RESUMEN
Duplication 15q (dup15q) syndrome is the most common genetic cause of autism spectrum disorder (ASD). Due to a higher genetic and phenotypic homogeneity compared to idiopathic autism, dup15q syndrome provides a well-defined setting to investigate ASD mechanisms. Previous bulk gene expression studies identified shared molecular changes in ASD. However, how cell type specific changes compare across different autism subtypes and how they change during development is largely unknown. In this study, we used single cell and single nucleus mRNA sequencing of dup15q cortical organoids from patient iPSCs, as well as post-mortem patient brain samples. We find cell-type specific dysregulated programs that underlie dup15q pathogenesis, which we validate by spatial resolved transcriptomics using brain tissue samples. We find degraded identity and vulnerability of deep-layer neurons in fetal stage organoids and highlight increased molecular burden of postmortem upper-layer neurons implicated in synaptic signaling, a finding shared between idiopathic ASD and dup15q syndrome. Gene co-expression network analysis of organoid and postmortem excitatory neurons uncovers modules enriched with autism risk genes. Organoid developmental modules were involved in transcription regulation via chromatin remodeling, while postmortem modules were associated with synaptic transmission and plasticity. The findings reveal a shifting landscape of ASD cellular vulnerability during brain development.
RESUMEN
The direct conversion of adult human skin fibroblasts (FBs) into induced neurons (iNs) represents a useful technology to generate donor-specific adult-like human neurons. Disease modeling studies rely on the consistently efficient conversion of relatively large cohorts of FBs. Despite the identification of several small molecular enhancers, high-yield protocols still demand addition of recombinant Noggin. To identify a replacement to circumvent the technical and economic challenges associated with Noggin, we assessed dynamic gene expression trajectories of transforming growth factor-ß signaling during FB-to-iN conversion. We identified ALK2 (ACVR1) of the bone morphogenic protein branch to possess the highest initial transcript abundance in FBs and the steepest decline during successful neuronal conversion. We thus assessed the efficacy of dorsomorphin homolog 1 (DMH1), a highly selective ALK2-inhibitor, for its potential to replace Noggin. Conversion media containing DMH1 (+DMH1) indeed enhanced conversion efficiencies over basic SMAD inhibition (tSMADi), yielding similar ßIII-tubulin (TUBB3) purities as conversion media containing Noggin (+Noggin). Furthermore, +DMH1 induced high yields of iNs with clear neuronal morphologies that are positive for the mature neuronal marker NeuN. Validation of +DMH1 for iN conversion of FBs from 15 adult human donors further demonstrates that Noggin-free conversion consistently yields iN cultures that display high ßIII-tubulin numbers with synaptic structures and basic spontaneous neuronal activity at a third of the cost.
