Effects of ABCD1 deficiency on endothelial function and permeability to leukocytes in Cerebral X-linked Adrenoleukodystrophy

X连锁肾上腺脑白质营养不良(ALD)是一种毁灭性的神经系统疾病，由基因突变引起 ABCD1基因的特征是积累超长链脂肪酸，影响1：17,000人 在美国，大约60%的男性ALD患者将转变为毁灭性的快速进展 炎性脱髓鞘的形式，导致2-3年内丧失能力或死亡(脑性ALD)。年龄 发病和表型甚至在具有相同突变但初始事件不变的个体之间也不同 脑性阿尔茨海默病是血脑屏障(BBB)的破坏，白细胞向脑内迁移。精准的 ALD疾病进展过程中控制血脑屏障功能的分子和细胞机制 仍然知之甚少，因为缺乏细胞或动物模型，忠实地概括了大脑ALD。 为了解决这一关键的知识差距，穆索里诺博士的实验室最近开发了一种ALD体外模型 使用基因编辑策略和人脑微血管内皮细胞的系统。穆索里诺博士的首字母 研究提供证据表明，ABCD1的丢失直接损害了脑内皮细胞屏障的完整性， 转化生长因子β-1水平与Claudin 5转录严重下调和上调相关 对小分子的渗透性。这些变化先于超长链脂肪酸的积累。 这表明血脑屏障功能障碍并不是血脑屏障功能障碍的直接后果。就像病人一样，在 在这个模型中，除了ABCD1缺乏之外，第二个事件是炎性细胞因子激活内皮细胞 或流动的纯粹压力，是必要的，以增加对白细胞的渗透性。在这些强大的基础上 穆索里诺博士的初步数据假设脑微血管内皮细胞中ABCD1的表达水平 通过转化生长因子β1调节的途径控制紧密连接蛋白的转录调节，并确定 内皮激活过程中对白细胞的通透性呈剂量依赖性。 为了验证这一假说，穆索利诺博士将通过以下方式探讨ABCD1缺陷对血脑屏障的影响：(1) ABCD1-的紧密连接破坏和通透性增加的分子机制 脑内皮细胞缺陷(目标1)，(2)确定紧张型血管紧张素转换酶下调的功能后果 连接蛋白和主要调控通路(AIM 2)，以及(3)ABCD1基因的剂量效应 内皮屏障功能(目标3)。在成功完成这些研究后，穆索里诺博士将拥有 利用对单基因突变的影响进行建模的能力来揭示 ALD中通过血脑屏障的细胞流量提出了一种识别分子和细胞生物学的策略 转化为脑部疾病的潜在机制，开发功能分析以测试新的治疗方法 方法，并告知神经炎症领域。

X-linked adrenoleukodystrophy (ALD) is a devastating neurologic disorder caused by mutations in the ABCD1 gene characterized by the accumulation of very long-chain fatty acids that affects 1:17,000 individuals in the U.S. Approximately 60% of male patients with ALD will convert to a devastating rapidly progressive form of inflammatory demyelination that leads to incapacitation or death within 2-3 years (cerebral ALD). Age of onset and phenotype varies even among individuals with the same mutation but a constant initial event in cerebral ALD is blood brain barrier (BBB) disruption with migration of leukocytes to the brain. The precise molecular and cellular mechanisms controlling BBB function during the course of ALD disease progress remain poorly understood given lack of cellular or animal models that faithfully recapitulate cerebral ALD. To address this critical knowledge gap, Dr. Musolino’s laboratory recently developed an ALD ex-vivo model system using gene-editing strategies and human brain microvascular endothelial cells. Dr. Musolino’s initial studies provide evidence that loss of ABCD1 directly impairs brain endothelial barrier integrity by increasing TGFβ1 levels in a manner correlated with severe transcriptional downregulation of Claudin 5 and increased permeability to small molecules. These alterations precede the accumulation of very-long chain fatty acids suggesting that the BBB dysfunction is not a direct consequence of their accumulation. As with patients, in addition to ABCD1 deficiency in this model, a second event, endothelial activation by inflammatory cytokines or flow sheer stress, is necessary to increase the permeability to leukocytes. Building upon these strong preliminary data Dr. Musolino’s hypothesizes that levels of ABCD1 expression in brain microvascular endothelium controls transcriptional regulation of tight junction proteins via TGFβ1-regulated pathways and determines the permeability to leukocytes during endothelial activation in a dose-dependent manner. To test this hypothesis Dr. Musolino will probe the effect of ABCD1 deficiency upon the BBB by (1) Identifying the molecular mechanisms governing tight junction disruption and increased permeability of ABCD1- deficient brain endothelium (Aim 1), (2) Determining functional consequences of downregulation of tight junction proteins and main regulatory pathways (Aim 2), and (3) Quantifying ABCD1 gene-dose effect on endothelial barrier function (Aim 3). Upon successful completion of these studies Dr. Musolino will have leverage the ability to model the impact of a single-gene mutation to unravel the mechanisms governing the traffic of cells across the BBB in ALD setting forth a strategy to identify the molecular and cell biological mechanisms underlying the conversion to cerebral disease, develop functional assays to test novel therapeutic approaches, and inform the field of neuroinflammation.