Germline PTEN genotype-dependent phenotypic divergence during the early neural developmental process of forebrain...

PTEN germline mutations account for ~0.2-1% of all autism spectrum disorder (ASD) cases, as well as ~17% of ASD patients with macrocephaly, making it one of the top ASD-associated risk genes. Individuals with germline PTEN mutations receive the molecular diagnosis of PTEN Hamartoma Tumor Syndrome (PHTS), an inherited cancer predisposition syndrome, about 20-23% of whom are diagnosed with ASD. We generated forebrain organoid cultures from gene-edited isogenic human induced pluripotent stem cells (hiPSCs) harboring a PTEN^G132D (ASD) or PTEN^M134R (cancer) mutant allele to model how these mutations interrupt neurodevelopmental processes. Here, we show that the PTEN^G132D allele disrupts early neuroectoderm formation during the first several days of organoid generation, and results in deficient electrophysiology. While organoids generated from PTEN^M134R hiPSCs remained morphologically similar to wild-type organoids during this early stage in development, we observed disrupted neuronal differentiation, radial glia positioning, and cortical layering in both PTEN-mutant organoids at the later stage of 72+ days of development. Perifosine, an AKT inhibitor, reduced over-activated AKT and partially corrected the abnormalities in cellular organization observed in PTEN^G132D organoids. Single cell RNAseq analyses on early-stage organoids revealed that genes related to neural cell fate were decreased in PTEN^G132D mutant organoids, and AKT inhibition was capable of upregulating gene signatures related to neuronal cell fate and CNS maturation pathways. These findings demonstrate that different PTEN missense mutations can have a profound impact on neurodevelopment at diverse stages which in turn may predispose PHTS individuals to ASD. Further study will shed light on ways to mitigate pathological impact of PTEN mutants on neurodevelopment by stage-specific manipulation of downstream PTEN signaling components.