Neuroanatomical phenotypes such as brain overgrowth and macrocephaly have previously been associated with autism spectrum disorder (ASD).1 The findings of a new study co-led by Salk Institute scientists that was published in the journal Molecular Psychiatry provide a novel insight into cellular and molecular mechanisms that underlie abnormal brain growth in individuals diagnosed with autism.2

Brain overgrowth is observed in approximately 9% of patients with autism, according to a recent systematic review and meta-analysis. In the same report, researchers identified macrocephaly in approximately 16% of individuals with autism. “The prevalence and extent of brain overgrowth,” however, “may be underestimated by brain imaging studies,” the authors noted. They concluded that the “identification of the mechanisms underlying macrocephaly … will be extremely useful in paving the path to targeted pharmacological intervention.”1

Significant advances in stem cell technology are beginning to unravel the mechanisms that underlie neuroanatomic abnormalities in individuals diagnosed with AUD.

In the present study,2 the investigators selected participants based on the total brain volume; brain scans (MRI) were performed previously when children were between the ages of 2 and 5 years. They used stem cell reprogramming techniques to model early human neurodevelopment and brain overgrowth. Skin fibroblasts were reprogrammed to generate induced pluripotent stem cells (iPSCs), neural progenitor cells (NPCs), and neurons. Fibroblasts were derived from a “carefully clinically characterized cohort of ASD patients who have an anatomical phenotypic trait that occurs in approximately 20% to 30% of idiopathic ASD: an early developmental enlargement of brain volume, including macrencephaly.”

It is known that the contribution of common genetic variants, or single nucleotide polymorphisms (SNPs), to risk of autism is relatively small, but the contribution of rare genetic variants, such as copy number variations (CNVs), is much larger, with estimates for increased risk of autism ranging from 20 to 60 fold.

Although the participants diagnosed with autism (n=8) had a significantly enlarged brain compared with the brain size of typically developing controls (n=5), whole blood DNA analysis, using high-confidence copy number variation (CNV) array, did not reveal the presence of any rare structural variant that was previously associated with ASD. (The structural variants that are considered to be rare are those that are observed in less than 1% of the population.)

The investigators then used exome sequencing to examine [fibroblast] DNA from 8 participants diagnosed with autism. They found stop gain mutations in the canonical Wnt pathway genes CTNNB1 and FZD6. These results are consistent with previous reports that implicate the canonical Wnt pathway in the process of brain overgrowth in ASD. “It is known that Wnt signaling controls NPCs proliferation and differentiation in a temporal and context-dependent manner during brain development,” the authors noted.

Next, they generated NPCs from iPSCs, which were generated from individuals with ASD and macrencephaly, or from nonaffected typically developing individuals. They hypothesized that “an alteration of the rates of NPC proliferation could result in early brain overgrowth.” The results indicate that NPCs derived from individuals with autism display rapid rates of proliferation in culture, and that “proliferation is robustly significant in its correlation with brain [size] volume, suggesting that it may be proliferation that specifically contributes to the large brain early on in ASD.”

Although LiCl (lithium chloride) treatment increased Wnt transcriptional activity and reversed the overproliferation that was observed in ASD-derived NPCs, it did not restore network defects in ASD-derived neurons. However, when ASD-derived neuronal cultures were treated with IGF-1 (insulin growth factor 1), deficiency in network formation and connectivity was partially rescued. (IGF-1 is a neurotrophic factor that is crucial for brain development and neural plasticity; it is currently being evaluated in clinical trials of autism.)

Further, compared with control neurons, ASD-derived neurons did not show significantly altered survival, but they did show significantly reduced synaptogenesis (ie, reduced density of synapsin and vesicular glutamate transporter-1).

“This is the first time that clinical data are significantly correlated to an iPSC-neural phenotype and open the doors for potential use of in vitro assays as diagnostic tools for complex multigenic disorders such as ASD,” the authors concluded in their publication.

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1. Sacco R, Gabriele S, Persico AM. Head circumference and brain size in autism spectrum disorder: a systematic review and meta-analysis. Psychiatry Res. 2015;234:239-251.

2. Marchetto MC, Belinson H, Tian Y, et al. Altered proliferation and networks in neural cells derived from idiopathic autistic individuals. Mol Psychiatry. 2016. doi: 10.1038/mp.2016.95. [Epub ahead of print]