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Additional file 1: Figure S1. Characterization of TREM2-sol in bone-marrow derived macrophages, brain and primary microglia. a Flow cytometry analysis of murine intracellular TREM2 in BMDM. MFI: median fluorescence intensity. Cells were either untreated or treated with the sheddase inhibitor DCP333 (5 µM overnight, DPC) or the sheddase activator PMA (50 ng/ml for 30 min). The gate for TREM2 positive cells was set based on the isotype control (threshold 0.2% positive cells in the isotype control). The dotted line marks the background signal which is observed in TREM2-KO. b Mouse Trem2 qRT-PCR of total RNA from WT and TREM2-sol brains (age 12 months). Normalized to Gapdh, 2^-ΔΔCt = 2^-(ΔCtTrem2-AvgΔCtControl/). n = 6 per group. Statistics: unpaired t test two-tailed. **: p < 0.01. c MIP1-β analysis in supernatants from BMDM for each genotype data in triplicates (n = 3) at days in vitro (DIV) 7. d In vitro phagocytosis of primary microglia over 24 h with 10 µg pHrodo-myelin per well. The WT AUC of the integrated fluorescence intensities was set to 100%. All genotypes were compared to the other three, and all significant differences are displayed. In both experiments, data for each genotype data were generated in triplicates (n = 3). Fluorescence measurements in wells without prey were used as controls (data not shown). Statistics: one-way ANOVA test with Tukey`s multiple comparisons test (***: p < 0.001, ****: p < 0.0001). Figure S2. T2-weighted MRI signal analysis in the CC region for the 5-week cuprizone model in WT, TREK2-KO, TREM2-sol and TREM2-IPD mice. Groups consisted of mice treated for 5 weeks with control food (normal food) or 0.2% cuprizone in food and then switched back to control food (normal food) for the 4-week recovery. MRI measurements were performed at week 0 (baseline), week 3 and week 5 of cuprizone intoxication, at week 7 (2 weeks of recovery on control food) and at week 9 (4 weeks of recovery on control food). Mice were culled at week 9 immediately after the last MRI measurement. EC: external capsule, CC: corpus callosum. Group sizes: n = 7–9 for all genotypes and at all timepoint. Statistics: ANOVA with random effects comparisons indicated significant differences with respect to WT mice: *: p < 0.05, ****: p < 0.0001. For each group examined, T2-weighted signals were significantly increased with respect to baseline values (significances not shown). Data are shown as means ± SEM. wt1 is the respective wild-type group for the study with TREM2-IPD and TREM2-sol, wt2 is the wild-type group for the TREM2-KO study. Figure S3. MTR analyses in the combined external capsule (EC) and corpus callosum (CC) region for the 5-week cuprizone and 4-week recovery model. Data (means ± SEM) presented as relative to baseline values for each genotype. Group sizes: n = 14 until week 5, n = 7 until week 9 for all genotypes. Statistics: ANOVA with random effects. The levels of significance ###: p < 0.001 correspond to comparisons betweenTREM2-KO and WT animals at the specified timepoints. WT: wild-type, TREM2-KO: TREM2 knockout. Figure S4. MRI analyses in external capsule (EC) and corpus callosum (CC) of TREM2-IPD and TREM2-sol mice for the 5-week cuprizone and 4-week recovery model. Data presented as means ± SEM. Group sizes: n = 7 for each genotype. Statistics: ANOVA with random effects. The levels of significance #: p < 0.05, ##: p < 0.01, ###: p < 0.001 correspond to comparisons betweenTREM2-sol and WT animals at the specified timepoints. WT: wild-type, TREM2-IPD: TREM2 cleavage-reduced, TREM2-sol: TREM2 soluble-only. Figure S5. Quantitative image analysis of histological stainings in CC. Luxol fast blue (LFB) optical density (OD) analysis of LFB (normalized to WT at control food), mature oligodendrocytes (GST-π soma area in %), myelin basic protein debris (dMBP-stained area in %), neurofilament (SMI312-stained area in %). Iba1-positive soma numbers (normalized to WT at week 5 cuprizone), LAMP-1 (lysosomal-associated membrane protein 1)-stained area in %, TMEM119 (homeostatic marker, TMEM119-stained area in %), astrocytes (GFAP-stained area in %). Group sizes: n = 2-7 for all genotypes and timepoints. Data shown as means ± SEM. WT: wild-type, TREM2-IPD: TREM2 cleavage-reduced, TREM2-sol: TREM2 soluble-only, TREM2-KO: TREM2 knockout. Ctrl: control food, cpz: cuprizone food for 5 weeks, rec: recovery on control food for 4 weeks. CC: corpus callosum. Scale bars: 300 µm (overview), 50 µm (close-up). Statistics: Holm–Sidak`s multiple comparison test one-way ANOVA (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001). Comparisons not indicated are non-significant. Wt ctrl1, wt cpz1 and wt rec1 are the respective wild-type groups for the study with TREM2-IPD and TREM2-sol, wt ctrl2, wt cpz2 and wt rec2 are the wild-type groups for the TREM2-KO study. Only statistical analysis within a study was performed. The analyses of dMBP and TMEM119 for the respective wild-type groups for TREM2-KO were omitted as no obvious signals were observed. Figure S6. Brain histological analysis for WT, TREM2-IPD, TREM2-sol and TREM2-KO mice receiving control normal food showed no difference between groups. Quantitative image analysis is shown in Figs. 3 and 5. Luxol Fast Blue (LFB), mature oligodendrocytes (GST-π), myelin basic protein debris (dMBP), neurofilament (SMI312), Iba1, LAMP-1 (lysosomal-associated membrane protein 1), TMEM119 (homeostatic marker), astrocytes (GFAP). WT: wild-type, TREM2-IPD: TREM2 cleavage-reduced, TREM2-sol: TREM2 soluble-only, TREM2-KO: TREM2 knockout. ec: external capsule, cc: corpus callosum. Scale bars: 300 µm. Figure S7. Image analysis of Iba1-positive microglia activation in the EC and CC (normalized to WT in %) from the staining shown in Fig. 5. Microglia activation is calculated as microglia and proximal processes area normalized to non-soma-associated processes area. Data are shown as means ± SEM. WT: wild-type, TREM2-IPD: TREM2 cleavage-reduced, TREM2-sol: TREM2 soluble-only, TREM2-KO: TREM2 knockout. Ctrl: control food, cpz: cuprizone food for 5 weeks, rec: recovery on control food for 4 weeks. ec: external capsule, cc: corpus callosum. Statistics: Holm–Sidak`s multiple comparison test one-way ANOVA (*: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001). Comparisons not indicated are non-significant. wt ctrl1, wt cpz1 and wt rec1 are the respective wild-type groups for the study with TREM2-IPD and TREM2-sol, wt ctrl2, wt cpz2 and wt rec2 are the wild-type groups for the TREM2-KO study. Only statistical analysis within a study was performed. Figure S8. MRI analyses of TREM2-IPD mice in the chronic cuprizone model. Data presented as means ± SEM. Group sizes: n = 7 for each genotype. Statistics: ANOVA with random effects. The levels of significance #0.01 < p < 0.05, ##0.001 < p < 0.01 correspond to comparisons between TREM2-IPD and WT animals at the specified timepoints. WT: wild-type, TREM2-IPD: TREM2 cleavage-reduced. Control refers to TREM2-IPD mice receiving normal food throughout the study. Figure S9. Microglia (Iba1), astrocytes (GFAP) and lysosomal marker LAMP-1 were quantified in WT and TREM2-IPD in the chronic cuprizone model (12-week cuprizone treatment and 3-week recovery). Group sizes: n = 5–7. Data are shown as means ± SEM. WT: wild-type, TREM2-IPD: TREM2 cleavage-reduced. Ctrl: control food, rec: recovery on control food for 3 weeks. EC: external capsule, CC: corpus callosum. Scale bars: 500 µm. Statistics: Holm–Sidak`s multiple comparison test one-way ANOVA (*: p < 0.05, **: p < 0.01, ****: p < 0.0001, n.s.: not significant).

The drimane sesquiterpenoids drimenin, cinnamolide, dendocarbin A, and polygodial were purified from the Canelo tree (Drimys winteri) and chemically characterized by spectroscopic methods. The pharmacological activity of these natural compounds were determined on hα4β2, hα3β4, and hα7 nicotinic acetylcholine receptors (AChRs) by Ca2+ influx measurements. The results established that drimane sesquiterpenoids inhibit AChRs with the following selectivity: hα4β2 > hα3β4 > hα7. In the case of hα4β2 AChRs, the following potency rank order was determined (IC50’s in μM): drimenin (0.97 ± 0.35) > cinnamolide (1.57 ± 0.36) > polygodial (62.5 ± 19.9) ≫ dendocarbin A (no activity). To determine putative structural features underlying the differences in inhibitory potency at hα4β2 AChRs, additional structure–activity relationship and molecular docking experiments were performed. The Ca2+ influx and structural results supported a noncompetitive mechanism of inhibition, where drimenin interacted with luminal and nonluminal (TMD-β2 intrasubunit) sites. The structure–activity relationship results, i.e., the lower the ligand polarity, the higher the inhibitory potency, supported the nonluminal interaction. Ligand binding to both sites might inhibit the hα4β2 AChR by a cooperative mechanism, as shown experimentally (nH > 1). Drimenin could be used as a molecular scaffold for the development of more potent inhibitors with higher selectivity for the hα4β2 AChR.