Fig 2: INPP5D expression in the human brain is restricted to microglia in the human brain.Human brain sections (25 μm) (a) and cultured iMGs (b) were immunostained for IBA1 and INPP5D. Nuclei are visualized with DAPI and preparations were imaged using confocal microscopy. Images representative of 16 human brain samples and over three iMG differentiations analyzed. Scale bars = 50 μm. c–e UMAP plots of sNucRNAseq of iMGs combined with snRNAseq of dorsolateral prefrontal cortex (dlpfc) from 12 human postmortem brain samples29 using Harmony35 to integrate across datasets. Single nucleus data from human brain samples and iMGs are depicted separately in (c) and (d), respectively. Relative INPP5D expression (after applying the SCTransformation in the Seurat package) across the harmonized iMG and human brain single nucleus samples shown in (e). Data are from 7121 iMG nuclei, 55,671 nuclei from the postmortem human brain (39,239 glutamatergic neurons, 14,958 astrocytes, 1474 microglia). f–i Data from microglia subcluster from the snRNAseq in c and d were isolated and re-clustered to examine microglial subsets. UMAP plots are shown in (f–h). Relative INPP5D expression (z-score of log-transformed, normalized data) across the subclusters is shown in (h). INPP5D was significantly lower in cluster 13 (adj. p-val = 8.0 × 10−226, Wilcoxon rank-sum test, with FDR correction). A dot plot showing a subset of the genes that define cluster 13 are shown in (i). See also Supplementary Fig. 1 and Supplementary Data 1 showing enrichments in all microglial clusters.

Fig 3: Chronic reduction in INPP5D levels results in inflammasome activation and reduction in autophagic flux.a–c Volcano plot (a) of adjusted p-values showing 71 DEPs from the 7868 quantified via TMT-MS (two-way t-test with Benjamini–Hochberg multiple comparisons test; FDR < 0.05. Heatmap (b) of relative expression levels of proteins involved in immune signaling that are differentially up and downregulated in INPP5D WT vs HET iMGs. Asterisks indicate proteins that also were differentially up or downregulated (q < 0.05) with 3AC treatment (Fig. 3). GO analysis (c) of DEPs; Fisher’s exact test, Bonferroni multiple comparison’s test, adj p-value as shown by size of circles. d, e Representative western blot and quantification across three differentiations of MRC1, PLA2G7, COLEC12, IBA1, and GAPDH in INPP5D WT and HET iMGs. n = 3 independent experiments, mean ± SEM; two-sided t-test. f Heatmap of TMT-MS data (z-score) for lysosomal DEPs in INPP5D HET vs WT iMGs. g Representative western blot of INPP5D iMGs treated with either vehicle (DMSO) or bafilomycin (baf, 100 nM) for either 24 or 6 h. Western blots are probed for INPP5D and LC3 with GAPDH as a loading control. h Quantification of autophagic flux (baf - veh of LC3-II/GAPDH) in iMGs with treatment of 24 h of baf. n = 6 wells vehicle, n = 6 wells of 6 h baf. Mean ± SEM, two-sided t-test with Welch’s correction. i, j Secreted IL-1ß and IL-18 measured by ELISA of media collected from INPP5D HET and WT iMGs. Media were concentrated tenfold in order to be above the limit of detection and normalized to WT mean. n = 14 wells WT, n = 21 wells HET. Mean ± SEM. Two-sided Mann–Whitney test. k, l Secreted IL-1ß and IL-18 measured by ELISA of media collected from INPP5D HET iMGs treated with either vehicle (DMSO) or MCC950 (10 μM) for 24 h. Media were concentrated tenfold for detection. n = 10 wells vehicle, n = 10 wells MCC950-treated. Mean ± SEM. Mann–Whitney test. For e, h–l *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Supplementary Fig. 9, Supplementary Data 4 and 5 for full datasets.

Fig 4: Acute INPP5D inhibition results in inflammasome activation and an increase in the secretion of IL-1ß and IL-18 in iMG cultures.a Fold change of secreted cytokines from iMGs treated with LPS (100 ng/mL) or 3AC (1.25 μM) for 6 h across iMGs derived from four iPSC lines, assayed by ELISA. Fold change was calculated compared to vehicle-treated cells. b Levels of IL1B following 6 h treatment with vehicle, LPS (100 ng/mL), or 3AC (1.25 μM, 2.5 μM) treatment as measured by quantitative real-time PCR (qPCR). Data are normalized to GAPDH expression then values were normalized to vehicle treatment within each experiment. n = 3 differentiations with well-level data shown as dots. One-way ANOVA with Dunnett’s T3 multiple comparisons test. c Secreted levels of IL-1ß measured for the same experiments as (b) following 6 h treatment as measured by ELISA. IL-1ß levels were normalized to 1.25 μM 3AC-treated conditions within each experiment. n = 3 differentiations with well-level data shown as dots. d Representative western blot of protein expression in iMGs with biallelic loss-of-function mutations generated with CRISPR targeting. Loss of INPP5D protein observed repeatedly over 3 differentiations. e, f WT INPP5D iMGs and KO INPP5D iMGs were treated with 3AC for 6 h and the levels of IL-1ß and IL-18 were measured by ELISA. Multiple two-sided t-tests, Two-stage step-up (Benjamini, Krieger, and Yekutieli), **q < 0.01; ***q < 0.005; ****q < 0.001; LLOD= lower limit of detection. n = 3 biological replicates. g, h Treatment of iMGs with either vehicle or 3AC (5 μM) with either VX-765 (25 μM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 measured via ELISA following treatments and normalized to 3AC-treated samples in each experiment. One-way ANOVA with Sidak’s multiple comparison test. 3 differentiations, n = 10 per condition. i, j iMGs were treated with either vehicle, primed with LPS (100 ng/mL) for 3 h and then treated with nigericin (10 μM) for 1 h, or treated with 3AC (1.25 μM or 5 μM) for 2 h. Cells were immunostained for ASC and IBA1, DNA is stained with DAPI (i) and imaged using confocal microscopy. Scale bars = 100 μm. j Quantification of ASC specks. Number of cells analyzed: n = 226 (vehicle); 151 (1.25 μM 3AC); 173 (5.0 μM 3AC) over three experiments and 10 images per condition. Kruskal–Wallis test to determine significance. k, l Treatment of iMGs with either vehicle or 3AC (5 μM) with either MCC950 (10 μM) or its vehicle (DMSO). Levels of secreted IL-1ß and IL-18 in the media were measured and normalized to 3AC-treated samples in each experiment. One-way ANOVA with Sidak’s multiple comparison test. n = 4 differentiations with well-level data shown as dots. m A summary figure of experiments performed to interrogate inflammasome activation, created using BioRender.com. For all graphs, data are presented as mean values ± SEM. For b, c, g, h, j, l: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Cell lines used for experiments are detailed in Supplementary Data 8. See also Supplementary Figs. 7 and 8.

Fig 5: Complex post-translational regulation of INPP5D in the AD brain.a–c Representative images of immunostaining of human brain sections for INPP5D, IBA1, and Aβ. DNA is stained with DAPI. Scale bars = 10 μm. b, c Magnified image from white box outlined in (a). d–h Quantifications from brain immunostaining from 8 NCI and 8 AD individuals. d Quantification of intensity of INPP5D immunostaining across human subjects, two-sided t-test, p = 0.0009. e Quantification of INPP5D intensity across all microglia quantified between the 16 human subjects. Two-sided Wilcoxon test, p < 2.2e−16. f Quantification of INPP5D levels in microglia that were either plaque-associated (plaque) or not plaque-associated (none). Two-sided Wilcoxon test, p < 2.2e-16. For d–f, boxes mark the 1st and 3rd quartiles of data (25th and 75th percentiles), surrounding the median. Whiskers extend to furthest values up to 1.5xIQR (inter-quartile range or distance between 1st and 3rd quartiles). Data beyond the whiskers are “outlying” points for the purpose of plotting and extend to the min and max of individual datasets. g Correlations between form factor and INPP5D intensity across categories of microglia; regression lines for individual datasets as well as the r- and p-value statistics, represent the output of linear model calculation in R. Shaded regions represent 95% CI limits for each dataset. h Histogram of the relative frequency of IBA1+ microglia containing diffuse versus punctal INPP5D immunostaining intensity, determined by the difference between the upper quartile of INPP5D intensity and the lowest quartile of INPP5D intensity. Two-way, unpaired t-tests. i Representative WB of TBS extracts from postmortem human brain, and INPP5D WT and KO iMGs for INPP5D and GAPDH. Vertical line shows INPP5D bands quantified. GAPDH portion of the blot containing iMG samples were digitally over-exposed for presentation purposes. j, k Western blot quantification of INPP5D and GAPDH in TBS postmortem brain (prefrontal cortex, PFC) brain extracts using two antibodies to INPP5D. Kruskal–Wallis test with Dunn’s multiple comparisons test. Quantification of peptides mapping to INPP5D (Q92835) in ROSMAP brain samples extracted with urea (l–n) or TBS (o) and quantified using TMT-MS, urea TMT-MS data generated and reported in34. For l, n = 99 AD, 121 HP-NCI, and 101 LP-NCI human subjects. For o, n = 10 AD, 8 HP-NCI, and 12 LP-NCI human subjects. Kruskal–Wallis test with Dunn’s multiple comparisons tests performed in (l–o). p. Schematic summarizing data presented in this figure. For all panels: ns = not significant, *p < 0.05, ***p < 0.001, ****p < 0.0001. LP = no neuropathological diagnosis of AD, HP = neuropathological diagnosis of AD, NCI = not cognitively impaired, AD = clinical and neuropathological diagnosis of AD. n = number of human subjects and is represented by dots in j, k, m, n For all graphs, Data are presented as mean values ± SEM. See also Supplementary Data 9–13 and Supplementary Data 2, 3 for full datasets. See also Supplementary Figs. 2–4.