Establishment of a novel amyotrophic lateral sclerosis patient (TARDBP N345K/+)-derived brain microvascular endothelial cell model reveals defective Wnt/β-catenin signaling: investigating diffusion barrier dysfunction and immune cell interaction

Abstract

Amyotrophic lateral sclerosis (ALS) is a major neurodegenerative disease for which there is currently no curative treatment. The blood-brain barrier (BBB), multiple physiological functions formed by mainly specialized brain microvascular endothelial cells (BMECs), serves as a gatekeeper to protect the central nervous system (CNS) from harmful molecules in the blood and aberrant immune cell infiltration. The accumulation of evidence indicating that alterations in the peripheral milieu can contribute to neurodegeneration within the CNS suggests that the BBB may be a previously overlooked factor in the pathogenesis of ALS. Animal models suggest BBB breakdown may precede neurodegeneration and link BBB alteration to the disease progression or even onset. However, the lack of a useful patient-derived model hampers understanding the pathomechanisms of BBB dysfunction and the development of BBB-targeted therapies. In this study, we differentiated BMEC-like cells from human induced pluripotent stem cells (hiPSCs) derived from ALS patients to investigate BMEC functions in ALS patients. TARDBP ^N345K/+ carrying patient-derived BMEC-like cells exhibited increased permeability to small molecules due to loss of tight junction in the absence of neurodegeneration or neuroinflammation, highlighting that BMEC abnormalities in ALS are not merely secondary consequences of disease progression. Furthermore, they exhibited increased expression of cell surface adhesion molecules like ICAM-1 and VCAM-1, 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 BMEC dysfunction compared to the isogenic control. Interestingly, transactive response DNA-binding protein 43 (TDP-43) was mislocalized to cytoplasm in TARDBP ^N345K/+ carrying model. Wnt/β-catenin signaling was downregulated in the ALS patient (TARDBP ^N345K/+)-derived BMEC-like cells and its activation rescued the leaky barrier phenotype and settled down VCAM-1 expressions. These results indicate that TARDBP ^N345K/+ carrying model recapitulated BMEC abnormalities reported in brain samples of ALS patients. This novel patient-derived BMEC-like cell is useful for the further analysis of the involvement of vascular barrier dysfunctions in the pathogenesis of ALS and for promoting therapeutic drug discovery targeting BMEC.

Keywords: TDP-43; Wnt/β-catenin signaling; amyotrophic lateral sclerosis; blood-brain barrier; human induced pluripotent stem cells.

FIGURE 1

Establishment and characterization of EECM-BMEC-like cells and SMLCs. (A) An overview of the donors of hiPSCs and the origin of TARDBP mutated gene editing hiPSC clones were shown. hiPSCs were generated from two distinct healthy donors (HC1 and HC2) and one ALS patient carrying heterozygous TARDBP mutation, TARDBP ^N345K/+. HC2 is the isogenic control of TARDBP ^K263E/K263E and TARDBP ^G295S/G295S. hiPSC: human induced pluripotent stem cells, HC: healthy control, ALS: amyotrophic lateral sclerosis. Figure created with BioRender.com. (B) The Locus of each TARDBP mutation was shown. NTD: N-terminal domain, RRM: RNA recognition motif, IDR: intrinsically disordered regions; CR: conserved region. Figure created with BioRender.com. (C) The overview of EECM protocol. EPC: endothelial progenitor cell, MACS: magnetic activated cell sorting, EC: endothelial cell, mTeSR1/LaSR/CHIR/hECSR: specific name of the medium and compound. Recipes were shown in Supplementary Table S2. EECM: extended endothelial cell culture method, BMEC: brain microvascular endothelial cell, SMLC: smooth muscle-like cell. (D) Representative immunostaining images of EECM-BMEC-like cells and SMLCs. Cells from HCs were grown in an 8-well chamber plate and EECM-BMEC-like cells were stained for junctional protein VE-cadherin, claudin-5, or occludin (red). SMLCs were stained for smooth muscle cell markers αSMA, calponin, or SM22α (red). Nuclei were stained using DAPI (blue). Scale bar = 50 μm.

FIGURE 2

ALS patient-derived EECM-BMEC-like cells exhibit tight junction discontinuation. EECM-BMEC-like cells were cultivated onto a 0.4 um pore size Transwell filter for 6 days. Junctions were stained for VE-cadherin, claudin-5, or occludin (red), and nuclei were stained with DAPI (blue). Yellow arrowheads indicate visible discontinuations of the claudin-5 and occludin. Each staining represents results from at least five independent experiments, each performed with five separate differentiation batches. Scale bar = 50 μm.

FIGURE 3

EECM-BMEC-like cells derived from ALS patients exhibited impaired diffusion barrier properties. EECM-BMEC-like cells derived from HCs (black), ALS clone (red), or gene-editing clones (orange) were grown on 0.4 μm pore size Transwell filters for 6 days to form a confluent monolayer in monoculture. Permeability was measured on day 6. The permeability was calculated from the fluorescence intensity of 0.37 kDa sodium fluorescein (NaFl) across the EECM-BMEC-like cell monolayers. Each symbol showed the permeability of independent filters with at least three independent differentiations, each performed in at least triplicate. Data were shown as mean ± SD. Statistical analysis: unpaired t-test. P-values are indicated in the corresponding figures.

FIGURE 4

ALS patient-derived EECM-BMEC-like cells showed enhanced endothelial adhesion molecules. EECM-BMEC-like cells were seeded onto type Ⅳ collagen-coated well plates and treated with conditioned medium from the same hiPSC clone, with or without proinflammatory cytokine stimulation (1 ng/mL TNF-α + 20 IU/mL IFN-γ). A flow cytometer was used to detect cell surface immunostaining of EECM-BMEC-like cells for the adhesion molecules ICAM-1 and VCAM-1. Gray, blue, and red lines indicate isotype control, non-stimulated (NS), and 16-h proinflammatory cytokine-stimulated conditions, respectively. (A) Representative histograms for ICAM-1 and VCAM-1 were shown for each clone. At least three replicates were analyzed for each clone. (B) The change in the geometric mean of cell surface ICAM-1 and VCAM-1 of EECM-BMEC-like cells was analyzed by flow cytometer. Each symbol represents an experiment using an independently differentiated sample. left side: non-stimulated condition (NS), right side: stimulated condition with 1 ng/mL TNF-α and 20 IU/mL IFN-γ(Stim). Statistical analysis: unpaired t-test. P-values were indicated in the corresponding figures.

FIGURE 5

ALS patient-derived EECM-BMEC-like cells enhanced PBMCs adherent EECM-BMEC-like cells were seeded onto type Ⅳ collagen-coated 96-well plates and treated with conditioned medium from the same hiPSC clone, with or without 16 h proinflammatory cytokines stimulation (0.1 ng/mL TNF-α + 2 IU/mL IFN-γ). Fluorescently labeled PBMCs from allogeneic healthy donors interacted with the EECM-BMEC-like cell monolayer for 30 min. After washing, phase contrast and fluorescence images from the center of each well were photographed automatically. The number of adherent PBMCs was automatically counted using the hybrid cell count software BZ-X analyzer. (A) The heat map of the number of adherent PBMCs in the wells. A1-12 were TARDBP ^K263E/K263E-derived clone under non-stimulated conditions. B1-6 were TARDBP ^K263E/K263E-derived clone under cytokine-stimulated conditions. B7-12 were TARDBP ^G295S/G295S-derived clone under non-stimulated conditions. C1-12 were TARDBP ^G295S/G295S-derived clone under cytokine-stimulated conditions. D1-12 were ALS patient (TARDBP ^N345K/+)-derived clone under cytokine-stimulated conditions. E1-6 were ALS patient (TARDBP ^N345K/+)-derived clone under non-stimulated conditions. E7-12 were HC1 under non-stimulated conditions. F1-12 were HC1 under cytokine-stimulated conditions. G1-12 were HC2 under cytokine-stimulated conditions. H1-12 were HC2 under cytokine-stimulated conditions. Black-colored wells were excluded because the cell layers in the center of the wells were detached. (B) Representative images of each well. Green fluorophore-labeled adherent PBMCs and monolayers of EECM-BMEC-like cells on phase contract image. D3, H6, and B10 were the locations of the wells representing a high, middle, and low number of adherent PBMCs, respectively. (C) Number of adherent PBMCs onto EECM-BMEC-like cells derived from each clone in the presence or absence of proinflammatory cytokines stimulation. A dot represented the number of adherent PBMCs in the FOV at the center of each well. Left side: non-stimulated condition (NS), right side: stimulated condition with 0.1 ng/mL TNF-α and 2 IU/mL IFN-γ (Stim). Data are shown as mean ± SD. The Statistical analysis: unpaired t-test. P-values were indicated in the corresponding figures.

FIGURE 6

ALS patient-derived EECM-BMEC-like cells showed cytoplasmic mislocalization of TDP-43. (A) Representative images of triple staining TDP-43 (red), VE-cadherin (green), and DAPI (blue). TDP-43 proteins colocalized in nuclei were stained purple, whereas TDP-43 proteins mislocalized in the cytoplasm remained red (yellow arrow). Each staining represents results from at least three independent experiments, each performed with three separate differentiation batches. Scale bar = 30 μm (B) TDP-43 staining in the cytoplasm were counted in a field of view of 40x images. Each dot represents the mean of the counts made by three independent examiners, each investigating 10 pictures. The Statistical analysis: unpaired t-test. P-values are indicated in the corresponding figures.

FIGURE 7

Activation of downregulated Wnt/beta-catenin signaling restored impaired barrier properties in ALS patient-derived EECM-BMEC-like cells. (A) AXIN2, TTYH2, CLDN5, LEF1, FZD3, FZD4, FZD6, SPOCK2, and FABP4 mRNA expression was measured via quantitative real-time polymerase chain reaction (RT-qPCR) in HC1, HC2, and ALS patient (TARDBP ^N345K/+)-derived EECM-BMEC-like cells. The mean value of the HC1-derived sample was set to 100% and used as a normalization factor for each value. Each dot represents one sample, and qPCR was performed at least three times for each sample. The black bar represented the mean of mRNA expression. (B) ALS-patient (TARDBP ^N345K/+)-derived EECM-BMEC-like cells were cultivated onto collagen type Ⅳ coated plate for 3 days in the presence of 4 μM CHIR99021. Junctions were stained for claudin-5 (red), and nuclei were stained with DAPI (blue). Each staining represents results from at least three independent experiments, each performed with three separate differentiation batches. Scale bar = 40 μm (C) Left: The impedance of monolayered EECM-BMEC-like cells was shown. Cells derived from HC1 (blue) or ALS patient (TARDBP ^N345K/+) (red) were seeded on E-plate 16. Some wells were treated with 4 μM CHIR99021 (green). The black arrow indicates the timing when CHIR99021 or DMSO control was added to each well. Each condition had at least three replicates. Higher normalized cell index values indicate increased barrier integrity. Right: Area under the curve of the cell index in each condition. (D) The images showed representative ICAM-1 and VCAM-1 expression histograms of TARDBP ^N345K/+ carrying ALS patient-derived EECM-BMEC-like cells in the presence or absence of CHIR99021 treatment. EECM-BMEC-like cells were treated with conditioned medium from the same hiPSC clone-derived SMLCs with proinflammatory cytokine stimulation (1 ng/mL TNF-α + 20 IU/mL IFN-γ) in the presence or absence of 4 μM CHIR99021 treatment. The expression of ICAM-1 or VCAM-1 was detected by flow cytometry analysis. Each staining represented results from three independent experiments. The below dot graph showed the change in the mean (ΔMFI = MFI staining–MFI isotype) of cell surface ICAM-1 and VCAM-1 of TARDBP ^N345K/+ carrying ALS patient-derived EECM BMEC-like cells. Each symbol represented an experiment using an independently differentiated sample. Bars showed the mean ΔMFI value of three experiments. Statistical analysis: unpaired t-test. P-values are indicated in the corresponding figures.