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. 2023 Apr;71(4):974-990.
doi: 10.1002/glia.24318. Epub 2022 Dec 8.

Alzheimer's disease-associated R47H TREM2 increases, but wild-type TREM2 decreases, microglial phagocytosis of synaptosomes and neuronal loss

Affiliations

Alzheimer's disease-associated R47H TREM2 increases, but wild-type TREM2 decreases, microglial phagocytosis of synaptosomes and neuronal loss

Alma S Popescu et al. Glia. 2023 Apr.

Abstract

Triggering receptor on myeloid cells 2 (TREM2) is an innate immune receptor, upregulated on the surface of microglia associated with amyloid plaques in Alzheimer's disease (AD). Individuals heterozygous for the R47H variant of TREM2 have greatly increased risk of developing AD. We examined the effects of wild-type (WT), R47H and knock-out (KO) of human TREM2 expression in three microglial cell systems. Addition of mouse BV-2 microglia expressing R47H TREM2 to primary mouse neuronal cultures caused neuronal loss, not observed with WT TREM2. Neuronal loss was prevented by using annexin V to block exposed phosphatidylserine, an eat-me signal and ligand of TREM2, suggesting loss was mediated by microglial phagocytosis of neurons exposing phosphatidylserine. Addition of human CHME-3 microglia expressing R47H TREM2 to LUHMES neuronal-like cells also caused loss compared to WT TREM2. Expression of R47H TREM2 in BV-2 and CHME-3 microglia increased their uptake of phosphatidylserine-beads and synaptosomes versus WT TREM2. Human iPSC-derived microglia with heterozygous R47H TREM2 had increased phagocytosis of synaptosomes vs common-variant TREM2. Additionally, phosphatidylserine liposomes increased activation of human iPSC-derived microglia expressing homozygous R47H TREM2 versus common-variant TREM2. Finally, overexpression of TREM2 in CHME-3 microglia caused increased expression of cystatin F, a cysteine protease inhibitor, and knock-down of cystatin F increased CHME-3 uptake of phosphatidylserine-beads. Together, these data suggest that R47H TREM2 may increase AD risk by increasing phagocytosis of synapses and neurons via greater activation by phosphatidylserine and that WT TREM2 may decrease microglial phagocytosis of synapses and neurons via cystatin F.

Keywords: Alzheimer's disease; R47H variant; TREM2; cystatin F; iPSC-derived microglia; phosphatidylserine; synapses.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Overexpression of R47H hTREM2 increases BV‐2 phagocytic uptake of phosphatidylserine beads and synaptosomes compared with WT hTREM2 and TREM2 KO. (a, b, c) Microscopy images of BV‐2 microglia expressing eGFP (green) co‐cultured with (a) Carboxylated beads (red). (b) Rhodamine‐phosphatidylserine beads (red) and (c) pH‐rodo labeled synaptosomes (red). (d, e, f) Phagocytic uptake of three phagocytic targets was measured using flow cytometry. (d) Phagocytic uptake of sky‐blue 5 um carboxyl‐latex beads analyzed after 2 h co‐culture and shown as % uptake. (e) Rhodamine labeled phosphatidylserine beads analyzed after 1.5 h co‐culture shown as % uptake (f) synaptosomes isolated from rat and stained with pH‐rodo analyzed after 1.5 h co‐culture and shown as mean fluorescence intensity (MFI). (d, e, f) Cells were pre‐treated with the phagocytosis inhibitor cytochalasin D (CytoD) as a negative control. The CytoD negative control values have been removed from the data points presented. Each data point is the mean result from an independent experiment with three technical repeats. Co‐localization of phagocytic target in red with green BV‐2 cells shows target that has most likely been phagocytosed. Phagocytosis was quantified using flow‐cytometry and appropriate controls. Paired data points from the same experiment are represented by the same shape and color within a figure. Error bars are SEM and Tukey's multiple comparisons one‐way ANOVA was performed (d) KO Control versus WT hTREM2 **P = .0038, KO Control versus R47H hTREM2 **P = .0066 (e) KO Control versus R47H hTREM2 **P = .0066, WT hTREM2 versus R47H hTREM2 *P = .0333, KO Control versus WT hTREM2 ns = 0.3663 (f) KO Control versus R47H hTREM2 *P = .0127, WT hTREM2 versus R47H hTREM2 *P = .0209, KO Control versus WT hTREM2 ns = 0.9352.
FIGURE 2
FIGURE 2
Co‐culturing primary cerebellar mouse neuronal‐glial co‐cultures with KO Control or R47H hTREM2 BV‐2 microglia induces significant neuronal loss after 24 h which is rescued by treatment with annexin‐V, an inhibitor of the eat‐me signal phosphatidylserine. Neuronal loss is not observed when co‐cultured with WT hTREM2 BV‐2 microglia. Primary neuronal‐glial co‐cultures, primarily consisting of neurons, were prepared from mouse cerebellum and seeded at 250,000 cells/well. 50,000 BV‐2 microglia (KO Control, WT hTREM2 or R47H hTREM2) or media control were added to these mixed primary neuronal‐glial cultures and left for 24 h ± 100 nM annexin V, an inhibitor of phosphatidylserine. After 24 h, cells were stained and imaged and density of microglia and of necrotic, apoptotic and healthy neurons was counted. (a–i) Representative images taken after 24 h of neuronal‐glial cerebellar cultures from mouse being (a, f) treated with media only control (b, g) co‐cultured with KO Control BV‐2 microglia (c, h) co‐cultured with WT hTREM2 BV‐2 microglia (d, i) co‐cultured with R47H hTREM2 BV‐2 microglia. (f–i) were treated with 100 nM annexin V immediately before addition of BV‐2 microglia. (a–i) Images show primary microglia (IB4, green), BV‐2 microglia (eGFP, green) necrotic cells (PI, red) and nuclei (Hoechst, blue) staining. Neurons were distinguished from astrocytes and microglia through their nuclear morphology and absence of IB4 staining. (e, j) Percentage of neuronal loss, which refers to loss of healthy neurons. The numbers of necrotic & apoptotic cells did not vary between conditions and were very low, less than 1% of cell population (data not shown). Data shown is from three biological repeats with each data point representing the mean result from one independent experiment. Each independent experiment was made up of three technical repeats and four 20x images were analyzed per technical repeat. Error bars are SEM and Tukey's multiple comparisons one‐way ANOVA was performed on raw, un‐normalized values for neuronal counts. “Media only” versus “+ KO Control BV‐2” **P = .0070, “media only” versus “+ R47H hTREM2 BV‐2” **P = .0072, “+ KO Control BV‐2” versus “+ WT hTREM2 BV‐2” *P = .0396, “+ WT hTREM2 BV‐2” versus “+ R47H hTREM2 BV‐2” *P = .0414.
FIGURE 3
FIGURE 3
Overexpression of WT TREM2 in CHME‐3 cells reduces phagocytosis of synaptosomes and phosphatidylserine coated beads. Phagocytosis of (a) Synaptosomes isolated from rat and stained with pH‐rodo analyzed after 1 h of treatment and shown as mean fluorescence intensity (MFI) and (B) rhodamine‐labeled phosphatidylserine‐coated beads analyzed after 1 h of treatment and shown as MFI. Overexpression of WT TREM2 decreased uptake of both targets compared to control. R47H expression also decreased uptake of synaptosomes and phosphatidylserine‐coated beads but did so less than WT TREM2. Repeats done on the same day with the different cell lines are depicted by the same color and symbols. (a & b) N = 3, statistics: repeated measures, one way ANOVA, followed by Tukey's post hoc test.*p < .05, **p < .01.
FIGURE 4
FIGURE 4
TREM2 induces an increase in CST7 mRNA expression and knock down of CST7 increases phosphatidylserine coated bead uptake in TREM2 overexpressing CHME‐3 cells. (a) Data is expressed as fold change over non‐target control and all samples were normalized to 18 S rRNA housekeeping gene. CST7 mRNA level is increased when TREM2 is present and when treated with RNAi against CST7, CST7 is successfully reduced in all cell lines by at least 50%. RNA was extracted from CHME‐3 microglia 48 h after treatment with non‐target (scrambled) or CST7 targeting RNAi. N = 5. Repeats done on the same day with the three variant cell lines are depicted by the same color and symbols. Statistics: repeated measures, one way ANOVA with Tukey's post hoc test. **p < .01, ***p < .001. (b) Uptake of rhodamine‐labeled, phosphatidylserine‐coated 3 micron beads by control, WT and R47H TREM2 overexpressing CHME‐3 cells 48 h after treatment with non‐target (scrambled) or CST7 targeting RNAi. Phagocytosis was analyzed after 1 h treatment of CHME‐3 cells with phosphatidylserine‐coated beads. N = 5. Statistics: repeated measures, one way ANOVA with Tukey's post hoc test. * p < .05, *** p < .001.
FIGURE 5
FIGURE 5
Overexpression of R47H TREM2 mutant in CHME‐3 microglia leads to increased neuron (LUHMES) loss in co‐culture with neurons. (a) Image of differentiated LUHMES cells. (b) Image of CHME‐3 cells. (c) Image of LUHMES (blue) and CHME‐3 (green) co‐culture. (d) Percentage neuron loss in LUHMES:CHME‐3 (100,000: 100,000) co‐culture after treatment ±2 μM Aβ for 48 h. Percentage loss is compared to neuron only control. Number of neurons was identified via blue CellTrace Violet staining and counted using automated software. N = 4. Statistics: two‐way ANOVA, with Sidak's post hoc test. *p < .05, ***p < .001, ****p < .0001.
FIGURE 6
FIGURE 6
Heterozygous R47H expression in microglia‐like iPSCs results in increased uptake of rat cortical synaptosomes. (a) Uptake of pH‐Rodo stained synaptosomes by control (BIONi010‐C) and heterozygous R47H (ADRC8.6 and 26.15) microglia‐like hiPSCs. R47H heterozygous derived hiPSCs increased uptake of synaptosomes compared to control line. Data is normalized to mean of control and has had cytochalasin D control subtracted to show active phagocytosis. N = 6 of control (BIONi010‐C), from two different differentiations and N = 3 from both ADRC8.6 and 26.15 (R47Hhet clones). Repeats done on the same day with the different cell lines are depicted by the same color and symbols. Statistics: unpaired student's t‐test. ***p < .001. (b) Representative flow plots of pH‐Rodo stained synaptosomes by control and R47H heterozygous microglia‐like hiPSCs. Black histogram = untreated cells (no cargo), red histogram = cells with cargo (Ph‐Rodo stained synaptosomes) and blue histogram = cells with cargo +1 h pre‐treatment with 10uM cytochalasin D.
FIGURE 7
FIGURE 7
pSYK activation by liposomes & antibodies in hiPSC‐derived microglia. hiPSC‐derived microglia expressing homozygous wild‐type or R47H TREM2 were exposed to the indicated concentrations of (a) phosphatidylserine‐containing liposomes, (b) phosphatidylserine‐containing liposomes or (c) anti‐TREM2 antibodies, and phosphorylated SYK (pSYK) measured by alphaLISA, 2 min after adding liposomes or 5 min after adding antibodies. pSYK is normalized to the level before addition of liposomes or antibodies. The experiment was repeated on 3 separate occasions, of which means and SEM are represented. Significant differences between WT and R47H TREM2 expressing cells were tested by unpaired t test at each concentration, *p < .05.

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