly disposed of them than wild-type. Together, the findings support the idea that TREM2 and PLCG2 variants influence AD risk via lipid metabolism.

Lest a reader be tempted to tie a neat little bow on this set of results, here comes the twist: PLC2 also takes marching orders from toll-like receptors. This was previously reported in peripheral immune cells. The Denali researchers found the same in iMGs, as PLCG2 knockouts failed to mount a pro-inflammatory response to the TLR2 ligand zymosan.

Interestingly, the same pro-inflammatory cytokines that were down in response to zymosan in PLCG2 knockout iMGs were up in TREM2 knockout iMGs. For example, compared with wild-type iMGs treated with zymosan, PLCG2 knockouts secreted 50 percent less IL-1, while TREM2 knockouts secreted 64 percent more.

The same pattern emerged when the researchers used the TLR4 ligand LPS to trigger the microglial NLRP3 inflammasome, which itself has been tied to AD (Nov 2019 news). Loading up the microglia with myelin prior to triggering the inflammasome dramatically enhanced the inflammatory response in TREM2 KO iMGs, the scientists report. This implies that intracellular lipid accumulation may exacerbate damaging inflammatory pathways. The findings dovetail with those of a recent study that tied lipid droplet-accumulating microglia (LAM) in the aging hippocampus to neuroinflammation (Aug 2019 news).

Overall, the findings cast PLC2 as a two-faced player in microglia. When triggered via TREM2, this phospholipase facilitates processing of lipids and microglial survival. When tripped off by TLRs, it ramps up potentially damaging pro-inflammatory responses. And when lipids build up, as might occur in the aging brain, they exacerbate the pro-inflammatory pathway, Andreone told Alzforum. He believes the balance between these two PLC2 signaling pathways could dictate whether microglia help or harm.

The findings lend support to a therapeutic strategy of agonizing TREM2 signaling, Lewcock told Alzforum. That the protective PLC2 variant enhances lipid processing in microglia fits with the idea that even people whose TREM2 functions normally could stand to benefit from a boost in this pathway. Activating PLC2 is also a potential strategy, Lewcock said, although it would come with the risk of rousing its pro-inflammatory side. More work is needed to dissect how the PLC2 protective variant influences signaling downstream of TREM2 versus TLRs.

This is a very important paper, wrote Christian Haass at the German Center for Neurodegenerative Diseases in Munich. Haass noted that its findings fit with fresh data from his and other groups, but also cautioned that the molecular signature of a protective subpopulation of microglia needs to be defined in much greater detail (full comment below).

Denali is collaborating with Haass group to develop an activating antibody for TREM2, which will come with a blood-brain barrier transport vehicle to shuttle it into the brain (May 2019 conference news;May 2020 news).AL002, a TREM2-activating antibody developed by Alector and Abbvie, entered early clinical trials last year (see clinicaltrials.gov).Jessica Shugart

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Janus-Faced PCL2? Alzheimer's Risk Protein Toggles TREM2 and TLR Pathways - Alzforum