Establishment of a novel amyotrophic lateral sclerosis patient-derived blood-brain barrier model: Investigating barrier dysfunction and immune cell interaction

Amyotrophic lateral sclerosis (ALS) is a major neurodegenerative disease for which there is currently no effective treatment. The main feature of the disease is the loss of motor neurons in the central nervous system (CNS), but other CNS components and the immune system also appear to be involved in the pathogenesis of the disease. The blood-brain barrier (BBB) is formed by specialized brain microvascular endothelial cells (BMECs) and plays a crucial role in maintaining homeostasis within the central nervous system (CNS). Autopsy studies have reported BBB abnormalities in ALS patients, and studies in animal models suggest that BBB breakdown may precede neurodegeneration. However, the difficulty of obtaining patient samples has hampered further studies. We used the method of differentiating BMEC-like cells from human induced pluripotent stem cells (hiPSCs), which have robust barrier functions and express adhesion molecules necessary for immune cell adhesion, to investigate BBB functions in ALS patients. We also established a method using automated analysis techniques to analyze immune cell adhesion to the BBB in multiple samples simultaneously and with minimal bias. BMEC-like cells derived from ALS patients carrying TARDBP N345K/WT mutations exhibited increased permeability to small molecules due to loss of tight junction, as well as increased expression of cell surface adhesion molecules, leading to enhanced immune cell adhesion. BMEC-like cells derived from hiPSCs with other types of TARDBP gene mutations (TARDBP K263E/K263E and TARDBP G295S/G295S) introduced by genome editing technology did not show such BBB dysfunction compared to the isogenic control. These results indicate that our model can recapitulate BBB abnormalities reported in ALS patients using patient (TARDBP N345K/WT) -derived hiPSCs and that two types of missense mutation, TARDBP K263E/K263E and TARDBP G295S/G295S, do not contribute to the BBB dysfunction. This novel ALS patient-derived BBB model is useful for the challenging analysis of the involvement of BBB dysfunction in the pathogenesis and to promote therapeutic drug discovery.