Neuronal Hyperactivity in Neurons Derived from Individuals with Grey Matter Heterotopia

Periventricular heterotopia (PH), the most common form of grey matter heterotopia, represents a cortical malformation that is often associated with developmental delay and drug-resistant seizures1,2. The detailed neurophysiological underpinnings of PH symptoms in humans remain, however, elusive. Human cerebral organoids (hCOs) derived from patients with causative mutations in FAT4 or DCHS1 exhibit key features of PH3, but neuronal activity in these 3D models has not yet been investigated. Here, using silicon probe recordings, we detected exaggerated spontaneous spike activity in FAT4 and DCHS1 hCOs, suggesting functional changes in neuronal networks. Patch-clamp recordings revealed a decreased spike threshold exclusively for DCHS1 neurons, which presumably results from an enhanced density of somatic voltage-gated sodium channels. Furthermore, single-cell morphological reconstructions and immunostainings demonstrated greater morphological complexity of neurons and synaptic alterations, rather than an imbalance of excitatory-inhibitory neuron number, as contributing to the hyperactivity observed in FAT4 and DCHS1 hCOs. The morphological phenotype was rescued by an expression of wild-type DCHS1 in DCHS1 neurons. In addition, transcriptome and proteome analyses uncovered changes in GO terms associated with neuronal morphology and synaptic function. Overall, we provide detailed new insights into cellular alterations likely contributing to the emergence of symptoms in grey matter heterotopia. Overall design: Comparative gene expression profiling analysis of RNA-seq data for neuronal nuclei sorted from FAT4, DCHS1 and controls human cerebral organoids (hCOs)

室管膜下灰质异位（Periventricular heterotopia, PH）是最常见的灰质异位类型，属于一类皮质发育畸形，常与发育迟缓及耐药性癫痫发作相关[1,2]。然而，人类PH症状背后具体的神经生理学机制仍不明晰。源自携带FAT4或DCHS1致病性突变患者的人类脑类器官（human cerebral organoids, hCOs）可呈现PH的关键特征[3]，但目前尚未针对这类三维模型中的神经元活动开展研究。本研究通过硅探针记录，在FAT4及DCHS1突变型hCOs中检测到过度增强的自发放电活动，提示神经元网络存在功能异常。膜片钳记录结果显示，仅DCHS1突变神经元的放电阈值出现降低，推测该现象源于胞体电压门控钠通道密度的上调。此外，单细胞形态重建与免疫染色实验表明，神经元形态复杂度提升及突触改变，而非兴奋性-抑制性神经元数量失衡，是FAT4与DCHS1突变型hCOs中过度兴奋表型的潜在诱因。在DCHS1突变神经元中过表达野生型DCHS1可逆转该形态学表型。另外，转录组与蛋白质组分析显示，与神经元形态及突触功能相关的基因本体（Gene Ontology, GO）条目出现表达变化。综上，本研究为灰质异位症状出现相关的细胞改变提供了全新的详细见解。整体实验设计：针对从FAT4突变型、DCHS1突变型及对照组人类脑类器官中分选得到的神经元细胞核的RNA测序（RNA-seq）数据开展比较基因表达谱分析。