PDF(Pubmed)
Abstract：
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder with minimally effective treatment options. An important hurdle in ALS drug development is the non-invasive therapeutic access to the motor cortex currently limited by the presence of the blood-brain barrier (BBB). Focused ultrasound and microbubble (FUS+ MB) treatment is an emerging technology that was successfully used in ALS patients to temporarily open the cortical BBB. However, FUS+ MB-mediated drug delivery across ALS patients\' BBB has not yet been reported. Similarly, the effects of FUS+ MB on human ALS BBB cells remain unexplored.
METHODS: Here we established the first FUS+ MB-compatible, fully-human ALS patient-cell-derived BBB model based on induced brain endothelial-like cells (iBECs) to study anti-TDP-43 antibody delivery and FUS+ MB bioeffects in vitro.
RESULTS: Generated ALS iBECs recapitulated disease-specific hallmarks of BBB pathology, including reduced BBB integrity and permeability, and TDP-43 proteinopathy. The results also identified differences between sporadic ALS and familial (C9orf72 expansion carrying) ALS iBECs reflecting patient heterogeneity associated with disease subgroups. Studies in these models revealed successful ALS iBEC monolayer opening in vitro with no adverse cellular effects of FUS+ MB as reflected by lactate dehydrogenase (LDH) release viability assay and the lack of visible monolayer damage or morphology change in FUS+ MB treated cells. This was accompanied by the molecular bioeffects of FUS+ MB in ALS iBECs including changes in expression of tight and adherens junction markers, and drug transporter and inflammatory mediators, with sporadic and C9orf72 ALS iBECs generating transient specific responses. Additionally, we demonstrated an effective increase in the delivery of anti-TDP-43 antibody with FUS+ MB in C9orf72 (2.7-fold) and sporadic (1.9-fold) ALS iBECs providing the first proof-of-concept evidence that FUS+ MB can be used to enhance the permeability of large molecule therapeutics across the BBB in a human ALS in vitro model.
CONCLUSIONS: Together, this study describes the first characterisation of cellular and molecular responses of ALS iBECs to FUS+ MB and provides a fully-human platform for FUS+ MB-mediated drug delivery screening on an ALS BBB in vitro model.
METHODS: Here we established the first FUS+ MB-compatible, fully-human ALS patient-cell-derived BBB model based on induced brain endothelial-like cells (iBECs) to study anti-TDP-43 antibody delivery and FUS+ MB bioeffects in vitro.
RESULTS: Generated ALS iBECs recapitulated disease-specific hallmarks of BBB pathology, including reduced BBB integrity and permeability, and TDP-43 proteinopathy. The results also identified differences between sporadic ALS and familial (C9orf72 expansion carrying) ALS iBECs reflecting patient heterogeneity associated with disease subgroups. Studies in these models revealed successful ALS iBEC monolayer opening in vitro with no adverse cellular effects of FUS+ MB as reflected by lactate dehydrogenase (LDH) release viability assay and the lack of visible monolayer damage or morphology change in FUS+ MB treated cells. This was accompanied by the molecular bioeffects of FUS+ MB in ALS iBECs including changes in expression of tight and adherens junction markers, and drug transporter and inflammatory mediators, with sporadic and C9orf72 ALS iBECs generating transient specific responses. Additionally, we demonstrated an effective increase in the delivery of anti-TDP-43 antibody with FUS+ MB in C9orf72 (2.7-fold) and sporadic (1.9-fold) ALS iBECs providing the first proof-of-concept evidence that FUS+ MB can be used to enhance the permeability of large molecule therapeutics across the BBB in a human ALS in vitro model.
CONCLUSIONS: Together, this study describes the first characterisation of cellular and molecular responses of ALS iBECs to FUS+ MB and provides a fully-human platform for FUS+ MB-mediated drug delivery screening on an ALS BBB in vitro model.
摘要：
背景：肌萎缩侧索硬化症（ALS）是一种快速发展的神经退行性疾病，具有最低限度的有效治疗选择。ALS药物开发中的一个重要障碍是目前受血脑屏障(BBB)存在限制的运动皮质的非侵入性治疗性通路。聚焦超声和微泡（FUS+MB）治疗是一项新兴技术，已成功用于ALS患者暂时打开皮质BBB。然而,FUS+MB介导的跨ALS患者BBB的药物递送尚未报道。同样，FUS+MB对人ALSBBB细胞的影响仍未被探索。
方法：在这里，我们建立了第一个FUS+MB兼容，基于诱导的脑内皮样细胞（iBECs）的完全人ALS患者细胞衍生的BBB模型，以研究体外抗TDP-43抗体递送和FUSMB生物效应。
结果：生成的ALSiBECs概括了BBB病理的疾病特异性标志，包括降低BBB完整性和渗透性，和TDP-43蛋白病。结果还确定了散发性ALS和家族性（携带C9orf72扩增）ALSiBECs之间的差异，反映了与疾病亚组相关的患者异质性。这些模型中的研究揭示了在体外成功的ALSiBEC单层开放,没有FUS+MB的不利细胞作用,如通过乳酸脱氢酶(LDH)释放活力测定所反映的,并且在FUS+MB处理的细胞中缺乏可见的单层损伤或形态变化。这伴随着ALSiBECs中FUSMB的分子生物学效应，包括紧密和粘附连接标记表达的变化，以及药物转运蛋白和炎症介质，零星和C9orf72ALSiBECs产生瞬时特异性反应。此外,我们证明,在C9orf72(2.7倍)和散发性(1.9倍)ALSiBECs中,FUS+MB有效增加了抗TDP-43抗体的递送量,这首次提供了概念证据,证明FUS+MB可用于增强大分子疗法在人ALS体外模型中跨BBB的通透性.
结论：一起，这项研究首次描述了ALSiBECs对FUS+MB的细胞和分子反应,并为ALSBBB体外模型上FUS+MB介导的药物递送筛选提供了完全人类的平台.
方法：在这里，我们建立了第一个FUS+MB兼容，基于诱导的脑内皮样细胞（iBECs）的完全人ALS患者细胞衍生的BBB模型，以研究体外抗TDP-43抗体递送和FUSMB生物效应。
结果：生成的ALSiBECs概括了BBB病理的疾病特异性标志，包括降低BBB完整性和渗透性，和TDP-43蛋白病。结果还确定了散发性ALS和家族性（携带C9orf72扩增）ALSiBECs之间的差异，反映了与疾病亚组相关的患者异质性。这些模型中的研究揭示了在体外成功的ALSiBEC单层开放,没有FUS+MB的不利细胞作用,如通过乳酸脱氢酶(LDH)释放活力测定所反映的,并且在FUS+MB处理的细胞中缺乏可见的单层损伤或形态变化。这伴随着ALSiBECs中FUSMB的分子生物学效应，包括紧密和粘附连接标记表达的变化，以及药物转运蛋白和炎症介质，零星和C9orf72ALSiBECs产生瞬时特异性反应。此外,我们证明,在C9orf72(2.7倍)和散发性(1.9倍)ALSiBECs中,FUS+MB有效增加了抗TDP-43抗体的递送量,这首次提供了概念证据,证明FUS+MB可用于增强大分子疗法在人ALS体外模型中跨BBB的通透性.
结论：一起，这项研究首次描述了ALSiBECs对FUS+MB的细胞和分子反应,并为ALSBBB体外模型上FUS+MB介导的药物递送筛选提供了完全人类的平台.
