Understanding Convergent Pathobiology in Idiopathic Autism Using Human iPSCs

Background: Autism spectrum disorders (ASDs) are a phenotypically and genetically complex group of neurodevelopmental conditions characterized by deficits in social interaction and communication, as well as the presence of repetitive and stereotyped behavior. To date, hundreds of genetic loci have been implicated in ASD risk. Recent studies have shown that genes harboring ASD risk loci are highly enriched in sets of genes expressed during early neocortical development and genes encoding proteins that function in specific biological pathways involving regulation of transcription, chromatin remodeling, cell adhesion, signaling complexes, and synapse function. However, the impact that these genetic variants have on ASD pathophysiology remains largely unknown. This is due in large part to a lack of genetically-relevant human disease models. The advent of human induced pluripotent stem cell (iPSC) technology and advances in neural differentiation techniques, have made it possible to study the molecular mechanisms that underlie ASD pathology.

Objective: Patient-specific induced pluripotent stem cells (iPSCs) present a unique opportunity to examine the hypothesis that heterogeneous ASD loci converge on specific molecular pathways during early neural development.

Methods: iPSC lines were derived from individuals with idiopathic autism and differentiated into cortical neurons. We examined the transcriptional differences between iPSC-derived cortical neurons from individuals with ASD and cognitively normal control individuals over a 135 day neuronal differentiation approach using RNAseq analysis. Bioinformatic analysis was performed by weighted gene coexpression network analysis (WGCNA) and Ingenuity Pathway Analysis (IPA). This RNA-seq analysis was complemented by functional studies of these developing neurons using electrophysiological, morphological, and biochemical analyses.

Results: Transcriptional analyses of ASD and control neurons at culture days 35 and 135 of their in vitro development showed ASD-specific transcriptional signatures, including differences in coding genes, alternative splicing, and non-coding RNAs. These changes in transcription mainly affecting pathways/networks involved in neuronal differentiation, the cytoskeletal matrix structure formation (i.e. axon guidance and cell migration), regionalization, patterning, and DNA and RNA metabolism. Additionally, developing networks of neurons were analyzed using multi-electrode array (MEA) recordings, measurements of calcium transients, and cell migration assays. Neurons from ASD individuals demonstrated significantly decreased network spiking activity and decreased numbers of calcium transients. Additionally, ASD lines showed significant differences in neurite morphology and decreased cell migration at early neuronal differentiation times.

Conclusions: The results of this study suggest that iPSC-derived neurons from individuals with ASD may have early deficits in network activity and morphology based on a combination of cell based assays, including spontaneous action potentials, calcium transients, and neurite outgrowth complementing the transcriptomic analyses. Taken together, these data suggest that, although there is significant genetic diversity in ASD, there is a convergence of pathophysiological processes that effect neuronal functionality.