Use of IPSC to define role of astrocytes in specifying risk for onset of cerebral adrenoleukodystrophy

项目摘要/摘要 遗传性X连锁男性致死性表型神经炎的发病机制 肾上腺脑白质营养不良(X-ALD)病尚不清楚。60%的男性X-ALD患者发生致命的脑部疾病 神经炎症(CALD)而其余的发展成较轻的肾上腺髓神经病(AMN)，其特征是 无神经炎症的轴索病变。X-ALD的原发遗传缺陷(ABCD1基因突变/缺失) 而生化缺陷(超长链脂肪酸在血浆和组织中的积累；C&gt；22：0)不能 预测AMN或CALD的发病。我们的长期目标是剖析 X-ALD的不同表型发育。该应用程序的目标是识别代谢途径 这是AMN和CALD人类星形胶质细胞不同的神经炎性反应的基础。这些星形胶质细胞 是从诱导的多能干细胞(IPSCs)分化而来的，而IPSCs又是通过重新编程产生的 人类对照、AMN和CALD患者来源的成纤维细胞。新陈代谢重新编程正在作为一种新的 炎症反应的调节因子。星形胶质细胞的代谢依赖线粒体呼吸(OXPHOS) 需要在神经炎性环境下改用糖酵解来促进生物合成途径 炎症介质。我们的初步概念验证数据，以及非靶向代谢组学，确定了 死后脑组织的代谢产物在健康对照组和CALD表型之间发生了变化。在卡尔德的脑白里 物质，独特的代谢物变化被记录在遥远的正常区域和邻近的区域之间 斑块表明与疾病的进展有关。我们发现了氧磷酶和糖酵解 尽管炎症反应较高，但人Cald星形胶质细胞减少(低代谢状态)。这么低 代谢状态提示新的替代燃料来源(S)在推动神经炎性反应中的作用 卡尔德星形胶质细胞。我们的中心假设是，Cald星形胶质细胞的代谢重新编程推动了他们的 前炎性转变是CALD神经炎性疾病进展的基础。来检验我们的假设 我们提出了两个特定的目标：1)阐明与炎症有关的代谢重编程 Cald星形胶质细胞的反应。2)确定功能障碍的线粒体是否在炎症性质中起作用 卡尔德星形细胞？我们将利用来自IPSC的控制、AMN和CARD星形胶质细胞在我们的 这些研究的实验室。这一提议是创新的，因为它通过确定 首次发现代谢途径对AMN和CALD炎症反应的不同调节作用 星形胶质细胞。这项拟议的研究意义重大，因为导致不那么严重的细胞机制(S) 即使在40年后，AMN或致死性CALD表型对相同ABCD1突变的反应仍不清楚 X-ALD基因缺陷的鉴定影响：随着X-ALD后新诊断病例的增加 已于2016年被列入联邦新生儿筛查名单，迫切需要确定新的目标来开发 没有令人满意的治疗方法的AMN和CALD的有效治疗。

PROJECT SUMMARY/ABSTRACT The mechanism of onset of neuroinflammation in fatal phenotypes in males with inherited X-linked adrenoleukodystrophy (X-ALD) disease remains unknown. 60% of male X-ALD patients develop fatal cerebral neuroinflammation (cALD) while remaining develop milder adrenomyeloneuropathy (AMN) characterized by axonopathy without neuroinflammation. The primary genetic defect in X-ALD (mutation/deletion in ABCD1 gene) and the biochemical defect (accumulation of very long chain fatty acid; C>22:0 in plasma and tissues) cannot predict the onset of AMN or cALD. Our long-term goal is to dissect the molecular mechanism underlying differential phenotype development in X-ALD. The objective of this application is to identify metabolic pathways that underlie the differential neuroinflammatory response in AMN and cALD human astrocytes. These astrocytes were differentiated from induced pluripotent stem cells (iPSCs), which in turn were generated by reprogramming of human control, AMN and cALD patient-derived fibroblasts. Metabolic reprogramming is emerging as a novel regulator of inflammatory response. Astrocytes rely on mitochondrial respiration (OXPHOS) for their metabolic needs but switch to glycolysis under neuroinflammatory environment to boost biosynthetic pathways to produce inflammatory mediators. Our preliminary proof-of-concept data, with untargeted metabolomics, identified metabolites altered between healthy-control and cALD phenotype postmortem brain. Within the cALD brain white matter, unique metabolite changes were recorded between distant normal looking areas and areas adjacent to the plaque suggesting an association with disease progression. We found both OXPHOS and glycolysis decreased (low metabolic state) in human cALD astrocytes despite higher inflammatory response. This low metabolic state suggests role of novel alternative source(s) of fuel driving the neuroinflammatory response in cALD astrocytes. Our central hypothesis is that metabolic reprogramming in cALD astrocytes drives their proinflammatory shift that underlies the neuroinflammatory disease progression in cALD. To test our hypothesis we propose two specific aims: 1) To elucidate the metabolic reprogramming responsible for inflammatory response in cALD astrocytes. 2) To determine if dysfunctional mitochondria play a role in inflammatory nature of cALD astrocytes? We will take advantage of control, AMN and cALD astrocytes generated from iPSC’s in our laboratory for these studies. This proposal is innovative, because it departs from the status quo by identifying for the first time, metabolic pathways differentially regulating inflammatory response in human AMN and cALD astrocytes. The proposed research is significant because the cellular mechanism(s) that lead to less severe AMN or fatal cALD phenotype in response to same ABCD1 mutation remain unknown even four decades after the identification of gene defect in X-ALD. Impact: With the rising rate of newly diagnosed cases after X-ALD was added to the federal newborn screening list in 2016, there is urgent need to identify novel targets to develop effective therapies for AMN and cALD for which no satisfactory therapy exists.