Impact of LAMA5 Variants on Immune Cell Infiltration Using 2D Blood Brain Barrier Culture Model of Multiple Sclerosis
Aksel Siva1, Umut Voyvoda2, kerem cotuk2, Hanbike Efe2, Sevim Naz Karisik2, Meziyet Reda2, Betul Cakici2, Elif Celik3, Ahmet Can Timucin2, Umut İnci Onat2, Bade Gulec1, Melih Tutuncu1, Ugur Uygunoglu1, Eda Turanli2
1Istanbul University Cerrahpasa School of Medicine, 2molecular biology and genetics, 3Translational Medicine, Acibadem University
Objective:
To investigate the impact of LAMA5 variants identified in multiple sclerosis (MS) families on immune cell infiltration across the blood–brain barrier (BBB) using a 2D in vitro co-culture model.
Background:
Disruption of BBB integrity plays a central role in MS pathogenesis by facilitating peripheral immune cell entry into the central nervous system, leading to neuroinflammation and neurodegeneration. Laminin subunit alpha-5 (LAMA5), a key extracellular matrix component of the BBB, has been implicated in regulating endothelial–immune cell interactions. Variants in LAMA5 may therefore alter adhesion molecule expression and promote lymphocyte migration across the BBB.
Design/Methods:
The LAMA5 p.Thr3126Ile missense mutation, identified in MS families, was introduced into human umbilical vein endothelial cells (HUVECs) using cytosine base editing. Wild-type and mutant HUVECs were co-cultured with peripheral blood mononuclear cells (PBMCs) in a Transwell 2D co-culture system. PBMC migration was induced with TNFα and IFNγ stimulation. Quantitative PCR was performed to assess the expression of ICAM1, VCAM1, CLDN5, ITGB1, and ITGAL—genes involved in lymphocyte adhesion and transmigration across the BBB.
Results:
PBMC migration through the mutant HUVEC monolayer was 25% higher compared to wild-type cells. Expression of ICAM1, VCAM1, and CLDN5 was significantly reduced in mutant HUVECs, whereas ITGB1 and ITGAL expression was significantly increased (p < 0.05 for all). These molecular changes indicate compromised BBB integrity and enhanced lymphocyte adhesion and migration in the presence of LAMA5 variants.
Conclusions:
The LAMA5 p.Thr3126Ile variant increases lymphocyte infiltration across the BBB, potentially contributing to MS pathogenesis through impaired endothelial barrier function and dysregulated adhesion molecule expression. Ongoing studies using prime editing technology will extend this investigation to additional LAMA5 variants identified in familial MS, providing translational insights into the genetic regulation of BBB integrity in neuroinflammatory diseases.
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