Quantifying Differences in mTOR Activity and Tumor Development Between Neural Stem Cell Microdomains

 描述（由申请人提供）：多发性硬化症（TSC）是一种常染色体显性遗传疾病，由分别编码蛋白质hamartin和tuberin的两种基因（TSC 1或TSC 2）中的任一种突变引起。这些蛋白质通常作为雷帕霉素（mTOR）通路的哺乳动物靶点的负调节剂，但在这些基因之一突变后，患者表现出通路的过度活化，导致细胞翻译、大小和增殖的显著增加。TSC症状包括在脑中形成两种类型的良性肿瘤：室管膜下结节（SENs）和室管膜下巨细胞星形细胞瘤（SEGA）。这两种肿瘤类型都存在于侧脑室周围的脑室-脑室下区（V-SVZ），但SEGA仅限于该神经源性小生境的特定腹侧亚区。SENS通常是无症状的，但SEGA如果不治疗可能是致命的。TSC领域研究和治疗的关键障碍是真正的起源细胞和SEGA发展的机制仍有待阐明。目前尚不清楚为什么SEGA存在于腹侧V-SVZ中，以及为什么有些人会发展这些肿瘤，而其他人则不会。据认为，TSC肿瘤的起源细胞是放射状胶质细胞，因为它们在成人脑中产生干细胞，并且在发育期间是神经元、神经胶质细胞和少突胶质细胞的主要祖细胞。最近对V-SVZ内干细胞的研究表明，它们在这个小生境中的位置可以预测它们将产生的后代的类型。因此，神经干细胞不是一个同质的混合等效塑料细胞，而是一个空间组织的一组有限的和不同的群体。这引入了这样的想法，即某些细胞群体比其他细胞群体更容易发生突变，并且源自这些细胞类型的肿瘤可能反映了它们起源的位置的特性。这一发现将为确定治疗SEGA的新治疗策略提供新的途径，这些治疗策略也可能具有治疗存在过度活跃的mTOR通路活性的其他肿瘤类型的潜力。因此，该项目的重点是确定细胞的位置特性如何有助于mTOR通路活性和脑肿瘤形成。我们将使用mTOR通路活性的单细胞测量、培养的干细胞和急性解剖的V-SVZ祖细胞来剖析驱动mTOR通路活化的亚区域特异性差异的机制。然后，我们将研究空间上不同的干细胞如何有助于在大脑内形成位置特异性肿瘤，使用神经发育障碍的神经性硬化症复合体（TSC）的新小鼠模型。最后，我们将使用这种小鼠模型和我们的单细胞分析方法来鉴定V-SVZ内mTOR通路突变的非细胞自主效应。

 DESCRIPTION (provided by applicant): Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder that results from a mutation in either of two genes, TSC1 or TSC2, that encode the proteins hamartin and tuberin respectively. These proteins normally serve as negative regulators of the mammalian target of rapamycin (mTOR) pathway but upon mutation of one of these genes, patients exhibit hyper-activation of the pathway resulting in significant increases in cell translation, size and proliferation. TSC symptoms include the formation of two types of benign tumors in the brain: subependymal nodules (SENs) and subependymal giant cell astrocytomas (SEGAs). Both of these tumor types present in the ventricular-subventricular zone (V-SVZ) that surrounds the lateral ventricles, but SEGAs are confined to a specific ventral subregion of this neurogenic niche. SENs are typically asymptomatic, but SEGAs can be lethal if left untreated. A critical barrier to research and treatment in the TSC field is that the true cll of origin and the mechanism of SEGA development remain to be elucidated. It is currently unknown why SEGAs present in the ventral V-SVZ and why some individuals develop these tumors while others do not. It is thought that the cells of origin for TSC tumors are the radial gla since they give rise to the stem cells in the adult brain and are the primary progenitors of neurons, glia and oligodendrocytes during development. Recent work on the stem cells within the V-SVZ showed that their location within this niche can predict the type of progeny they will create. Thus, neural stem cells are not a homogenous mix of equivalently plastic cells, but rather are a spatially organized set of restricted and diverse populations. This introduces the idea that certain cell populations are more susceptible to mutations than others and that tumors derived from these cell types may reflect the properties of the location from which they originated. This finding would provide a new avenue for identifying novel therapeutic strategies to treat SEGAs that could also have the potential to treat other tumor types that present with hyperactive mTOR pathway activity. Thus, the focus of this project is to determine how a cell's positional identity contributes to mTOR pathway activity and brain tumor formation. We will use single-cell measurements of mTOR pathway activity, cultured stem cells, and acutely dissected V-SVZ progenitors to dissect the mechanisms driving subregion-specific differences in mTOR pathway activation. We will then investigate how spatially distinct stem cells contribute to the formation of location-specific tumors within the brain using a novel mouse model of the neurodevelopmental disorder Tuberous Sclerosis Complex (TSC). Finally, we will use this mouse model and our single-cell analytical methods to identify non-cell-autonomous effects of mTOR pathway mutations within the V-SVZ.