Understanding human lysosomal-autophagic dysfunction in CLN6 with IPSC-derived neural cells

摘要 神经元蜡样脂褐质沉积症（NCL）是 最常见的遗传性小儿神经退行性疾病 疾病它们是一组异质性疾病，具有进行性临床病程，包括癫痫发作， 失明痴呆脑萎缩和死亡CLN 6是一种NCL疾病，由于CLN 6基因突变， 编码内质网（ER）跨膜蛋白（CLN 6p）。CLN 6p功能差 然而，它的功能障碍导致细胞内储存物质的积累， 该组分是线粒体ATP合酶蛋白的亚基C（SUBC）。存储材料被认为 可能是由于自噬-溶酶体系统的破坏，但病理机制仍不清楚。 目前，CLN 6无法治愈，治疗干预受到对其机制认识不足的限制。 CLN 6的动物模型再现了具有进行性神经功能衰退和沉积的人类疾病 SUBC+储存物质。小鼠和绵羊原代神经培养物的研究 提供了一些关于这种疾病的见解，常见的变化包括SUBC+积累，减少 溶酶体酸化和自噬功能障碍。不幸的是，初级神经元可以从一个 准备另一个，以及难以操纵基因。此外，虽然主要动物 神经元可能与人类神经元相似，但它们仍然可能无法充分概括人类遗传疾病。 目前，没有可再生来源的人细胞模型可用于研究人CLN 6病理学。 非神经元患者来源的细胞（例如成纤维细胞）不积累显著的储存材料。因此，我们认为， 我们从受CLN 6影响的受试者中产生了患者来源的诱导多能干细胞（IPSC）， 在常规分化方案后，在混合神经培养物中存在显著的SUBC储存。可惜 存在于这些培养物中的混合型群体（例如祖细胞、神经元）以及 但是，利用存储产生细胞限制了这些方法的有效应用能力。因此，我们执行了 使用诱导型神经生成素-2表达（I3 N）系统的初步工作。该I3 N系统被设计成 CLN 6-和CTL-IPSC系各自产生同质和同步的皮质神经元培养物， 多西环素诱导的天数。I3 N-CLN 6-神经元具有增加的SUBC+储存物质，证实了I3 N-CLN 6-神经元的SUBC+储存物质增加。 系统对CLN 6建模的价值。我们目前的项目旨在增加CLN 6-和CTL-I3 N-的数量， IPSC系，整合额外的可诱导转基因用于调节CLN 6p的表达，并研究如何 CLN 6 β功能障碍影响细胞器的形态和定位沿着ER溶酶体自噬 系统.还将评价ER应激和溶酶体应激标志物的活化，以确定潜在的 未来激活或耗尽的目标。这些初步研究将评估CLN 6如何影响细胞器 功能包括ER应激、溶酶体应激和自噬功能。这项工作旨在建立适当的 和可再生模型的机制研究和潜在的治疗目标的识别。

ABSTRACT The neuronal ceroid lipofuscinoses (NCLs) are the most common type of inherited pediatric neurodegenerative diseases. They are a group of heterogenous disorders with a progressive clinical course that includes seizures, blindness, dementia, brain atrophy and death. CLN6 is an NCL disorder due to mutations in the CLN6 gene that encodes a resident endoplasmic reticulum (ER) transmembrane protein (CLN6p). CLN6p’s function is poorly understood; however its dysfunction leads to the intracellular accumulation of storage material whose prominent component is the Subunit C (SUBC) of the mitochondrial ATP synthase protein. Storage material is thought to be due to disruption of the autophagic-lysosomal system, but the pathologic mechanism has remained unclear. Currently, CLN6 has no cure and therapeutic intervention is limited by inadequate knowledge of its mechanism. Animal models of CLN6 recapitulate the human disease with progressive neurological decline and deposition of SUBC+ storage material within the brain. Investigations with primary neural cultures from mice and sheep have provided some insight into the disorder, with common changes including SUBC+ accumulation, decreased acidification of lysosomes, and autophagic dysfunction. Unfortunately, primary neurons can vary from one preparation to another, as well as being difficult to manipulate genetically. Furthermore, although primary animal neurons can be similar to human neurons, they still may not adequately recapitulate human genetic disease. Currently, there are no renewable sources of human cell models available to investigate human CLN6 pathology. Non-neuronal patient-derived cells (e.g. fibroblasts) do not accumulate significant storage material. Therefore, we generated patient-derived induced pluripotent stem cells (IPSCs) from CLN6-affected subjects, that exhibited significant SUBC storage in mixed neural cultures after conventional differentiation protocols. Unfortunately, the mixed-type populations present in these cultures (e.g. progenitors, neurons) and the length of time required to generate cells with storage has limited these methods’ ability to be applied effectively. Therefore, we performed preliminary work using an inducible neurogenin-2 expression (I3N) system. This I3N system was engineered into CLN6- and CTL-IPSC lines each and produced homogeneous and synchronous cortical neuron cultures within days of doxycycline induction. I3N-CLN6-neurons had increased SUBC+ storage material, confirming the system’s value for modeling CLN6. Our current project seeks to increase the number of CLN6- and CTL-I3N- IPSC lines, integrate additional inducible-trangenes for regulated expression of the CLN6p, and investigate how CLN6p dysfunction affects the morphology and localization of organelles along the ER to lysosomal-autophagic systems. Activation of ER stress and lysosomal stress markers will also be evaluated to determine potential targets for future activation or depletion. These preliminary studies will evaluate how CLN6 affects organellar function including ER stress, lysosomal stress, and autophagic function. This work seeks to establish adequate and renewable models for mechanistic studies and the potential identification of therapeutic targets.