幼年形式的神经元类脂菌病(JNCL)是该组罕见的溶酶体贮积症中最常见的形式,导致小儿神经变性.遗传性疾病,这是由影响CLN3基因的隐性突变引起的,以渐进性视力丧失为特征,认知和运动衰退以及其他精神疾病,癫痫发作,导致过早死亡。传统上,动物模型有助于理解疾病机制和病理学,并且与生物标志物研究和治疗测试非常相关。然而,有必要建立可靠和预测的人类细胞模型来研究这种疾病。因为病人的材料,特别是儿童,稀缺且难以获得,我们产生了一个工程化的CLN3突变体等基因人类诱导多能干细胞(hiPSC)系,携带c.1054C→T病理变异,使用最先进的CRISPR/Cas9技术。为了证明等基因对模型JNCL的适用性,我们在非神经元二维细胞培养模型以及大脑类器官中筛选了疾病特异性表型.我们的数据表明,仅引入致病性变体会在体外产生JNCL的经典标志。此外,我们发现了由这种特殊突变引起的剪接改变。接下来,我们得出了脑类器官,并将其用作神经发育模型,以研究CLN3Q352X突变在疾病背景下脑形成过程中的特殊作用.大约一半的携带突变的脑类器官完全无法正常发育。另一半,摆脱了这种严重缺陷的方法被用于分析更微妙的改变。在这些逃脱者中,全转录组分析显示早期疾病特征,影响与发展相关的途径,皮质发生和突触。补充代谢组学分析证实脑组织代谢物水平降低,一些与突触形成和神经传递特别相关的,例如γ-氨基丁酸(GABA)。我们的数据表明,CLN3的突变严重影响大脑发育。此外,在疾病发作之前,疾病相关的神经发育变化,特别是关于突触的形成和功能,发生。
The juvenile form of neuronal ceroid Lipofuscinosis (JNCL) is the most common form within this group of rare lysosomal storage disorders, causing pediatric neurodegeneration. The genetic disorder, which is caused by recessive mutations affecting the CLN3 gene, features progressive vision loss, cognitive and motor decline and other psychiatric conditions, seizure episodes, leading to premature death. Animal models have traditionally aid the understanding of the disease mechanisms and pathology and are very relevant for biomarker research and therapeutic testing. Nevertheless, there is a need for establishing reliable and predictive human cellular models to study the disease. Since patient material, particularly from children, is scarce and difficult to obtain, we generated an engineered a CLN3-mutant isogenic human induced pluripotent stem cell (hiPSC) line carrying the c.1054C → T pathologic variant, using state of the art CRISPR/Cas9 technology. To prove the suitability of the isogenic pair to model JNCL, we screened for disease-specific phenotypes in non-neuronal two-dimensional cell culture models as well as in cerebral brain organoids. Our data demonstrates that the sole introduction of the pathogenic variant gives rise to classical hallmarks of JNCL in vitro. Additionally, we discovered an alteration of the splicing caused by this particular mutation. Next, we derived cerebral organoids and used them as a neurodevelopmental model to study the particular effects of the CLN3Q352X mutation during brain formation in the disease context. About half of the mutation -carrying cerebral organoids completely failed to develop normally. The other half, which escaped this severe defect were used for the analysis of more subtle alterations. In these escapers, whole-transcriptome analysis demonstrated early disease signatures, affecting pathways related to development, corticogenesis and synapses. Complementary metabolomics analysis confirmed decreased levels of cerebral tissue metabolites, some particularly relevant for synapse formation and neurotransmission, such as gamma-amino butyric acid (GABA). Our data suggests that a mutation in CLN3 severely affects brain development. Furthermore, before disease onset, disease -associated neurodevelopmental changes, particular concerning synapse formation and function, occur.