Extended Data Fig. 5: The LOAD risk-allele of PICALM impairs MG phagocytosis.

(a) Representative fluorescence images of iMG showing time-dependent phagocytosis of Aβ-pHrodo (green). RFP+, iMG infected with pLenti-CRISPRa. Time indicates minutes after adding Aβ-pHrodo. Scale bar, 100 μm. (b) Representative images showing time-dependent iMG phagocytosis of Aβ-pHrodo (red) for a second clone of both isogenic pairs CD04 and CD09. Scale bar, 100 μm. (c) Quantification of Aβ-pHrodo phagocytosis in iMG of CD04 and CD09 lines. Data are from 2 experiments, each with 3 wells of differentiations, one clone (clone 2) per line. LMM was used with experimental round as the random factor; two-sided test, nominal p-value; (d) Schematics of CRISPRa design to increase PICALM expression in risk-allele iMG (non-risk and risk control iMG also contain pLenti-CRISPRa encoding RFP). Illustrations were created using BioRender. Sudwarts, A. (2025) https://BioRender.com/vveaczo. (e) qPCR results showing normalized PICALM expression in iMGs with non-risk, risk, and risk-CRISPRa lines. Expression was normalized to GAPDH. Each datapoint represents a single-well measurement from one experiment; for each line (CD04 and CD09) and each condition (non-risk, risk, and risk-CRISPRa), data are from one clone, collected from 2 experiments each with 3 wells of differentiations (n = 6). (f) Schematics of CRISPRoff to knockdown PICALM expression for CD04 and CD09 lines with PICALM non-risk alleles. (g) qPCR results show CRISPRoff-induced reduction of PICALM in iMG for both lines. Expression was normalized to GAPDH. Each datapoint represents a single-well measurement from one experiment; for each line (CD04 and CD09) and each condition (risk, non-risk, and non-risk-CRISPRoff), data are from one clone from one experiment with 3 wells of differentiations (n = 3). (h) Representative fluorescence images of iMG showing time-dependent phagocytosis of myelin-pHrodo (red). Cell mask stains for the plasma membrane and NucBlue stains the nucleus in live cells. Time indicates minutes after adding myelin pHrodo. Scale bar, 100 μm. (i) Quantifying myelin-pHrodo phagocytosis in iMG carrying non-risk, risk, and non-risk-CRISPRoff for CD04 and CD09 lines. Scale bar, 100 μm. (j) Representative images showing time-dependent iMG phagocytosis of Aβ-pHrodo (red). (k) Quantification of Aβ-pHrodo phagocytosis in iMG carrying non-risk, risk, and non-risk-CRISPRoff for both CD04 and CD09 lines. (l) Cytochalasin D (10 µM) nearly completely inhibits phagocytosis. Cell mask stains for the plasma membrane and NucBlue stains nuclei in live cells. Time indicates minutes after adding myelin pHrodo. Scale bar, 100 μm. From (h) to (k), for each donor line (CD04 and CD09) and each condition (non-risk, risk, and non-risk-CRISPRoff), data are from one clone of one experiment with 3 wells of differentiations (each with 2-3 FOV). For all comparisons except in (c), one-way ANOVA was used; Dunnett’s correction. * P < 0.05, ** P < 0.01, ***, P < 0.001; mean ± s.e.m.