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.

PTEN基因的突变与自闭症谱系障碍（autism spectrum disorders, ASD）密切相关，尤其在伴有巨头畸形共病的个体中更为显著。在我们的既往研究中，已证实PTEN p.Ile135Leu变异会以ASD相关遗传背景依赖的方式，同时破坏皮质神经发生与胶质发生进程。尽管小脑发育异常已被公认是ASD的特征之一，但小脑分化与发育过程中受扰动的特定细胞靶点及时序机制仍尚不明确。
为探究上述问题，我们采用既往建立的小脑类器官培养方案，并使用携带该PTEN变异的同基因人类诱导多能干细胞（induced pluripotent stem cell, iPSC）系。我们在分化至22周前检测了浦肯野细胞、颗粒细胞、中间神经元及胶质细胞的表达特征，在分化8周时评估了基因表达水平，并在分化11周后检测了浦肯野细胞的自发放锋电位活动。
研究结果显示，在对照与ASD遗传背景中，PTEN p.Ile135Leu变异的细胞类型特异性表达模式存在显著差异。但在两种遗传背景下的PTEN敲除系中，此类背景相关差异均有所减弱。我们的单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）数据集表明，PTEN p.Ile135Leu变异会增加中间神经元祖细胞的数量，而PTEN敲除则会在两种遗传背景下扩增脑膜样细胞群体。此外，在两种遗传背景（包括经PTEN校正的患者来源细胞系）中，PTEN p.Ile135Leu变异与PTEN敲除均会消除浦肯野细胞的自发放简单锋电位活动。综上，本研究结果为PTEN功能异常与遗传背景可通过改变人类小脑类器官中的小脑分化进程及神经元网络活动提供了直接实验证据。
### 整体实验设计
针对10X单细胞RNA测序，我们采用Chromium Nest GEM Single Cell 3' Kit v3.1（10x Genomics, 1000268）完成所有scRNA-seq文库制备。将来自Chap WT/WT、Chap WT/I135L、Chap KO/KO、Apex WT/WT、Apex WT/I135L及Apex KO/KO的单细胞悬液加载至Chromium Next Chip G Single Cell Kit（10x Genomics, 1000120），并通过Chromium Controller（10x Genomics）处理以生成单细胞凝胶珠乳液。随后在NovaSeqX平台上采用双端测序策略对文库进行测序，第一读长为28个碱基，第二读长为90个碱基。原始fastq文件上传至10x Genomics云平台，reads通过Cell Ranger比对至人类参考基因组GRCh38。最终生成的包含条形码、特征及表达矩阵的过滤后原始数据上传至Seurat v5进行后续分析。