Human tau mutations in cerebral organoids induce a progressive dyshomeostasis of cholesterol

Abstract

Mutations in the MAPT gene that encodes tau lead to frontotemporal dementia (FTD) with pathology evident in both cerebral neurons and glia. Human cerebral organoids (hCOs) from individuals harboring pathogenic tau mutations can reveal the earliest downstream effects on molecular pathways within a developmental context, generating interacting neurons and glia. We found that in hCOs carrying the V337M and R406W tau mutations, the cholesterol biosynthesis pathway in astrocytes was the top upregulated gene set compared with isogenic controls by single-cell RNA sequencing (scRNA-seq). The 15 upregulated genes included HMGCR, ACAT2, STARD4, LDLR, and SREBF2. This result was confirmed in a homozygous R406W mutant cell line by immunostaining and sterol measurements. Cholesterol abundance in the brain is tightly regulated by efflux and cholesterol biosynthetic enzyme levels in astrocytes, and dysregulation can cause aberrant phosphorylation of tau. Our findings suggest that cholesterol dyshomeostasis is an early event in the etiology of neurodegeneration caused by tau mutations.

Keywords: MAPT mutation; astrocyte; brain organoid; cholesterol; disease model; neurodegeneration; single-cell RNA sequencing; tau; tauopathy.

Graphical abstract

Figure 1

Composition of the human cerebral organoid (hCO) single-cell RNA dataset (A) iPSCs from 6 donors with MAPT mutations and 5 isogenic controls were grown into hCOs and subjected to drop-seq. (B) Overview of single-cell (sc) dataset and numbers of cells sequenced. Entries with more than one number indicate replicates from independent differentiation experiments, each number representing the number of cells sequenced for a replicate. For full line names, see supplemental experimental procedures. (C) Cell numbers sequenced per sample (1,228 cells, mean ± 239.6 SD). Box and whiskers plots represent median (line in box center), first and third quartile (lower and upper box border, respectively), and minimum and maximum values (whiskers). (D) UMAP of entire dataset, 62 samples and 76,111 cells. Colors represent cell types. (E) Expression of selected canonical markers. (F) UMAP subsetted to ages in months. (G) Relative abundances (n cells in a cluster divided by total n of cells in a sample) of cell populations over hCO development. Box and whiskers plots represent median (line in box center), first and third quartile (lower and upper box border, respectively), and minimum and maximum values (whiskers). Ast, astrocytes; Glia, glia expressing markers of choroid plexus, ependymal cells and Wnt/Bmp signaling molecules; EN, excitatory neurons; EN-P, pyramidal neurons; GPC, glial progenitor cells; IN, inhibitory neurons; IN-GE, inhibitory neurons derived from ganglionic eminences; Mes, mesenchymal cells; N, neurons; NPC1, neuronal progenitors 1; NPC2, neuronal progenitors 2; NPC/RGC-cycling, cycling neural progenitors and radial glia; RGC, radial glia; OPC, oligodendrocyte progenitors. See also Figure S1.

Figure 2

Effects of MAPT mutations on pyramidal glutamatergic neurons (A) UMAP and subclustering of EN-Ps. (B) Expression of selected subcluster markers. (C) Volcano plot of differential gene expression (DGE) results of MAPT mutant versus control mature neurons, combining isogenic pairs with ≥30 mature neurons in each sample (n = 10 isogenic pairs from 5 hCO batches, 4,044 cells). Differentially expressed genes (DEGs) were identified using the MAST test (Finak et al., 2015) with a logFC cutoff of 0.05 and Benjamini-Hochberg (BH)-corrected p values <0.05. DEGs involved in glycolysis are highlighted. (D) Gene Ontology (GO) enrichment of genes up- (top) and downregulated (bottom) in MAPT mutant mature EN-Ps. (E) Heatmap of logFC of glycolysis-related genes identified in (C) across different ages. (F) Heatmap of logFC of glycolysis-related genes identified in (C), and logFC in deep- and upper-layer neurons. (G) Downregulation of GABA receptor genes GABRA2 and GABRG2, and the immediate-early gene NPAS4 in upper-layer neurons. Negative logFC indicates decreased gene expression in heterozygous mutant compared with control, positive logFC the opposite. ^∗p ≤ 0.05, ^∗∗∗p ≤ 0.001. See also Figures S2 and S3.

Figure 3

Effects of MAPT mutations on astrocytes (A) Volcano plot of DGE in astrocytes of isogenic pairs with >5% EN-Ps and <50% INs, ENs, and Ns in each sample (n = 6 isogenic pairs from 4 hCO batches, 1,802 cells) using the MAST test with a logFC cutoff of 0.05 and BH-corrected p values <0.05. Cholesterol-related DEGs are highlighted. (B) GO enrichment of genes up- (top) and downregulated (bottom) in MAPT mutant astrocytes including isogenic pairs with at least 30 mature astrocytes in each sample (n = 11 isogenic pairs from 7 hCO batches, 3,398 cells). (C) Expression levels (Z scores) of cholesterol-related genes in astrocytes from control, heterozygous R406W and V337M mutant, homozygous R406W mutant. (D) Heatmap of logFC of cholesterol-related genes identified in (A) across hCO ages. Positive logFC indicates increased gene expression in mutant (heterozygous and homozygous combined) compared with control, negative logFC the opposite. (E) Violin plots showing gene signature enrichment scores of a cholesterol biosynthetic gene set (“superpathway of cholesterol biosynthesis” deposited in the Human Cyc database, 25 genes) across ages (4 months: n = 247 control cells, 254 mutant cells; 6 months, n = 286 control cells, 424 mutant cells; 8 months, n = 375 control cells, 375 mutant cells) and genotypes (mut, homozygous and heterozygous combined; ctrl, control cells). Statistical significance was determined using two-sided Wilcoxon rank-sum test and BH-corrected p values for 3 comparisons. (F) Representative image of HMGCS1 and GFAP co-labeling in 6-month-old V337M mutant hCOs and isogenic control. (G) Percentages of HMGCS1-positive astrocytes (GFAP+) from two R406W and three V337M isogenic pairs across three hCO prep dates at 6 months. Bars and error bars represent means ± SD. (H) Percentages of HMGCS1+ astrocytes (GFAP+) from all three isogenic MAPT mutant and control lines. Box and whiskers plots represent median (line in box center), first and third quartile (lower and upper box border, respectively), and minimum and maximum values (whiskers). Student’s t test was used to determine significance. ^∗p ≤ 0.1, ^∗∗p ≤ 0.01, ^∗∗∗∗p ≤ 0.0001. See also Figure S4.

Figure 4

Sterol quantification in MAPT mutant hCOs (A) Mutant and control hCOs from three isogenic lines were subjected to sterol analysis with liquid chromatography-mass spectrometry (LC-MS). Five individual hCOs from each line were analyzed at 4 and 7 months hCO age. (B and C) LC-MS data of all three isogenic lines combined. Metabolites detected in >50% hCO samples at each time point were analyzed. n = 15 from 3 hCO batches for each control and mutant at each time point except for 7-DHC (n = 10 from 2 hCO batches for each group at each time point), T-MAS (n = 10 from 2 hCO batches for each group at each time point), and Dihydro T-MAS (4 months control n = 14 from 3 hCO batches, 4 months mutant n = 15 from 3 hCO batches, 7 months n = 10 from 2 hCO batches for each group); see also Figure S5C. Mutant values were normalized to control. Box and whiskers plots represent median (line in box center), first and third quartile (lower and upper box border, respectively), and minimum and maximum values (whiskers). Significance was determined using Wilcoxon rank-sum test. BH-corrected p values (for 22 multiple comparisons) <0.05 are reported as statistically significant. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01. (D) Heatmap of log2FC for the three isogenic pairs. Positive log2FC indicates increased concentrations in mutant hCOs compared with control, negative log2FC the opposite. See also Figure S5.

Figure 5

Summary of changes in cholesterol biosynthesis caused by MAPT mutations Boxed/italic, genes encoding cholesterol biosynthetic enzymes; red/boxed/italic, genes with increased expression in MAPT mutant astrocytes, determined by scRNA-seq; black/boxed/italic, genes with unaltered expression; unboxed, metabolites (cholesterol and its precursors); red/arrow up, metabolites with increased levels in MAPT mutant hCOs determined by LC-MS; blue/arrow sideways, metabolites with unaltered levels; black, metabolites not detected or not included in the sterol panel.