Evaluation of Convergent Molecular Pathways during Neuronal Development Using Autism-Specific Induced Pluripotent STEM CELLS

Background:

Autism is a phenotypically and etiologically complex group of neurodevelopmental conditions that are behaviorally defined based on the presence of social-communicative problems and restricted and repetitive behaviors. Recent studies have shown that autism risk loci are highly enriched in genes expressed during early neocortical development and genes which encode proteins that function in biological pathways involved in regulating transcription, chromatin remodeling, cell adhesion, signaling complexes, and synapse function. However, there has been a considerable lag in understanding the relationship between suspected ASD risk variants and how these variants result in the cell-based phenotypes with which they are associated.

Objectives: The aim of this study is to use induced pluripotent stem cell (iPSC)-based approaches to identify key differences in important neurobiological processes by evaluating the expression of gene networks that regulate these processes, both in a temporal and neural-specific manner.

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

Results: Transcriptional analyses of autism and control neurons in culture at days 35 and 135 of their in vitro development showed autism-specific transcriptional signatures mainly affecting pathways/networks involved in neuronal differentiation, the cytoskeletal matrix structure formation (i.e. axon guidance and cell migration), regionalization, patterning, DNA and RNA metabolism. Additionally, developing networks of neurons were analyzed using multi-electrode array (MEA) recordings, measurements of calcium transients, immunocytochemical analyses for important morphological markers, and cell migration assays at time-points aligning with the transcriptional analyses. Neurons from autism individuals demonstrated significantly decreased network spiking activity from MEA recordings (40-80% decrease, p<0.05) as well as decreased numbers of calcium transients (30-60% decrease, p<0.01). Additionally, autism lines showed significant differences in neurite morphology and decreased cell migration (p<0.05) 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 MEA, calcium transients, and quantification of neurite outgrowth that complement their transcriptomic profiles. Taken together, these data suggests a convergence of pathophysiological processes that effect neuronal functionality in autism.