Molecular Characterization of Neural Stem Cell Apoptosis

描述(申请人提供)：神经干细胞对大脑形态发生和神经系统对神经病理侮辱的适应起着关键的调节作用。它们可能是儿童和成人脑肿瘤的起源细胞，并代表着潜在的丰富的可移植细胞来源，用于治疗广泛的人类神经疾病。神经干细胞可以从胚胎和成人脑中分离和扩增，也可以从胚胎干细胞中获得。最近的研究表明，DNA修复酶和端粒酶在大脑早期发育中是至关重要的，神经干细胞中染色体异常和凋亡的频率出人意料地高。鉴于神经干细胞的巨大生物学意义和细胞凋亡死亡途径的细胞特异性，我们对DNA损伤诱导神经干细胞凋亡的分子调控进行了一系列的研究。我们的初步研究，使用靶向基因中断的小鼠，以及体内和体外的神经干细胞死亡模型，揭示了一种涉及P53、促凋亡的多域Bcl-2家族成员、APAF-1和caspase-9的凋亡途径。神经干细胞死亡需要新的基因转录和蛋白质翻译，但令人惊讶的是，不需要激活caspase-3。这种凋亡途径在成纤维细胞生长因子-2扩增的神经干细胞中重现，并可能涉及BH3结构域仅限BH3结构域的Bcl-2家族成员Noxa。我们推测，神经干细胞不可修复的DNA损伤触发了依赖于P53的NoxA的上调，从而促进了Bax/Bak介导的细胞色素c的释放，caspase-9的激活和死亡。为了验证这一假设，我们将使用来自野生型和基因干扰小鼠的FGF-2扩增端脑神经干细胞，基因转染和反义寡核苷酸修饰的mRNA表达水平。我们将确定Noxa的表达是否是p53依赖的神经干细胞死亡的必要条件和充分条件，以及caspase家族对DNA损伤诱导的神经干细胞凋亡的要求。这些研究将确定在这一重要细胞群体中调节死亡的关键分子，并为神经干细胞生物学提供新的见解。

DESCRIPTION (provided by applicant): Neural stem cells critically regulate brain morphogenesis and nervous system adaptation to neuropathological insults. They are the likely cells of origin for both childhood and adult brain tumors and represent a potentially rich source of transplantable cells for treatment of a wide range of human neurological diseases. Neural stem cells can be isolated and expanded from both embryonic and adult brain or be derived from embryonic stem (ES) cells. Recent studies have demonstrated a critical requirement for DNA repair enzymes and telomerase in early brain development and an unexpectedly high frequency of chromosomal abnormalities and apoptosis in neural stem cells. Because of the tremendous biological significance of neural stem cells and the cell specificity of apoptotic death pathways, we have performed a series of studies on the molecular regulation of DNA damage-induced neural stem cell apoptosis. Our initial investigations, using mice with targeted gene disruptions and in vivo and in vitro models of neural stem cell death, have revealed an apoptotic pathway involving p53, pro-apoptotic multi-domain Bcl-2 family members, Apaf-1, and caspase-9. Neural stem cell death requires new gene transcription and protein translation, but surprisingly, not caspase-3 activation. This apoptotic pathway is recapitulated in fibroblastic growth factor (FGF)-2 expanded neural stem cells and may involve Noxa, a BH3 domain-only Bcl-2 family member. We hypothesize that irreparable DNA damage to neural stem cells triggers p53-dependent upregulation of Noxa which promotes Bax/Bak mediated cytochrome c release, caspase-9 activation and death. To test this hypothesis, we will use FGF-2 expanded telencephalic neural stem cells derived from wild-type and gene disrupted mice, gene transfections, and antisense oligonucleotide modification of mRNA expression levels. We will determine if Noxa expression is necessary and sufficient for p53-dependent neural stem cell death and the caspase family requirements for DNA damage-induced neural stem cell apoptosis. These studies will define the key molecules regulating death in this important cell population and provide new insights into neural stem cell biology.