Idiopathic ASD Patient Derived Neural Precursor Cells (NPCs) Exhibit Dysregulated Proliferation and Altered Response to bFGF Stimulation

Background: Autism spectrum disorder (ASD) is a complex, heterogeneous disorder exhibiting abnormalities in social communication and repetitive behaviors. Despite being highly heritable, the majority (~80%) of cases are genetically undefined. Postmortem and genetic studies suggest convergence on early neurodevelopmental processes in ASD pathologenesis. Further, ~20% of ASD individuals exhibit macrocephaly while ~10% exhibit microcephaly. One mechanism implicated in altered brain size is neural precursor cell (NPC) proliferation.

Objectives: Utilizing a cohort of three families, each containing a male with idiopathic autism and his unaffected brother, we investigated proliferation phenotypes in NPCs derived from induced pluripotent stem cells (iPSC). We hypothesized autism NPCs would exhibit proliferation defects compared to controls.

Methods: NPCs expressed markers Sox2, Pax6, and Nestin, and differentiated into neurons and glia. Assays examined 2 to 5 iPSC clones/individual and for each, ≥2 NPC lines were derived. A minimum of three experiments/NPC line was conducted per assay. Cells were cultured in control media +/- Fibroblast Growth Factor (bFGF) and labeled with tritiated-thymidine to assess DNA synthesis. Cultures were dissociated at 2, 4 and 6 days and quantified via hemocytometer. To measure apoptosis, NPCs plated for 24h were fixed, stained for cleaved caspase-3, and quantified. Protein levels were visualized via western blot.

Results: All ASD individuals exhibited altered NPC proliferation with either hypoproliferation or hyperproliferation. The hypoproliferative group, comprised 2 I-ASD individuals, exhibited a reduction in DNA synthesis (ASD-1072-65%, p <0.0001; ASD-1012-20%, p =0.0055) and cell numbers after 6 days (ASD-1072-60%, p <0.0001; ASD-1012-40%, p =0.0144). The hyperproliferative group (1 ASD) displayed increased DNA synthesis (ASD-1077-60%, p <0.0001) and proliferation (ASD-1077-60%, p <0.0001). Though all NPCs displayed changes in proliferation, each displayed person-specific defects in S-phase entry and apoptosis. To determine whether signaling pathways that regulate proliferation were affected, NPCs were stimulated with developmentally relevant mitogen, bFGF. ASD NPC proliferation was inversely correlated with mitogenic response. Hypoproliferative NPCs exhibited ~20% increased sensitivity (ASD-1072 p= 0.0059; ASD-1012 p= <0.0001) to bFGF, while hyperproliferative NPCs displayed diminished response, (~30%) (ASD-1077, p<0.0001). To begin defining mechanisms, we assessed a key mitogenic pathway, ERK signaling. In hypoproliferative NPCs, p-ERK1 levels were increased; whereas hyperproliferative NPCs showed no difference. Lastly, we wondered, do ASD proliferation differences emerge only in the neural lineage? Interestingly, there were no differences in iPSC proliferation in ASD subjects regardless of NPC proliferation phenotypes.

Conclusions: Using iPSC technologies, we observe dysregulation of ASD NPC proliferation and altered responsiveness to bFGF compared to sex-matched, sibling controls. In individuals exhibiting hypoproliferation, increased sensitivity to bFGF correlated with increased levels of p-ERK1, suggesting dysregulation of a key developmental signaling pathway in some ASD individuals. More broadly the expression of dysregulated proliferation in NPCs specifically, and not iPSCs from which they were derived, may suggest why the CNS is a major disease target. In aggregate, our findings suggest that alterations in neurogenesis may be a common feature of autism, while more nuanced differences underscore the need for personalized medicine in this heterogeneous disorder.