Fig 2: shRNA-mediated knockdown of LRP6 expression inhibits Schwann cell differentiation within injured sciatic nerve.(A) Images show morphology of GFP+ OPCs transduced in vitro with lentiviral-mediated control and LRP6-specific shRNA, scale bar 20 µm (B) FACS sorting strategy, numbers show percent of cells of a specified phenotype (C) Western blot analysis of LRP6 and tRFP protein level revealed efficient gene expression knockdown. (D) FACS-sorted GFP+ OPCs transduced with LRP6shRNA differentiate normally into MBP+ OLs, scale bar 20 µm (E) Differentiation of OPCs transduced with LRP6-specific shRNA into SCs is defective. t-Students test **p<0.01 (F) Confocal images show grafted GFP+/tRFP+ cells differentiated within host sciatic nerves at 4 weeks after implantation. Co-localization of GFP (in green), with MPZ or periaxin (in red) indicates SC differentiation of OPCs transduced with non-targeting control shRNA but not with LRP6 shRNA (scale bar 20 µm).

Fig 3: Accumulation of myelin debris in Grn KO microglia is exacerbated by the partial loss of Ctsd(A–C) Representative images of myelin (Mbp), pHrodo-myelin, and microglia marker (Iba1) co-staining in WT microglia fed with pHrodo-myelin (A), and quantitative analyses of relative phagocytosed pHrodo intensity (B) and Mbp intensity (C) in a time-dependent manner after adding pHrodo-myelin into medium (B) or after washing away extra myelin after incubation with microglia (C).(D and E) Representative images (D) and quantitative analysis (E) of Mbp staining in pons of 21-month-old WT, Ctsd HET, Grn KO, or Grn KO/Ctsd HET mice (n = 10 per group). Box indicates the region enlarged in the panels below. Arrows indicate Mbp deposits. Scale bar, 20 µm (A) and 50 µm (D).(F) Quantitative analysis of Ctsd staining in microglia of pons of 21-month-old WT, Ctsd HET, Grn KO, or Grn KO/Ctsd HET mice (n = 300 per group). Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA (B, C, and E) or one-way ANOVA (F), Tukey’s multiple-comparison test.

Fig 4: Myelin debris accumulates in Grn KO mice and in patients with GRN-associated FTD(A–D) Representative images (A and C) and quantitative analysis (B and D) of myelin staining (LFB) in the pons of 21-month-old WT or Grn KO mice (n = 10 per group) (A and B) and in the pons of patients with sporadic FTD and patients with GRN-associated FTD (n = 8–9 per group) (C and D). Arrows indicate myelin debris. Scale bar, 50 μm. Boxes in (A) and (C) indicate the region enlarged below. Data in (B) and (D) are presented as mean ± SEM. *p < 0.05, ****p < 0.0001, unpaired Student’s t tests.(E) Representative images of myelin (MBP) and microglial lysosome (CD68) co-staining in pons of patients with GRN-associated FTD. Arrow indicates myelin debris. Scale bar, 5 μm.

Fig 5: Brain cell type differentiation and characterization. (a) Schematics for the excitatory neuron, astrocyte, microglia, and oligodendrocyte differentiation from hiPSCs. (b) Representative immunofluorescent images of cell type specific markers. iPSC-induced excitatory neurons (iNeurons) were stained for neuron-specific markers (MAP2, NeuN, and SYP; synaptophysin). Scale bar, 75 and 20 µm (high magnification). iPSC-derived microglia-like cells (iMGLs) were stained for microglia-specific markers (TREM2 and P2RY12). Scale bar, 30 µm (upper) and 10 µm (lower). iPSC-derived astrocytes (iAstrocytes) were stained for astrocyte-specific markers (GFAP, Vimentin, and APOE). Scale bar, 75 and 20 µm (high magnification). iPSC-derived oligodendrocyte-like cells (iOligos) were stained for oligodendrocyte-specific markers (O4 and MBP). Scale bar, 75 µm. (c) qRT-PCR of cell type-specific markers for iNeuron (MAP2, NeuN and SYT1), iMGL (IBA1, PU.1 and TREM2), iAstrocyte (GFAP, S100B and ALDH1L1), and iOligo (MOG, MBP and CNP) showing the relative mRNA levels of these genes normalized by the specified cell type. Data are presented as mean ± SEM. n.d., non-determined; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, determined by one-way ANOVA with Dunnett's multiple comparisons. (d) IL-6 secretion following 24 h of treatment with poly(I:C) and lipopolysaccharide in iAstrocytes (n = 3). Data are presented as the mean ± SEM using one-way ANOVA with Tukey multiple comparisons. ***p < 0.001 and ****p < 0.0001. (e) Phagocytosis of pHrodo-tagged Zymosan A over 24 h (representative images at 24 h) in iMGLs determined by fluorescence intensity. Inhibition of actin polymerization with Cytochalasin D (10 µM, 30 min pre-treatment) attenuated phagocytosis. n = 4 wells (four images per well, imaged hourly on Incucyte S3). Scale bar, 200 µm. (f) Stimulation of iMGLs with 100 ng/ml LPS for 24 h increased pro-inflammatory cytokine secretion (n = 4). Data are presented as mean ± SEM using two-way ANOVA with Sidak's multiple comparisons. n.s., no significance, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (g) Venn diagram showing the number of differentially identified proteins in cell lysates from iNeuron, iMGL, iAstrocyte, and iOligo. (h) Heatmap illustrating the top 30 enriched proteins across the four cell types based on their protein intensity as determined from mass spectrometry. Some common cell type-specific protein markers were identified in iNeuron (e.g., MAPT), iMGL (e.g., AIF1, PU.1), iAstrocyte (e.g., AQP4), and iOligo (e.g., OLIG2). MAP2, microtubule associated protein2; NeuN, RNA binding Fox-1 homolog 3; SYP, synaptophysin; TREM2, Triggering Receptor Expressed On Myeloid Cells 2; P2RY12, purinergic receptor P2RY12; GFAP, glial fibrillar acidic protein; APOE, apolipoprotein E; MBP, myelin basic protein; SYT1, synaptotagmin 1; IBA1/AIF1, ionized calcium binding adaptor molecule 1/allograft inflammatory factor 1; PU.1, transcription factor PU.1; S100B, S100 calcium-binding protein B; ALDH1L1, 10-formyltetrahydrofolate dehydrogenase; MOG, myelin oligodendrocyte glycoprotein; MBP, myelin basic protein; CNP, 2',3'-cyclic nucleotide 3'-phosphodiesterase; SEM, standard error of the mean; ANOVA, analysis of variances; LPS, lipopolysaccharide; MAPT, microtubule associated protein tau; AQP4, aquaporin 4; OLIG2, oligodendrocyte transcription factor 2