Fig 2: Blockage of MFG-E8 on the expression of AMPK and Src in microglia treated with propofol. BV2 cells were treated with or without an MFG-E8 antibody (5 μg/ml)/IgG antibody (5 μg/ml) for 45 min after treatment with propofol (12.5 μM) for 4 h. The expression of p-AMPK, AMPK, p-Src, Src, and GAPDH was measured by western blotting (a). The bands were quantitatively analyzed (b, c, d, e). The data are presented as the mean ± SD. Pro, propofol; M8 Ab, MFG-E8-neutralizing antibody. *P < 0.05 versus the control

Fig 3: Effects of AMPK and Src pathway modulation on MFG-E8-mediated microglial phagocytosis. BV2 cells were treated with dasatinib (100 nM, 30 min) and compound C (10 μM, 1 h), treated with propofol, and then incubated with MFG-E8 (100 ng/ml) and fluorescence-labeled latex beads. Flow cytometry (a, b) and immunofluorescence (c) were used to assess the microglial phagocytosis of latex beads. Immunofluorescence staining: phalloidin-red, latex beads-green, DAPI-blue. The data are presented as the mean ± SD. Pro, propofol; M8, MFG-E8; CC, compound C; DAS, dasatinib. *P < 0.05 versus the control; #P < 0.05 versus propofol; ^P < 0.05 versus propofol+MFG-E8

Fig 4: pFTAA/HT7+ DRG Neurons Are Preferentially Removed by Phagocytosis(A) pFTAA+ DRG neuronsfrom 5-month-old P301S mice are depleted after contact co-culture with BV2 cells for 4 days (***p < 0.001), but not if BV2 cells are in transwells. No loss of HT7+ neurons when BV2 cells are contact co-cultured with neurons from 2-month-old P301S mice or 5-month-old Alz17 mice. Mean ± SD, n = 3 independent ex- periments,two-wayANOVA, Bonferroni corrected.(B) pFTAA+ DRG neuronsfrom 5-month-old P301S mice are removed from cultures in contact with primary microglia from C57 mice for 4 days but not those from MFGE8 KO mice. Mean ± SD, n = 3 independent experiments, *p < 0.05, one-way ANOVA, Bonferroni corrected.(C) Endogenous phagocytes mediate the slow removal of cultured pFTAA+ neurons from 5-month-old P301S mice. Representative image of a pFTAA+ neuron (green) surrounded by an IB4+ phagocyte (red). Nuclei (DAPI) in white. Arrow marks pFTAA+ inclusions inside the phagocyte.(D) Removal of pFTAA+ neurons over 14 days is halted by elimination of phagocytes using LME (50 mM). x axis, days in vitro beginning 1 day after phagocyte elimination. Each point shows mean ± SD, n = 3 independent experiments. Lines denote linear regressions depicting rates of neuronal loss; slopes, ****p < 0.0001.(E) Insoluble pFTAA+ tau is transferred from pFTAA+ DRG neurons to BV2 cells in co-contact cultures. FACS sorting of IB4–594 pre-labeled BV2 cells collected 4 days after co-culture with pFTAA+ neurons from 5-month-old P301S-tau or C57 mice. Note double-positive population (4.9%, P6) of BV2 cells present only in cultures from P301S mice.(F) Insoluble tau in BV2 cells. Single IB4-labeled (P4) and double-labeled (P6) populations of BV2 cells shown in (E), extracted in 5% SDSand filtered through cellulose nitrate membrane, which traps insoluble tau. Sarkosyl-insoluble tau fibrils from 5-month-old P301S-tau brains are the positive control.(G) Microglia in the facial nucleus of 5-month-old P301S-mice engulf pFTAA+ neurons with tau. Total fluorescent staining of (i) pFTAA+ neurons, (ii) Iba+ microglia (red), (iii) human (HT7) P301S-tau (white), (iv) nuclei (DAPI, blue). Enlarged image: confocal z section shows the entire pFTAA+ neuron encased inside a microglial cell. The neuronal nucleus (arrow) is also engulfed. Scale bar, 5 μm.(H) Iba-1 staining of microglia in the FN from 5-month-old C57, 5-month-old P301S-tau, 5-month-old P301S-tau/MFGE8 KO with tau pathology, or MFGE8 KO mice. FN neurons are closely apposed by globoid-like microglia. Brown, Iba-1/DAB; blue, cresyl violet. Scale bar, 10 mm.(I) Relative positions of motor neurons and microglia in representative examples of the FN. Microglia and neurons are more closesly distributed in mice with P301S-tau pathology.(J) Quantification of proximity; significant increases in interaction strength between P301S-tau+ neurons and microglia in P301S-tau and P301S-tau/MFGE8 KO mice compared with C57 or MFGE8 KO controls. *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA, Bonferroni corrected.

Fig 5: MFGE8 and NO Are Produced and Secreted by Co-cultured BV2 Microglia Only When Co-cultured in Contact with pFTAA+ Neurons(A and B) Representative blot (A) ofMFGE8 in BV2 cells lysates cultured alone or co-cultured in direct contact, or via a transwell, with DRG neurons from 5-month-old P301S-tau, 5-month-old C57, 5-month-old Alz17 mice, or 2-month-old P301S mice. Asterisk: either another isoform ofMFGE8 or a breakdown product. (B) Densitometry of MFGE8 expression normalized to β-actin; significantly more MFGE8 is produced only in contact co-cultures containing pFTAA+ neurons. Mean ± SD, n = 3 independent experiments (*p < 0.05), twoway ANOVA, Dunnett’s correction.(C and D) Elevated MFGE8 (C) or NO (D) in medium conditioned by BV2 cells co-cultured in contact with DRG neurons from 5-month-old P301S mice compared with 5-month-old Alz17, 5-month- old C57, or 2-month-old P301S mice (MFGE8: ****p < 0.0001 versus all; NO: ****p < 0.0001 versus 5-month-old C57 or 2-month-old P301S mice; ***p < 0.001 versus 5-month-old Alz17 mice). N.D., none detected. Mean ± SD, n = 3 independent experiments, two-way ANOVA, Bonferroni corrected.(E and F) Elevated MFGE8 (E) or NO (F) in medium conditioned by primary microglia from C57 mice co-cultured in contact with DRG neurons from 5-month-old P301S mice; microglia from MFGE8 KO mice are negative controls. Mean ± SD, n = 3 independent microglial preparations, one-way ANOVA, **p < 0.01 (MFGE8), *p < 0.05 (NO).(G) Increased MFGE8 immunostaining intensity in frontal motor cortex of 5-month-old P301S mice compared with 5-month-old C57 mice; MFGE8 KO mouse is negative control; 25 μm section at inter- aural 5.12 mm, bregma 1.32 mm; brown, DAB; blue, cresyl violet. Scale bar, 130 mm. Inset, 65 μm.(H and I) Elevated MFGE8 (H) in 5-month-old P301S-tau brains. Lysatesfrom cortex(Ctx), brain stem (BS), and cerebellum (Cb) of 5-month-old C57, 5-month-old P301S-tau, and 5-month-old Alz17 mice probed with anti-MFGE8; MFGE8 KO brain lysate is negative control. rec, recombinant mouse MFGE8. (I) Densitometry of MFGE8 expression normalized to β-actin. Significantly higher MFGE8 expression in P301S versus C57BU6 Ctx (**p < 0.01), BS (****p < 0.0001), Cb (**p < 0.01), and P301SversusALz17BS (****p < 0.0001). Mean ± SD, n = 3 independent preparations, two-way ANOVA, Bonferroni corrected.(J) Elevated MFGE8 expression in brain extracts from cortex (Ctx) of FTDP-17T patients with two different MAPT mutations (P301L, +3) or sporadic Pick’s disease but not in extracts from patients with C9orf72 hexanucleotide expansions and TDP-43 aggregates. No expression is found in the cerebellum, where tau pathology is absent in all cases. Human milk MFGE8 is a positive control.