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

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. [Display omitted] •5hmC is dynamically regulated during forebrain organoid development•AD organoids recapitulate cellular and molecular pathologies seen in patient brains•Aberrant 5hmC in AD organoids could subtly disrupt early neuronal networks•5hmC crosstalk with histone marks could affect enhancer activity in AD organoids Kuehner et al. use forebrain organoids derived from healthy controls to study the dynamics of 5hmC across early brain development. In addition, organoids derived from several AD patients reveal aberrant 5hmC patterns that could disrupt early neuronal networks and contribute to the onset of AD later in life.