Brain organoids with Neanderthal genes: The genomes of Neanderthals and modern humans are overall very similar. To understand the impact of genetic variants that are specific to modern humans, Trujillo et al. performed a genome-wide analysis to identify 61 coding variants in protein-coding genes. Identifying the gene encoding the RNA-binding protein NOVA1 as a top candidate for functional analyses, they introduced the archaic gene variant into human pluripotent stem cells and generated brain organoids. These organoids showed alterations in gene expression and splicing as well as morphology and synaptogenesis, suggesting that this method could be used to explore other genetic changes that underlie the phenotypic traits separating our species from extinct relatives. Science , this issue p. eaax2537

Many cellular models aimed at elucidating cancer biology do not recapitulate pathobiology including tumor heterogeneity, an inherent feature of cancer that underlies treatment resistance. Here we introduce a cancer modeling paradigm using genetically engineered human pluripotent stem cells (hiPSCs) that captures authentic cancer pathobiology. Orthotopic engraftment of the neural progenitor cells derived from hiPSCs that have been genome-edited to contain tumor-associated genetic driver mutations revealed by The Cancer Genome Atlas project for glioblastoma (GBM) results in formation of high-grade gliomas. Similar to patient-derived GBM, these models harbor inter-tumor heterogeneity resembling different GBM molecular subtypes, intra-tumor heterogeneity, and extrachromosomal DNA amplification. Re-engraftment of these primary tumor neurospheres generates secondary tumors with features characteristic of patient samples and present mutation-dependent patterns of tumor evolution. These cancer avatar models provide a platform for comprehensive longitudinal assessment of human tumor development as governed by molecular subtype mutations and lineage-restricted differentiation.

Myotonic dystrophy type 1 (DM1) is a multisystem, autosomal-dominant inherited disorder caused by CTG microsatellite repeat expansions (MREs) in the 3′ untranslated region of the dystrophia myotonica-protein kinase ( DMPK ) gene. Despite its prominence as the most common adult-onset muscular dystrophy, patients with congenital to juvenile-onset forms of DM1 can present with debilitating neurocognitive symptoms along the autism spectrum, characteristic of possible in utero cortical defects. However, the molecular mechanism by which CTG MREs lead to these developmental central nervous system (CNS) manifestations is unknown. Here, we showed that CUG foci found early in the maturation of three-dimensional (3D) cortical organoids from DM1 patient–derived induced pluripotent stem cells (iPSCs) cause hyperphosphorylation of CUGBP Elav-like family member 2 (CELF2) protein. Integrative single-cell RNA sequencing and enhanced cross-linking and immunoprecipitation (eCLIP) analysis revealed that reduced CELF2 protein–RNA substrate interactions results in misregulation of genes critical for excitatory synaptic signaling in glutamatergic neurons, including key components of the methyl-CpG binding protein 2 (MECP2) pathway. Comparisons to MECP2 ^(y/−) cortical organoids revealed convergent molecular and cellular defects such as glutamate toxicity and neuronal loss. Our findings provide evidence suggesting that early-onset DM1 might involve neurodevelopmental disorder-associated pathways and identify N -methyl- d -aspartic acid (NMDA) antagonists as potential treatment avenues for neuronal defects in DM1.

It is well established that inflammation leads to the creation of potent DNA damaging chemicals, including reactive oxygen and nitrogen species. Nitric oxide can react with glutathione to create S-nitrosoglutathione (GSNO), which can in turn lead to S-nitrosated proteins. Of particular interest is the impact of GSNO on the function of DNA repair enzymes. The base excision repair (BER) pathway can be initiated by the alkyl-adenine DNA glycosylase (AAG), a monofunctional glycosylase that removes methylated bases. After base removal, an abasic site is formed, which then gets cleaved by AP endonuclease and processed by downstream BER enzymes. Interestingly, using the Fluorescence-based Multiplexed Host Cell Reactivation Assay (FM-HCR), we show that GSNO actually enhances AAG activity, which is consistent with the literature. This raised the possibility that there might be imbalanced BER when cells are challenged with a methylating agent. To further explore this possibility, we confirmed that GSNO can cause AP endonuclease to translocate from the nucleus to the cytoplasm, which might further exacerbate imbalanced BER by increasing the levels of AP sites. Analysis of abasic sites indeed shows GSNO induces an increase in the level of AP sites. Furthermore, analysis of DNA damage using the CometChip (a higher throughput version of the comet assay) shows an increase in the levels of BER intermediates. Finally, we found that GSNO exposure is associated with an increase in methylation-induced cytotoxicity. Taken together, these studies support a model wherein GSNO increases BER initiation while processing of AP sites is decreased, leading to a toxic increase in BER intermediates. This model is also supported by additional studies performed in our laboratory showing that inflammation in vivo leads to increased large-scale sequence rearrangements. Taken together, this work provides new evidence that inflammatory chemicals can drive cytotoxicity and mutagenesis via BER imbalance.

Over two decades ago, increased levels of RNA oxidation were reported in postmortem patients with ALS, Alzheimer’s, Parkinson’s, and other neurodegenerative diseases. Interestingly, not all cell types and transcripts were equally oxidized. Furthermore, it was shown that RNA oxidation is an early phenomenon, altogether indicating that oxidative RNA damage could be a driver, and not a consequence, of disease. Despite all these exciting observations, the field appears to have stagnated since then. We argue that this is a consequence of the shortcomings of technologies to model these diseases, limiting our understanding of which transcripts are being oxidized, which RNA-binding proteins are interacting with these RNAs, what their implications are in RNA processing, and as a result, what their potential role is in disease onset and progression. Here, we discuss the limits of previous technologies and propose ways by which advancements in iPSC-derived disease modeling, proteomics, and sequencing technologies can be combined and leveraged to answer new and decades-old questions.