Oral Presentation International Congress on Neuronal Ceroid Lipofuscinoses 2025

Pieces of a puzzle: How does CLN3 disease impact the blood-brain barrier? (127750)

Adelene SL Chiam 1 , Sueanne Chear 1 , Natalie E King 1 , Jana Talbot 1 , Elizabeth Read 1 , Emma Wilkinson 1 , Richard Wilson 1 , Brad A Sutherland 1 , Anthony L Cook 1
  1. University of Tasmania, Hobart, TAS, Australia

BACKGROUND: CLN3 disease is a lysosomal storage disorder that causes childhood dementia. Previous literature suggests blood-brain barrier (BBB) compromise in CLN3 disease. However, cell-based models exploring this are limited. Here, we investigated the effects of pathological CLN3 genetic variants on the function of BBB cells (astrocytes, pericytes, endothelial cells).

METHOD: Fibroblasts from an individual with protracted CLN3 disease harbouring a compound heterozygous mutation in the CLN3 gene (1kb-deletion / E295K) were reprogrammed into induced pluripotent stem cells (iPSCs). An isogenic pair of cell lines (i.e. CLN3 and Corrected) was generated using CRISPR/Cas9 gene editing. The iPSCs were differentiated into astrocytes, pericytes, and endothelial cells. Immunofluorescent staining for marker expression as well as functional assays were performed for each cell type. For astrocytes, glutamate uptake and/or release in response to exogenous glutamate (100µM and 500µM) was examined by measuring extracellular glutamate using a luminescence-based assay. For pericytes, cells were treated with PDGF-BB (100 ng/mL) and proliferation was determined based on EdU incorporation. For endothelial cells, barrier permeability was quantified by culturing endothelial monolayers on cell culture inserts and quantifying the transport of FITC-dextran (MW 4000) across the apical-to-basolateral compartments.

RESULTS: Successful iPSC differentiation into all 3 cell types was verified by positive expression of canonical markers. Corrected astrocytes demonstrated higher glutamate uptake compared to CLN3 astrocytes. Furthermore, the Corrected endothelial barrier was less permeable to FITC-dextran compared to CLN3. Interestingly, there was no significant difference between the CLN3 and Corrected pericytes in terms of proliferative capacity.

CONCLUSION: The functional activity of CLN3 and Corrected iPSC-derived astrocytes, pericytes and endothelial cells is similar to that expected of these cell types in vivo. Significant functional differences between CLN3 and Corrected astrocytes and endothelial cells – but not pericytes – suggest that CLN3 disease differentially impacts the blood-brain barrier in a cell-specific manner.

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