Rescue of Impaired Blood-Brain Barrier in Tuberous Sclerosis Complex Patient Derived Neurovascular Unit

Abstract

Tuberous sclerosis complex (TSC) is a multi-system genetic disease that causes benign tumors in the brain and other vital organs. The most debilitating symptoms result from involvement of the central nervous system and lead to a multitude of severe symptoms including seizures, intellectual disability, autism, and behavioral problems. TSC is caused by heterozygous mutations of either the TSC1 or TSC2 gene. Dysregulation of mTOR kinase with its multifaceted downstream signaling alterations is central to disease pathogenesis. Although the neurological sequelae of the disease are well established, little is known about how these mutations might affect cellular components and the function of the blood-brain barrier (BBB). We generated disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies. Using these microphysiological systems, we demonstrate that the BBB generated from TSC2 heterozygous mutant cells shows increased permeability which can be rescued by wild type astrocytes and with treatment with rapamycin, an mTOR kinase inhibitor. Our results further demonstrate the utility of microphysiological systems to study human neurological disorders and advance our knowledge of the cell lineages contributing to TSC pathogenesis.

Keywords: BBB; astrocytes; human stem cells; mTOR; microfluidics; rapamycin; tissue chips.

Figure 1.. Schematic overview of NVU.

The vascular chamber (1, Pink) into which BMECs are loaded and the media for the vascular chamber is perfused. Porous PET membrane (2, Grey) with 3 μm pores supports a layer of BMECs on one side and a layer of astrocytes on the opposite “brain” neural cell chamber side. Neural chamber (4, Yellow) contains neurons and additional astrocytes within a hydrogel. Perfusion channels through the neural cell chamber indicated as Purple.

Figure 2.. Comparison of barrier function of control and TSC2-mutant BMEC monocultures in Transwell plates.

A) TSC patient-derived (TSP8-15) BMEC transwell cultures showed a statistically non-significant trend towards larger permeability for the 3000 DA, but not 300 Da dextrans when compared to control (CC3) BMEC cultures. Pericytes (Peri) formed a minimal barrier (N = 2-4). B) Transendothelial electrical resistance (TEER) of CC3 and TSP8-15 BMECs cultured on membranes of varying pore sizes were measured. A two-way ANOVA analysis of the data indicated a genotype-dependent F (1,16) = 18.87 (P < 0.0005), but membrane pore-size independent F(2,16) = 1.91 (P = 0.1791) effect on TEER (overall P < 0.0001, N= 2-4).

Figure 3.. Disruption of BBB in an NVU co-culture system.

(A) TSC patient-derived (TSP8-15) BMEC Transwell cultures 8 days in culture show a small, but not statistically significant higher permeability with 3 kD FITC dextran as compared to CC3 (p< 0.06, N=5). (B) In the NVU system, after 8 days co-culture with astrocytes and neurons of matching genotype, BBB permeability for the TSP8-15 was significantly increased compared to CC3 (p < 0.01, N=5).

Figure 4.. Expression of junctional proteins in BMEC cultures.

(A) BMEC monolayers derived from CC3 and TSP8-15 iPSC lines express BMEC markers occludin, claudin-5, and ZO-1 and VE-cadherin. Scale bar for all images = 100 μm. (B) Phase images of seeded CC3 and TSP8-15 NVU vascular BMEC-containing compartments (top panels) and the neuron and astrocyte-containing neural cell chambers (bottom panels) are shown. Scale bar in all images = 400 μm.

Figure 5.. Increased BBB permeability in NVU generated from TSC2 heterozygous mutant cells compared to isogenic control cells.

(A) TSP77+/+ and TSP77+/− derived BMECs express the BMEC marker proteins occluding, claudin-5, ZO-1 and Glut-1. Scale bar for all images 100 μm. (B) The BBB permeability in TSP77+/− NVUs is significantly increased compared to isogenic control TSP77 +/+ NVUs (p < 0.01, N = 5).

Figure 6.. Control astrocytes rescue TSC BBB function.

A) The BBB permeability measured on days 6-8 in vitro (DIV) in TSC2 mutant (TSP8-15) NVUs is significantly higher than the one in control (CC3) NVUs (p < 0.01, N=5). B) The BBB permeability in TSC2 (TSP8-15) mutant NVUs seeded with control (CC3) astrocytes is not significantly different from the BBB permeability measured in control (CC3) NVUs (N=5, p>0.05). C) The BBB permeability in NVUs seeded with BMECs, neurons and astrocytes derived from hiPSC derived from a different TSC patient (TSP23-9) carrying a distinct TSC2 loss of function mutation is also significantly higher compared to control NVUs (CC3 = 2.95 x 10⁻⁷ cm/s). (p < 0.001, N=5). D) The presence of control (CC3) astrocytes in otherwise TSC2 mutant (TSP23-9) NVUs rescued BBB permeabilities to levels statistically indistinguishable from that measured in control (CC3) NVUs. (p= N.S., N=5).

Figure 7.. Rapamycin rescues TSC BBB permeability.

A) On day 5 in vitro, BBB permeability of TSP8-15 NVUs is significantly higher than that seen in CC3 NVUs (p< 0.01; N=5). Perfusion of the vascular compartment with rapamycin results in TSC BBB permeabilities statistically significantly reduced compared to vehicle-treated TSC NVUs and statistically indistinguishable from controls (CC3). B) As observed for the TSP8-15 NVUs, the BBB permeability of TSP23-9 NVUs is significantly higher than in CC3 NVUs on 5 day in culture (p < 0.001, N=5). BBB permeabilities were significantly smaller in rapamycin perfused TSP23-9 NVUs than their vehicle treated counterparts and at levels statistically indistinguishable from CC3 levels. The error bar for the TSP23-9 sample is too small to be resolved on this graph (p N.S., N=5).

Figure 8.. Effect of genotype and rapamycin on neural cell and BBB NVU exometabolomes.

A) Principal Component Analysis indicates a pronounced metabolic difference between tissue types (vascular versus brain). In addition, rapamycin treatment for brain and vascular compartments causes a shift in the metabolome. The genotype (wild type, CC3 versus TSC2 heterozygous mutation, TSP8-15) appeared to affect the metabolome to a much lesser degree. B) Hierarchical clustering maps provide global comparisons for differences of metabolite levels > 2-fold with a p value of less than 0.05. Different individual metabolite levels (rows) are clustered by genotype, tissue type and rapamycin treatment (columns). Metabolites are colored according to relative feature abundance across all samples ranging from low (green) to high (red).

Figure 9.. Metabolic pathways altered by rapamycin, genotype, and tissue type in control (CC3) and TSC (TSP8-15) NVUs.

The top 25 significant response pathways are shown. In each pathway listed, at least one variable (rapamycin treatment, genotype, tissue type) showed a significant difference. Significance was defined as p-values less than or equal to 0.05 in pathways that had four or more metabolites with altered expression levels at a minimal two-fold change.