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）是一种破坏性的神经系统疾病， ABCD 1基因的特征是积累非常长链的脂肪酸，影响1：17，000的个体 在美国，大约60%的男性ALD患者会转化为毁灭性的快速进展性疾病。 炎性脱髓鞘，导致2-3年内失能或死亡（脑ALD）。年龄 即使在具有相同突变但初始事件恒定的个体中， 脑ALD是血脑屏障（BBB）破坏，白细胞迁移到脑中。的精确 在ALD疾病进展过程中控制BBB功能的分子和细胞机制 由于缺乏忠实地再现脑ALD的细胞或动物模型，因此仍然知之甚少。 为了解决这一关键的知识差距，Musolino博士的实验室最近开发了一种ALD离体模型， 系统使用基因编辑策略和人脑微血管内皮细胞。Musolino医生的姓名首字母 研究提供证据表明，ABCD 1的缺失通过增加脑血管内皮细胞的存活率， TGFβ1水平与Claudin 5的严重转录下调相关， 对小分子的渗透性。这些改变先于超长链脂肪酸的积累 提示BBB功能障碍不是它们积累的直接结果。与患者一样，在 在该模型中，除了ABCD 1缺陷外，第二个事件是炎症细胞因子引起的内皮活化， 或流动剪切应力是增加白细胞渗透性所必需的。建立在这些强大的 初步数据Musolino博士假设，脑微血管内皮细胞中ABCD 1的表达水平 通过TGFβ1调节途径控制紧密连接蛋白的转录调节，并决定 在内皮活化过程中以剂量依赖性方式增加对白细胞的渗透性。 为了验证这一假设，Musolino博士将通过以下方式探讨ABCD 1缺乏对BBB的影响：（1）鉴定 控制紧密连接破坏和ABCD 1 - 1通透性增加的分子机制 脑内皮细胞缺陷（目标1），（2）确定紧张性神经元下调的功能后果 连接蛋白和主要调控途径（目标2），和（3）定量ABCD 1基因剂量效应， 内皮屏障功能（Aim 3）。成功完成这些研究后，Musolino博士将 利用模拟单基因突变影响的能力来解开控制基因突变的机制。 ALD中细胞穿过BBB的运输提出了一种策略，以确定分子和细胞生物学特征， 转换为脑疾病的潜在机制，开发功能测定来测试新的治疗方法， 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.