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Autism Human Neural Precursor Cells (NPCs) Display Common As Well As Personalized Developmental and Molecular Phenotypes
Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by impaired social interaction and communication and the presence of repetitive behaviors. Human neuropathological and MRI studies of ASD reveal changes in brain size, neuronal-connectivity, and brain-architecture, which suggest dysregulation of multiple developmental processes. However, inability to directly study human neurons, limitations of animal-models, and disorder heterogeneity have thwarted discovery of underlying mechanisms. Now, induced- pluripotent-stem-cell (iPSC) technology has allowed the generation of neural precursor cells (NPCs) from individuals with neuropsychiatric disorders thereby allowing study of human development and disease. Using iPSCs we have generated NPCs from 8 boys with severe idiopathic autism (I-ASD) and their unaffected-brothers (Sib) as controls.
Objectives:
Our goals are to study developmental processes such as neurite-outgrowth and cell-migration in our ASD patient-derived NPCs. Our studies employ developmentally-relevant extracellular-factors (EFs) such as PACAP, 5-HT, and NGF to uncover both common and personalized developmental abnormalities. We also aim to identify signaling-pathway defects that contribute to the developmental abnormalities and could serve as targets for personalized medicine.
Methods:
NPCs were derived from 3 iPSC clones per individual. Two separate NPC derivations were conducted per clone. At least 2 experiments were conducted per NPC derivation per clone (minimum 12 expts/individual/assay).
Neurite Assay: 48 hrs after plating, the percentage of cells with neurites≥2 cell diameters were counted blind in 1 cm rows
Neurosphere Migration: Neurospheres generated from NPCs were plated for 48h on Matrigel. Migration = total neurosphere area-inner mass area.
Results:
Our studies indicate common reductions in neurite-outgrowth and cell-migration in two I-ASD individuals. Additionally, while Sib-NPCs exhibited 2-fold increases in neurite-outgrowth in response to PACAP, NGF, and 5-HT, both I-ASD NPCs failed to increase neurites in response to these EFs. Preliminary studies in I-ASD-NPCs from a 3rd patient also showed similar defects in EF response. Likewise, Sib neurospheres exhibited increased migration with PACAP yet I-ASD neurospheres were unresponsive. I-ASD NPCs in Family-1, which did unresponsive to PACAP, exhibited 4x-lower PACAP induced P-CREB levels compared to Sib-NPCs. Conversely, increasing I-ASD P-CREB levels using db-cAMP restored neurite-outgrowth and migration to levels of PACAP-treated Sib-NPCs. Surprisingly, Family-2 ASD-NPCs exhibited different signaling abnormalities. We next compared these I-ASD profiles to a genetically-defined subtype of ASD (three 16p11.2-deletion patients). Remarkably, both neurite-outgrowth and migration were also reduced, however, all three 16p11.2-del NPCs responded normally to EFs.
Conclusions:
Using iPSC technology, our studies identified common defects in neurite-outgrowth and migration in two distinct subtypes of ASD (I-ASD; 16p11.2-Del-ASD), suggesting the disorder may have a common underlying mechanism. Interestingly, while NPCs from I-ASD patients failed to respond to EFs, 16p11.2-del-ASD NPCs had typical EF responses, suggesting there are autism subtype-based differences. Within the I-ASD subtype, both NPCs showed defects in developmental phenotypes yet, only one depended on PKA pathway dysfunction, suggesting that common abnormalities may reflect personalized causative mechanisms. Thus, our studies illustrate the benefit of studying ASDs using iPSC techniques to obtain a personalized understanding of both common and unique neurodevelopmental abnormalities in affected individuals. Ultimately, this information could help develop personalized treatments for ASD.