PTEN variant and genetic backgrounds combine to modify cerebellar neuronal differentiation in autism spectrum disorder
Mutations in the PTEN gene have been implicated in autism spectrum disorders (ASD), particularly among individuals with comorbid macrocephaly. In our previous study, we demonstrated that the PTEN p.Ile135Leu variant, in an ASD-related genetic background dependent fashion, disrupts both cortical neurogenesis and gliogenesis. While abnormal cerebellar development is a recognized feature of ASD, the specific cellular targets and timing of disruptions during cerebellar differentiation and development remain poorly understood. To investigate these aspects, we applied our previously established cerebellar organoid protocol and used isogenic human iPSC lines harboring this PTEN-variant. We examined the expression of Purkinje cells, granule cells, interneurons, and glial cells prior to 22 weeks of differentiation, assessed genes expression at 8 weeks, and evaluated spontaneous spikes activity in Purkinje cells after 11 weeks. We observed that cell-type-specific expression patterns differed between the PTEN p.Ile135Leu variant in control versus ASD-genetic backgrounds. However, these background differences were diminished in PTEN knockout lines across both backgrounds. Our single-cell RNA sequencing (scRNA-seq) dataset revealed that the PTEN p.Ile135Leu variant increased the number of interneuron progenitor cells, whereas PTEN knockout led to an expansion of meningeal-like cells in both genetic contexts. Moreover, both the PTEN p.Ile135Leu variant and PTEN knockout abolished spontaneous simple spikes activity in Purkinje cells across both backgrounds, including PTEN-corrected patient-derived lines. Together, these findings provide direct evidence linking PTEN dysfunction and genetic background to altered cerebellar differentiation and neuronal network activity in human cerebellar organoids. Overall design: For 10X scRNA-seq, we used the Chromium Nest GEM Single Cell 3' Kit v3.1 (10x Genomics, 1000268) for all scRNA-seq library preparations. Single-cell suspensions from Chap WT/WT, Chap WT/I135L, Chap KO/KO, Apex WT/WT. Apex WT/I135L and Apex KO/KO were loaded onto the chromium Next Chip G Single Cell Kit (10x Genomics, 1000120) and processed with Chromium Controller 10v Genomics (10x Genomics) to generate single-cell gel beads in emulsion. The libraries were sequenced on the NovaSeqX platform using paired-end sequencing, with the first read having a length of 28 bases and the second read having a length of 90 bases.  Raw fastq files were uploaded into the 10x Genomics cloud and reads were aligned to Human (GRCh38) with Cell Ranger. The generated filtered raw matrix including barcodes, features and matrix were uploaded into Seurat v5.