5-hydroxymethylcytosine is dynamically regulated during forebrain organoid development and aberrantly altered in Alzheimer's disease

Abstract

5-hydroxymethylcytosine (5hmC) undergoes dynamic changes during mammalian brain development, and its dysregulation is associated with Alzheimer's disease (AD). The dynamics of 5hmC during early human brain development and how they contribute to AD pathologies remain largely unexplored. We generate 5hmC and transcriptome profiles encompassing several developmental time points of healthy forebrain organoids and organoids derived from several familial AD patients. Stage-specific differentially hydroxymethylated regions demonstrate an acquisition or depletion of 5hmC modifications across developmental stages. Additionally, genes concomitantly increasing or decreasing in 5hmC and gene expression are enriched in neurobiological or early developmental processes, respectively. Importantly, our AD organoids corroborate cellular and molecular phenotypes previously observed in human AD brains. 5hmC is significantly altered in developmentally programmed 5hmC intragenic regions in defined fetal histone marks and enhancers in AD organoids. These data suggest a highly coordinated molecular system that may be dysregulated in these early developing AD organoids.

Keywords: 5-hydroxymethylcytosine; Alzheimer’s disease; forebrain organoids; neurodevelopment.

Figure 1.. Genome-wide profiling of 5hmC in forebrain organoids during development

(A) Schematic of the collection time points of forebrain organoids derived from controls and patients with Alzheimer’s disease (AD) for genome-wide 5hmC and RNA sequencing: day 8 embryoid bodies (EBs), day 56 (D56), day 84 (D84), day 112 (D112), and AD organoid at day 84. (B) Number of 5hmC peaks identified across developmental stages. (C) Distribution of 5hmC peaks across genomic features in the human genome. (D) Enrichment of 5hmC peaks at 3′ and 5′ untranslated regions (3′ UTR and 5′ UTR), promoters, exons, introns, transcription termination sites (TTSs), and intergenic regions. (E, G, I, and K) Average normalized 5hmC read counts across 5hmC peaks for EBs (E), D56 (G), D84 (I), and D112 (K). (F, H, J, and L) Normalized 5hmC counts at peak regions identified in ANK1 (F), DRD2 (H), NTRK3 (J), and TUBB2B (L) in forebrain organoids across developmental stages. Red boxes indicate where on the gene the displayed peak region(s) originated.

Figure 2.. Dynamics of 5hmC regulation during forebrain organoid development

(A) Number of established and disappeared 5hmC peaks at D56, D84, and D112. (B–D) Heatmaps of developmental-stage-specific DhMRs, where the color scale represents normalized 5hmC read counts. (B) DhMRs that were enriched in developmental stages. (C) DhMRs that were depleted in developmental stages. (D) DhMRs with continual 5hmC accumulation (top) and continual 5hmC depletion (bottom) during organoid development. (E and F) Average normalized 5hmC read counts per stage with continual 5hmC accumulation (E) and continual 5hmC depletion (F). (G) Heatmap of RPKM (reads per kilobase per million) for genes that concomitantly increase in 5hmC and gene expression (top) or concomitantly decrease in 5hmC and gene expression (bottom). (H and I) Gene Ontology (GO) analysis of the genes that concomitantly increase in 5hmC and gene expression (H) and concomitantly decrease in 5hmC and gene expression (I). Reg., regulation; Dev., development; Morph., morphogenesis. (J and K) Normalized 5hmC read count and transcriptome across the concomitantly increasing SOX11 gene (J) or the concomitantly decreasing FGF8 gene (K). (L) Average normalized 5hmC read counts per stage across enhancer regions. (M and N) Enrichment of histone modifications at enhancer regions from fetal brains overlapped with DhMRs that continually accumulated (M) or lost (N) 5hmC.

Figure 3.. AD organoids recapitulate hallmark pathologies of human AD brains

(A) Representative phosphorylated Tau immunostaining of fAD organoids at day 84 and controls; scale bar = 50 μm. (B) Representative amyloid-beta (Aβ) immunostaining of fAD organoids at day 84 and controls; scale bar = 50 μm. (C) Immunoblot of phosphorylated and total Tau protein derived from independent control organoid lines (n = 2 biological replicates done in triplicate) and independent fAD patient organoid lines (n = 3 biological replicates done in triplicate) (*p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test; data are represented as mean ± SEM). (D) ELISA quantification of Aβ1–40 and Aβ1–42 peptide levels in control and fAD organoids (**p < 0.01, unpaired t test, n = 3 biological replicates; data are represented as mean ± SEM). (E) Average normalized 5hmC read counts across the whole genome show that 5hmC is significantly depleted (p = 2.513 × 10⁻⁷ by unpaired t test) in AD versus control organoids. (F) 5hmC dot blot showing whole-organoid 5hmC enrichment in controls versus AD organoids at day 84 (top). Methylene blue staining confirms equal amounts of DNA were loaded per sample (bottom). (G) Quantification of 5hmC dot blot in controls and AD organoids (p < 0.05, unpaired t test, n = 3 biological replicates; data are represented as mean ± SEM). (H) Proportions of 5hmC peaks across genomic features in control and AD organoids.

Figure 4.. Aberrant alteration of 5hmC in AD organoids

(A and B) Average normalized 5hmC read counts at AD-enriched and AD-depleted DhMRs. (C) Differentially expressed genes (n = 7,976 downregulated genes and n = 10,458 upregulated genes in AD organoids). Green dots: AD-depleted DhMRs with decreasing gene expression. Purple dots: AD-enriched DhMRs with increasing gene expression. (D and E) GO analysis of genes annotated to AD-enriched DhMRs with increasing gene expression (D) and AD-depleted DhMRs with decreasing gene expression (E) in AD organoids. (F and G) Normalized 5hmC read counts and transcriptome of GRIN3A, which depicts AD-enriched DhMRs with increasing gene expression (F), or CENPO, which depicts AD-depleted DhMRs with decreasing gene expression (G). Red boxes indicate where on the gene the displayed peak region(s) originated from. C, control; M, merge. (H–K) Venn diagrams and corresponding GO analysis results with respect to overlapped AD-enriched and -depleted DhMRs and continual 5hmC accumulation (H and I) or continual 5hmC depletion (J and K) during development. (L) AD-depleted DhMRs are most similar to H3K4me3 regions, whereas AD-enriched DhMRs are most similar to H3K27ac and H3K4me1. (M) Enrichment of fetal brain histone modifications in the overlapped regions between fetal brain enhancer regions and AD-specific DhMRs (n = 3,688 enhancers).